Selected antibodies and duramycin peptides binding to anionic phospholipids and aminophospholipids and their use in treating viral infections and cancer.

(54) Title: PEPTIDES THAT LINK TO PHOSFATIDYLETHANOLAMINE AND ITS USES IN THE TREATMENT OF VIRAL AND CANCER INFECTIONS.

(54) Title: SELECTED ANTIBODIES AND DURAMYCIN PEPTIDES BINDING TO ANIONIC PHOSPHOLIPIDS AND AMINOPHOSPHOLIPIDS AND THEIR USE IN TREATING VIRAL INFECTIONS AND CANCER.

(57) Summary

The present invention relates to discoveries concerning the role anionic phospholipids and aminophospholipids play in tumor vasculature and viral entry and spread, and compositions and methods for using these discoveries in the treatment of cancer and viral infections. Antibody, immunoconjugate, and duramycin-based compositions, and combinations that bind and inhibit anionic phospholipids and aminophospholipids, are also described for use in the safe and effective treatment of cancer, viral infections, and related diseases.

(57) Abstract

Disclosed are surprising discoveries concerning the role of anionic phospholipids and aminophospholipids in tumor vasculature and in viral entry and spread, and compositions and methods for utilizing these findings in the treatment of cancer and viral infections. Also disclosed are advantageous antibody, immunoconjugate and duramycin-based compositions and combinations that bind and inhibit anionic phospholipids and aminophospholipids, for use in the safe and effective treatment of cancer, viral infections and related diseases.

Institute

Mexican Property

Industrial

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PATENT TITLE NO. 337052

Cellular (s):

Home:

BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM

201 West7th Street, Austin, Texas, 78701, E.UX '/.

Name: PEPTIDES THAT LINK TO PHOSFATIDYLETHANOLAMINE AND ITS USES IN THE TREATMENT OF VIRAL AND CANCER INFECTIONS

Classification: lnt.CI.8: A61K45 / 06; A61K47 / 48; A61P31 / 12; A61P35 / 00

Inventor (s): PHILIP E. THORPE; MELINA M. SOARES; JIN HE

Date Sheet »15 co

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1st ustrial articles.

I articulate

Regulation of the I

7/2002, 07/15/2004, 28 / lyclela og ables, mtes los lulo lo hi Acial of the Mexican Institute of July 2003 and Patent Number: 291602

PRIORITY

Date:

July 200

60 / 396,263 of 2023

The «reference tente is &lt;irga with D &lt;informidad with the day 123 of the ada as of the pre-dated date of the log

Qi * n subscribes to the present

Industrial P'abiedad (Daily

1/2004. 06/16/2005, 1/25/2006, 0®5 / 2009,08 / 01/2010, icon of the Federation (D.

and will be subject to pe in &lt;® a). 4th and 12 * fracaora I and III di '----- - ...... / 2004 and i&gt; 9/2007); 1 * items. 3 * 4 * opiety I iiistrial (DOF 12/27/1998, amended delegate faWNNEMMSMMNI9l &lt;roÍMMMMN | N bis 2 of the ey of 12/26/1997, 1 05/1999, 12); articles Γ, 3^to action V (DOF 12/14/1999, re-signed I and III and 30 of the Organic Statute 002, 07/29/2004, 08/04/2004 and 13 / 0S / 2 &lt;7); 1, 3 »NHBNNNanMMMMMMNNMinNNBes of the

Regio nal Offices, Divisional Deputy Directors, Departmental Coordinators and other subordinates of the Mexican Institute of Industrial Property. (DOF 12/15/1999, amended on 02/04/2000, 07/29/2004, 08/04/2004 and 09/13/2007).

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MX / 2016/11970 lUiW'-WU.

FIELD OF THE INVENTION

The present invention relates to the fields of biology of aminophospholipids and anionic phospholipids, tumor blood vessels, and viral infections. Surprisingly it provides new compositions, methods and combinations to be targeted to the tumor vasculature and for the treatment of cancer, and to inhibit viral entry and spread and for the treatment of viral infections. The invention further provides a number of preferred compositions based on antibodies, immunoconjugates and duramycin, which bind and inhibit aminophospholipids and anionic phospholipids for use in the treatment of cancer, viral infections and related diseases.

DESCRIPTION OF THE RELATED TECHNIQUE

The resistance of tumor cells to chemotherapeutic agents represents a significant problem in clinical oncology. Another main problem that must be addressed in the treatment of tumors is the desire to achieve total annihilation of cells, that is, to kill all malignant cells.

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. grow in an uncontrolled way and any tumor mass that can be removed by therapy. Despite certain advances in the field, there are two main reasons why many prevalent forms of human cancer still resist effective chemotherapeutic intervention.

Due to the goal of developing treatments that focus on total cell annihilation, certain types of tumors have been more susceptible to therapy than others. For example, soft tissue tumors, for example lymphomas, and tumors of the blood and blood-forming organs, for example leukemias, have generally been more sensitive to chemotherapeutic therapy than solid tumors such as carcinomas.

One reason for the susceptibility of soft tumors and blood-based tumors to chemotherapy is the increased ease of access of lymphoma and leukemic cells to chemotherapeutic intervention. Put simply, it is much more difficult for most chemotherapeutic agents to reach all cells in a solid tumor mass than soft tumors and blood-based tumors, and therefore much more difficult to achieve total annihilation of the

1'-crfíTUTC · MEXICAN PROPERTY cells. Increasing the dose of chemotherapeutic agents most of the time results in toxic side effects, which generally limits the effectiveness of conventional antitumor agents.

Another strategy for treating tumors is the use of an immunotoxin, in which an anti-tumor cell antibody is used, to deliver a toxin to the tumor cells. However, in common with chemotherapeutic approaches, immunotoxin therapy also suffers from some significant disadvantages when applied to solid tumors. For example, antigen negative or antigen deficient cells may survive and repopulate the tumor or lead to further metastasis. An additional reason for resistance to solid tumors to antibody-based therapies is that the tumor mass is generally impermeable to macromolecular agents such as antibodies and immunotoxins. Both the distances for physical diffusion and the interstitial pressure within the tumor are significant limitations to this type of therapy.

An improved treatment strategy is to target the vasculature of solid tumors. Targeting the blood vessels of tumors, rather than the tumor cells themselves, has certain advantages because it is unlikely to lead to resistant tumor development, and because the cells reached are easily accessible. In addition, the destruction of blood vessels leads to an amplification of the antitumor effect, since many tumor cells depend on a single vessel to obtain their oxygen and nutrients. Exemplary vasculature directed agents (VTAs) are described in United States Patent Nos. 5,855,866, 5,965,132, 6,261,535, 6,051,230, and

6,451,312, which describe the targeted delivery of anti-cellular agents and toxins to markers of the tumor vasculature.

Another effective version of the vasculature targeting approach is to target a clotting factor in a marker expressed or adsorbed within the vasculature of the tumor or stroma (Huang et al., 1997; US Patent Nos. 6,093,399, 6,004,555. , 5,877,289, and 6,036,955). The provision of coagulants, rather than toxins, to the tumor vasculature has the additional advantages of reduced immunogenicity and even fewer toxic side risks. As described in the patent of the

A preferred clotting factor for use in those tumor-specific coaguligands, United States of America No. 5,877,289 is a truncated version of the ation of the ü- JLVJL Τ '1

INSTITUTO MEXJCANU human coagulation-inducing protein,

Tissue (TF), the main initiator of blood.

Recently the aminophospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) were identified as specific markers for tumor vasculature (Ran et al., 1998). This led to the development of new anti-PS and anti-PE immunoconjugates to deliver anti-cellular agents, toxins, and clotting factors to tumor blood vessels (United States Patent No. 6,312,694). Further, unconjugated antibodies to PS and PE were found to exert an anticancer effect without binding to a therapeutic agent, which became known as the 'naked aminophospholipid antibody approach to vascular targeting and treatment of tumors (US Patent United States of America No. 6,406,693).

Although the preceding vasculature targeting methods, with immunoconjugates and aminophospholipids, represent significant advances in the treatment of tumors, certain peripheral tumor cells can survive the widespread destruction of the tumor caused by these therapies. Anti-angiogenic strategies that inhibit the development of new vasculature from pre-existing blood vessels and / or circulating endothelial totipotent cells,

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then for use in combination with VTA, coaguligand and aminophospholipid targeting methods of United States of America Patent Numbers

5,855,866, 6,093,399, 6,312,694 and 6,406,693.

Angiogenesis plays an important role in physiological processes, such as in embryogenesis, wound healing, and menstruation, but it is also involved in certain pathological events such as the growth of tumors, arthritis, psoriasis, and diabetic retinopathy (Ferrara, 1995) . As applied to the treatment of tumors, antiangiogenic strategies are based on inhibiting the proliferation of gemin vessels, generally on the periphery of a solid tumor. These therapies are applied primarily to reduce the risk of micrometastasis or to inhibit further growth of a solid tumor after more conventional intervention (such as surgery or chemotherapy).

United States Patent Nos. 6,342,219, 6,524,583, 6,342,221, and 6,416,758 describe antibodies and immunoconjugates that bind to vascular endothelial growth factor-A (VEGF, formerly known as vascular permeability factor, VPF), a primary stimulant of the angiogenesis. These antibodies have the important advantage of inhibiting binding of primary receptors.

VEGF only

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of the VEGF. By blocking VEGF binding to VEGFR2, but not VEGFR1, these antibodies have an improved safety profile, maintaining beneficial effects mediated by VEGFR1, for example on macrophage, osteoclast, and chondroclast functions.

Although the preceding methods have advanced the technique of treating tumors, the development of therapies directed at the vasculature, additional or alternative, is still being sought. Identification of new markers for tumor vasculature is necessary to expand the number of therapeutic options. The development of new naked antibodies with anticancer properties would be a particularly important advance as this allows the same targeting portion to be used both as a single agent therapeutic product and as a vasculature targeting agent for delivery of other drugs. Therapeutic agents that have both anti-angiogenic and anti-vascular properties, that is, tumor-destroying properties, within the same molecule, would be of great value. An even more important advance would be identification

I of a class of therapeutic agents with anticancer properties and therapeutic effects in other systems. The development of agents capable of treating both cancer and viral infections, two of the

MEXICAN INSTITUTE most significant of this era, would be a remarkable and important artisan.

SUMMARY OF THE INVENTION

The present invention addresses the foregoing needs as well as other needs of the prior art, by providing compositions for safely and effectively reaching tumor vasculature, antiangiogenesis, and tumor destruction, where the methods and compositions are also surprisingly effective in inhibit viral entry and spread and for the treatment of viral infections and diseases. The invention is based, in part, on surprising discoveries concerning the expression and role of anionic phospholipids in tumor vasculature and the involvement of aminophospholipids and anionic phospholipids in viral entry and spread. The present invention further provides particularly advantageous antibodies and immunoconjugates, which bind to aminophospholipids and anionic phospholipids, and to a new class of peptide-based derivatives that bind phosphatidylethanolamine.

General review: In a first global modality, the invention provides new methods to reach the discovery of the tumor vasculature, treatment of tumors, in the unexpected that anionic phospholipids, such as phosphatidylinositol (Pl), phosphatidic acid (PA ) and phosphatidylglycerol (PG), (as well as phosphatidylserine, PS), are accessible and stable markers that can be targeted to the tumor vasculature. This modality arose from the unexpected discovery that antibodies against PA, Pl and PG, and other anionic phospholipid components, are specifically located in the vasculature of solid tumors.

Additional aspects within this modality were developed from the unexpected discovery that naked antibodies against anionic phospholipids, such as PA, Pl, and PG (as well as PS), specifically inhibit angiogenesis of tumor blood vessels and induce destruction of tumor vasculature and tumor necrosis in vivo in the absence of conjugation for effector molecules, such as toxins or coagulants. The invention then provides safe and effective methods for targeting the vasculature, anti-angiogenesis, and treatment of tumors using single-component antibody-based therapeutics that bind to anionic phospholipids.

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the vasculature of tumors occurs, at least to a significant extent, independently of cell damage and apoptotic or other mechanisms of cell death. The expression of anionic phospholipids in the tumor vasculature is therefore not a consequence of, or a trigger for, cell death and destruction, but occurs in vascular, morphologically intact endothelial cells. This means that the expression of anionic phospholipid in the vasculature of tumors is not transient, but rather stable enough to provide a target for therapeutic intervention.

Given the discovery that anionic phospholipids are stably induced in the tumor vasculature, the invention further provides a variety of new methods and compositions for imaging and destroying the tumor vasculature, using immunoconjugates of antibodies against phospholipids. anionic. These immunoconjugates comprise antibodies against anionic phospholipids that are functionally linked to therapeutic agents, such as toxins and coagulants, and are useful in the supply

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of tumors. Those in intimate contact with the specific therapeutic products to the endothelial surface of the therapeutic vasculature are 'supplied with endothelial cell membrane of the vasculature of tumors, allowing either rapid entry into the target cell or rapid association with effector cells , components of the coagulation cascade, and the like.

In a second global embodiment, the invention provides a number of preferred antibodies that bind to aminophospholipids and anionic phospholipids (and immunoconjugates and related compositions), where those antibodies have structures and properties that provide advantages over those known in the art. These antibodies known as second generation or improved antibodies, will preferably be used in anti-angiogenic, anti-cancer and antiviral treatment methods, as well as in other treatment methods, described herein.

The new classes of antibodies that bind to aminophospholipids and anionic phospholipids provided by the present invention, exceed several

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therapeutic without previous,

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provide antibodies

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pathogenic properties usually associated with antibodies

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The invention was developed, in part, using new immunization and selection techniques developed from unique observations of the inventors, in the behavior of phospholipids in endothelial cells of the tumor vasculature, distancing the antibodies generated from associated antiphospholipid antibodies with the disease. These antibodies not only have unique properties and improved safety, but are equally or more effective than existing antibodies in comparative studies. The compositions and methods of those aspects of the invention also extend to the use of immunoconjugates and combinations, using the specific category of antibodies provided.

Prior to the present invention, antibodies that bind to anionophospholipids and anionic phospholipids and that have the properties of the novel antibodies described here were not known. However, in view of the invention described herein, the methodology is now provided to the art to generate new candidate antibodies and, with the techniques to analyze those antibodies, to identify additional useful antibodies from the pool of candidates. In view of this invention, a variety of

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Notorious disadvantages and side effects associated with the prior art antibodies. Those antibodies can then be used in a variety of modalities, including in the inhibition of angiogenesis and in the treatment of cancer and viral infections.

In addition to the new immunization and selection techniques provided herein, antibodies that bind to aminophospholipids and anionic phospholipids, and that have a number of advantageous properties, can now be identified by competition assays and / or functional assays, using the antibodies monoclonal 1B9, 1B12, 3B10, 2G7, 7C5, 9D2 or 3G4. Antibodies 1B12, 3B10, 9D2 and 3G4 are currently preferred, and these antibodies do not require serum for binding to the phospholipid. 9D2 and 3G4 monoclonal antibodies are most preferred, and 3G4 monoclonal antibody (ATCC 4545) is currently the most preferred. To identify additional antibodies that compete with any of the foregoing antibodies, preferably 3G4, the preferred assays are currently ELISA based proficiency assays, some of which are described herein as well as working examples thereof.

In a third global embodiment, the present cell-impervious peptides, which aminophospholipid, phosphatidylethanolamine derived from peptides that bind to the at least one first

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bind to (PE). These

PE comprise PE-binding peptide, preferably duramycin, which has been modified to substantially prevent non-specific toxicity, preferably by modification of the PE-binding peptide, preferably duramycin, to form a PE-binding construct Substantially impermeable to the cell or substantially non-porous.

Generation of a binding construct to the

PE substantially impervious to the cell or duramycin, is preferably achieved by attaching the binding peptide to the PE or duramycin, at least to a first group impermeable to the cell. The synthesis of a number of exemplary duramycin derivatives is described herein. The cell group (s) may be small molecules, inert carriers, or they may themselves be targeting agents that impart an additional targeting function to the resulting construct, such as targeting to the tumor vasculature. In this way the PE-binding peptide may be the sole inert agent, or it may be targeting

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be one of two agents each of the

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addressing to which it imparts a construct function. Additionally, the PE-binding peptides, preferably duramycin, are functionally linked to the effectors, such that the PE-binding peptide or duramycin provides the targeting function, and the bound agent has a therapeutic effect substantial, once delivered to the target cell. Preferred examples are PE binding peptides or duramycin bound to antiviral agents, such as nucleosides.

Since PE is essentially absent from the surface of normal cells under normal conditions, the substantially cell-impermeable PE-binding peptides of the present invention function to selectively bind PE to the surface of aberrant cells. or cells associated with the disease, such as tumor vascular endothelial cells, proliferating and / or virally infected cells. By binding to those aberrant target cells, the PE binding constructs or derivatives inhibit or disrupt the functions of PE in those cells, thus resulting in an overall therapeutic benefit, for example in the treatment of or

tumors. and / or viral diseases. The PE-binding peptides, substantially impervious to cells, in inhibiting viral entry and spread are described herein. In modalities where PE binding peptides are bound to antiviral agents, such as cidofovir, an improved and safer antiviral treatment is provided.

In a fourth global embodiment, the invention further provides an important new class of compositions and methods for inhibiting viral replication, infection, and spread, for use in treating viral infections and diseases. These methods are based on the surprising introspective that antibodies and peptides that bind to aminophospholipids and anionic phospholipids, such as PS, PE, Pl, PA and PG, particularly PS and PE, would be safe and effective antiviral agents. This instrospective has not only proven to be correct, but the present invention provides data showing the unexpectedly effective uses of antibodies and peptides that bind aminophospholipids and anionic phospholipids, to combat viral spread, which means that these agents can be widely applied in 1 treatment of a variety of

Λ discoveries include not only viral infections

These new categories games and methods of use in which an antibody to an aminophospholipid or anionic phospholipid, particularly PS and PE, is functionally linked to an antiviral agent. Derivatives of PE binding peptides, substantially impervious to the cell, such as derivatives of duramycin peptide, can also be linked to antiviral agents. Each of these agents then provides novel antiviral drugs uniquely targeted at virally infected cells.

The development of new, safe, effective therapeutic agents in the treatment of aberrant angiogenesis, cancer, and viral infections is thus a watershed in the art.

Although uniquely effective, the different methods and compositions of the present invention can also be used to take advantage in combination with other therapies and agents to provide combined treatment methods, and related compositions, pharmaceuticals, and kits of the invention. In a fifth global embodiment, the invention then further provides particular, combined compositions, methods and

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been selected and found

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and surprisingly well together, as explained in greater detail here.

Second Generation Antibodies: Certain methods discovered to work well in the generation of antibodies with the desired properties are described herein in Example IV and are incorporated in the pending claims. These methods allowed the generation of the advantageous antibodies of the invention, as exemplified by monoclonal antibodies 1B9, 1B12, 3B10, 2G7, 7C5, 9D2 and 3G4, particularly 3G4 (ATCC 4545).

The present invention then provides purified antibodies, antigen binding fragments, and immunoconjugates thereof, which bind to at least one aminophospholipid or anion phospholipid, preferably PS, and effectively compete with monoclonal antibody 1B9, 1B12, 3B10, 2G7. , 7C5, 9D2 and 3G4, preferably with 9D2 or 3G4 (ATCC 4545), and most preferably with 3G4, for binding to aminophospholipid or anionic phospholipid, preferably the PS.

As used throughout the application, the terms a, one, ones and ones are used in the sense meaning at least one, first, one or more or a plurality

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components or steps to which reference is made, except in cases where an upper limit is specifically established later. Therefore an antibody as used in the present, means at least a first antibody. The limits and functional combination parameters, as well as the amounts of any individual agent, will be known to those of ordinary skill in the art in view of the present disclosure.

In certain respects the antibodies will effectively compete with monoclonal antibody 1B9, 1B12, 3B10, 2G7, 7C5, 9D2 and 3G4, preferably with 9D2 or 3G4 and most preferably with 3G4 (ATCC 4545), for binding to a aminophospholipid or anionic phospholipid, preferably PS, or will have an aminophospholipid or anionic phospholipid binding profile of monoclonal antibody 1B9, 1B12, 3B10, 2G7, 7C5, 9D2 and 3G4, preferably 9D2 or 3G4 and in the most preferred form of 3G4, as presented in Table 4; and they will not be serum-dependent, that is, they will not require i * serum to bind the aminophospholipid or anionic phospholipid; nor be derived from a patient with a disease, and will not significantly inhibit coagulation reactions in vitro,

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significant in vivo or will have anticoagulant activities of lupus.

Preferably these antibodies will also show improvement in structural properties or in the range or degree of advantageous functional properties, in controlled studies, compared to an antibody in the literature, such as IgG, which has a higher affinity or exhibits binding. enhanced to activated endothelial cells, increased inhibition of endothelial cell proliferation or angiogenesis, improved localization of tumor blood vessels, anticancer and / or antiviral effects.

Particular aspects of the invention are then based on the surprising and original generation of antibodies, achieved by the inventors, which has the foregoing properties, other described properties and inherent advantageous properties. Now that a panel of preferred antibodies has been provided, and a number of particularly preferred antibodies, the present invention further encompasses a class of antibodies of defined epitope specificity, wherein those antibodies, or antigen-binding fragments thereof, compete effectively with monoclonal antibody 1B9, 1B12, 3B10, 2G7, 7C5, 9D2 or 3G4, /

preferably with 9D2 or 3G4,

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preferably with 3G4 (ATCC 4545), for binding to the antigen, such that they bind to essentially the same epitope as monoclonal binding 1B9, 1B12, 3B10, 2G7, 7C5, 9D2 or 3G4, preferably with 9D2 or 3G4 and most preferably with 3G4 (ATCC 4545).

The invention as claimed is enabled in accordance with the present specification and technological references, operating procedures and starting materials, readily available. However, on behalf of the present Applicant, the Board of

Reagents, The University of Texas System, samples of the hybridoma cell line that produces the 3G4 monoclonal antibody were submitted for deposit at the American Type Culture Collection (ATCC), 10801 University

Blvd., Manassas, VA 20110-2209, United States of America. The samples were submitted by Avid Bioservices, Inc. 14272 Franklin Avenue, Tustin, CA 92780, United States of America, a licensee subsidiary, Peregrine Pharmaceuticals, Inc., during the week beginning July 8, 2002, and were received on July 10 and July 12, 2002, proving to be viable, and being provided with the ATCC access number, PTA 4545 on July 30, 2002.

This deposit was made under the

International Depository

Purposes of Procedures thereof (Treaty of

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of Microorganisms for Patent and regulations

Budapest). The hybridoma will be put

Treaty

States made available by the ATCC under the terms of the Budapest with the issuance of a patent of the

United States of America with relevant claims. The availability of the deposited hybridoma should not be considered a license to practice the invention, contrary to the rights granted under the authority of any government, in accordance with its patent laws.

In view of the panel of antibodies, the preferred antibodies and techniques described herein and known in the art, those of ordinary skill in the art are now provided with a new class of antibodies that bind to anionophospholipids or anionic phospholipids and that have advantageous properties. These antibodies are similar or based on monoclonal antibodies 1B9, 1B12, 3B10, 2G7, 7C5, 9D2 or 3G4. Preferably the antibodies of the invention are antibodies based on 9D2 or 9D2-like, and most preferably, the antibodies of the invention are antibodies based on 3G4 or 3G4-like. The following description of similar antibodies is provided in terms of the antibody

H / 'DUi'i'UiAL

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for simplicity, but are specifically incorporated herein by reference as applicable to each of antibodies 1B9, 1B12, 3B10, 2G7, 7C5 and 9D2.

A 3G4-like antibody is an antibody, or antigen-binding fragment thereof, that binds to substantially the same epitope as the 3G4 monoclonal antibody (ATCC 4545) or binds to at least a first aminophospholipid or anionic phospholipid, preferably the PS, essentially the same epitope as the 3G4 monoclonal antibody (ATCC 4545). Preferably the antibody, or an antigen-binding fragment thereof, will bind to the same epitope as the 3G4 monoclonal antibody (ATCC 4545).

The terms "roughly" substantially or essentially the same, or the same, epitope as the 3G4 monoclonal antibody (ATCC 4545) means that an antibody cross-reacts with the 3G4 monoclonal antibody (ATCC 4545). Cross-reacting antibodies are those that recognize, bind to, or have immunospecificity for substantially or essentially the same, or the same, epitope, epitopic or aminophospholipid site, or anionic phospholipid epitope, as monoclonal antibody 3G4 (ATCC 4545). such that it can effectively compete with the 3G4 monoclonal antibody (ATCC^INSTÍSW ^ a &amp; o (| 3, industrial binding to at least one anionic aminophospholipid or phospholipid, rather than an anionic phospholipid or aminophospholipid, or to all anionic phospholipids or phospholipids to which the monoclonal antibody binds

3G4 (ATCC 4545). Cross-reactive 3G4 antibodies are briefly referred to as 3G4-like antibodies and 3G4-based antibodies, and those terms are used interchangeably herein and apply to compositions, uses, and methods.

Identification of one or more antibodies that binds, approximately, substantially, essentially, or the same epitope, such as the 3G4 monoclonal antibody (ATCC 4545) is a direct technical issue known as 3G4 which with its advantageous properties has been provided. Since the identification of cross-reacting antibodies is determined compared to a reference antibody, it will be understood that the actual determination of the epitope to which the reference antibody (3G4) and the test antibody binds does not require any way to identify an antibody that binds to the same or substantially the same epitope as the 3G4 monoclonal antibody. However considerable information on the epitope bound by 3G4 is included herein and can be carried out

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Antibodies to be further determined by mapping the epitope.

Identification of cross-react can easily be done using any of a variety of immunological selection assays in which competition for the antibody can be assessed. All such tests are routine in the art and are further described herein in detail. Each of the United States of America Patents Nos. 6,342,219 and 6,342,221 are specifically incorporated herein by reference, for the purpose of including a still further complement to the present disclosure, concerning how to produce antibodies that bind thereto or substantially or essentially the same epitope as a given antibody, such as 3G4, or which effectively compete with a given antibody for binding to an antigen.

For example, when the test antibodies to be examined are obtained from different source animals, or even are of a different isotype, a simple competence assay can be employed in which the control (3G4) and test antibodies are tested. mix (or pre-adsorb) and apply to an antigenic composition of aminophospholipid or anionic phospholipid, preferably PS. Due to the antigenic composition of aminophospholipid or phospholipid

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any composition containing a 3G4 binding antigen, as described herein, and as described in Table 4. Thus, protocols based on ELISAs and Western blotting are convenient for use in those simple competence studies. .

In certain embodiments, the control (3G4) antibodies could be pre-mixed with varying amounts of test antibodies (eg 1:10 or 1: 100) for a period prior to application of an antigenic composition. In other modalities, control and varying amounts of test antibodies can simply be mixed during exposure to the antigenic composition. In any case, by using secondary isotype species or antibodies, only bound control antibodies can be detected, and the binding of which will be reduced by the presence of a test antibody that recognizes substantially the same epitope.

When conducting an antibody competition study, between a control antibody and any test antibody (regardless of species or isotype), control (3G4) can first be labeled with a detectable marker, such as for example biotin or a enzyme marker (or even subsequent identification.

previously or they would incubate the labeled control antibodies, with the antibodies of examined in various proportions or 1: 1000) and (optionally after proof that they would be (for example 1:10, 1: 100 of an appropriate period) would then be tested for reactivity of the labeled control antibodies and these would be compared to a control value where a potentially competing test antibody had not been included in the incubation.

The assay may again be any of a variety of immunological assays based on antibody hybridization, and control antibodies would be detected through detection of its marker, for example using streptavidin in the case of biotin-labeled antibodies or by using a chromogenic substrate relative to an enzyme marker (such as the 3,3'5,5'-tetramethylbenzidine (TMB) substrate with the peroxidase enzyme) or simply by detecting a radioactive marker. An antibody that binds to the same epitope as control antibodies will be able to effectively compete for binding and will thus significantly reduce binding to the control antibody, as evidenced by a reduction in the linked marker.

<img file="MX337052B_D0031.tif" />

The reactivity of the anti

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(marked) in the absence of a completely irrelevant antibody, would be the high value of the control. The low value of the control would be obtained by incubating the labeled antibodies (3G4) with unlabelled antibodies of exactly the same type (3G4), when competition occurs and binding of the labeled antibodies is reduced. In a test assay, a significant reduction in the reactivity of the labeled antibodies, in the presence of a test antibody, indicates a test antibody that recognizes the same epitope, i.e. one that cross-reacts with the labeled antibody ( 3G4).

A significant reduction is a reproducible reduction that is, consistently observed, in binding. A significant reduction in terms of the present application is defined as a reproducible reduction (in 3G4 binding to one or more aminophospholipids or anion phospholipids, preferably PS, in an ELISA) of at least about 70%, about 75% or about 80% in any ratio that is between about 1:10 and about 1: 1000. Antibodies with even more stringent cross-blocking activities will exhibit a reproducible reduction (in 3G4 binding to one or more aminophospholipids or anionic phospholipids, τ

preferably PS, in an ELISA of at least about 82%,

<img file="MX337052B_D0033.tif" />

another root apr

<img file="MX337052B_D0034.tif" />

about 85%, about 88%, about 90%, about

92%, or approximately 95% or similar value, in any ratio between approximately 1:10 and approximately 1: 1000. Complete or near-complete cross-blocking, such as exhibiting a reproducible reduction in 3G4 binding to one or more aminophospholipids or anion phospholipids of approximately 97% or approximately 96% or similar value, although in no way required to lead to the invention practices it, it is not truly excluded.

As for the second generation antibodies as a whole, competition can be measured with reference to an antibody that binds at least phosphatidylserine, where the second generation antibody effectively competes for binding to phosphatidylserine; with reference to an antibody that binds at least phosphatidic acid, wherein the second generation antibody effectively competes for binding to phosphatidic acid; referring to an antibody that at least binds to phosphatidylinositol; wherein the second generation antibody effectively competes for binding to phosphatidylinositol; with reference to an antibody that at least binds to phosphatidylglycerol, competing generation

<img file="MX337052B_D0035.tif" />

effectively by binding with reference to an antibody that is phosphatidylglycerol;

binding to at least cardiolipin, where the second generation antibody effectively competes for binding to cardiolipin;

and optionally with reference to an antibody that binds at least phosphatidylethanolamine, wherein the second-generation antibody effectively competes for binding to phosphatidylethanolamine.

In certain embodiments, second generation antibodies can be measured with reference to an antibody that binds to at least a first or second anionophospholipid or phospholipid, and where the second generation antibody effectively competes for binding to the first and second aminophospholipids or anionic phospholipid; with reference to an antibody that binds to at least a first, second and third anionic phospholipid or aminophospholipid, and wherein the second generation antibody effectively competes for binding to the first, second and third anionophospholipid or anionic phospholipid; with reference to an antibody that binds to at least a first, second, third and fourth aminophospholipid or anionic phospholipid, and wherein the second generation antibody competes

<img file="MX337052B_D0036.tif" />

effectively because of the link to the pritór // se^! third and fourth aminophospholipid with reference to an antibody that binds to at least a first, second, third, fourth, and fifth aminophospholipid or anionic phospholipid, and wherein the second generation antibody effectively competes for binding to the first, second, third, fourth and fifth aminophospholipid or anionic phospholipid.

In additional embodiments, a second generation antibody can be characterized as an antibody that exhibits significant binding to at least one aminophospholipid or anionic phospholipid, an undetectable binding to a choline-containing neutral phospholipid and that effectively competes with a monoclonal antibody of the invention, preferably 3G4 (ATCC 4545).

In particular embodiments, the antibody exhibits significant binding to the anionic phospholipids PS, PA, Pl, PG, and CL; has a phospholipid binding profile of PS = PA = PI = PG&gt; CL &gt;&gt; PE, where&gt; indicates a difference of at least 2 times in 1 binding and &gt;&gt; indicates a difference of at least 10 times in binding to those phospholipids; exhibits an undetectable binding to phosphatidylcholine or sphingomyelin; and it effectively competes with the antibody

<img file="MX337052B_D0037.tif" />

3G4 (ATCC 4545)

<img file="MX337052B_D0038.tif" />

phospholipids

<img file="MX337052B_D0039.tif" />

Preferably the second generation antibodies will have the preceding characteristics and will also exhibit significant binding to at least one anionic phospholipid present on the cell surface of activated, divided, damaged, apoptotic, or virally infected cells. More preferably the antibody also significantly inhibits the proliferation of dividing endothelial cells, without significantly altering the inactive cells, and more preferably, has no significant anticoagulant activities of lupus.

Functionally the second generation antibodies will preferentially suppress angiogenesis, have an antitumor effect and an antiviral effect, preferably in vivo and more preferably will do so without causing significant thrombotic complications in animals or patients. Thus, preferred antibodies possess the combined properties of an anti-angiogenic, vascular anti-tumor, anti-tumor, and antiviral agent.

The invention is exemplified by the 3G4 monoclonal antibody produced by the ATCC hybridoma

4545, or an antigen-binding fragment, of that monoclonal antibody. A monoclonal antibody hybridoma that binds the same epitope as the 3G4 monoclonal antibody (ATCC 4545) is another aspect of the invention.

In the following descriptions of the compositions, immunoconjugates, pharmaceuticals, combinations, cocktails, sets, first and second medical uses and all methods according to the invention, the terms antibodies and immunoconjugates, or an antigen binding region thereof , unless specifically mentioned otherwise or clarified from scientific terminology, Refers to a variety of anti-aminophospholipid or anti-anion phospholipid antibodies, as well as specific antibodies that cross-react with 3G4.

The terms antibodies and immunoglobulin, as used herein, broadly refer to any immune binding agent, including antibodies. monoclonal and polyclonal. Depending on the type of constant domain in heavy chains, the antibodies are assigned to one of five main classes: IgA, IgD, IgE, IgG, and IgM. Several of these are further divided into subclasses or isotypes, such as IgGl, IgG2, IgG3, IgG4, and the like.

The heavy chain constant domains, ^^ ¿^^ Kr ^ sponde ^ T. The different classes of immunoglobulins are named a, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

Generally, where antibodies are used in the invention rather than antigen binding regions, IgG and / or IgM are preferred, because they are the most common antibodies in the physiological situation and because they are most easily produced in an array from laboratory. Mammalian antibody light chains are assigned to one of two clearly distinct types: kappa (k) and landa (λ), based on the amino acid sequences of their constant domains. There is essentially no preference for the use of κ or λ light chains in the antibodies of the present invention.

The use of monoclonal antibodies (MAbs) or derivatives thereof is much more preferred. MAbs are recognized for having certain advantages, for example reproducibility and large-scale production, which makes them suitable for clinical treatment. The invention then provides monoclonal antibodies of murine, human, monkey, rat, hamster, rabbit and even frog or chicken origin. Taurine, human or humanized monoclonal antibodies are generally preferred.

How will the experfftidhüad.os understand in

<img file="MX337052B_D0040.tif" />

Technique, the immunological binding reagents encompassed by the term "antibody" extend to all antibodies of all species, and antigen binding fragments thereof, including dimeric, trimeric, and muitimeric antibodies;

bispecific antibodies;

chimeric antibodies, humanized human antibodies;

Recombinant antibodies, engineered camelized antibodies, and fragments thereof.

The term antibody is then used to refer to any molecule similar to that has an antibody binding region, which antigen, and this term includes antibody fragments such as

Fab ', Fab, F (ab)'₂, single domain antibodies (DABs), diabodies, camelized antibodies, and the like. Procedures for preparing and using various antibody and fragment based constructs are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404,097 and WO 93/11161, each specifically incorporated herein by reference; while linear antibodies are further described in

<img file="MX337052B_D0041.tif" />

Zapata et al. (1995), incorporated specifically as reference.

In certain embodiments the compositions of the invention comprise at least a first anti-aminophospholipid or anti-anionic phospholipid antibody, which comprises at least a first variable region that includes a region of an amino acid sequence of at least about 75%, more preferably of at least about 80%, more preferably at least about 85%, more preferably of at least about 90% and most preferably of at least about 95% or the like of amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; wherein that anti-aminophospholipid or anionic antiphospholipid antibody maintains at least substantially the biological properties of the anti-aminophospholipid or anti-anionic phospholipid antibodies of the present invention, as exemplified by the 3G4 antibody.

The identity or homology with respect to these anti-aminophospholipid or anionic antiphospholipid antibody sequences of the present invention is defined herein as the percentage of amino acid residues in a candidate sequence that are identical to the sequences of SEQ ID NO: 2 or SEQ ID NO: 4; or to

<img file="MX337052B_D0042.tif" />

i hlnl. - sequence of another anti-aminophospholipid or anionic antiphospholipid antibody, of the invention, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percentage identity of the sequences. Maintaining substantially the same, or even more effective, biological properties of the anti-amino phospholipid or anti-anion phospholipid antibody, used for sequence comparison, is particularly important. Those comparisons are easily carried out, for example using one or more of the different tests described in detail herein.

In certain preferred embodiments, the anti-aminophospholipid or anti-anion phospholipid antibodies of the invention comprise at least a first variable region that includes an amino acid sequence region having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, exemplified by variable regions including an amino acid sequence region encoded by the nucleic acid sequences of SEQ ID NO: 1 or SEQ ID NO: 3. Those sequences are the Vh and Vk sequences of the 3G4 ScFv that comprise the CDR1-3 (complementarity determining regions) of the variable regions of heavy and light chains.

In other preferred embodiments, second generation antibodies having improved or superior properties are provided, in comparison to original anti-aminophospholipid or anti-phospholipid antibody, such as 3G4 (ATCC 4545).

In certain modalities the antibodies used will be humanized, part human or human antibodies. Humanized antibodies are generally chimeric monoclonal antibodies from mouse, rat or other non-human species, which contain human constant and / or variable region domains (chimeric antibodies in part human).

Various humanized monoclonal antibodies for use in the present invention will be chimeric antibodies wherein at least a first antigen-binding region, or complementarity determining region (CDR), of a mouse, rat, or other non-human monoclonal antibody is bound functionally a, or grafted to a constant region or structure of the human antibody.

Humanized monoclonal antibodies for use herein can also be monoclonal antibodies from non-human species where one or more selected amino acids have been exchanged for amino acids most commonly observed in human antibodies. This can be easily accomplished through the use of routine recombinant technology, particularly site-specific mutagenesis.

_h 1 i ' ^! Fully human antibodies, in ygz

Humanized IND-jj.AiAL can also be prepared and used in the present invention. Those human antibodies can be obtained from their healthy groups, simply by obtaining a population of mixed peripheral blood lymphocytes from a human subject, including antigen presenting cells and antibody producing cells, and stimulating the cell population in vitro by mixing with an immunogenically effective amount of a sample of aminophospholipid or anionic phospholipid. Human cells producing anti-aminophospholipid or anti-anionic phospholipid antibody, once obtained, are used in the production of the hybridoma and / or the recombinant antibody.

Additional techniques for the production of human monoclonal antibodies include immunizing a transgenic animal, preferably a transgenic mouse, comprising a library of human antibodies with an immunogenic amount of an aminophospholipid or anionic phospholipid sample. This also generates human cells producing anti-aminophospholipid or anti-anion phospholipid antibodies, for further manipulation in the production of the hybridoma and / or the recombinant antibody, with the advantage that spleen cells, rather than peripheral blood cells pu den

<img file="MX337052B_D0043.tif" />

be easily obtained from the uanimal or D3 i. ·. . ·. &lt;. ··. · 'Transgenic.

Antibodies according to the invention can be easily prepared by selecting an antibody that substantially cross-reacts or competes with the 3G4 monoclonal antibody (ATCC PTA 4545). Appropriate preparation processes and methods include:

(a) prepare candidate antibody-producing cells; and (b) selecting from the candidate antibody-producing cells an antibody that substantially cross-reacts or competes with the 3G4 monoclonal antibody (ATCC PTA 4545). ·

A process to prepare appropriate antibody producing cells and obtain antibodies from them, can be carried out in situ in a given patient. That is, simply providing an immunogenically effective amount of an immunogenic anionic phospholipid or aminophospholipid sample to a patient will result in an appropriate generation of the antibody. In this way, the antibody is still obtained from the antibody-producing cell, but does not have to be isolated from a host and subsequently provided to a patient, and can be localized to modalities not currently preferred.

Appropriate antibody-producing cells can be stimulated and can also be obtained subsequently isolated and the antibodies and / or purified, peripheral blood lymphocytes with aminophospholipid or anionic phospholipid in vitro.

Other animal methods comprise an anionic phospholipid and immunogenic immunizing first component composition to select, which from administering to at least one comprises aminophospholipid or immunized animal, an antibody that substantially cross-reacts or competes with the monoclonal antibody (ATCC PTA 4545 ).

animal at least dose of a

These methods generally comprise:

immunize a dose, immunogenically effective composition an animal by administering and optionally more than that of immunogenic anionic phospholipid;

(b) obtaining an appropriate antibody from the animal

3G4 at one comprises an amount an immunized aminophospholipid producing cell, such as an antibody producing cell, that produces an or competes with the monoclonal antibody 3G4 (ATCC PTA 4545).

antibody that substantially cross-reacts

ΙΜΒ '5 ¡NST1

A composition comprising ^Luija immunogenically effective amount ^ of an aminophospholipid o. Preferred immunogenic anionic phospholipid, as used herein, is a composition comprising activated endothelial cells. Activated endothelial cells are preferably prepared by placing endothelial cells under at least one first condition, or in contact with at least one first factor, that activates endothelial cells, and / or that simulates the environment of a tumor, for an effective time to maintain substantially cell viability and stimulate the expression of at least one anionic phospholipid on the surface of endothelial cells. Examples of effective conditions for preparing activated endothelial cells are hypoxic and / or acidic environments. Examples of effective factors for preparing activated endothelial cells are effective concentrations of H2O2, thrombin, inflammatory cytokine (s), such as IL-la,

<td>IL-Ιβ, interferon</td><td>0 TNFa,</td><td>and generally</td><td>combinations</td><td>of</td>

<td>conditions and / or</td><td>factors</td><td>that simulate the</td><td>environment of</td><td>a</td>

<td>tumor.</td><td></td><td></td><td></td><td></td>

<td>Without</td><td>to import</td><td>nature</td><td>of process</td><td>of</td>

immunization, or the type of immunized animal, appropriate antibody-producing cells are obtained from the immunized animal, and preferably are

<img file="MX337052B_D0044.tif" />

further manipulated by the hand d ^ Sc / ¿fibiiabré. _x A? Immunized animal, as used herein, is a non-human animal, unless expressly mentioned otherwise. Although an antibody-producing cell can be used, most preferably, spleen cells are obtained as the source of antibody-producing cells. Antibody-producing cells can be used in a preparative process comprising:

(a) fusing an appropriate anti-aminophospholipid or anti-phospholipid antibody-producing cell with an immortal cell to prepare a hybridoma that produces a monoclonal antibody in accordance with the present invention; and (b) obtaining an appropriate anti-aminophospholipid or anti-phospholipid antibody, according to the invention, from the hybridoma.

Cells producing anti-aminophospholipid or anti-anionic phospholipid antibodies, hybridomas and appropriate antibodies are those that produce, or exist as, anti-aminophospholipid or anionic antiphospholipid antibodies, preferably antibodies that substantially cross-react or compete with the monoclonal antibody'3G4 (ATCC PTA 4545).

Hybridoma-based monoclonal antibody preparation methods then include an amount comprising those comprising:

(a) immunizing animal at least one dose, dose, of an immunogenically effective composition of an immunogenic anionophospholipid or anionic phospholipid, preferably a composition comprising activated endothelial cells;

(b) preparing a collection of monoclonal antibody-producing hybridomas, from the immunized animal;

(c) selecting from the collection at least a first hybridoma that produces at least a first monoclonal anti-aminophospholipid or anionic antiphospholipid antibody, according to the invention, optionally an anti-aminophospholipid or anionic antiphospholipid antibody, which substantially reacts in cross-form or compete with monoclonal antibody 3G4 (ATCC PTA 4545); and (d) culturing the at least one first antibody-producing hybridoma, to provide the at least one first anti-aminophospholipid or anionic antiphospholipid monoclonal antibody; and preferably (e) obtaining the at least one anti-aminophospholipid or anti-anionic phospholipid monoclonal antibody, from the at least one first hybridoma

<img file="MX337052B_D0045.tif" />

identification of an antio anti-anion phospholipid antibody that cross-reacts with the culture.

<img file="MX337052B_D0046.tif" />

In aminophospholipid substantially 3 monoclonal antibody 3G4 (ATCC PTA 4545), the selection step may comprise:

(a) contacting an aminophospholipid or anion phospholipid sample, preferably a PS sample, with effective amounts of the 3G4 monoclonal antibody (ATCC PTA 4545) and a candidate antibody; and (b) determining the ability of the candidate antibody to substantially reduce the binding of the 3G4 antibody to the aminophospholipid or anion phospholipid, preferably the PS sample; wherein the ability of a candidate antibody to substantially reduce the binding of the 3G4 antibody to the aminophospholipid or anion phospholipid, preferably the PS sample, is indicative of an anti-aminophospholipid or anionic antiphospholipid antibody that binds to substantially the same epitope as the antibody. monoclonal 3G4 (ATCC PTA

4545).

The selection step may further comprise:

(a) contacting a first sample of aminophospholipid or anionic phospholipid, preferably

PS, with an effective binding amount of

<img file="MX337052B_D0047.tif" />

3G4 monoclonal antibody (ATCC PTA 454 amount of 3G4 that binds to the aminophospholipid or anionic phospholipid, preferably PS;

(b) Contacting a second sample of an aminophospholipid or anionic phospholipid, preferably PS, with an effective binding amount of monoclonal antibody 3G4 (ATCC PTA 4545) in combination with an effective amount of competition, of a candidate antibody, and determining the amount of 3G4 that binds to the anionic phospholipid or aminophospholipid, preferably PS, in the presence of the candidate antibody; and (c) identifying an anti-aminophospholipid or anti-anionic phospholipid antibody that binds to substantially the same epitope as the 3G4 monoclonal antibody (ATCC PTA 4545) by selecting a candidate antibody that reduces the amount of 3G4 that binds to the aminophospholipid or anionic phospholipid, preferably

PS, preferably at least about 80%.

All selection criteria, as used herein, are preferably carried out in the absence of serum, to avoid the disadvantages of generating antibodies that could simulate pathological patient antibodies, which bind to aminophospholipids or anionic phospholipids. along with protein.

Since animals are not used for immunization

one. -OR.·'.!!. ³ J. human, monoclonal antibodies obtained ^^ to a hybridoma will often have a non-human constitution.

Those antibodies can optionally be subjected to a humanization, grafting or mutation process, as is known to those skilled in the art and as further described herein. Alternatively, transgenic animals, such as mice, comprising a genetic library of human antibodies can be used. Immunization of these animals will then directly result in the generation of appropriate human antibodies.

After production of an appropriate antibody-producing cell, most preferably a hybridoma, whether it produces human or non-human antibodies, nucleic acids encoding the monoclonal antibody can be cloned to prepare a recombinant monoclonal antibody. Any recombinant cloning technique can be used, including the use of "PCR" * to prime the synthesis of the nucleic acid sequences encoding the antibody. Therefore, methods for the preparation of appropriate, still further monoclonal antibodies include those that comprise using the antibody-producing cells as follows:

(a) obtain at least a first molecule of

FT nucleic acid encoding a

1:

Appropriate anionic anti-phospholipid or aminophospholipid, or segment of an appropriate anti-aminophospholipid or anti-phospholipid antibody-producing cell, preferably a hybridoma; and (b) expressing the nucleic acid molecule or segment in a recombinant host cell to obtain a recombinant anti-aminophospholipid or anionic antiphospholipid monoclonal antibody, in accordance with the present invention.

However, other powerful recombinant techniques are available that are ideally convenient for the preparation of recombinant monoclonal antibodies. Those recombinant techniques include preparative methods of monoclonal antibodies based on one comprise:

(a) immunizing animal at least one dose, dose, of an immunogenically effective phagemid library composition, than an animal administering to and optionally more than one comprising a quantity of an immunogenic anionophospholipid or phospholipid, preferably a composition comprising cells activated endothelials;

(b) preparing a library of combinatorial immunoglobulin phagemids expressing the RNA isolated from the antibody-producing cells, spleen, of the immunized animal;

(c) select from

<img file="MX337052B_D0048.tif" />

the phagemid library, at least a first clone expressing at least a first anti-aminophospholipid or anti-anion phospholipid antibody, optionally one that substantially cross-reacts or competes with the 3G4 monoclonal antibody (ATCC PTA 4545);

(d) obtaining nucleic acids encoding the anti-aminophospholipid or anti-anionic phospholipid antibody, from at least one first selected clone and that expresses the nucleic acids in a host cell

<td>recombinant</td><td>to provide the</td><td>to the</td><td>minus a first</td>

<td>antibody</td><td>anti-aminophospholipid</td><td>or</td><td>anti-phospholipid</td>

<td colspan="2">anionic; and preferably</td><td></td><td></td>

<td></td><td>(e) get the at least</td><td>a</td><td>first antibody</td>

anti-aminophospholipid or anti-anionic phospholipid, expressed by nucleic acids obtained from the at least one first selected clone.

Again, in these phagemid library-based techniques, transgenic animals carrying genetic libraries of human antibodies can be employed, thereby producing recombinant human monoclonal antibodies.

Regardless of the way of preparing a first nucleic acid segment

<img file="MX337052B_D0049.tif" />

Aminophospholipid or anionic anti-phospholipid, q-acid segments, additional, appropriate antibody nuclei can be readily prepared by standard molecular biology techniques. In order to confirm that some second-generation anti-aminophospholipid or anti-phospholipid antibody nucleic acid variant, mutant, or segment is appropriate for use in the present invention, the nucleic acid segment will be analyzed to confirm expression. of an anti-aminophospholipid or anti-anion phospholipid antibody, according to the present invention. Preferably the second generation nucleic acid variant, mutant, or segment will also be tested to confirm hybridization under standard conditions, more preferably stringent standard hybridization conditions.

Exemplary appropriate hybridization conditions include hybridization in approximately 7% sodium dodecyl sulfate (SDS), approximately 0.5 M NaPO₄, about 1 mM EDTA at about 50 ° C; and washed with about 1% SDS at about 42 ° C.

As a variety of recombinant monoclonal antibodies, whether of human or non-human origin, any of the treatment methods of the invention can be readily prepared.

<img file="MX337052B_D0050.tif" />

• providing the animal or patient with at least a first nucleic acid segment that expresses a biologically effective amount of at least a first anti-aminophospholipid or anti-anionic phospholipid antibody, in 1 patient. The nucleic acid segment expressing an anti-aminophospholipid or anti-anionic, 3G4-like or 3G4-based antibody will generally be in the form of at least one expression construct and can be in the form of a comprised expression construct within a virus or within a recombinant host cell. The preferred gene therapy vectors of the present invention will generally be viral readers, such as those comprised of a recombinant retrovirus, herpes simplex virus (HSV), adenovirus, adeno-associated virus (AAV), cytomegalovirus (CMV), and Similar.

Duramicin Derivatives Impervious to

Cells: The invention further provides substantially cell-impermeable phosphatidylethanolamine (PE) binding peptide constructs and derivatives, comprising at least one first PE-binding peptide, which has been modified to form a binding eosynthesis. to PE, substantially impervious to the cell.

Preferably the

<img file="MX337052B_D0051.tif" />

Pharmaceutical compositions comprising, in a pharmaceutically acceptable carrier, a biologically or therapeutically effective amount of at least a first PE-binding construct, substantially cell impermeable, comprising at least a first PE-binding peptide that has been modified to form a PE-binding construct, substantially impermeable to the cell. Thus, the PE-binding constructs, substantially impermeable to the cell, are constructs for pharmaceutical, pharmacological and therapeutic uses, i.e. for medical uses, preferably use in the treatment of viral infections. In certain embodiments the invention provides a PE-binding construct, substantially impermeable to the cell, different from biotin-linked cinnamycin.

Most preferably, the derivatives of the cell-binding PE peptides, substantially impermeable to the cell, of the invention are derivatives of duramycin peptides, substantially impermeable to the cell, and pharmaceutical compositions thereof. The duramycin peptide is typically modified to form a duramicin derivative substantially impermeable to the cell,. by union operates

<img file="MX337052B_D0052.tif" />

first cell impermeable group. The oporative binding gives a cell impermeable group could be through the lysine residue at position 2 of the amino acid in SEQ ID NO: 9.

The impermeable cell group can be a positive or negative charge at physiological pH or it can be polar. Exemplary groups include sulfate, sulfonate, phosphate, carboxyl, phenolic, quaternary ammonium ion, and amine groups. A pharmaceutical composition comprising biotin-linked duramycin is a particular example within the invention.

Duramicins substantially impervious to the cell can also be operably linked to a sugar, oligosaccharide or polysaccharide, amino acid, peptide, polypeptide, protein or to a polyol group. Certain cell impervious duramycins are those operably linked to an inert carrier protein, such as neutravidin, streptavidin, albumin, or an inert immunoglobulin carrier protein, of which duramycin bound to human IgG (HIgG) is particularly preferred. Other examples of cell impervious duramycins are those linked to targeting agents, preferably those that bind to a tumor cell, the tumor vasculature, or stroma.

11 ^ WO

TESTING the tumor or a cell virally inf ect ^ ds.s ^ jpjen ^^^ í ^ ® Targeting agents that target a component of a tumor cell, tumor vasculature, or tumor stroma are described in U.S. Patent Nos. 6,093,399, 6,004,555, 5,877,289, and

6,036,955, each of which is specifically incorporated herein by reference.

Tumor Treatment: The invention further provides compositions comprising at least a first purified anti-aminophospholipid or anti-phospholipid antibody, or antigen binding or immunoconjugate fragment thereof, optionally one that binds to essentially the same epitope as the 3G4 monoclonal antibody ( ATCC PTA 4545), or a PE-binding peptide derivative, substantially cell impermeable, preferably a duramycin derivative substantially impermeable to the cell. Those compositions are preferably pharmaceutically acceptable compositions, including those formulated for parenteral administration, such as for intravenous administration, or for administration as a liposome or as an aerosol.

The present invention provides a number of methods and uses for anti-3G4-like or 3G4-based antibodies, and duramycin derivatives substantially impervious to the cell. With respect to all methods, the terms one, one, ones and ones are used to mean at least one, at least a first, one or more or a plurality of steps in the methods described, except where specifically mentioned. This is particularly relevant to the administration steps in the treatment methods. In this way not only different doses can be used with the present invention, but different numbers of doses can be used, for example injections or inhalations, including injections or

<td>inhalations</td><td>multiple. Products can be used</td>

<td>therapeutic</td><td>combined, administered before, after or</td>

<td>during the</td><td>administration of the anti-antibody</td>

<td>aminophospholipid</td><td>or anti-phospholipid</td><td>anionic or</td>

<td>immunoconj ugado,</td><td>or the derivative</td><td>duramycin</td>

<td>substantially</td><td>impermeable to the cell.</td><td></td>

<td>I know</td><td>provide methods in</td><td>I saw other useful and</td>

uses that have important biological implications. Methods of and uses are first provided in the binding of aminophospholipids or anionic phospholipids, preferably PS or PE, which generally comprise effectively contacting a compos? / ·;

aminophospholipid or anionic phospholipid, preferably PS or PE, with at least a first anti-aminophospholipid or anti-anionic phospholipid antibody, or an antigen-binding fragment thereof, optionally an antibody that binds substantially to the same epitope as the 3G4 monoclonal antibody (ATCC PTA 4545), or with a duramycin derivative substantially impermeable to the cell. Contact is under conditions effective to allow the formation of a binding complex, and any of the complexes thus formed are detected. Detection methods and uses can be used in relation to biological samples, for example in the diagnosis of apoptosis, tumors and virally infected cells, and diagnostic kits based thereon are also provided.

Methods and uses for inhibiting proliferation are provided, preferably using the antibodies, antigen binding fragments and immunoconjugates of the invention. Methods of inhibiting endothelial cell proliferation and / or migration generally comprise contacting a population of cells or tissues including a population of endothelial cells, with a composition comprising a biologically effective amount of at least · a first anti57 antibody.

<img file="MX337052B_D0053.tif" />

one that binds to substantially the same epitope as the 3G4 monoclonal antibody (ATCC PTA 4545) or an antigen-binding fragment thereof, under conditions effective to inhibit proliferation and / or migration of the endothelial cell.

The foregoing methods and uses can be carried out in vitro and in vivo, in the latter case, where the tissues or cells are placed in an animal and the anti-aminophospholipid or anti-anionic phospholipid antibody is administered to the animal. In both cases, the methods and uses become methods and uses for inhibiting angiogenesis, which comprise contacting tissue containing, or a population of, potentially angiogenic blood vessels with an antiangiogenic composition comprising a biologically effective amount of at least a first anti-aminophospholipid or anti-anionic phospholipid antibody, optionally one that binds to substantially the epitope as the 3G4 monoclonal antibody (ATCC PTA 4545), or an antigen-binding fragment thereof, under conditions s effective to inhibit angiogenesis.

Where potentially angiogenic blood vessel populations are maintained ex vivo, the present invention has utility in üUSMxMIt programs.

INS7 drug discovery. In trials of

<img file="MX337052B_D0054.tif" />

Vitro with positive controls and non-reliable npnaUvpg are useful as a first step in the development of drugs to inhibit or promote angiogenesis, as well as in the delineation of additional information regarding the angiogenic process. Where the angiogenic blood vessel population is potentially localized in an animal or patient, the composition. Anti-angiogenic is administered to the animal as a form of therapy.

Anti-angiogenic and anti-vascular therapies are provided in terms of animals and patients who have, or are at risk of developing, any disease or disorder characterized by unwanted, inappropriate, aberrant, excessive and / or pathological vascularization. It is well known to those skilled in the art that since aberrant angiogenesis occurs in a wide variety of diseases and disorders, a given anti-angiogenic therapy, once it has been shown to be effective in some acceptable model system, may be used to treat the full range of angiogenesis related diseases and disorders.

The methods and uses of the present invention particularly serve for use in animals and patients who have, or are at risk of developing, and and. and ό ί any form of vascularized tumor; g.eii macular, including age-related macular degeneration; arthritis, including rheumatoid arthritis; atherosclerosis and atherosclerotic plaques; diabetic retinopathy and other retinopathies; thyroid hyperplasias, including Grave's disease; hemangioma; neovascular glaucoma; and psoriasis.

The methods and uses of the invention further serve for the treatment of animals and patients who have, or are at risk of developing, arteriovenous malformations (AVM), meningioma, and vascular restenosis, including restenosis followed by

<img file="MX337052B_D0055.tif" />

angioplasty. Other intended purposes of therapeutic methods and uses are animals and patients who have, or who are at risk of developing, angiof ibroma, dermatitis, endometriosis, hemophilic joints, hypertrophic scars, inflammatory diseases and disorders, pyogenic granuloma, scleroderma, synovitis, trachoma and vascular adhesions.

As described in U.S. Patent No. 5,712,291, specifically incorporated herein by reference, each of the preceding treatment groups is by no means exhaustive of the types of conditions to be treated by the present invention. The patent of the

<img file="MX337052B_D0056.tif" />

INSTITUI D £ 1 United States of America No.5,712,291 is hereby presented as reference for specific cleiLUüprupúsiLu »including the purpose of identifying a number of other conditions that can be effectively treated by an anti-angiogenic therapeutic product; the purpose of showing that the treatment of all angiogenic diseases represents a unified concept, once a defined category of angiogenesis inhibiting compounds has been described and claimed (in the present case anti-aminophospholipid or anti-anionic phospholipid antibodies, optionally those that bind to substantially the same epitope as the 3G4 monoclonal antibody (ATCC PTA 4545)); and the purpose of showing that the treatment of all angiogenic diseases is enabled by data only from a single model system in addition to the treatment of angiogenic and vascular diseases, important and unified aspects of the present invention are compositions and methods for the treatment of animals and patients who have, or are at risk of developing, cancer. All · the methods and uses of cancer treatment comprise the administration or use of at least a first purified anionic anti-aminophospholipid or antiphospholipid antibody, or an antigen binding fragment or immunoconjugate thereof,

<img file="MX337052B_D0057.tif" />

<img file="MX337052B_D0058.tif" />

epitope such as monoclonal antibody (ATCC PTA 4545), or a PE-binding peptide derivative substantially impermeable to the cell, preferably a duramycin derivative substantially impermeable to the cell. Those constructs are administered to animals or cancer patients in therapeutically effective amounts.

The cancer treatment methods of the invention, including those using the antibodies, are not based solely on exerting anti-vascular and / or anti-angiogenic effects. The cancer treatment methods and uses of the invention are suitable for the treatment of all forms of cancer, including animals and patients, that have, or are at risk of developing, a vascularized solid tumor, a metastatic tumor, or metastasis of a primary tumor.

Both unconjugated antibodies or naked antibodies and fragments thereof, as well as immunoconjugates in which the antibody, or antigen-binding fragment thereof, is operably linked to a therapeutic agent, can be used in aspects against the cancer of the invention. Unless specifically mentioned otherwise or clarified in scientific terms, the terms antibody and fragment thereof, as used

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INSTITUTO I at Dil / a

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They then mean an unconjugated or naked antibody or fragment, which is not bound to another agent, particularly a therapeutic or diagnostic agent. These definitions do not exclude modifications of the antibody, such as, by way of example, modifications to improve the biological half-life, affinity, avidity or other properties of the antibody, or combinations of the antibody with other effectors.

In immunoconjugate-based methods of treating cancer, the antibody, or an antigen-binding fragment thereof, is functionally linked to any one or more of a variety of biological, therapeutic, and / or so-called second agents anti-cancer (the anti-amino phospholipid or anti-anion phospholipid antibody itself is the first anti-angiogenic agent), which may have a direct or indirect anti-cancer effect.

Accordingly, the invention further provides methods and uses for administering selected therapeutic or diagnostic agents to tumors. Those modalities comprise administering to a tumor-bearing animal or patient, a biologically effective amount of a composition comprising at least a first immunoconjugate in which an anionic antibody diagnostic or therapeutic agent, or ± 1V1JT £ is functionallyf§^YOUOF^ORL £ ¿O, dÓÑ INDUSTRIAL

<img file="MX337052B_D0061.tif" />

or anti-aminophospholipid or anti-phospholipid antigen-binding fragment thereof, optionally one that binds to substantially the same epitope as the 3G4 monoclonal antibody (ATCC PTA

4545) .

The compositions, as well as the methods and uses, of the invention then include compositions comprising an anti-aminophospholipid or anionic antiphospholipid antibody, optionally one that binds substantially to the same epitope as the 3G4 monoclonal antibody (ATCC PTA 4545), bound functionally to at least one first biological, therapeutic or diagnostic agent. Antibodies are preferentially bound to radiotherapeutic agents, anti-angiogenic agents, apoptosis-inducing agents, anti-tubulin drugs, anti-cellular agents, cytotoxic agents, or cytokines (or to antiviral drugs, as discussed below).

Certain preferred agents for binding are in vivo diagnostic agents, which allow, for example, the conjugate to be used as a surrogate marker for chemotherapy.

Preferred agents for use in anti-aminophospholipid or anti-anion phospholipid antibody or therapeutic conjugates based on 3G4 are

<img file="MX337052B_D0062.tif" />

those who

INSTiTUTi / complete or enhance the effects of iWWfciuc and / or those selected for a type d »*» · »» »» · parj-Íreiiar or patient.

Therapeutic agents that complement or enhance the effects of the antibody include radiotherapeutic agents, agents that improve vascular permeability, certain cytokines, anti-angiogenic agents, apoptosis-inducing agents, and antitubulin drugs, any or more of which may be used herewith.

Currently preferred agents are the cytotoxic agent, gelonin; cytokines, such as TNFa, yl-12 and LEC (chemokine expressed in the liver); anti-cancer agents with anti-angiogenic effects, as in Table E; anti-cancer agents that induce apoptosis, as in Table F; and anti-tubulin drugs of the combretastatin family. A particularly preferred agent is docetaxel.

The compositions and methods for cancer treatment of the present invention can be used. also in combination with other therapeutic and diagnostic products. The combined uses in terms of anti-aminophospholipid or anionic antiphospholipid antibodies or 3G4-based antibodies, in combination with therapeutic agents, also including combined compositions, pharmaceuticals, INSTITUTO MEXICANO cocktails, games, methods, where the agerite ^^ g ^ p

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it is found in the form of a prodrug.

Combined methods for the treatment of cancer are those in which at least a first anti-aminophospholipid or anti-anion phospholipid antibody, antigen to be purified, or an immunoconjugate or binding fragment thereof, optionally binding essentially monoclonal antibody 3G4 (ATCC peptide binding to

PTA the same

4545),

PE, cell impermeable, preferably epitope that he or a derivative of substantially a duramycin derivative substantially impermeable to administered to an animal or patient the in combination with a therapeutically cell amount, cancer effective at least one second therapeutic or anticancer agent.

The invention further provides compositions, optionally containerized therapeutics, a pharmaceutical compositions, medicinal cocktail sets, comprising at least a first composition biologically effective amount less a first anti-aminophospholipid or anionic phospholipid antibody, optionally substantially the same epitope n, of to the anti-one that binds as the antibody binding to the antigen or immunoconjugate, thereof, or monoclonal 3G4 (ATCC PTA

4545), or a peptide derivative fragment of

<img file="MX337052B_D0064.tif" />

INDUSTRIAL substantially cell impermeable, preferably a substantially cell impermeable duramycin derivative; and a biologically effective amount of at least one second biological agent, component, or system, preferably at least one second therapeutic or anti-cancer agent.

The at least one second biological agent, component, or system will often be a therapeutic or diagnostic agent, component, or system but may not be. For example, the at least one second biological agent, component, or system may comprise components for modification of the antibody and / or for binding other agents to the antibody. Certain preferred second biological agents, components, or systems are prodrugs or components to produce and use prodrugs, including components to make the prodrug itself and components to tailor the antibodies of the invention to work in those prodrug or ADEPT modalities.

Where the disease to be treated is cancer, at least one second therapeutic or anticancer agent will be included in the therapeutic kit or cocktail. The term at least a second anti-cancer agent is selected with reference to the anti-aminophospholipid or anti-anion phospholipid antibody, construct 3G4, or a peptide derivative to i

PE, substantially waterproof

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preferably a derivative of duramycin

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first cell impermeable agent, which is the anticancer.

The antibodies of the invention can then be combined with chemotherapeutic agents, radiotherapeutic agents, cytokines, anti-angiogenic agents, apoptosis-inducing agents or immunotoxins or anticancer coaguligands. Chemotherapeutic agents also include genes, vectors, antisense constructs, and ribozymes.

The currently preferred second anti-cancer agents are anti-cancer agents with anti-angiogenic effects, as in Table E; anti-cancer agents that induce apoptosis, as in Table F; and anti-tubulin drugs of the combretastatin family. A particularly preferred agent is docetaxel.

In terms of compositions, kits and / or drugs of the invention, the combined effective amounts of the therapeutic agents can be comprised within a single container or container element or comprised within different containers or container elements. The cocktails will generally be mixed together for combined use. Agents Formulated for Administration ^ TintSpaMefebb^c INDUSTRIAL often preferred. Components for imaging can also be included. The kits may also comprise instructions for using the at least one first antibody and the one plus more different biological agents included.

Generally speaking, the at least one second anti-cancer agent can be animal or patient substantially simultaneously with that of the anti-aminophospholipid or anti-anionic phospholipid antibody, a 3G4-based therapeutic product or a duramycin derivative · substantially impervious to the cell; such as from an individual pharmaceutical composition or from two pharmaceutical compositions administered closely to each other.

Alternatively, the at least one second anticancer agent can be administered to the animal or patient, at a time sequential to the administration of the anti-aminophospholipid or anti-anionic phospholipid antibody, 3G4-based therapeutic product or duramycin derivative substantially impermeable to the cell . At a sequential time as used herein, it means staggered, such that at least one second anticancer agent is administered to the animal or patient at a time other than administration of the anti-aminophospholipid antibody.

<img file="MX337052B_D0067.tif" />

anionic, 3G4-based or duramycin-derived therapeutic product substantially impermeable to the cell. The two agents are administered at effectively separate times to allow the two agents to exert their respective therapeutic effects, that is, they are administered at biologically effective time intervals. The at least one second anti-cancer agent can be administered to the animal or patient, in a biologically effective time before the anti-aminophospholipid or anti-anionic phospholipid antibody, 3G4-based therapeutic product or duramycin derivative substantially impermeable to the cell, or biologically effective subsequent to that therapeutic product.

Tumor imaging can also preferably be carried out using an anti-aminophospholipid or anti-anion phospholipid antibody. or 3G4-based antibody construct, detectably labeled. Imaging according to the invention can detect pre-apoptotic and apoptotic cells in such a way that it can be used after therapy as a surrogate marker. Alternatively, since the image formed will be predictive of the binding sites of the therapeutic product to be used,

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[OM © ajQQ A INDUSTRIAL PROPERTY imaging can be carried out ^ treatment. Cancer treatment can. then be carried out by:

(a) imaging a tumor by administering to a tumor-bearing animal or patient, a diagnostically minimal amount of at least a first tumor-binding agent, detectably labeled, preferably an anti-aminophospholipid or anionic antiphospholipid antibody or a 3G4-based antibody construct, comprising a diagnostic agent operably linked to the tumor binding agent or anti-aminophospholipid or anti-anionic phospholipid antibody, or 3G4-based antibody, thereby forming a detectable image of the tumor; and (b) subsequently administering, to the same animal or patient, a therapeutically optimized amount of at least a first naked anti-aminophospholipid or anti-phospholipid antibody, or 3G4 antibody or antibody-therapeutic agent construct, using that antibody and causing thereby an antitumor effect.

Therefore imaging or treatment formulations or medications are provided, generally comprising:

(a) a first pharmaceutical composition comprising a diagnostically effective amount of a

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p .1VJL χ f¡

INSTITUTO MEXICANO tumor-binding agent, detectable if ^ ^ ^ preferably an anti-amino £ ® &amp; £ Qj_ípidQ_Q_anti phospholipid antibody or a 3G4 antibody comprising antibody construct based on a detectable agent functionally linked to the tumor binding agent or anti-aminophospholipid or anti-anion phospholipid antibody or 3G4-based antibody; and (b) a second pharmaceutical composition comprising a therapeutically effective amount of at least one anti-aminophospholipid or anionic antiphospholipid antibody, nude, or 3G4 antibody, or antibody-therapeutic agent construct, using that antibody.

Treatment of Viral Infections: The particularly important and surprising developments of the invention concern compositions, combinations, games, methods, uses and medications for the treatment or prevention of viral infections. The antiviral treatment methods of the invention concern the administration or use of any or more of the preceding and additional therapeutic agents of the invention.

In the first instance, the antiviral compositions and treatment method of the invention,

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concern the administration or use of at least a first

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anti-aminophospholipid antibody or^{, NSll}™ ^^^ go

INDUSTRIAL anionic, purified, or an antigen-binding fragment thereof, optionally one which binds to the same epitope as the 3G4 monoclonal antibody (ATCC PTA 4545), or a PE-binding peptide derivative, substantially impervious to cell, preferably a duramycin derivative substantially impermeable to the cell, as described above in terms of the compositions and in terms of cancer treatment. Of the PE-binding peptide derivatives, substantially cell-impermeable, those preferred for use will be substantially cell-impermeable duramycin derivatives, such as biotin-bound duramycin or HIgG-linked duramycin.

Given the surprising connection between the antibodies and peptides of the invention and viral infections, the present invention further provides a range of new therapeutic treatment agents for use in viral infections. In particular the invention provides an antibody to an anionic aminophospholipid or phospholipid, particularly PS and PE, functionally linked to at least one first ahtiviral agent. The invention further provides a derivative of PE binding peptide, substantially impermeable i

to the cell, preferably a duramycin peptide, functional linked!

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i a first antiviral agent. Appropriate antiviral agents for binding to the antibodies and peptides of the invention include those presented in Table G.

Therefore, in general, the antiviral compositions and treatment methods of the invention concern the administration to an animal or patient with a viral infection, of a composition comprising a therapeutically effective amount of at least a first anti-aminophospholipid antibody or purified anionic anti-phospholipid, or a fragment of antigen binding or antiviral immunoconjugate thereof, optionally one that binds to essentially the same epitope as the 3G4 monoclonal antibody (ATCC PTA

4545), or a peptide derivative of linkage to the

PE, substantially impermeable to the cell, preferably a duramicin derivative substantially impermeable to the cell, or an antiviral immunoconjugate thereof.

The antiviral treatment methods and uses of the invention are appropriate for the treatment of all viruses in animals and patients, and even plants. The therapeutic agents of the invention can inhibit viral entry, but preferably inhibit viral replication, egress and spread of cells.

<img file="MX337052B_D0074.tif" />

infected host. The invention treats all viruses that infect vertebrates, as listed herein in Table H, particularly humans, and particularly viruses that are pathogenic in humans. Viral infections and associated diseases that can be treated by the invention include those viruses and diseases presented in Table J, exemplified for the treatment of CMV, RSV, and arenavirus infections, as well as hepatitis, influenza, pneumonia, Lassa fever, and AIDS. .

The compositions and methods for antiviral treatment of the present invention can also be used in combination with other therapeutic and diagnostic products. These combined uses are combined with separate antiviral agents in combined compositions, pharmaceuticals, cocktails, kits, and treatment methods.

The foregoing methods and uses for cancer and antiviral treatment often involve administering the pharmaceutically effective composition to the animal or patient systemically, such as by transdermal, intramuscular, intravenous injection, and the like. For the treatment of viral infections, particularly respiratory viral infections, delivery to the lung is preferred, using an aerosol. However,

<img file="MX337052B_D0075.tif" />

Administration that allows the therapeutic agent to locate the tumor or site of nation will be acceptable. Therefore other appropriate routes of administration include oral, rectal, nasal, topical, and vaginal. For uses and methods of treatment employ intrasynovial administration, as described for other immunological agents in the US Patent

United States of America No. 5,753,230 specifically incorporated herein by reference. For conditions associated with it;

s eyes, the formulations and ophthalmic administration are contemplated.

Administration, as used herein means the provision administration of anti-aminophospholipid or anti-anionic phospholipid antibody or 3G4-based therapeutic products, or derivatives of PE-binding peptide, substantially impermeable to. the cell, preferably duramycin derivatives in an amount (s) and for an effective period to exert a therapeutic effect. Passive administration of proteinaceous therapeutics is generally preferred, in part, for its simplicity and reproducibility.

However, the term administration is

Λ -.ΙΌ. X 1_Ί χ. _Λ .a-yjp-NQ is used herein to refer to any means by which therapeutic products are supplied. Therefore administration includes the provision of cells that produce the anti-aminophospholipid or anti-anionic phospholipid antibodies, therapeutics derived from duramycin or based on 3G4, in an effective manner. In those modalities it may be desired to formulate or package the cells according to the selectively permeable membrane, implantable structure or device, generally one that can be removed to stop therapy. Exogenous administration will generally continue to be preferred, as this represents a non-invasive method that allows close monitoring and control of the dose.

The therapeutic methods and uses of the invention also extend to the provision of nucleic acids encoding the anti-aminophospholipid or anti-anionic phospholipid antibody, therapeutics derived from duramycin or based on 3G4 in an effective manner so as to result in their expression in vivo. . Any gene therapy technique such as delivery of naked DNA, recombinant genes and vectors, cell-based delivery, including, ex vivo manipulation of patient cells, and the like can be employed. Sneaky liposomes and liposomes are

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IM-Pi will prefer for use in some modalities. ^INdu¡¡tri_TOl

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Pharmaceutical compositionsm ^^ p ^ ns d *¹»¹The inventive treatment employs therapeutically effective amounts of an anti-aminophospholipid or anti-anionic phospholipid antibody, optionally one that binds to substantially the same epitope as the 3G4 monoclonal antibody (ATCC PTA 4545), or an antigen-binding fragment or immunoconjugate thereof. antibody, or a PE-binding peptide derivative, substantially impermeable to the cell, preferably a duramycin derivative substantially impermeable to the cell. The therapeutic effects and consequently therapeutically effective amounts are measured through different parameters in cancer treatment versus antiviral treatment.

In the treatment of cancer, the amounts of the agents are effective in specifically killing at least a portion of the tumor cells,. vascular tumor or intratumoral endothelial cells; to specifically induce apoptosis in at least a portion of tumor cells, of, tumor or intratumoral vascular endothelial cells; to specifically promote coagulation in at least a portion of the tumor or intratumoral blood vessels;

<img file="MX337052B_D0078.tif" />

1NSTITUT to specifically occlude or destroy the menoe of the blood-carrying vessels of the tumor: to specifically induce necrosis in at least a portion of a tumor; and / or to induce regression or remission of the tumor or

with administration to an animal or a patient.

In the treatment of viral infections and related diseases, the amounts of the agents are effective in inhibiting one or more requirements for ongoing viral infection, such as viral entry, and preferably in viral replication, egress, and spread of cells. infected host. The amounts can kill or remove at least a portion of the virally infected cells in a way that counteracts viral replication, spread, and ongoing infection. In general, the amounts of the agents are effective to reduce, significantly or eradicate, the viral infection, with the administration to an animal or patient.

The terms preferentially and specifically, as used herein, means that anti-aminophospholipid or anionic antiphospholipid antibodies, 3G4-based therapeutics, or derivatives of PE-binding peptides, substantially impervious to the cell, preferably derived from duramycin, they achieve anticancer effects or

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antivirals that are substantially confined ^ p. ^ j ^? - ^? ·

DELAPROHcÓáÓ INDUSTRIAL does not cause clotting, tissue disease, and substantially destruction and / or necrosis of tissue in normal, healthy, animal or subject. The structure and function of healthy cells and tissues are then maintained substantially undamaged through practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are part of the present description and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by referring to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. The United States of America file for this patent contains at least one color drawing. Copies of this patent with the color drawing (s) will be provided by the Patent and Trademark Office upon request (S) and when paying the necessary rights.

Figure 1. Localization of anti-PS antibody (3SB) in vascular endothelial cells in L540 human Hodgkin lymphoma, 3LL murine lung carcinoma and B16 murine melanoma tumors in mice. SCID mice containing tumors were injected intravenously with 20 g of anti-PS mouse IgM (3SB) or

INSTITUTO MEXICANO DS THE PROPERTY V blood circulation was perfused with soluClW *¹ It's an hour later. The mice were afterwards and the tumor and organs were collected and frozen. Mouse IgM was detected in frozen sections using goat anti-mouse IgM-peroxidase conjugate. The anti-PS antibody was found specifically located in the blood vessels (indicated by arrows) in all tumors. Localization was not observed in mice injected with the control, IgM anti-CL.

Figure 2A and Figure 2B. Binding of the 9D2 antibody and annexin V to phospholipids absorbed in plastic. Phospholipids were absorbed into the plastic of microtiter plates. After blocking with 10% serum, antibody 9D2 (Figure 2A) or annexin V (Figure 2B) were added at concentrations ranging from 6.66 nM to 0.005 nM in the presence of 10% serum. Plates were washed and bound 9D2 antibody and annexin V were detected using goat anti-rat IgM-HRP and rabbit anti-annexin IgG, followed by anti-rabbit or HRP, respectively.

Figure 3. Inhibition of binding of 9D2 antibody and annexin V to anionic phospholipids in endothelial cells treated with H₂0₂ with competing phospholipid liposomes. Antibody 9D2 and annexin V

<img file="MX337052B_D0080.tif" />

IMPI® (6.66 nM) were pre-incubated with vari © 8'S'¿3 ^^ goil »INDUSTRIAL phospholipids (200 g / ml) DPBS buffer containing 10% serum. Bound 9D2 antibody and annexin V were detected using goat anti-rat IgM-HRP and rabbit anti-annexin V IgG, followed by anti-rabbit HRP, respectively. Binding in the presence or absence of competing liposomes was determined. The standard deviations of triplicate measurements were less than 10% of the average values.

Figure 4. Localization of biotin and annexin V-labeled 9D2 antibody to vascular endothelial cells and tumor cells in orthotopic MDA-MB-231 human breast tumors in mice. Nu / nu mice that

<img file="MX337052B_D0081.tif" />

they had MDA-MB-231 tumors in their mammary fat pads, they were injected intravenously with 50 g of antibody

Biotin-labeled 9D2 or 100 g biotin-labeled annexin V. An hour later, his blood circulation was perfused with saline. Tumors and organs were removed and frozen under pressure. Localized 9D2 and annexin V 20 were detected on frozen sections using streptavidin-HRP conjugate. Tumor sections derived from mice injected with saline or the rat IgM control served as negative controls.

Figure 5. Combined effects of hypoxia and

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inflammatory cytokines on exposure to bEnd.3 cells for 24 hours with IL-la

<img file="MX337052B_D0083.tif" />

hypoxia (gray normal bars (white bars)). The cell monolayers remained intact and viable under these conditions. The externalization of the PS was determined by measuring the binding of the¹²⁵I-annexin

V. The level of exposure to PS was expressed as a percentage of that value in cells treated with a combination of actinomycin D and TNFa.

Figure 6A and Figure 6B. Antitumor effects of the anti-PS antibody (3SB) in animals with syngeneic and xenogeneic tumors. 1 x 10⁷ Colonic murine colo 26 carcinoma cells Colo 26 (Figure 6A) or human Hodgkin lymphoma L540 (Figure 6B) were injected subcutaneously into the right flank of male BALB / c mice (Figure 6A) or SCID CB17 mice (Figure 6B), respectively. Tumors were allowed to grow to a size of approximately 0.6 to 0.9 cm³ and then the mice (4 animals per group) were injected intraperitoneally with 20 g of naked anti-PS antibody (open squares) or saline solution (open circles). The treatment was repeated 3 times with an interval of 48 hours. Animals were monitored daily for tumor and body weight measurements. Mice were euthanized when tumors reached 2 signs of necrosis

<img file="MX337052B_D0084.tif" />

<img file="MX337052B_D0085.tif" />

or ulceration. Control IgM from mouse gave similar results to saline.

Figure 7. Antitumor effects of the 9D2 antibody in mice possessing L540 human Hodgkin lymphoma. Groups of mice bearing tumors were injected with 100 g of the 9D2 antibody (closed circles) intraperitoneally 3 times per week, instead of the control (open squares). Tumor size was measured by calipers twice a week. The tumor volume was plotted against the number of days after injections of the tumor cells. The numbers in parentheses indicate the number of mice with regressing tumors / total number of mice per group.

Figure 8A, Figure 8B, Figure 8C, Figure 8D, Figure 8E, Figure 8F, and Figure 8G. Antitumor effects of the anti-PS antibody, 3G4, in animals with syngeneic and xenogeneic tumors. Murine Meth A tumor cells (Figure 8A), MDA-MB-231 human breast cancer (Figure 8B and Figure 8E), L540 human Hodgkin lymphoma (Figure 8C and Figure 8D) and MDA-MB-231 cancer. (Figure 8F and Figure 8G) were injected into mice. Tumors were allowed to grow to the sizes shown before treatment. Human Hodgkin lymphoma cells were left to form large tumors.

<img file="MX337052B_D0086.tif" />

mice were injected intraperitoneally 3 times a week with 100 g of 3G4 antibody instead of the control (3G4 is mentioned in figure 8A, figure 8B, figure

8C; and is shown by open circles in Figure 8D, Figure 8E, Figure 8f). Animals were monitored twice weekly for tumor measurements. The volume of the tumors was plotted against the number of days after inoculation of the tumors (Figure 8A) or against the days of treatment (Figure 8B and Figure 8C for a time of 20 to 30 days (Figure 8A, Figure 8B and Figure 8C); numbers in parentheses indicate 1 number of mice with regressing tumors / total number of mice per group) or 60 days (Figure 8D, Figure 8E and Figure

8F). The 3G4 antibody and the chimeric 3G4 antibody (ch3G4) were used to treat MDA-MB231 cancer cells, rather than the control (Figure 8G).

Figure 9A and Figure 9B. Inhibition of CMV replication in vitro by the 3G4 antibody.

HHR-R2 cells infected with CMV were treated with 3G4 (two panels above). Control wells were either left untreated (bottom two panels) or treated with isotype matched control, IgG antibody₃ GV39G (two central panels). The cells were observed at different time points: day 3 (left column) and day 9 (right column). Greens under the microscope antibody to 100 g / ml cells inf ectatdasroMiaparXá ^ ení

FROM THE PROÍ ftDAO VWZSaZj INDUSTRIAL fluorescent. Treatment with (figure 9A) and 50 g / ml (figure

Figure

10.

Concentration-dependent inhibition of CMV replication in vitro.

Cells

HHF-R2 infected with CMV were treated with different concentrations of 3G4 (panels above). Control wells were left untreated (bottom panel) or were treated with the lgG antibody₃ control equal to the GV39G isotype (intermediate panels). Cells were observed on day 9. Infected cells appeared green under the fluorescent microscope.

Figure 11A, Figure 11B and Figure 11C. Quantification of CMV viral load in antibody-treated cells and inhibition of replication at a late stage of the viral replication cycle. Monolayers of human fibroblasts were infected with CMV at a low moi of 0.01 pfu / cell and were treated with the indicated concentrations of the 3G4 antibody; the control antibody, GV39G; or the anti-colchicine control antibody, C44 (Figure 11A; untreated, untreated control). Monolayers of human fibroblasts were infected with CMV at a high moi of 3 pfu / cell and treated with 50 g / ml or 100 g / ml of the 3G4 antibody or 1 control antibody, GV39G (figure

ÍÑDUSTRIÁL were infected with CMV at a τη.oi

high human fibroblast antibody

3G4 or the control antibody, GV39G were added at the indicated time points after infection

11A, Figure 11B as in (Figure 11C). In both Figure 11C, viral load in cells and supernatants was quantified using a standard plate assay *.

Figure 12. Inhibition of RSV replication in vitro by antibodies 3G4, 1B9 and 3SB. , RSV-infected A-549 cells were treated with 3G4, 1B9 or 3SB or were left untreated as a control. Treatment with 1B9 (green) and 3SB (red) resulted in a logarithmic reduction in viral replication (versus the blue control). The even more pronounced antiviral effect of 3G4 is shown in pink.

Figure 13A, Figure 13B, Figure 13C, Figure 13D, Figure 13E, Figure 13F, Figure 13G, Figure 13H, Figure 131, Figure 13J, Figure 13K, Figure 13L, Figure 13M, Figure 13N, Figure 130, Figure 13P, Figure 13Q and figure 13R. Duramycin derivative structures. Chemical structures for exemplary duramycin derivatives, from Example XV, are depicted. In each of the compounds of Figure 13A to Figure 130, the PE-binding peptide, duramycin, has been bound to a significant exerting construct construct group. The

<img file="MX337052B_D0087.tif" />

INDUSTRIAL NON-SPECIFIC TOXIC EFFECTS, Schematic structure of the related parent cyclic duramycin peptide, is shown in Figure 13P. The linear sequence is represented by the

SEQ ID NO: 9, and the structures of the modified amino acids in the sequences are depicted in Figure 13Q. Figure 13R depicts an exemplary antiviral duramycin construct in which duramycin is linked to cidofovir.

Figure 14A, Figure 14B, Figure 14C and Figure 14D. Binding specificities of duramycin derivatives. Duramycin derivatives were prepared as described in Example XV and their specificities were determined using ELISAs and competing ELISAs, as described in Example XVI. Fig. 14^to shows the phospholipid binding profile of duramycin derivatives, against a panel of phospholipids, shows specificity for PE. In Figure 14B, serum has no significant effect on PE binding; Figure 14C and Figure 14D show the result of competition ELISAs confirming the specificity of duramycin derivatives for PE.

Figure 15. Inhibition of replication of the

In vitro CMV by duramycin derivatives. HHF88 cells

R2 infected with duramycin (DLB) ₄NA

INSTITUTE and (DIM)_nHIgG. The wells were left untreated. Cells were observed ™ · different time points: day 4 (left panels) and day 6 (right panels). Infected cells appear green under the fluorescence microscope. The (DLB)₄NA and (DlM)_nHlgG inhibit viral spread from individually infected cells.

Figure 16. Selective inhibition of endothelial cell division by anti-PS antibodies.

Anti-PS antibodies 3SB, 9D2 and 3G4 were analyzed for inhibitory effects on endothelial cells in vitro as in Example XVIII. Each of the 3SB, 9D2, and 3G4 antibodies exhibit selective inhibition of endothelial cell division (subconfluents) rather than inactive (confluent) cells. Antibodies 9D2 and 3G4 both have a greater inhibitory effect than 3SB.

Figure 17A and Figure 17B. Antiangiogenic and vascular targeting effects of the antibody

3G4 in mice with tumors. Naked mice bearing MDA-MB-231 orthotopic tumors were treated 3 times per week with 100 g / dose of 3G4 antibody (treated, right panels) or with the same dose of a control antibody matched to the isotype (control, panels of the left). Tumors were frozen, cut, and antibody to the

Murine CD31 (rat CD31), a murine pan-endothelial marker (Figure 17A), under tension with Η YE

<img file="MX337052B_D0088.tif" />

anti-mouse, vasculature-encrusted or paraffin-embedded and tumor-bearing animals of which administration of the anti-angiogenic (vascular figure (figure 17B).

Figure 18A Complementarity amino acids weighing sequences (figure sites the (CDRs)

DNA and (Figure 18A; SEQ

18B; SEQ ID NO: 3 restriction control

3G4 gives

17A) and comparing sections and treatises is observed by result addressing effects Figure 18B. DNA and sequences of determining regions of the 3G4 antibody. Amino acids are presented for chains

ID NO: 1 and SEQ ID in and SEQ ID NO: 2) and light

N0: 4), and the DNA sequences are shown. The leader sequence is distinguished from the mature protein, which begins as shown by the first arrow in both Figure 20 18A and Figure 18B. Exemplary means are presented for grafting each variable sequence with a human constant region, where the first part of the respective human constant region sequences (SEQ ID

NO: 7 and SEQ ID NO: 8) are shown by the second arrow in both Figure 18A and Figure 18B.

Figure 19A PS binding of the IgG anti-PS antibody, 3G4, with the IgM anti-PS antibody, 3SB. Binding to the PS of the IgM antibody, 3SB (♦) and two

IgG, 3G4 (A) and 3B10 (), antibodies were determined by ELISA using antibody concentrations of up to 3,375 nM (Figure 19A). Antibody binding to PS

3SB (♦), 3G4 (A) and 3B10 () at concentrations up to 0.06 nM are shown separately (Figure 19B).

Figure 20. Inhibition of binding of the 3G4 antibody to immobilized PS, using competing phospholipid liposomes. The 3G4 antibody (0.1 g / ml) was preincubated for 30 minutes with various liposomes produced from pure phospholipids (PS-L, PE-L, PIL, PC-L, CL-L, PA-L · and PG-L ) or regulatory solution alone (control). The mixtures were then added to PS-coated ELISA plates, washed, and bound antibodies detected using secondary antibodies and OPD. Binding in the presence of the listed liposomes is shown and compared to binding of the 3G4 antibody in the absence of liposomes.

Figure 21. Linkage of chimeric 3G4 to phospholipid. The chimeric 3G4 antibody (ch3G4) was prepared as described in Example XIX. The phospholipids (PS, Pl, PE, PC, SM, CL, PG and PA) were

------

<img file="MX337052B_D0089.tif" />

adsorbed to the plastic of

After blocking, the 3G4 antibody cpi-i ™ ¿-v - ¡ _cradded to the concentrations shown. The plates were washed and the bound 3G4 chimeric antibody was detected through binding of the secondary antibody and development.

Figure 22. Location of the chimeric 3G4 for the vascular endothelium of tumors, in vivo. Biotin-labeled Ch3G4 (top panels) and control IgG (bottom panels) were administered to mice bearing MD-MBA-435S tumors. The tumor sections were stained with Cy3-streptavidin conjugate to detect biotin-labeled antibodies (left panels). Staining with MECA 32 antibody followed by FITC-labeled anti-rat IgG secondary antibody was carried out to detect the vascular endothelium (intermediate panels). Red and green images emerged (right panels), whereby the biotin-labeled proteins, linked to the vascular endothelium of the tumors, appeared yellow. The coincident staining of the localized 3G4 antibody and the MECA 32 marker of the vascular endothelium is shown by the yellow color on the superimposed images (upper right).

Figure 23. Increase of macrophage phagocytosis of PS positive cells by 3G4. Cells

<img file="MX337052B_D0090.tif" />

of tumors

HL-60 were the exposure to the PS was

<img file="MX337052B_D0091.tif" />

fluorescent-induced CFDA green harvested, and

200 M of H₂OR₂.

and opsonized or a target cell control antibody were

The treated cells were for 1 hour using 5 g / ml of isotype matching (BBG3).

added later

3G4

Macrophages, which were isolated from mouse marrow and cultured on chamber slides by 5 media containing 5 ng / ml GM-CSF. After bone from days to hours the slides were fixed and phagocytosis was counted visually under the fluorescent microscope. Results are presented as the percentage of macrophages that exhibited phagocytosis (macrophages that had carried out phagocytosis in at least one tumor cell).

Figure 24A and Figure 24B. Induction of exposure to PS in endothelial cells by docetaxel. Human umbilical vein endothelial cells (HUVEC) and human microvessel endothelial cells (HMVEC) were treated with 10 nM docetaxel for 24 hours. The cells were collected, washed with PBS and incubated with 3G4 at a rate of 10 g / ml for 30 minutes on ice. The cells were then washed twice, FITC-labeled goat anti-mouse IgG was added, and the cells were incubated for an additional 30 minutes on

<img file="MX337052B_D0092.tif" />

ice. The cells were washed and

INDUSTRIAL PROPERTY by FACS using a FACSCalibur cytometer (BectonDickinson, San José, CA) with CellQuest acquisition software. Both treated HUVEC (Figure 24A) and HMVEC (Figure 24B) show significant increases in 3G4 binding compared to untreated cells.

Figure 25A, Figure 25B and Figure 25C. Induction of PS exposure in tumor cell lines by docetaxel. 3LL mouse Lewis lung carcinoma, Colo26 mouse colon carcinoma and MDA-MB-435 human breast cancer cells were treated with 10 nM docetaxel for 24 hours. The cells were collected, washed with PBS and incubated with 3G4 at a rate of 10 g / ml for 30 minutes on ice. The cells were then washed twice, and FITC-labeled goat anti-mouse IgG was added and the cells were incubated for an additional 30 minutes on ice. The cells were then washed and analyzed by FACS using a FACSCalibur cytometer (Becton-Dickinson, San José, CA) using the Cell-Quest acquisition software. The 3LL cells (Figure 25A), Colo26 (Figure 25B) and MDA-MB-435, (Figure 25c), treated, show significant increases in 3G4 binding, compared to untreated cells.

Figure 26.

Induction of

<img file="MX337052B_D0093.tif" />

L. '·. tKOFihÜALJ expo si cióil'a

<img file="MX337052B_D0094.tif" />

in MDA-MB-231 human breast cancer cells by docetaxel. MDA-MB-231 human breast cancer cells were treated with 10 nM docetaxel for 24 hours. Cells were harvested, washed with PBS and incubated with chimeric 3G4 (ch3G4) or control, human IgG for 30 minutes on ice. The cells were then washed twice, FITC-labeled anti-IgG was added and the cells were analyzed by FACS, as above. There is a significant increase in ch3G4 binding compared to the control, human IgG.

Figure 27. Treatment with anti-PS antibodies increases the survival of mice infected with mCMV. Balb / C mice were infected with mCMV and treated with 3G4 or ch3G4, as described in Example XXI. Mice were monitored for survival 90 days after infection.

Figure 28. Treatment with the duramycin-biotin derivative, DLB increases the survival of mice infected with mCMV. Balb / C mice were infected with mCMV and treated with DLB as described in Example XXII. Mice were monitored for survival 90 days after infection.

Figure 29A and Figure 29B. Linkage of chimeric 3G4 to cells infected with Vaccinia virus. Cells

U937

<img file="MX337052B_D0095.tif" />

chimeric 3G4 antibody (ch3G4) or IgGi-human de-oenterol (HIgG) on day 2 after infection. Figure 29A, U-937 cells not infected. Figure 29B, U937 cells infected with Vaccinia virus. The peaks in Figure 29A and Figure 29B are: left peak (red), control-only secondary antibody; intermediate peak (blue), control HIgG; right beak (green), ch3G4.

Figure 30A, Figure 30B, Figure 30C, and Figure 30D. Inhibition of Pichinde virus replication in vitro by the 3G4 antibody. Vero cells were infected with Pichinde virus at a moi of 0.01 pfu / cell. Infected cells were treated with 100 g / ml of 3G4 (Figure 30A) or with isotype-matched control antibody, GV39G (Figure 30B). On day 2 after infection the cells were harvested with trypsin and allowed to adhere to the slides. Cells were fixed with acetone, and stained with polyclonal rabbit anti-PIC serum followed by biotin-conjugated goat anti-rabbit secondary antibody. Infected cells stain red-brown. The secondary antibody alone did not produce staining (Figure 30C). Also shown is the% of cells infected in 3G4 versus the treated control cells (Figure 30D).

Figure 31. The duramycin-IgG i conjugate

Human (HIgG) inhibits the growth of the

JCANC) alive.

BALB / c mice bearing cells. deX-JbuxnoxkJíethA were treated with the duramycin-HIgG conjugate (DSIAB)_nHIgG, in which duramycin is conjugated to HIgG using the SIAB linker or with control HIgG as described in Example XXV.

Figure 32. The duramycin conjugate is not cytotoxic. The natural duramycin compound duramycin and the biotin-labeled construct DLB were analyzed for their cytotoxic effects on human umbilical vein endothelial cells (HUVEC) using an MTT assay.

Figure .

The duramycin-antibody conjugate increases apoptopic cells.

duramycin-antibody IgG antibody_2nd phagocytosis of

A mouse duramycin linker was constructed to create macrophages, conjugated to C44, of a duramic ina-C4 4 (DuC44). Apoptopic marrow-derived macrophage HL-60 cells were incubated with mouse bone in the presence of DuC44, to a control mouse antibody,

BBG3 and the 3G4 antibody.

Phagocytosis was evaluated as the percentage of positive phagocytes per uptake. Data are mean values ​​± SE

DESCRIPTION OF ILLUSTRATIVE MODALITIES

<img file="MX337052B_D0096.tif" />

INDUSTRIAL more than 90% of cancers in man. Although the use of monoclonal antibodies and immunotoxins has been investigated in the therapy of lymphomas and leukemias (Vitettaa et al., 1991), these agents have been unpleasantly ineffective in clinical trials against carcinomas and other solid tumors (Abrams and Oldham, 1985). A primary reason for the ineffectiveness of antibody-based treatments is that macromolecules are not easily transported to solid tumors. Even once inside a tumor mass these molecules fail to distribute evenly due to the presence of tight junctions between tumor cells, fibrous stroma, interstitial pressure gradients, and binding site barriers (Denekamp, ​​1990; Dvorak et al., 1991 ).

In developing new strategies for treating solid tumors, methods that involve targeting the tumor vasculature, rather than tumor cells, offer several advantages. An effective destruction or blockage of the tumor vessels detracts from the blood flow through the tumor, resulting in an avalanche of tumor cell death. Antibody-toxin and antibody-coagulant constructs, examples of VTAs that selectively destroy and / or occlude blood vessels of

<img file="MX337052B_D0097.tif" />

to produce a great effect in the specific location and destruction of the tumor vasculature, resulting in tumor necrosis (Burrows et al., 1992;

Burrows and Thorpe, 1993; WO 93/17715; WO 96/01653; Huang et al., 1997; each incorporated herein by reference).

VTAs exert their primary action on the pre-existing blood vessels of solid tumors, and differ from anti-angiogenic agents in that they prevent new blood vessel formation. There are numerous advantages to VTA over other cancer therapies. First, a single vessel provides nutrition and facilitates removal of metabolic waste products from hundreds or thousands of tumor cells, and only needs to be damaged at one point to block blood flow upstream and downstream. VTAs are then particularly effective in established tumors. Second, the annihilation of endothelial cells, although s

<td colspan="2">a useful mechanism there</td><td>is required.</td><td colspan="2">A change of</td><td>the shape o</td>

<td>local start</td><td>of the</td><td>coagulation</td><td colspan="2">of the blood</td><td>can be</td>

<td>enough.</td><td>Third,</td><td>the cell</td><td>endothelial</td><td>I know</td><td>find</td>

<td>adjacent to</td><td>torrent</td><td>blood,</td><td>ensuring</td><td>a</td><td>supply</td>

<td>suitable of</td><td>drug.</td><td>Fourth, the</td><td>objective</td><td>is</td><td>a cell</td>

<td colspan="3">normal diploid that is unlikely</td><td colspan="2">that you acquire</td><td>mutations</td>

<img file="MX337052B_D0098.tif" />

blood flow, it is measurable.

Sixth, the temporal effects on vascular function may be sufficient to represent significant antitumor effects. Studies indicate that more than 99% of tumor cells in vivo can be killed during a two-hour period of ischemia.

<td>Finally, to</td><td>difference</td><td>of the</td><td>inhibitors</td><td>of</td><td>the</td>

<td>angiogenesis,</td><td>the VTA</td><td>require</td><td>only</td><td>of</td><td>the</td>

<td>administration</td><td>intermittent</td><td>to enter</td><td>in synergy</td><td>with</td><td>the</td>

conventional treatments, instead of chronic administration for months or years.

Cytotoxic VTAs are described in the following patents: U.S. Patent Nos. 5,660,827, 5,776,427, 5,855,866, 5,863,538 5,965,132, 6,004,554, 6,051,230, 6,261,535, and 6,451,312, each of which is incorporated herein by reference. Where antibodies, growth factors, or other binding ligands are used to specifically deliver a coagulant to the tumor vasculature, those agents are called coaguligands. Coaguligand VTAs are described in the following patents:

North America Numbers 6,093,399, 6,004,555, 5,877,289 and

100

6,036,955, each of which is presented as a reference.

<img file="MX337052B_D0099.tif" />

A currently preferred coagulant for use in coaguligands is Truncated Tissue Factor (tTF) (Huang et al., 1997; WO 96/01653; US Patent No. 5,877,289). TF is the main initiator of blood clotting (Ruf et al., 1991; Edgington et al., 1991). At sites of injury, factor VlI / VIIa in the blood comes in contact with and binds to TF found on cells in perivascular tissues. The TF: VIIa complex in the presence of the phospholipid surface activates factors IX and X. This in turn leads to the formation of thrombin and fibrin and ultimately to a blood clot (Ruf and Edgington, 1994).

<td></td><td>The</td><td>shape</td><td colspan="3">recombinant, trickled</td><td colspan="2">factor</td>

<td>tissue</td><td>(tTF)</td><td>, than</td><td>lacks the</td><td>domains</td><td colspan="2">cytosolic</td><td>and</td>

<td colspan="2">transmembrane,</td><td colspan="2">it's a protein</td><td>soluble</td><td>than</td><td colspan="2">has</td>

<td colspan="2">approximately</td><td>five</td><td>orders from</td><td>magnitude</td><td>less</td><td>in</td><td>the</td>

<td>capacity</td><td colspan="2">to induce</td><td>coagulation,</td><td colspan="2">compared</td><td>with</td><td>the</td>

<td>Native TF</td><td>(Stone</td><td>et al.</td><td>, nineteen ninety five; Huang</td><td colspan="2">et al., 1997).</td><td>This</td><td>I know</td>

it is because TF needs to be associated with phospholipids for the Vlla complex to activate IXa or Xa efficiently. However, when tTf is supplied to the endothelium of the tumor vasculature by a

101

<img file="MX337052B_D0100.tif" />

targeting agent or antibody, is brought back into proximity to a lipid surface and regains thrombogenic activity (Huang et al., 1997; US Patent Nos. 6,093,399, 6,004,555, 5,877,289, and 6,036,955). In this way a coaguligant is created that selectively causes thrombosis in the tumor vasculature.

Truncated TF has several advantages that recommend its use in coaguligands targeting the vasculature: human tTF is readily available, and the human protein will have low or negligible immunogenicity in man; Human tTf is fully functional in experimental animals, including mice; and directed tTF is very powerful because it triggers the activation of a coagulation protein cascade, which provides a greatly amplified effect (United States Patent Nos. 6,093,399, 6,004,555, 5,877,289, and 6,036,955).

A wide variety of appropriate target molecules have been described that are available in the endothelium of the tumor but are largely absent from the normal endothelium. For example, expressed targets can be used, such as endoglin, Eselectin, P-selectin, VCAM-1, ICAM-1, PSMA, a TIE, a LAM-1 reactive ligand, a VEGF / VPF receptor, an FGF receptor ,

102

<img file="MX337052B_D0101.tif" />

(United States of America Patents Numbers

5,855,866 5,877,289; Burrows et al., 1992; Burrows and

Thorpe, 1993; Huang et al., 1997; Liu et al., 1997; Ohizumi et al., 1997; each incorporated herein by reference.

Adsorbed targets are another appropriate group, such as VEGF, FGF, ΤΘΡβ, HGF, PF4, PDGF, TIMP, a ligand that binds to a TIE, or a fibronectin isoform associated with a tumor (US Patent Numbers 5,877,289, 5,965,132,

6,051,230 and 6,004,555). Fibronectin isoforms are ligands that bind to the integrin receptor family. Fibronectin isoforms associated with tumors are objective components of both the tumor vasculature and the tumor stroma. The monoclonal antibody BC-1 (C RNA emolia et al., 1989) binds specifically to the tumor associated fibronectin isoforms.

Other targets inducible by the natural tumor environment or following human intervention, are those target entities, as described in United States Patents Nos. 5,776,427, 5,863,538, and 6,036,995). When used in conjunction with pre-suppression in normal tissues and induction

103

<img file="MX337052B_D0102.tif" />

<img file="MX337052B_D0103.tif" />

vascular in tumors, the MHC antigens class' ^ ISAT ^^

INb'JSTRÍAL also used as targets (US patents

United States of America Numbers 5,776,427, 5,863,538,

6,004,554 and 6,036,995).

A currently preferred target for clinical applications is the vascular, endothelial addition-1 molecule (VCAM-1) (US Patent Nos. 5,855,866, 5,877,289,

6,051,230, 6,004,555 and 6,093,399). VCAM-1 is an adhesion molecule that is induced by inflammatory cytokines IL-Ία, IL-4 (Thornhill et al., 1990) and TNFa (Munro, 1993) and whose role in vivo is to recruit leukocytes to sites of acute inflammation (Bevilacqua, 1993).

VCAM-1 is present in vascular endothelial cells in a number of human malignancies including neuroblastoma (Patey et al., 1996), renal carcinoma (Droz et al., 1994), non-small lung carcinoma (Staal-van den Brekel et al., 1996), Hodgkin's disease (Patey et al., 1996), and angiosarcoma (Kuzu et al, 1993), as well as in benign tumors, such as angioma (Patey et al., 1996) and hemangioma (Kuzu t al., 1993). The constitutive expression of VCAM-1 in man is confined to a few vessels in the

Dinklo, 1993), and in the mouse in the vessels in the heart and

<img file="MX337052B_D0104.tif" />

thyroid, thymus and kidney (Kuzu et al., 1993; Bruijn and

104 lung (Fríes et al., 1993). J1M [] P Γ

I - ÓT11 UTO MEXICANO

Certain of the data presented¹¹-¹ ^ These moreover, in a suitable way, 1 complement those provided in US Patent Nos. 5,855,866, 5,877,289, 6,051,230, 6,004,555 and 6,093,399, and show the selective induction of thrombosis and infarction of tumors resulting from the administration of a coaguligando anti-VCAM-l * tTF. The presented results were generated using mice that had L540 human Hodgkin lymphoma. When grown as a xenograft in SCID mice, this tumor shows close similarity to human disease with respect to the expression of inflammatory cytokines (Diehl et al., 1985) and the presence of VCAM-1 and other cell activation molecules. endothelial in its vasculature.

Using a covalently linked anti-VCAM-l * tTF coaguligand, in which the tTF was directly linked to the anti-VCAM-1 antibody, it is shown herein that the coaguligand is selectively localized to the tumor vessels, inducing thrombosis in those vessels, which causes necrosis to develop throughout the tumor and slows tumor growth in mice that have solid Hodgkin L540 tumors. Tumors generally need to be at least about 0.3 cm in diameter to respond to coaguligando, because VCAM-1

105 tumor cells or tumor cells found absent from tumors in small tumors, too low

VCAM-l. This agrees with the levels

<img file="MX337052B_D0105.tif" />

of secreted host cytokines that infiltrate in to cause induction studies¹ of the patents of

<img file="MX337052B_D0106.tif" />

the United States of America Numbers 5,855,866,

5,877,289, 6,051,230, 6,004,555 and 6,093,399, where the inventions proved to be most useful in larger solid tumors.

Although initially the staining of the

VCAM-l, more in the periphery of the tumor, the coaguligando s evidently linked to the occluded blood transport vessels, while it was able to reduce the blood flow in all the tumor regions. Furthermore, one of the inventors 15 contemplates that the generation of thrombin caused by the initial administration of the coaguligand probably leads to. an induction of additional VCAM-1, in the central vessels (Sluiter et al., 1993), resulting in an amplified signal and the evident destruction of the intratumoral region. That type of coagulant-induced expression of additional target markers, and hence also signal amplification, is also described in the United States patent.

No. 6,036,955.

As shown here, although the

106 localization in the VCAM-1 expressing vessels&gt;&gt; In the heart and lungs of the mice, it was observed with the administration of coaguligando anti-VCAM-1, this construct did not induce thrombosis in those sites without tumor. Furthermore, the anti-VCAM-1 coaguligant was no more toxic to mice than a control coaguligant of irrelevant specificity, again indicating that constitutive expression of VCAM-1 in the vessels of the heart and lungs did not lead to toxicity. These data are important for the immediate clinical progress of coaguligand therapy, since VCAM-1 is a natural marker of the vascular endothelium of tumors in humans. However this phenomenon also provided the inventors with a unique insight that led to a totally different approach to the destruction of the tumor vasculature.

<td>A. Treatment of</td><td colspan="2">Tumors with Antibodies</td><td>Nude</td><td>for</td>

<td>Aminophospholipids</td><td></td><td></td><td></td><td></td>

<td>THE</td><td>inventors</td><td>they searched</td><td>understand</td><td>the</td>

<td>mechanism that</td><td>found</td><td>behind the</td><td>capacity</td><td>of the</td>

coaguligando anti-VCAM-1 to bind to VCAM-1 constitutively expressed in the blood vessels in the heart and lungs, without causing thrombosis in those vessels. There are numerous scientific possibilities for this empirical observation, generally connected with nature «ΤΒΤΓτπηίΓΠ'ΊΐΊ ι ........

107 prothrombotic of the tumor environment

<img file="MX337052B_D0107.tif" />

fibrinolytic predisposition in the heart and lungs.

Generally, there is a biological balance between the coaguligando system (fibrin deposition) and the fibrinolytic system (degradation of fibrin by enzymes). However, in a malignant disease, particularly carcinomas, this balance is disrupted resulting in abnormal activation of coagulation (hypercoagubility or the prothrombotic state). Despite extensive research, a clear molecular explanation for the prothrombotic nature of the tumor environment could not be discerned until recently.

After detailed analyzes of many possible options, the inventors reasoned that the failure of the anti-VCAM-1 coaguligant to cause thrombosis in vessels of normal tissues was due to the absence of the aminophospholipid, phosphatidylserine (PS) from the luminal surface of those glasses. To complete the theory, then not only would phosphatidylserine have to be shown to be absent from these normal vessels, but its presence on the luminal side of the vessels associated with the tumor would also have had to be demonstrated.

Therefore, the inventors used immunohistochemical staining to evaluate the distribution of a

108

<img file="MX337052B_D0108.tif" />

W [anti-phosphatidylserine monoclonal antibody (anti intravenously, in mice that had tumors. These studies revealed that the vessels that expressed VCAM-l in the heart and lungs lacked PS, while the vessels that expressed VCAM-l in the tumor expressed PS. The need for surface expression of PS in coaguligand action is further indicated by the inventors' discovery that annexin V, which binds to PS, blocks the action of anti-VCAM-l coaguligand * tTF, both in vitro and in vivo.

The lack of the thrombotic effect of coaguligando anti-VCAM-l in the vessels of the heart and lungs, normal, was explained in this way at least in part: the absence of the aminophospholipid, phosphatidylserine, means that the normal vessels lack a procoagulant surface in which coagulation complexes can be assembled. In the absence of surface VCAM-l, anti-VCAM-l «tTF binds to the vessels of the heart and lungs that express VCAM-l, but cannot induce thrombosis. In contrast, the vessels that express VCAM-l in the tumor exhibit a coincident expression of surface PS. The coaguligand thus binds to the tumor vessels and activates clotting factors locally to form an occlusive thrombus.

In addition to outlining the thrombotic effects

109 specific to expression ^: ..i i., coaguligandob tumor

INDUSTRIAL aminophospholipid, specific for phosphatidylserine, on the luminal surface of the tumor's blood vessels also allowed the inventors to explain the prothrombotic phenotype observed, but not understood, in previous studies. The expression of PS plays a significant role in the prothrombotic state of the tumor vasculature.

Following their discovery that the representative aminophospholipid, phosphatidylserine, was specifically expressed on the luminal surface of tumor blood vessels, but not in normal blood vessels, the inventors reasoned that other aminophospholipids had potential as targets for therapeutic intervention. The inventors then developed methods of treatment and action directed at the tumor vasculature, based on the action directed towards the aminophospholipids phosphatidylserine and phosphatidylethanolamine (PE).

A particularly surprising aspect of the inventors' studies was that the administration of an unconjugated anti-aminophospholipid antibody was effective in the treatment of tumors. This gave rise to important new routes for the treatment of tumors using unconjugated or naked antibodies that were i

110 j j- -ny

<img file="MX337052B_D0109.tif" />

bind to aminophospholipids.

These treatments and actions directed at the tumor vasculature are described in the United States patent of

North America No. 6,406,693, incorporated herein by reference. Although antitumor effects in animal models accepted in the art, are demonstrated in United States Patent No.

6,406,693, and expanded herein, the ability of aminophospholipids to act as safe, effective and objective markers of tumor vasculature could not have been predicted from pre-patent studies of the United States of America. No.

6.406.693.

Once the discovery of aminophospholipids as specific markers for tumor vasculature was demonstrated, the inventors began to develop a variety of aminophospholipid-targeted immunotoxins and coaguligands for use in treating tumors. As explained 'in United States Patent No. 6,406,693, this led to the unexpected discovery of naked anti-aminophospholipid antibodies, for use in the treatment of tumors. In investigating the potential target aminophospholipids in the context of supplying a toxin or coagulant to the tumor vasculature, the

<img file="MX337052B_D0110.tif" />

111 Inventors in a stroke of luck nude anti-PS antibodies had an effect on tumor tumor vasculature in vivo in the absence of any additional effector moieties. The ability of aminophospholipid antibodies to both specifically localize the tumor vasculature and to exert a concomitant killing effect, which led to tumor necrosis, was most unexpected.

The present invention provides surprising and improved second generation anti-PS antibodies, among other modalities, as naked antibodies in the treatment of tumors. A panel of second generation anti-PS antibodies is described herein, of which • · 9d2 and 3G4 monoclonal antibodies (ATCC 4545) are currently preferred, along with particular immunization and selection techniques for generation and selection of additional antibodies with these advantageous properties. It is also shown herein that vascular damage to tumor vessels due to anti-PS antibodies is mediated, at least in part, by host effectors. These and other insights from the inventors herein made it possible to optimize treatment with bare antibodies, both as when used alone and in combination with other anticancer agents, as described herein.

Anionic phospholipids

North America

112

<img file="MX337052B_D0111.tif" />

The patent of the States

United of

No.

6,406,693 explains that aminophospholipids, phosphatidylserine and phosphatidylethanolamine are normally secreted on the inner surface of the bilayer of the plasma membrane in different cells (Gaffet et al.,

nineteen ninety five; Julien et al.,

1995) and that this lipid segregation creates an asymmetric translayer. Although the existence of membrane asymmetry has been disputed for some time, the reason for its existence and the mechanisms for its generation and control are poorly understood (Williamson and

Schlegel, 1994) particularly in cells other than platelets.

The inventors previously demonstrated that the

PS is translocated on the surface of the vascular endothelial cells of the tumor and this occurs, at least in significant part, independently of apoptotic mechanisms or other mechanisms of cell death (United States Patent

North American.

6,406,693). In this way, the superficial expression of the

PS in the tumor environment is not a consequence of cell death and does not trigger immediate destruction

113

<img file="MX337052B_D0112.tif" />

Consistently detected in intact vascular enaolellcLltJü cells in various solid tumors, the vascular endothelium of the tumors is not frankly

<img file="MX337052B_D0113.tif" />

apoptotic, but is morphologically healthy (although different from that of normal tissues) and metabolically active. This is important for therapeutic methods.

<td>based</td><td>in the PS</td><td>objective, which</td><td>it means</td><td>than</td><td>the</td>

<td colspan="2">translocation of the</td><td>PS in the membrane</td><td>Exterior,</td><td>in</td><td>the</td>

<td>10 cells</td><td>endothelial</td><td>vascular</td><td>tumors,</td><td>is</td><td>the</td>

Stable enough for PS to serve as an objective entity, for successful therapy (using either nude antibodies or therapeutic conjugates).

Despite the important discoveries of United States Patent No.

6,406,693 (and 6,312,694, see below), the suggestions for phospholipid-based action of vascular endothelial cells of tumors were confined to targeted actions of aminophospholipids, such as PS and PE. Through the development of biological tools with exquisite specificity for different phospholipids and aminophospholipids, the invent ors of the present have now identified a new category of phospholipids that are surprisingly overregulated in vascular ara ndothelial cells.

tumors. These are the anionic phospholipids, los ^ © «alea, presented here as specific markers and ootublag of the tumor vasculature, allowing therapeutic intervention using both nude and immunoconjugated antibodies, which bind to anionic phospholipids.

Anionic phospholipids are largely absent from the surface of resting mammalian cells under normal conditions. Phosphatidylserine, which is the most abundant anion phospholipid, in the plasma membrane, is secreted closely to the inner lamella of the plasma membrane in most cell types, under normal conditions (Williamson and Schlegel, 1994; Zwaal and Schroit, 1997). Phosphatidylinositol (Pl), another major anion phospholipid, is also located predominantly in the inner lamella of the plasma membrane (Calderón and DeVries, 1997). Minority anionic phospholipids, phosphatidic a cid (PA), and phosphatidylglycerol (PG) have been examined in only a few cell types; but they also seem to be located mainly in the inner lamella of the plasma membrane (Hinkovska-Galcheva t al., 1989). Cardiolipin (CL), another anionic phospholipid, is present in the mitochondrial membrane and is absent from the plasma membrane (Daum,

115

1985).

.. J lL too

Asymmetrically distributed neutral phospholipids are found in the plasma membrane.

The neutral aminophospholipid, phosphatidylethanolamine (PE) is found predominantly on the inner lamella. Choline-containing neutral phospholipids, phosphatidylcholine (PC), and sphingomyelin (SM) are found predominantly on the outer lamella.

The asymmetry of PS, along with that of PE, is maintained by an ATP-dependent transporter, the translocase aminophospholipid (Mg²⁺ ATPase), which catalyzes the transport of aminophospholipids from the outer lamella to the inner lamella of the plasma membrane (Seigneuret and Devaux, 1984). The loss or collapse of the asymmetry of PS and PE results from the outward movement of these phospholipids in the plasma membrane and is caused either by inhibition of the translocase (Bitbol et al., 1987; Comfurius et al., 1990) , activation of PS transporters and / or activation of escramblase enzymes, Ca-dependent enzymes²⁺ that transport all lipids bidirectionally (Zhao et al., 1998).

Loss of PS asymmetry is observed under different pathological conditions and

116 physiological, including cell damage,

<img file="MX337052B_D0114.tif" />

programmed and apoptosis (Blankenberg et al., 1998;

Bombeli et al., 1997), cellular aging (Herrmann and Devaux, 1990), platelet activation (Rote et al., 1993; Zwaal et al., 1989), damage (Boyle et al., 1996) and malignant transformation (Sugimura et al., 1994). PS exposure also plays a role in the intercellular fusion of myoblasts (Sessions and Horwitz, 1981) and trophoblasts (Adler et al., 1995), cell migration (Vogt et al., 1996) and cell degranulation (Demo et al ., 1999). Endothelial cells exteriorize PS in response to increased Ca fluxes²⁺ induced by thrombin (Qu et al., 1996), calcium ionophore or phorbol esters (Julien et al., 1997), hyperlipidemia (Lupu et al., 1993), and non-lytic concentrations of C5b-9 complementary proteins ( Christiansen et al., 1997). Spontaneous exposure to PS has also been observed in malignant cells in the absence of exogenous activators or cell damage (Utsugi et al., 1991).

Several major consequences follow exposure of the membrane to PS. Phagocytic macrophages recognize, bind, and kill PS-positive senescent and apoptopic cells (McEvoy et al., 1986; Tait and Smith, 1999). PS also mediates the binding of T lymphocytes to activated endothelial cells

117 by thrombin (Qu et al., 1996). He is activated by the PS and contributes! ΊΓΧΊ- ··. Mess.! ·.¹ complem ^ n system

<img file="MX337052B_D0115.tif" />

to the lysis of cells positive to PS (Test and Mitsuyoshi, exposure to PS contributes to

1997). Finally, a procoagulant displacement on the endothelium (Williamson and Schlegel,

1994; Bombeli et al., 1997) by providing a negatively charged lipid surface for the assembly and activation of coagulation complexes (Bevers et al., 1985; Dachary-Prigent et al., 1996). The prothrombotic character of the endothelium of tumors has long been recognized (Donati and Falanga, 2001).

Despite the fact that the scientific literature focuses on PS, and that the inventors' previous work was confined to aminophospholipids such as PS and PE (United States Patent Nos. 6,406,693 and 6,312,694), the inventors of the present they hypothesized that a broader category of phospholipids could become exposed in the vasculature of tumors. Due to the increased stress conditions of the tumor microenvironment, the inventors reasoned that a variety of anionic phospholipids could be upregulated in the tumor vasculature, providing new, potential opportunities for therapeutic intervention.

118 endothelium of tumors are caused activation of the

1) Cytoeins derived from tumors, such as

<img file="MX337052B_D0116.tif" />

that interleukin-1 and tumor necrosis factor activate the endothelium and induce the expression of cell adhesion molecules (Shaughnessy et al., 1989; Orr et al., 2000); 2) reactive oxygen species (ROS) generated by leukocytes that adhere to the endothelium (Orr et al., 2000); and 3) ROS generated by tumor cells themselves as a by-product of metabolism (Shaughnessy et al., 1989; Soares et al., 1994) or as a result of exposure to hypoxia followed by reoxygenation (Zulueta et al., 1995). These observations suggested that Ca fluxes²⁺ they could be generated by these tensions within the endothelium of the tumor, which, in turn, cause the exposure of PS and PE, through the activation of escramblase or the inhibition of the aminophospholipid translocase.

However, the inventors extended these insights to the hypothesis that phospholipids

<td>anionic,</td><td>not only</td><td colspan="2">aminophospholipids</td><td>PS and</td><td>PE,</td>

<td colspan="3">would be up-regulated in</td><td colspan="2">the vasculature</td><td>of the</td>

<td colspan="2">tumor. To detect</td><td>phospholipids</td><td>anionic</td><td>in</td><td>the</td>

<td>surface</td><td>cell phone</td><td>inventors</td><td>generated</td><td colspan="2">a new</td>

<td>antibody</td><td>monoclonal,</td><td>9D2,. than</td><td>reaction to</td><td>with</td><td>the</td>

119 anionic phospholipids but not with neutral ones. The bonding

<img file="MX337052B_D0117.tif" />

9D2 thus differs from and to general aminophospholipids in that it binds to anionic aminophospholipid, PS, but not to neutral aminophospholipid, PE. The 9D2 antibody is also more specific for anionic phospholipids than the natural ligand, annexin V, which binds strongly to PE, in addition to anionic phospholipids (Blankenberg et al ,, 1998).

As detailed in the present application, the inventors found that 9D2 and annexin V are specifically located in the endothelium of tumors after intravenous injection into mice containing

<img file="MX337052B_D0118.tif" />

various types of solid tumors. This discovery validates the inventors' hypothesis that anionic phospholipids routinely become exposed on the endothelium surface of the vasculature of tumors and that they can be used as target molecules for tumor therapy (and for imaging of tumors). the 20 themselves). The present invention thus provides a variety of new antibody-based methods and compositions for use in targeting anionic phospholipids and treating tumors, both in terms of naked antibodies and in the administration of cytotoxic drugs, cytokines, coagulants, and Similar. In addition to

120 anionic phospholipids the targeted localization of PS, US patents

United

6,406,693

6,312,694, those currently preferred for targeted localization by the present invention are Pl, a major anion phospholipid, PA, and

PG, but also the targeted location of the

CL in certain modalities.

One of the main discoveries arising from the present invention is that anionic phospholipids are exposed on the surface of the tumor endothelium (Example VI). This phenomenon was demonstrated using two independent reagents that selectively bind to anionic phospholipids: a monoclonal antibody, 9D2, developed particularly to validate this point, by the inventors and annexin V. The antibody

9D2 and competing antibodies are preferred additional components of the present invention.

with high anionics

Antibody

9D2 and annexin

V affinity and specificity bind adsorbed to plastic, to phospholipids as liposomes, or presented on the membrane surface of activated or apoptotic endothelial cells in vitro. 9D2 binds strongly to PS, PA, and CL, but in the form

121 weaker to Pl and PG. Annexin V binds to PE in addition to PS, CL, PA, Pl, and PG, as previously discovered (Andree et al., 1990; Schlaepfer et al., 1987; Boustead et al., 1993; Blackwood and Ernst, 1990). The recognition of anionic phospholipids by the 9D2 antibody was identical in the presence and absence of serum, indicating that binding does not require serum cofactors. Binding of 9D2 to anionic phospholipids did not require Ca ions²⁺, while binding of annexin V required Ca²⁺.

Cross-blocking studies on PS-coated plates showed that 902 and annexin V do not block each other for binding to PS. This indicates that the two reagents recognize different epitopes on the PS molecule, or, more likely, differently packaged forms of PS. Annexin V is believed to bind to the flat surfaces of PS, whereas anti-PS antibodies are believed to bind to hexagonally packed PS (Rauch and Janoff, 1990). Both forms are probably present on PS coated plates. These practical cross-blocking studies (Example VI) also serve to demonstrate that antibodies that effectively compete for binding to anionic phospholipids, that is, binding to essentially the same epitope, can be

122 easily identified once reference antibody (for example 9D2)

<img file="MX337052B_D0119.tif" />

The present application also demonstrates that the 9D2 antibody and annexin V are located specifically in tumor vessels, and in tumor cells, in and around necrotic regions of all tumors examined in vivo (Example VI). Between 15 and 40% of the blood vessels in the tumors had an anionic phospholipid positive endothelium. In contrast, none of the blood vessels in normal tissues had detectable, exteriorized anionic phospholipids.

The specificity of staining of the tumor vasculature by 9D2 was demonstrated by: 1) lack of staining of tumor vessels by control rat IgM; 2) blocking binding of 9D2 or annexin V to H-treated endothelial cells₂OR₂ in vitro by liposomes prepared from anionic phospholipids, but not neutral phospholipids; 3) the discovery that extraction of phospholipids from tumor sections, with detergents or organic solvents, eliminated staining; and 4) the lack of localization of either 9D2 or annexin V to the inactive endothelium in normal organs.

<td></td><td>The main</td><td>phospholipid</td><td>anionic</td><td>than</td><td>is</td>

<td>located</td><td colspan="2">by 9D2 or annexin V in</td><td colspan="2">the vasculature</td><td>of</td>

<td>tumors is</td><td>probably</td><td>the PS, already</td><td>That this</td><td>is</td><td>the</td>

123

<img file="MX337052B_D0120.tif" />

most abundant anionic phospholipid and its expo

<img file="MX337052B_D0121.tif" />

Cell surface is regulated by environmental influences or damage. However, other anionic phospholipids (eg PI, PA, PG) are also likely to be exposed, despite being less abundant.

Although not detected by 9D2, the main neutral phospholipid, PE, probably contributes, together with PS, to the location of annexin observed in tumor vessels. PE is also known to be exposed on the endothelium of tumors, and the position of PE in the plasma membrane is regulated in a similar way to that of PS (United States Patent No. 6,406,693). PE is secreted in the inner lamella of the plasma membrane, in part by the translocase aminophospholipid, although less rapidly than PS (Devaux, 1992), and is transported to the outer surface by escramblase (Zhou et al., 1997 ). PE, like PS, is also exposed during apoptosis and cell activation (Emoto et al., 1997; Umeda and Emoto, 1999).

To examine the mechanism of exposure of anionic phospholipids to tumor endothelial cells, a series of studies were conducted in which endothelial cells in vitro were treated with various factors and conditions known to exist.

124 ; IMPI Λ present in the tumor microenvironment (Ahem ¿&gt; Tó_VII) p ήά hypoxia followed by reoxygenation, acidity, and increased exposure of thrombin PS, in viable endothelial cells, is between 10 and 22% of the observed level when all cells are apoptotic. Inflammatory cytokines (TNFa and IL-1) also caused a weak but definite induction of PS exposure.

These findings are consistent with the possibility that, in tumors, exposure of anionic phospholipids or vascular endothelium is induced by hypoxia / reoxygenation in combination with inflammatory cytokines, thrombin, and acidity. Although the precise mechanism need not be known to practice the present invention, tumor cells can generate ROS as a by-product of metabolism or in response to hypoxia (Zulueta et al., 1995)

Cytokines released by tumor cells can induce leukocyte adhesion molecules on endothelium, which mediate the adhesion of activated macrophages, polymorphonuclear cells, and platelets, to the tumor endothelium, and further secretion of ROS. ROS can then induce translocation of PS through oxidation of thiol-containing transport molecules or lipid peroxidation (Herrmann and Devaux, 1990), ry / r · ρ&gt; if of Ca²⁺ or lib¿rab; i

LÁ1 Li \! - .GHiIOaO

Ikí) U $ TRIAL (Wang and Joseph, 2000).

<img file="MX337052B_D0122.tif" />

PS and other phospholipids

125 possibly causing an influx

AC²⁺ from intracellular deposits

Exposure of the anionics in part explains the long-recognized procoagulant status of the endothelium of tumors (Donati and Falanga, 2001). Anionic phospholipids provide the surface on which clotting factors are concentrated and assembled (Bevers et al., 1985; Dachary-Prigent et al., 1996). It also provides a binding site for circulating macrophages (McEvoy et al., 1986), T lymphocytes. (Qu et al., 1996) and polymorphonuclear cells that help leukocyte infiltration into tumors.

Antibodies and other ligands that bind to anionic phospholipids can then be used for targeted localization, imaging, and / or treatment of tumor blood vessels. Anionic phospholipids are attractive as target vessels for tumors for several reasons: They are abundant (PS is present at a ratio of 3 x 10⁶ molecules per cell); They are located on the luminal surface of the endothelium of the tumor, which is directly accessible for binding by the agents of

<img file="MX337052B_D0123.tif" />

<img file="MX337052B_D0124.tif" />

vascular targeted present

<img file="MX337052B_D0125.tif" />

<img file="MX337052B_D0126.tif" />

find

<img file="MX337052B_D0127.tif" />

126

TB / endothelial tumor rp in various tumors.

find essentially

<img file="MX337052B_D0128.tif" />

absent from the endothelium in all normal tissues.

Vascular targeting agents using drugs or coagulants have been shown to be slightly effective, and sometimes curative, in mice with large solid tumors (Huang et al., 1997;

Nilsson et al., 2001;

States patents

United d

North America Numbers

5,660,827,

5,776,427,

5,855,866,

5,863,538,

5,965,132,

6,004,554,

6,051,230,

6,261,535,

6,093,399,

6,004,555,

5,877,289, and

6,036,955)

The present invention provides nude antibodies and vascular targeting agents that are targeted against anionic phospholipids for use in targeting the vasculature of tumors in the diagnosis and treatment of cancer in man.

Although a precise molecular understanding of how naked antibodies directed against anionic phospholipids and the role of aminophospholipids in the treatment of tumors is not necessary in order to implement the treatment, the inventors have contemplated various mechanisms that may explain annihilation. of endothelial cell observed. The favored mechanisms (particularly for the 3G4 antibody described here) are Fe domain mediated immune effector functions,

127 such as cellular antibody cytotoxicity (ADCC), complement cytotoxicity (CDC) and phagocytosis

<img file="MX337052B_D0129.tif" />

antibody mediated. Cell-mediated cytotoxicity, complement-mediated lysis and / or apoptosis, antibody-induced cell signaling, and / or cytoskeletal disruptions may also be involved.

Binding of intact antibodies against anionic phospholipids and aminophospholipids, particularly 3G4, to the vascular endothelial cell surface means that the Fe portions of the antibodies protrude into the lumen of the vessels. Because the Fe fragments of the antibody activate the complement pathway, the observed cell destruction may be a result of complement-directed lysis. Antibody binding then activates the complement-dependent coagulation cascade, causing multi-component complexes to assemble and ultimately generate a lytic complex that permeabilizes the target cell. Complement-activated ADCC may also be operating in destruction, where the complement binds to the antibody-coated target cell, and in which cells, such as neutrophils, which have complement receptors, lyse the target cell.

128

<img file="MX337052B_D0130.tif" />

OF THE PROPERTY

INDUSTRIAL

Like deconjugated antibodies, which include fragments to antigen binding, thereof, bind to anionic phospholipids^-and aminophospholipids on the surface of the vascular endothelial cells of the tumor, will form an antibody coating on the luminal surface. This can function to attract immune effector cells, such as cytotoxic T cells and / or natural suppressor cells (NK), that will exert a cell-mediated cytotoxic effect, on vascular endothelial cells.

Binding of the antibody to anionic phospholipids and aminophospholipids can also induce apoptosis in vascular endothelial cells of tumors. Although there are no known reports of antibody binding to PS that actually induces apoptosis (rather than PS as a marker resulting from apoptosis), the inventors consider this as another possible mechanism of the observed antitumor effects.

It is also possible that binding of the antibody to anionic phospholipids and aminophospholipids, on the surface of the vascular endothelial cells of the tumor, may cause disturbances in the cytoskeletal organization of the cell · How the cytoskeleton plays a role in the organization of surface membranes , and like him

129 binding of an antibody may disturb- (or have

DE ¿AH; O1-Í = DAD,. . ,, INDUSTRIAL additionally) membrane, antibody binding

<img file="MX337052B_D0131.tif" />

Anionic phospholipids and aminophospholipids can transmit changes to cytoskeletal proteins that interact with the bilayer.

It is already known that the spatial organization of cytoskeletal proteins controls the stability of the membrane and the shape of the cell, and that it is possible that the disruption of a certain cytoskeletal balance may have far-reaching consequences on the integrity of the cell.

A further operating mechanism of the invention may be that binding of the antibody to anionic phospholipids and aminophospholipids, on the surface of the endothelial cell, may initiate signal transduction through hitherto undefined pathways. Antibody binding may also disrupt known signal transduction pathways, for example by altering the conformation and / or interactions of membrane receptors, signal transduction proteins, membrane channels, and the like. Signals for cell destruction (apoptosis) can be initiated or mimicked, and / or conservation and / or homeostatic signals can be inhibited.

Although of scientific interest, the

130

<img file="MX337052B_D0132.tif" />

determination of the exact nature of the deesti¿% ^ c

Tell the vascular PROPERTY achieved by the naked antibodies¹anionic phospholipids and aminophosph olip'tduy, --- not necessary to carry out the treatment. Since the administration of this category of antibodies turns out to be advantageous in the specific antitumor effects in vivo, the treatment can be used regardless of the molecular mechanism that supports this phenomenon. The use of naked antibodies that bind to anionic phospholipids and aminophospholipids, then represents an important advance in tumor therapy, providing advantages in preparation and cost.

C. Antibodies to Anionic Phospholipids and Aminophospholipids

As the present invention identifies a new category of tumor vasculature markers, anionic phospholipids, naked and immunoconjugated antibodies, which bind to one or more anionic phospholipids, optionally in combination with aminophospholipids, can now be used in diagnosis and treatment of tumors.

131

Cl. Anticu rpos Policlonal ¿T^ST D £ ¿a fÍos'i ^ DAD

INDUSTRIAL

Means for preparing and characterizing antibodies are well known in the art (see, for example, Antibodies: A Laboratory Manual, Coid Spring

Harbor Laboratory, 1988; incorporated herein by reference). To prepare polyclonal antisera an animal is immunized with a composition comprising an immunogenic, anionic and / or aminophospholipid phospholipid, including H-treated cells₂OR₂ and other agents, as described herein, and the antisera collected from that immunized animal. A wide range of animal species can be used for the production of antiserum. Typically the animal used for the production of antiserum is a rabbit, mouse, rat, hamster, guinea pig, or goat. Due to the relatively large volume of rabbits, a rabbit is the preferred choice for the production of polyclonal antibodies. .

The amount of immunogen composition used in the production of polyclonal antibodies varies according to the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen; subcutaneous, intramuscular, intradermal, intravenous, intraperitoneal, and intrasplenic. Polyclonal antibody production can be monitored by sampling the animal's blood

132 'vb jr IL ngg / immunized at various points after * ^;

A second booster injection may also be given. The reinforcement and titration process is repeated until a convenient titration is achieved. When a desired level of titration is obtained, the immunized animal can be bled and the serum isolated and stored. The animal can also be used to generate monoclonal antibodies.

As is well known in the art, the immunogenicity of a particular composition can be increased through the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary adjuvants include Freund's complete adjuvant, a non-specific stimulator of the immune response containing dead Mycobacterium tuberculosis; Freünd's adjuvant incomplete; and aluminum hydroxide adjuvant.

It may be desired to enhance the host immune system, which can be accomplished by associating anionic phospholipids and aminophospholipids with, or coupling them to, a carrier. Exemplary carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (ASB). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.

As is also known in the art, a

133 given composition may vary in its immunity

ΙΚ2ϊ; Ί'ϋ'ΓΟ MEXICAN

P2 LA FROHEDAO However, the generation of antibodies against anionic osfoIPlipids and aminophospholipids is not particularly difficult. For example, highly specific anti-phosphatidylserine antibodies were cultured in immunized mice by intramuscular injections of phosphatidylserine-containing polyacrylamide gels and with phosphatidylserine-cytochrome c vesicles (Maneta-Peyret et al., 1988; 1989; each incorporated herein. as reference). The use of acrylamide implants improved the production of antibodies (Maneta-Peyret et al., 1988; 1989). Anti-phosphatidylserine antibodies grown in this way can detect phosphatidylserine in situ in human platelets (Maneta-Peyret et al., 1988). The Inoue, Rote and Rauch groups have also developed anti-PS and anti-PE antibodies (see below).

Although the generation of antibodies against anionic phospholipids and aminophospholipids can be accomplished through various means, certain preferred methods are described here in Example IV.

C2. Monoclonal antibodies

Various methods of generating monoclonal antibodies (MAbs) are also well known in the art now. Antibody generation techniques

134 | monoclonal more standard generally;

along the same lines as those polyclonal antibodies

<img file="MX337052B_D0133.tif" />

(Antibodies:

<img file="MX337052B_D0134.tif" />

Coid Spring Harbor Laboratory, 1988; incorporated herein by reference). A polyclonal antibody response is initiated by immunizing an animal with an immunogenic, anionic phospholipid and / or aminophospholipid composition and, when a desired level of titration is obtained, the immunized animal can be used to generate Mabs. Preferably, the particular screening and screening techniques described herein are used to screen for antibodies with the desired subsequent properties.

Mabs can be easily prepared through the use of well-known techniques, such as those exemplified in US Patent No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with the selected immunogen composition. The immunizing composition is administered in an effective manner to stimulate the cells to produce antibodies. Rodents such as mice and rats are the preferred animals, however the use of rabbits, sheep and toads is also possible. The use of rats can provide certain

135 with the BALB / c mouse being most preferred, it is used more routinely and generally gives a higher percentage of stable fusions.

After immunization, somatic cells with the potential to produce the desired antibodies, specifically B lymphocytes (B cells), are selected for use in the protocol to generate Mabs. These cells can be obtained from spleens, tonsils, or lymph nodes, or from peripheral blood samples. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the plasmablast stage of division, and the latter because peripheral blood is readily accessible. Frequently, a panel of animals will have been immunized and the spleen of the animal with the highest antibody titer will be removed and the spleen lymphocytes will be obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5 X 10⁷ up to 2 X 10⁸ lymphocytes.

The B lymphocytes that produce antibodies from the immunized animal are then fused with cells from an immortal myeloma cell, usually one

136

<img file="MX337052B_D0135.tif" />

IMPI 77 of the same species as the animal that was infrtút¿t ^ ádbí¿: § i IRDCáriiiÁL Myeloma cell lines suitable for use in hybridoma-producing fusion procedures are preferably those that do not produce antibodies, have high fusion efficiency , and enzyme deficiencies that render them unable to grow the desired fused cells (hybridomas) in certain media.

Any one of a number of myeloma cells can be used, as known to those of skill in the art (Goding, pages

75-83, 1984;

each

65-66, 1986; Campbell, pages herein as incorporated reference).

For example, when an immunized animal is a mouse, one can use P3-X63 / Ag8,

X63-Ag8.653, NSl / l. Ag

1, Sp210-Agl4, FO, NSO / U, MPC-11,

MPC11-X45-GTG

1.7 and

S194 / 5XX0 Bul; for rats, one can use R210.RCY3,

Y3-Ag

1.2.3, IR983F, 4B210 or one of the mouse cell lines listed above; and

U-266, GM1500-GRG2, LICR-LONHMy2 and UC729-6, all are useful in connection with human cell fusions.

Methods for generating antibody-producing spleen and lymph node cells and myeloma cell hybrids usually comprise somatic cells with myeloma cells in a 4: 1 ratio, although the ratio may range from about

137 (

i ΊΓ D / IX

20: 1 to approximately 1: 1, respecti ^ i ^ nte,

From LA i. INuua of a substance or substances (chemical presence

<img file="MX337052B_D0136.tif" />

) that promote the fusion of cell membranes.

Fusion methods using the Rutai virus have been described by Kohler and Milstein (1975; 1976; each incorporated herein by reference), and those using polyethylene glycol (PEG), such as 37 percent (volume / volume) of polyethylene glycol, by Gefter et al., (1977;

incorporated herein by reference).

The use of electrically induced fusion methods is also appropriate (Goding pages 71-74,

nineteen ninety six; incorporated herein by reference).

Fusion procedures usually produce viable hybrids at low frequencies, approximately 1 x 10 '⁶ up to x 10 '⁸

However, this does not pose a problem, since viable, fused hybrids differ from non-fused parent cells (particularly non-fused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium.

The selective medium is generally one that contains a substance that blocks de novo nucleotide synthesis in tissue culture medium.

Exemplary and preferred substances are aminopterin, methotrexate, and azaserine.

Aminopterin and methotrexate

138

<img file="MX337052B_D0137.tif" />

block pyrimidines,

TWI de novo synthesis both of purinasiruoom

INDUSTRIAL whereas azaserine blocks only purine synthesis. When aminopterin or methotrexate is used, the media are supplemented with hypoxanthine and thymidine as a nucleotide source (HAT medium). When azaserine * is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT.

Only cells capable of operating recovered nucleotide pathways are able to survive in the HAT medium. Myeloma cells lack key enzymes of the recovery pathway, i.e. hypoxanthine phosphoribosyl transferase (HPRT), and cannot survive. B cells can operate this route, but they have a limited lifespan in culture and generally die within two weeks. Therefore, the only cells that can survive in the selective medium are those hybrids formed from B cells and myeloma.

This culture provides a population of hybridomas from which specific hybridomas are selected. Typically, hybridoma selection is performed by culturing cells by single-clone dilution in microtiter plates followed by testing the individual clonal supernatants (after approximately two to three weeks) for

139 desired anti-VEGF reactivity.

<img file="MX337052B_D0138.tif" />

Sensitive, simple and immunoassays from plaque assays, spot immuno-link assays, and the like.

Selected hybridomas would be diluted and cloned into cell lines that produce individual antibodies, the clones of which can be propagated indefinitely to provide Mabs. Cell lines can be exploited for Mabs production in two basic ways. A hybridoma sample can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors by secreting the specific monoclonal antibody produced by the fused cell hybrid. The animal's body fluids, such as serum or ascites fluid, can then be tapped to provide Mabs at high concentration. The individual cell lines could also be cultured in vitro, where the Mabs are naturally secreted into the culture medium from which they can easily be obtained in high concentrations.

Mabs produced by any means will generally be further purified, for example, using filtration, centrifugation, and ¹ chromatographic, such as chromatography

140

<img file="MX337052B_D0139.tif" />

performance or affinity chromatography, all of those purification techniques are well known to those skilled in the art. Purification techniques involve fractionation to separate the desired antibody from other components of a mixture. Particularly convenient analytical methods for antibody preparation include, for example, protein A-Sepharose and / or protein G-Sepharose chromatography.

D. Second Generation Antibodies to Anionic Phospholipids and Aminophospholipids

The present invention provides second generation antibodies that bind to aminophospholipids and anionic phospholipids, where the antibodies have improved properties and / or do not suffer from the disadvantage associated with the prior art antibodies. A panel of such antibodies is described herein, of which the 9D2 and 3G4 monoclonal antibodies are commonly preferred, and the 3G4 antibody (ATCC 4545) is particularly preferred. The invention also provides particular immunization and screening techniques that allow the production of similar or competing antibodies with advantageous and / or less disadvantageous properties.

141

<img file="MX337052B_D0140.tif" />

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX337052B_D0141.tif" />

GAVE. Anticuegpee Properties

The second generation antibodies of the invention bind to aminophospholipids and anionic phospholipids and. they do not yet have pathogenic properties usually associated with antibodies to these phospholipids. This was made possible, in part, by new immunization and screening techniques developed by the inventors.

Antiphospholipid syndrome (s) (APS) are associated with autoantibodies called anti-cardiolipin antibodies and lupus anticoagulant antibodies. These syndromes are associated with a predisposition to arterial and venous thromboembolism, thrombocytopenia, and a number of neurological syndromes. The anti-phospholipid antibodies in these patients are then pathogenic antibodies.

Although described for years as anti-phospholipid antibodies and anti-PS antibodies, these pathogenic antibodies do indeed recognize protein cofactors that bind to cardiolipin, PS, or both, and not the phospholipids themselves (Galli et al., 1990,1993 ; McNeil et al., 1990; Rote, 1996). Anti-cardiolipin antibodies recognize a particular region (between residue 281 and residue 288) on the

142

<img file="MX337052B_D0142.tif" />

Β92-glycoprotein I, while lupus anticoagulants similarly recognize, anti-PE antibodies that are present in disease state bind to PE in combination with proteins, such as e.1 high molecular weight kininogen (HK), precalicrein and factor XI (Sugi and Mclntyre, 1995; 1996^to; 1996b). Based on this type of protein recognition, anti-phospholipid antibodies in patients displace the protein cofactors of phospholipids, thereby creating symptoms of the disease.

The antibodies of the present invention have been particularly selected on the basis that they do not bind aminophospholipids and anionic phospholipids in combination with protein cofactors, but rather are true antiphospholipid antibodies. As such the antibodies of the invention do not bind to or displace the protein cofactors of phospholipids and are therefore safe to administer. Indeed, mice treated with the antibodies of the invention at high doses, for prolonged periods, showed no change in coagulation capacity, but the mice respond with APS when injected with anticardiolipin or with anticoagulant antibodies of *

Regardless of the fundamental mechanisms, lupus.

143 ; -χ .. ..i., the anti-phospholipid antibodies that are presented eri.

INDUS J'aiAL human population are correlated with autoimmune diseases, for example with systemic lupus erythematosus (Branch et al., 1987; Staub et al., 1989; Drouvalakis and Buchanan, 1998; Smirnov et al., 1995; Rauch et al., 1986; Rauch and Janoff 1990) and recurrent pregnancy loss (rote et al., 1995; Rote, 1996; Vogt et al., 1996; 1997;

Katsuragawa et al., 1997). Neither of these symptoms have been associated when the antibodies of the present invention are administered to mice or monkeys.

Also, the epitope recognized by the antibodies of the invention, such as antibodies 9D2 and 3G4 (ATCC 4545), is not the same as that recognized by annexin V. This is demonstrated herein as the agents do not block crossed the linkage with each other to phospholipids. The epitope recognized by the 3G4 and 9D2 antibodies is probably a hexagonally packed form of PS which is the immunogenic form. Annexin V probably binds to flat PS in addition to the hexagonal shape. The hexagonal form of PS concentrates in bumps on the plasma membrane associated with cell activation and in globules in apoptotic cells. The restricted distribution of the antibodies of the invention, such as the 9D2 and 3G4 antibodies (up to 45), then further contributes to the lack of

144 detectable toxicity and coagulation of antibodies.

<img file="MX337052B_D0143.tif" />

In order to generate antibodies to CPSar aminophospholipids and anionic phospholipids, with advantageous properties and / or without side effects or reduced side effects, the present invention provides immunization and screening methods. Other immunization and antibody techniques have been reported in the literature (Umeda et al., 1989; Igarashi et al., 1991; rote et al., 1990), including those with reported specificity for the type of fatty acid chains involved ( Levy et al., 1990, Qamar et al., 1990). However, the immunization techniques herein, and particularly the selection of antibodies that are not serum dependent, provide particular benefits.

Umeda et al., (1989) reported the production of monoclonal antibodies that recognize stereospecific phosphatidylserine epitopes. However, the Umeda system suffers from the disadvantage of using direct immunization of phosphatidylserine in the spleen of the mouse using a sample of aminophospholipid coated with Salmonella (Umeda et al., 1989). Many of the antibodies reported by Umeda et al., (1989) also exhibit anticoagulant activity, which is a disadvantage not

145 associated with

<img file="MX337052B_D0144.tif" />

binding profile of the antibody 3G4 CT ~^rria ± £ e.xtt.nLit al · of the PSC8 antibody from Umeda et al., (1989).

The antibodies of the present invention have the advantage of recognizing all or most of the anionic aminophospholipids, which may provide more targets for binding. Therefore, the second generation antibodies of the invention can be defined as those that have the same, or substantially the same, specificity for the phospholipid as the 9D2 or 3G4 antibodies (ATCC 4545), as described in the table 4 hereof, and as it is not dependent on serum.

Igarashi et al., (1991) also report the induction of anti-PS antibodies, but use intrabazal immunization again and only a slight increase in titer was observed when the antigen was re-injected intravenously. Most of the monoclonal antibodies of Igarashi et al., (1991) cross-reacted with DNA and many exhibited lupus anticoagulant activity, none of which disadvantages exist in the antibodies developed by the present inventors. The binding profile of the preferred 3G4 antibody of the invention is also different from that of the cr ~ antibodies.

146 Table 1 of Igarashi t al., (1991).

<img file="MX337052B_D0145.tif" />

Lupus anticoagulant activities of murine monoclonal antibodies that cross-react with more than one anion phospholipid have been reported by others (Alving et al., 1987; Rauch &amp; Janoff, 1990), but the inventors of the present have not experimented Difficulty obtaining antibodies free of lupus anticoagulant activity. This represents a distinct advantage of competing methods, antibodies, and antibodies in accordance with the present invention.

In addition to avoiding the use of patient antibodies, as described in Rauch et al., (1986), Hasegawa et al., (1994), Ravirajan et al., (1995) and Menon et al., (1997) The present application also demonstrates the advantageous properties of the antibodies provided by this invention in one-to-one comparisons with existing antibodies in the literature, such as the 3SB antibody described by Rote et al., (1993). Although 1 3SB antibody has properties appropriate for use in several of the methods described herein, the antibodies developed by the inventors herein nonetheless reproduce the 3SB antibody in comparative studies, for example as shown herein for increased antiviral effects. of the 3G4 antibodies as opposed to the 3SB antibody (example XIII).

147

<img file="MX337052B_D0146.tif" />

the present invention affinity. Before the literature they had one where it is reported) in

Antibodies can be characterized by their invention, antibodies at relatively weak affinity (in certain embodiments, the second generation antibodies of the invention and are then defined as those having an affinity for PS of at least equal affinity for 9D2 or 3G4 (ATCC 4545) antibodies, particularly when measured in an ELISA as described in the present one, described in Table 3, and for not being dependent on serum.

More preferably, the second generation antibodies of the invention are defined as those having an affinity for PS at least equal to the affinity of antibodies 9D2 or 3G4 (ATCC 4545) for PS, as described in Table 3, and because they have substantially the same, or the same, specificity for phospholipid, as antibodies 9D2 or 3G4 (ATCC 4545), as described in Table 4, and because they are not dependent on serum. Most preferably, second generation antibodies are those that have an affinity for PS at least equal to the affinity of the 3G4 antibody (ATCC 4545) for PS, as described in Table 3, and because they have the same specificity for phospholipid as the 3G4 antibody (ATCC 4545), as described in

148

<img file="MX337052B_D0147.tif" />

Table 4, and for not being dependent on serum.

<img file="MX337052B_D0148.tif" />

D. CDR technology

Antibodies are made up of variable and constant regions. The term "variable", as used herein, with reference to antibodies, means that certain portions of the variable domains differ extensively in sequence between antibodies, and are used in the binding and specificity of each particular antibody to its particular antigen . However, variability is not uniformly distributed across all variable antibody domains. It is concentrated in three segments called hypervariable regions, in the variable domains both «

light chain and heavy chain (different from the camelized antibodies analyzed later).

The most highly conserved portions of the variable domains are called the region of structure (FR). The variable domains of the native heavy and light chains each comprise four FRs (FR1, FR2, FR3, and FR4, respectively), which largely adopt a "leaf" configuration, connected by three hypervariable regions, which form curls that connect and in some cases are part of the leaf structure «o.

The regions of hypervariability in each

<img file="MX337052B_D0149.tif" />

chain are kept close together

<img file="MX337052B_D0150.tif" />

149

<img file="MX337052B_D0151.tif" />

FRs and, with the hypervariable regions -'lu other CTidenj = j contribute to the formation of the antigen binding site of the antibodies (Kabat et al., 1991, specifically incorporated herein by reference). Constant domains are not directly involved in binding an antibody to an antigen, but they do exhibit several effector functions, such as the involvement of the antibody in antibody-dependent cellular toxicity.

The term hypervariable region, as used herein, refers to amino acid residues of an antibody that are responsible for binding to the antigen. The hypervariable region comprises amino acid residues' of a complementarity determining region or CDR (i.e. residues 24-34 (Ll), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-56 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., 1991, specifically incorporated herein by reference) and / or those hypervariable curl residues (i.e. residues 26-32 (Ll), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain).

Structure residues or FR are those residues of the

150

<img file="MX337052B_D0152.tif" />

Variable domain other than the hypervariable already defined here.

<img file="MX337052B_D0153.tif" />

The DNA and deduced amino acid sequences of the VH and V chains of the 3G4 antibody (ATCC 4545) provided herein as SEQ ID NO: 1, 2, 3, and 4, respectively. These sequences span the CDR1-3 of the variable regions of the heavy and light chains of the antibody. In view of the sequence and other information provided herein, and knowledge of the art, a range of improved and improved 3G4-like antibodies and antigen binding regions can now be prepared and are thus encompassed by the present invention.

In certain embodiments the invention provides at least one CDR of the antibody produced by the deposited hybridoma as ATCC 4545. In other embodiments the invention provides a CDR, antibody or antigen binding region thereof, which binds at least to a first aminophospholipid or anionic phospholipid, preferably PS, and comprising at least one CDR of the antibody produced by the deposited hybridoma as ATCC 4545.

Important aspects of the invention concern at least one CDR having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or a variant

151 or mutagenized form of the invention concerns the same.

Others

<img file="MX337052B_D0154.tif" />

CDR, antibody, or antigen-binding region thereof, which binds at least a first aminophospholipid or anionic phospholipid, preferably PS, and which comprises at least one CDR with an amino acid sequence of SEQ ID NO: 2 or SEQ ID

NO: 4, or a variant or mutagenized form thereof, wherein that variant or mutagenized form maintains binding to the aminophospholipid or anionic phospholipid, preferably PS.

In a particular embodiment the invention provides an antibody, or an antigen binding region thereof, in which the regions of the 3G4 (ATCC 4545) antibody structure have been changed from mouse to human IgG, such as IgGi human or another IgG subclass to reduce immunogenicity in humans. In other embodiments, the sequence of the 3G4 antibody (ATCC 4545) is examined for the presence of T-cell epitopes, as is known in the art. The underlying sequence can then be changed to remove epitopes from T cells, that is, to deimmunize the antibody.

The availability of the DNA and amino acid sequences of the Vh and V chains of the 3G4 antibody (SEQ ID NO: 1, 2, 3 and 4) means that a

152

<img file="MX337052B_D0155.tif" />

<img file="MX337052B_D0156.tif" />

antibody range using CDR technology.^{go: s}T ^_n pá'Éfeí INÚ'iJSTRlAL random mutations are made in the CDR and the products are screened to identify antibodies with higher affinities and / or higher specificities. Such mutagenesis and selection is routinely practiced in antibody techniques. It is particularly convenient for use in the present invention, given the advantageous screening techniques described herein.

These techniques are used to generate antibody variants with improved biological properties relative to the original antibody from which they are prepared, such as antibodies 9D2 and 3G4 (ATCC 4545). Those variants, or second generation compounds, are typically substitution variants that involve one or more substituted, hypervariable region residues of an original antibody. A convenient way to generate those^- substitution variants is affinity maturation using the phage display.

In affinity maturation using phage display, - several sites in the hypervariable region (eg 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent form of filamentous phage particles, as fusions to the M13 gene III product packaged within each particle.

153

<img file="MX337052B_D0157.tif" />

phage display are selected later with respect ₍ to its biological activity (binding affinity) as described herein. In order to identify candidate hypervariable region sites, for modification, alanine scanning mutagenesis can be performed for identified hypervariable region residues that contribute significantly to antigen binding.

CDR intermingling and implant technologies can also be used with the antibodies of the present invention ·, preferably the 9D2 and 3G4 antibodies (ATCC 4545). CDR intermixing inserts CDR sequences into a specific region of the framework (Jirholt et al., 1998, specifically incorporated herein by reference. CDR implantation techniques allow the random combination of CDR sequences into a single master structure (Soderlind et al., 1999, 2000, each incorporated herein by reference). Using those techniques, the CDR sequences of the 3G4 antibody (ATCC 4545), for example, are mutagenized to create a plurality of different sequences, which are incorporated into a scaffold sequence and the resulting antibody variants selected with respect to the desired characteristics , for example a higher affinity.

154

M. r I

In view of the pres'giigg ^ information / description, the antigen binding fragment d 1 of the antibodies, preferably of the 9D2 3G4 antibodies (ATCC 4545), can also be minimized, providing improved stability. This can be accomplished by preparing single domain binding proteins based on the V domains._H and similar to V_H of immunoglobulin (Nuttall et al., 2000, specifically incorporated herein by reference).

Alternatively or additionally, the crystal structure of the antigen-antibody complex may be delineated and analyzed to identify contact points between the antibody and the target anionophospholipid or phospholipid, eg, PS. Those contact residues and neighboring residues are candidates for substitution. One such variant is generated, the variant panel is screened, as described herein, and antibodies with analogous but different or even superior properties, in one or more relevant assays, are selected for further development.

D3. Meat1 Antibodies

Additional examples of antibodies of the invention are camelized antibodies.

The exsSBiaaasj

155 camel and llama antibodies (camelildae; camel ^ s ^ i ^ includes a unique type of antibody, the &lt;-nai g &lt;=&gt; is devoid of light chains and is then made up of only heavy chains. These have been termed camelized antibodies. The antigen binding site of these antibodies is a single domain, referred to as Vhh (VHH).

As the DNA and amino acid sequences of the Vh and V chains of the 3G4 antibody (ATCC 4545) are provided herein (SEQ ID Nos: 1, 2, 3 and 4), camelized versions of the 3G4 antibody can be prepared. Mutations and structural adaptations can be performed to reshape a V_H of a pair V_H-V_L in a single domain Vhh with retention of sufficient variability (Muyldermans et al., 2001, specifically incorporated herein by reference). These Vhh constructs are small, robust, and efficient recognition units (Riechmann and Muyldermans, 1999) with potent antigen-binding ability, which can provide the additional advantage of interacting with novel epitopes that are inaccessible to V pairs._H-V_L conventional. In this way, camelized antibodies are related to the Fv fragments but may have additional benefits.

The United States Patent of

North America No. 5,800,988, the United States Patent

156

-AL «C- V

United States of America No. 6,005,079, sdT ^ ttud PCp No. 94/25591, Riechmann &amp;

WO 94/04678, PCT application WO

Muyldermans (1999) and Muyldermans et al., (2001) are each specifically incorporated herein by reference for the purpose of further describing and allowing the production of camelized antibodies.

Therefore, the

CDR of the 3G4 antibody can be inert on the Camelidae antibody heavy chain immunoglobulin variable domain structure.

D4. CDR sequences

Further aspects of the invention then concern

<img file="MX337052B_D0158.tif" />

Isolated DNA segments and recombinant vectors encoding CDR regions of antibody heavy and light chains, such as 9D2 and

3G4, and for example 3G4 (ATCC 4545), heavy and light chains, and the creation and use of recombinant host cells and phages through the application of DNA technology, which expresses those CDR regions.

The invention then provides an isolated polynucleotide that contains a nucleotide sequence encoding at least one CDR of the antibody produced by the deposited hybridoma as ATCC 4545. The invention further provides an isolated polynucleotide that

157

<img file="MX337052B_D0159.tif" />

it contains a nucleotide sequence that coolmf¿Reads a CDR, antibody, or antigen binding region thereof, which binds at least a first aminophospholipid or anionic phospholipid, preferably PS, and which comprises at least one CDR of the antibody produced by the hybridoma deposited as ATCC 4545.

Further aspects of the invention concern an isolated polynucleotide containing a nucleotide sequence encoding at least one CDR having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or a variant or mutagenized form of the same. Other aspects of the invention pertain to an isolated polynucleotide containing a nucleotide sequence encoding a CDR, antibody, or antigen-binding region thereof, which binds to at least a first anionophospholipid or phospholipid, preferably PS, and comprising a CDR with an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or a variant or mutagenized form thereof, where that variant or mutagenized form maintains the binding to the aminophospholipid or to the anionic phospholipid, preferably PS.

In other aspects of the invention, the isolated polynucleotide contains the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or a variant or form

158

Or ^ go mutagenized from it. In particular • the ^ j ^ fel'inüciéótldd¹ E lai-to :? V / · · - ^ 7 isolated contains the nucleotide sequence of í SÉQ ~ lb N0: lo SEQ ID NO: 3, or a variant or mutagenized form thereof, wherein the nucleotide sequence encodes a CDR, antibody, or antigen-binding region, thereof, that binds at least one anionic phospholipid or aminophospholipid, preferably PS, wherein any of these mutagenized variants or forms maintains binding to the anionic phospholipid or phospholipid, preferably PS.

The present invention then concerns a polynucleotide and DNA segments, which can be isolated from any mammal, preferably human or murine, which are free of total genomic DNA and which can express heavy and light chain CDR regions of an antibody. anti-anionic phospholipid or anti-aminophospholipid, such as 9D2 and 3G4, and preferably 3G4 (ATCC 4545), heavy and light chains. As used herein, the terms "polynucleotide segment" and "DNA segment" refer to polynucleotides and DNA molecules that have been isolated free of the total genomic DNA of a particular species. Included within the term polynucleotide segment and DNA segment are DNA segments and smaller fragments of those fragments, and also recombinant vectors,

159

Ha JLV. · '. .ri. ·. -.including, for example, plasmids, cosmids'^NSTíago.s, vi

<img file="MX337052B_D0160.tif" />

and the like.

Similarly, a DNA segment comprising an encoding segment or an isolated gene portion encoding purified CDR regions of heavy and light chains of anti-anionic phospholipid or anti-aminophospholipid antibodies, such as 9D2 and 3G4, and preferably the 3G4, heavy and light chains, refers to a segment of DNA that includes those coding sequences and, in certain respects, regulatory sequences, Isolated substantially far from other naturally occurring genes or protein coding sequences. In this regard the term gene is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be understood by those skilled in the art, this functional term includes the coding sequences of native antibodies and smaller engineered segments that express, or can be adapted to express, appropriate antigen-binding proteins, polypeptides, or peptides.

Isolated substantially away from other coding sequences means that the coding segment or portion of interest of the isolated gene forms the significant part of the coding region of the segment

160 of DNA, and that the DNA segment does not have portions

OF THE C; ) '- Large naturally occurring coding DNA JFs, such as large chromosomal fragments or other functional genes or coding regions of cDNA. Since this refers to the DNA segment as it was originally isolated and does not exclude genes or coding regions subsequently added to the segment by human hands.

In particular embodiments the invention relates to isolated coding segments or portions of isolated genes and recombinant vectors incorporating DNA sequences encoding CDR regions of heavy and light chains of anti-anionic phospholipid or anti-aminophospholipid antibodies, such as 9D2 and 3G4, ' and preferably 3G4, heavy and light chains, comprising at least a first sequence region that

<td colspan="4">includes an amino acid sequence region</td><td colspan="2">of at least</td>

<td>approximately</td><td> 75%,</td><td>plus</td><td>preferably</td><td>to the</td><td>less</td>

<td>approximately</td><td> 80%,</td><td>plus</td><td>preferably</td><td>to the</td><td>less</td>

<td>approximately</td><td> 85%,</td><td>plus</td><td>pre fe rentement e,</td><td>to the</td><td>less</td>

<td>approximately</td><td colspan="2"> 90%, 91%, 92%,</td><td>93%, 94%, and in</td><td colspan="2">the most</td>

<td>preferred to</td><td>less</td><td colspan="2">about 95%, 96%,</td><td> 97%,</td><td>98% or</td>

99% or similar identity value in the amino acid sequence with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; where the CDR regions keep the

161 less substantially the properties

<img file="MX337052B_D0161.tif" />

CDR regions

INDUSTIUAL amino acid sequence SEQ ID NO: 2 or SEQ

ID N0: 4.

sequences

As described herein, they can be comprised of certain amino acids equivalent in biological functionality or conservative substitutions.

Other sequences may comprise amino acids functionally substitutions deliberately conservative non-equivalent properties engineered to enhance CDR or CDR-containing antibody, as known to the art and described additional additional amino acids,

Also those of ordinary experience in addition to the present.

It will be understood that the nucleic acid sequences may include residues such as amino acids or 5 'or 3' sequences, a sequence sequence previously compliant, and maintenance of the invention, with which and improvement of the

N-terminal or C-terminal, and still correspond as long as the criteria presented preferably include the biological activity of the protein where the expression of the protein is involved.

The addition of terminal sequences includes several non-coding sequences that flank the portions either 5 'or

3 'from the coding region and

162

<img file="MX337052B_D0162.tif" />

nucleic

MEXICAN INSTITUTE

OF INDUSTRIAL PROPERTY also from the control regions.

The acid segments of the present invention can then be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their length Total may vary considerably. Therefore it is contemplated that a nucleic acid fragment of almost any length can be employed, and the overall length is preferably limited by the ease of preparation and use in the recombinant DNA protocol used.

Recombinant vectors form additional aspects of the present invention.

particularly useful are contemplated as then

Vectors those vectors in which the coding portion of the DNA segment is located under the control of a promoter.

Generally, although not exclusively, a recombinant or heterologous promoter will be used, that is, a promoter not normally associated with coding sequences in their natural environment. Those promoters may include bacterial, viral, eukaryotic, and mammalian promoters, as long as the promoter effectively directs expression of the DNA segment in the cell type, organism, or

163 inclusive animal

<img file="MX337052B_D0163.tif" />

<img file="MX337052B_D0164.tif" />

The use of promoter and cell type combinations for protein expression is known to those skilled in the art of molecular biology. The promoters employed can be constitutive, or inducible, and can be used under appropriate conditions to drive high-level expression of the introduced DNA segment, such as is advantageous in large-scale production of recombinant proteins or peptides.

Expression of the nucleic acid sequences of the invention may conveniently be through any of one or more standard procedures known to those of ordinary skill in the art and are further described herein. For example, the latest description of recombinant expression of fusion proteins applies equally well to antibodies and antibody fragments that are not functionally encoding sequence associated with other nucleic acids.

E. Additional Preparation Techniques

Antibodies

The. Phagemid Library Antibodies

164

<img file="MX337052B_D0165.tif" />

Recombinant technology now enables antibodies that have the specificity of recombinant genes encoding an antibody (Van Dijk et al., 1989;

this for reference). Certain recombinant techniques use the isolation of antibody genes by immunological selection of combinatorial immunoglobulin phage display libraries prepared from RNA isolated from the spleen of an immunized animal (Morrison et al., 1986; Winter and Milstein, 1991; Barbas et al. ., 1992; each incorporated herein by reference).

For these methods, the combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the spleen of the immunized animal, and phagemids expressing suitable antibodies are selected by panning, using cells that express the cell antigen. of control. The advantages of this approach over conventional hybridoma techniques are that approximately 10⁴ times »

so many antibodies can be produced and selected in a single round, and that new specificities are generated by the combination of the H chain and the L chain, which further increases the percentage of suitable antibodies generated.

165

<img file="MX337052B_D0166.tif" />

,,, ¡ΝΗΠΤΙΚΌ!: Ί \ '' ·

A method for generation- '_;;.^:from url.gran. Repertoire of various antibody molecules in bacteria use bacteriophage lambda as the vector (Huse et al., 1989; incorporated herein by reference). The production of antibodies using the lambda vector involves cloning heavy and light chain populations of the DNA sequences into separate initial vectors. The vectors are subsequently randomly combined to form a single vector that directs heavy and light chain co-expression to form antibody fragments. Heavy and light chain DNA sequences are obtained by amplification, preferably by polymerase chain reaction (PCR * ®) or a related amplification technique, of mRNA isolated from spleen cells (or hybridomas thereof) of an animal that has been immunized with a selected antigen. Heavy and light chain sequences are typically amplified using primers that incorporate restriction sites at the ends of the amplified DNA segment to facilitate cloning of the heavy and light chain segments into the initial vectors.

Another method for the generation and selection of large libraries of whole or partially synthetic antibody combination sites, or paratopes, uses display vectors derived from filamentous phage, such

166 like MI 3 fl or fd.

These filamentous vectors, known as

<img file="MX337052B_D0167.tif" />

Phagemids produce large monoclonal antibody libraries that have diverse and novel immunospecificities. The technology uses a filamentous phage coat protein membrane anchor domain as a means to link the gene product and gene during the assembly stage of filamentous phage replica, and has been used for the cloning and expression of antibodies from Combinatorial libraries (Kang et al., 1991; Barbas et al.

1991) each incorporated herein by reference).

This general technique for display of filamentous phage is described in US Patent No. 5,658,727, incorporated herein by reference. In a more general sense, the method provides a system for the simultaneous cloning and selection of preselected ligand binding specificities from antibody gene repertoires using a single vector system. Selection of isolated library members for a preselected ligand binding capacity allows correlation of the binding capacity of an expressed antibody molecule with a convenient means of isolating the gene encoding the library member.

<img file="MX337052B_D0168.tif" />

<img file="MX337052B_D0169.tif" />

MEXICAN INSTITUTE

The binding of the expression and carried out by combining the targeting of a fusion polypeptide in the periplasm of a bacterial cell to allow the assembly of a functional antibody, and the targeting of a fusion polypeptide on the coating of a particle of filamentous phage during phage assembly to allow convenient selection of the library member of interest. Periplasmic targeting is provided by the presence of a secretion signal domain in a fusion polypeptide. Targeting of a phage particle is provided by the presence of a filamentous phage coat protein membrane anchor domain (ie, a membrane anchor domain derived from cpIII or cpVIII) in a fusion polypeptide.

The diversity of a filamentous phage-based combinatorial antibody library can be increased by changing heavy and light chain genes, altering one or more of the complementarity determining regions of cloned library heavy chain genes, or introducing random mutations into the library by error-prone polymerase chain reaction. Other methods for selecting phagemid libraries are described in United States Patent Nos. 5,580,717;

168 the present for reference.

Another method for the selection of large s combinatorial antibody libraries has been developed, using the expression of diverse populations of heavy and light chain sequences on the surface of a filamentous bacteriophage, such as' M13, of the fl or fd (US Patent United States of America Number

5,698,426; incorporated herein by reference). Two populations of various heavy (He) and light (Le) chain sequences are synthesized by the polymerase chain reaction (PCR)¹¹). These populations are cloned into separate M13-based vectors containing the elements

<img file="MX337052B_D0170.tif" />

necessary for expression. The heavy chain vector contains a gene coat protein sequence

HIV (gvm), so that translation of the heavy chain sequence produces the gVIII-Hc fusion proteins. Populations of two vectors are randomly combined, so that only the portions of the vector containing the He and Le sequences are joined into a single circular vector.

The combined vector directs the co-expression of both the He and Le sequences to assemble the two polypeptides and the surface expression in M13 (US Patent 25, Number 5,698,426;

169

He and Le are different within two different populations in a single vector. The donated vector sequences of each independent vector are necessary for the production of a viable phage. Also, since the pseudo gVIII sequences contain only one of the two initial vectors, the co-expression of functional antibody fragments such as Le-associated gVlll-Hc fusion proteins cannot be carried out on the phage surface until the Vector sequences are linked into a single vector.

The surface expression of the antibody library is performed in a suppressor strain of amber. An amber stop codon between the He sequence and the gVIII sequence binds the two components into a non-suppressor strain. Isolating the phage produced from the non-suppressor strain and infecting a suppressor strain will bind the He sequences with the gVIII sequence during expression. Cultivating the suppressor strain after infection allows co-expression on the M13 surface of all antibody species within the library as gVIII fusion proteins (gVIII-Fab fusion proteins). Alternatively, DNA can be isolated from the non-suppressor strain and then introduced into

170

<img file="MX337052B_D0171.tif" />

a suppressor strain to carry out the same effect. ¿

The surface expression library is selected to determine specific Fab fragments that bind to preselected molecules by standard affinity isolation procedures. These methods include, for example, panning (Parmley and Smith, 1988; incorporated herein by reference), affinity chromatography, and solid phase staining procedures. Panning is preferred because high phage titers can be selected easily, quickly, and in small volumes. Furthermore, this procedure can select minor fragments of species Fab within the population, which would otherwise have been undetectable, and amplify to substantially homogeneous populations. Selected Fab fragments can be characterized by sequencing the nucleic acids encoding the polypeptides after amplification of the phage population.

Another method of producing different antibody libraries and selecting desirable binding specificities are described in United States Patent No. 5,667,988, and United States Patent No. 5,759,817, each incorporated herein by reference. The method involves preparation

171 τ ιχ + from heterodimeric molecule libraries in the form of phagemid libraries using degenerate oligonucleotides and primer extension reactions to incorporate degenerations into the 5 regions of DRC from variable immunoglobulin heavy and light chain domains, and display mutagenized polypeptides on the surface of the phagemid. After that, the deployment protein is selected for its ability to bind to a preselected 10 antigen.

lnm ^ pC [lpbuli4a

The method for producing a heterodimeric immunoglobulin generally introduces a molecule region coding gene for involves (1)

Heavy or light chain V of interest in the phagemid display vector; (2) introducing a phagemid randomized binding site by the oligonuleotide primer extension unfolding protein vector containing regions of homology

DRC gene vector encoding regions with a for a region V randomized generation of antibody and containing to produce sequences to form a large unfolding population, each capable of expressing different putative binding sites deployed (3) expressing the unfolding protein and the site of a phagemid surface unfolding protein;

172 bonding on the surface of a filamentous particle; and (4) isolate (select) the surface-expressed phage / strain using affinity techniques, such as panning of phage particles against a previously selected antigen, thereby isolating one or more phagemid species. containing a deployment protein that contains a binding site that binds to a preselected antigen.

Another variation of this method for producing various antibody libraries and selecting for desirable binding specificities is described in United States Patent No. 5,702,892, incorporated herein by reference. In this method, only heavy cad sequences are used, heavy chain sequences are randomized at all nucleotide positions, which encode either the RDCI or RDCIII hypervariable region, and genetic variability in RDCs is generated regardless of any biological process.

In the method, two libraries are technically designed to genetically change the oligonucleotide motifs within the framework of the heavy chain gene structure. In all the random mutation, either of the RDCI or RDCIII, the hypervariable regions in the heavy chain gn were reconstructed to give

173 result a collection

<img file="MX337052B_D0172.tif" />

of¹”™»

INDUSTRIAL

<img file="MX337052B_D0173.tif" />

diverse. Heavy chain proteins encoded by the collection of mutated gene sequences possess the potential to have all the binding characteristics of an immunoglobulin, while requiring only one of the two immunoglobulin chains.

Specifically, the method is practiced in the absence of the immunoglobulin light chain protein. A phage library that displays 10 modified heavy chain proteins is incubated with an immobilized ligand to select clones encoding recombinant proteins that specifically bind to the immobilized ligand. The bound phage dissociates from the immobilized ligand and is amplified by culture in bacterial host cells. The individual viral plates, each expressing a different recombinant protein, are spread, then the individual clones can be tested to determine their binding activity.

B7. Human Lymphocyte Antibodies

Antibodies against phospholipids are

Present in the Human Population. However these antibodies are typically associated with the disease and their use in the present invention should preferably be avoided. However, lymphocytes

174 Humans from healthy subjects may appropriate for starting materials to '1 ·' ', A. -'- r sgr used as:

In order to generate an antibody for use in the invention.

In vitro immunization, or stimulation

<img file="MX337052B_D0174.tif" />

of antigens, can be used to generate a human antibody for use in the present invention. These techniques can be used to stimulate the peripheral blood lymphocytes of normal, healthy subjects simply by stimulating cells that produce anionic phospholipid and aminophospholipid antibodies in vitro.

This in vitro immunization involves antigen-specific activation of unimmunized B lymphocytes, generally within a mixed population of lymphocytes (mixed lymphocyte cultures, MLC). The 15 in vitro immunizations can also be supported by B cell culture and differentiation factor and lymphokines. The antibodies produced by these methods are frequently IgM antibodies (Borrebaeck et al., 1986; incorporated herein by reference).

Another method has been described (Patent of the

United States of America Number 5,681,729, incorporated herein by reference), where human lymphocytes that primarily produce 25 IgG (or IgA) antibodies are obtainable. The method involves, in a

175

<img file="MX337052B_D0175.tif" />

generally speaking, transplanting immunodeficient human lymphocytes so that they take them into the animal's body; immunizing the animal with a desired antigen, so that it generates human lymphocytes that produce an antigen-specific antibody; and recovering human lymphocytes by producing the antibody from the animal. The human lymphocytes thus produced can be used to produce a monoclonal antibody by immortalizing the antibody-producing human lymphocytes, cloning the obtained immortalized human-derived lymphocytes that produce the antibody, and recovering a monoclonal antibody specific for the desired antigen from the human-derived lymphocytes. immortalized cloned.

The immunodeficient animals that can be used in this technique are those that do not exhibit rejection when human lymphocytes are transplanted into animals. These animals can be artificially prepared through physical, chemical or biological treatments. Any immunodeficient animal can be used. Human lymphocytes can be obtained from human peripheral blood, spleen, lymph nodes, tonsils, or the like.

Taking of transplanted human lymphocytes - in animals can only be achieved

176 administering human lymphocytes

<img file="MX337052B_D0176.tif" />

Route of administration is not restricted and may be, for example, subcutaneous, intravenous, or intraperitoneal. The human lymphocyte dose is not restricted, and can usually be 10⁶ up to 10® lymphocytes per animal. The immunodeficient animal is then immunized with the

VEGF desired.

After immunization, human lymphocytes are recovered from the blood, spleen, lymph nodes, or other lymphatic tissues by any conventional method. For example, mononuclear cells can be separated by the centrifugation method

<img file="MX337052B_D0177.tif" />

Ficoll-Hypaque (specific gravity: 1,077), and the monocytes recovered by the plastic plate 15 adsorption method. Contaminating cells originating from the immunodeficient animal can be removed using an antiserum specific to the animal's cells. Antiserum

<td>you can get for example</td><td colspan="2">immunizing</td><td>a</td><td>second</td>

<td>distinct animal with cells</td><td>of the</td><td>spleen</td><td>of the</td><td>animal</td>

<td>20 immunodeficient, and recovering</td><td>the</td><td>serum</td><td>of the</td><td>animal</td>

immunized differently.

antiserum

The treatment can be carried out at any stage. Human lymphocytes can also be recovered by an immunological method using a human immunoglobulin expressed on the cell surface as a marker.

177

Using these methods,

<img file="MX337052B_D0178.tif" />

INDUSTRIAL

<img file="MX337052B_D0179.tif" />

human lymphocytes that mainly produce antibodies

IgG and IgA specific to one or more selected anionic phospholipids and aminophospholipids. The monoclonal antibodies are then obtained from human lymphocytes by immortalization, selection, cell growth, and antibody production.

E3. Transgenic Mice Containing Human Antibody Libraries

Recombinant technology is now available for the preparation of antibodies. In addition to the combinatorial immunoglobulin phage display libraries described above, another approach to molecular cloning is to prepare antibodies from transgenic mice containing human antibody libraries. These techniques are described in United States Patent No. 5,545,807, incorporated herein by reference.

In a more general sense, these methods involve the production of a transgenic animal that has inserted into its germline genetic material that codes for at least part of a human-derived immunoglobulin, or that can rearrange to code for a repertoire of immunoglobulins. Genetic material

178 and 7.77. ^Λ··^; 'Χ7 inserted can be produced from a feⁱhuman source,: ^ or can be produced synthetically. The material can * encode at least part of a known SñunogTobul ina, or can be modified to encode at least part of an altered immunoglobulin.

The inserted genetic material is expressed in the transgenic animal, resulting in the production of an immunoglobulin derived at least in part from the inserted human immunoglobulin genetic material. It is found that the genetic material is rearranged in the transgenic animal, so that a repertoire of immunoglobulins with part or parts derived from the inserted genetic material can be produced, even if the inserted genetic material is incorporated into the germline in the wrong position, or with the wrong geometry.

The inserted genetic material may be in the form of cloned DNA in prokaryotic vectors, such as plasmids and / or cosmids. Larger fragments are inserted using yeast artificial chromosome vectors (Burke et al., 1987; incorporated herein by reference), or by introducing chromosome fragments (Richer and Lo, 1989; incorporated herein by reference). ). The inserted genetic material can be introduced into the host in a conventional manner, for example, by injection or other

179

ΙΤΧ / Τ Τ7 'Τ Μ' · 7 j; &lt;in 7 cells.

INSTITUI · ..: .. ·. .

OF THE totipotent embryonic.

In preferred aspects, an an ± ± lTcré§ped "that does not initially carry the genetic material encoding immunoglobulin constant regions is used, so that the resulting transgenic animal will use only the inserted human genetic material when producing immunoglobulins. This can be accomplished either by using a naturally occurring mutant host that lacks the relevant genetic material, or by artificially mutating, for example, in cell lines to eventually create a host, from which the relevant genetic material is has removed.

When the host animal carries genetic material encoding immunoglobulin constant regions, the transgenic animal will carry naturally occurring genetic material and inserted genetic material, and will produce immunoglobulins derived from naturally occurring genetic material, inserted genetic material, and mixtures of both types of genetic material. In this case, the desired immunoglobulin can be obtained by selecting hybridomas derived from the transgenic animal, for example, exploiting the phenomenon of allelic exclusion of the expression of the antibody gene or the loss of differential chromosome.

180

As soon as the animal has been prepaj ^ g 'Ιτίη

INSTITUTE \ ~ 't

FROM the convenient transgenic i- ... · ·, the animal is simply immunized with the desired immunogen. Depending on the ^ amaturaiefca of the inserted material, the animal can produce a chimeric immunoglobulin, eg, of mixed mouse / human origin, where the genetic material of foreign origin encodes only part of the immunoglobulin; or the animal may produce a completely foreign immunoglobulin, eg, of entirely human origin, where the genetic material of the foreign origin encodes an entire immunoglobulin.

Polyclonal antiserum can be produced from the transgenic animal after immunization. Immunoglobulin-producing cells can be removed from the animal to produce the immunoglobulin of interest. Preferably, monoclonal antibodies are produced from the transgenic animal, for example, by fusing spleen cells from the animal with myeloma cells and selecting the resulting hybridomas to choose those that produce the desired antibody. Appropriate techniques for these processes are described herein.

In an alternative approach, the genetic material can be incorporated into the animal, so that the desired antibody is produced in body fluids, such as serum or external animal growths, such

181 like milk, colostrum or saliva. By ejeiX

<img file="MX337052B_D0180.tif" />

in vitro genetic material that encodes at least part of a human immunoglobulin in a mammalian encoding gene for a milk protein and then introduces the gene into a fertilized mammalian egg, for example, by injection, the egg can be grown in an adult female mammal producing milk containing immunoglobulin derived at least in part from the inserted human immunoglobulin genetic material. The desired antibody can be harvested from milk. Convenient techniques for carrying out these processes are known to those skilled in the art.

The above transgenic animals are usually employed to produce human antibodies of a single isotype, more specifically an isotype that is essential for the maturation of B cells, such as IgM and possibly IgD. Another preferred method of producing human antibodies is described in US Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016 and 5,770,429; each incorporated herein by reference, where transgenic animals are described that are capable of changing from an isotype necessary for the development of B cells to other isotypes.

In the development of a B lymphocyte, the cell

182

<td>initially</td><td>T 7 \ T ' produces IgM with an efficiency of</td>

<td>linkage</td><td>productively determined by regions</td>

<td>rearranged</td><td>V_H and V_L. Subsequently, each B cell and its</td>

Progeny cells synthesize antibodies with the same V regions of L and H chains, but can change the H chain isotype. The use of mu or delta constant regions is largely determined by alternating separation, allowing IgM and IgD to co-express in one single cell. The other heavy chain isotypes (gamma, alpha, and epsilon) are only originally expressed after the gene rearrangement event deletes the mu C and delta C exons. This process of gene rearrangement, called isotype change, typically occurs by recombination between so-called segments of change located immediately upstream of each heavy chain gene (except delta). The individual exchange segments have between 2

<td>and 10 kb</td><td>of length,</td><td>and consist</td><td>mainly</td><td>in</td>

<td>sequences</td><td colspan="2">repeated short.</td><td></td><td></td>

<td></td><td>For these</td><td>reasons is</td><td>preferable that</td><td>the</td>

<td>transgenes</td><td>incorporate</td><td>sequences</td><td>regulatory</td><td>d</td>

transcript within approximately 1-2 kb upstream of each change region to be used for the isotype change. These transcriptional regulatory sequences preferably include a promoter and an enhancer element, and more preferably include the

183

<img file="MX337052B_D0181.tif" />

5 'flanking region (i.e. current above) that I know λ naturally associates (i.e. occurs in germline configuration) with a ^ region' of ^^-change. Although a 5 'franking sequence from one region of change can be operably linked to a different region of change for the transgene construct, in some embodiments it is preferred that each region of change incorporated into the transgene construct has a region of 5 'flanking occurring immediately upstream in the naturally occurring germline configuration. Sequence information related to the sequences of immunoglobulin change regions are known (Milis et al., 1990; Sideras et al., 1989; each incorporated herein by reference).

In the method described in US Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016 and 5,770,429, the human immunoglobulin transgenes contained within the transgenic animal function correctly through the pathway of B cell development, leading to the change of isotypes. Consistent with the above, in this method, these transgenes are constructed to produce isotype change and one or more of the following: (1) high level of cell type-specific expression, (2) rearrangement of

184 functional gene, (3) activation of and response to allelic exclusion, (4) expression of a sufficient primary repertoire, (5) signal transduction, 't ^ T ^ somatic permutation, and (7) antibody site domain of transgene 5 during the immune response.

An important requirement for transgene function is the generation of a primary antibody repertoire that is diverse enough to trigger a second immune response for a wide range of 10 antigens. The rearranged heavy chain gene consists of a signal peptide exon, a variable region exon, and a line array of regions of constant domains from multiple domains, each of which is encoded by or several exons. Each of the constant region genes 15 encodes the constant portion of a different class of immunoglobulins. During B cell development, the constant regions proximal to the V region are cleared leading to the expression of new classes of heavy chains. For each class of heavy chain, alternative RNA separation patterns give rise to both transmembrane and secreted immunoglobulins.

The human heavy chain site consists of approximately 200 V gene segments that span 2

Mb, approximately 30 D gene segments extending approximately 40 kb, six J segments branching with

185

J: a! 'YyL a 3 kb extension, and nine gene segments from region t. ,. and · '' constant scattered about 300 kb. The entire site extends approximately 2.5 Mb from the distal portion of the long arm of chromosome 14. The heavy chain transgene fragments containing members of the six V families_H Known gene segments D and J, as well as mu, delta, gamma 3, gamma 1 and alpha 1 constant regions are known (Berman et al. 1988; incorporated herein by reference). Genomic fragments containing all necessary gene segments and regulatory sequences from a human light chain site are similarly constructed.

Expression of successful rearranged immunoglobulin light and heavy transgenes usually has a dominant effect by suppressing rearrangement of endogenous immunoglobulin genes in the transgenic non-human animal. However, in certain embodiments, it is desirable to effect complete inactivation of endogenous Ig sites, such that hybrid immunoglobulin chains comprising a human variable region and a non-human (eg, murine) constant region cannot be formed, for example, by trans-switching between the 'transgene and endogenous Ig sequences. Using embryonic totipotent cell technology and homologous recombination, the immunoglobulin repertoire

186 Suppression of endogenous Ig immunoglobulin genes can be carried out using a variety of techniques, such as antisense technology.

In other aspects of the invention, it may be desirable to produce a trans-changed immunoglobulin. Antibodies comprising these chimeric switched immunoglobulins can be used for a variety of applications where it is desirable to have a non-human (eg, murine) constant region, eg, for retention of effector functions in the host. The presence of a murine constant region may have advantages over a human constant region, for example, to provide murine effector functions (eg, murine complement fixation, CCDA), so that this chimeric antibody can be tested in a disease model mouse. After evaluating the animal, the human variable region coding sequence can be isolated, for example, by PCR amplification or cloning of c from the source (hybridoma clone), and separated into a sequence encoding a Desired human constant region to encode a human sequence antibody more suitable for human therapeutic use.

E4. Humanized Antibodies

IΚ4 ΡI

Human antibodies generally

OF THE PROPERTY .·. 'INDUSTRIAL at least three potential advantages for its use

<img file="MX337052B_D0182.tif" />

human therapy. First, because the effector portion is human, it can better interact with the other parts of the human immune system, for example, to destroy target cells more efficiently by complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (CCDA). Second, the human immune system should not recognize the antibody as foreign. Third, the half-life in human circulation will be similar to naturally occurring human antibodies, allowing for smaller, less frequent doses.

Various methods of preparing anti-VEGF are provided herein. In addition to human antibodies, humanized antibodies have advantages. Humanized antibodies have many generally are chimeric or mutant monoclonal antibodies from mouse, rat, hamster, rabbit or other species, which carry human variable and / or constant region domains or specific changes. Techniques for generating a so-called humanized antiVEGF antibody are well known to those skilled in the art.

Humanized antibodies also share the following advantages. First, the effector portion

188 it is still human. Second, the human iW ^ iie 'system should not recognize the constant structure or region as foreign, and therefore, the antibody response against this injected antibody should be less than against a totally foreign mouse antibody. Third, injected humanized antibodies, as opposed to injected mouse antibodies, will presumably have a half-life more similar to naturally occurring human antibodies, also allowing for smaller and less frequent doses.

Various methods have been described to produce humanized antibodies. Controlled rearrangement of antibody domains linked through protein disulfide linkages to form new / artificial protein molecules, or chimeric antibodies can be used (Konieczny et al., 1981; incorporated herein by reference). Recombinant technology can also be used to construct gene fusions between sequences encoding variable light and heavy chain domains of mouse antibodies and constant light and heavy chain domains of human antibodies (Morrison et al., 1984; incorporated herein for reference).

The sequences of which encode the antigen binding portions or regions of

189 Complementarity Determination (RDC)

INSTITUTE '~'&gt; - - '' / murine monoclonal agents can be grafted by 'molecular means onto the sequences of human and light chain structures of human antibodies (Jones et al., 1986; Riechmann et al., 1988; each incorporated herein by reference). The expressed recombinant products are called humanized or reformed antibodies, and comprise the structure of a light or heavy chain of human antibodies and the antigen recognition portions, RDC, of ​​a murine monoclonal antibody.

Another method of producing humanized antibodies is described in United States Patent No. 5,639,641, incorporated herein by reference. The method provides, via way to renew the surface, humanized rodent antibodies that have improved therapeutic efficacy due to the presentation of a human surface in the variable region. In the method: (1) alignments of positions of a deposit of heavy and light chain variable regions of antibodies are generated to give a set of exposed portions of heavy and light chain variable region structure surface, where the positions of alignment for all variable regions are at least approximately 98 percent identical; (2) a

190 joint

<img file="MX337052B_D0183.tif" />

waste

<img file="MX337052B_D0184.tif" />

amino acids

<img file="MX337052B_D0185.tif" />

Heavy and light chain variable region structure is defined for a rodent antibody (or fragment thereof); (3) a set of surface exposed amino acid residues of the heavy chain variable region structure. and light that is almost identical to the set of amino acid residues exposed on the rodent's surface is identified; (4) the set of surface exposed amino acid residues of the heavy and light chain variable region structure defined in step (2) is replaced with the set of surface exposed amino acid residues of the variable region structure heavy and light chain identified in step (3), except for amino acid residues that are within 5Δ of any atom of any residue in the complementary determination regions of the rodent antibody;

and (5) humanized rodent antibody having binding specificity is produced.

A similar method for the production of humanized antibodies is described in US Pat.

United States of

5,693,761; 5,585,089 and

North America Numbers 5,693,762;

5,530,101, each incorporated herein by reference. These methods involve producing humanized immunoglobulins that have one or more

191 ^ι ΤΤ * jt. ,, r regions of determination of compleméntgj ^^ áíÍ 7ÍRDC) and possible additional amino acids to '' from a donor immunoglobulin and a region of structure from an acceptance human immunoglobulin. Each humanized immunoglobulin chain usually comprises, in addition to the complementarity determining region, amino acids from the donor immunoglobulin structure that are capable of interacting with CDRs to effect binding affinity, such as one or more amino acids that are immediately adjacent to a CDR in the

<td>immunoglobulin</td><td>donor</td><td>or</td><td>those</td><td>inside</td><td>of</td>

<td>approximately</td><td>3Δ predicted</td><td>by</td><td>modeling</td><td>molecular.</td><td>The</td>

<td>heavy chains</td><td>and light</td><td>I know</td><td colspan="3">can design using</td>

any, or any combination, or all of the distinct position criteria described in US Patent Nos. 5,693,762; 5,693 ·, 761; 5,585,089 and 5,530,101, each incorporated herein by reference. When combined into an intact antibody, humanized immunoglobulins are substantially non-immunogenic in humans and retain substantially the same affinity as the donor immunoglobulin for the original antigen.

A further method of producing humanized antibodies is described in the US Patents.

United States of America Numbers 5,565,332 and 5,733,743, each

192 one incorporated herein as a reference / This method combines the concept of humanizing antibodies with phagemid libraries also described herein.

In a general sense, the method uses sequences from the antigen binding site of an antibody or population of antibodies directed against an antigen of interest. Thus for a single rodent antibody, sequences comprising part of the antigen binding site of the antibody can be combined with various repertoires of human antibody sequences that can, in combination, create a complete antigen binding site.

The antigen binding sites created by this process differ from those created by CDR grafting, because only the sequence portion of the original rodent antibody is likely to make contacts with the antigen in a similar way. The selected human sequences probably differ in sequence and make alternative contacts with the antigen from those of the original binding site. However, the restrictions imposed by the binding of the portion of the original sequence to the antigen and the forms of the antigen and their antigen binding sites are likely to lead to further contacts of the human sequences with the same region or epitope of the antigen. This process so • m-tKAn-tnrr.

193 So much has been called (SIE).

selection

<img file="MX337052B_D0186.tif" />

give epitope

<img file="MX337052B_D0187.tif" />

Starting with an animal antibody, a process results in the selection of antibodies that are partially human antibodies. These antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or after alteration of some key studies. Sequence differences between the rodent component 10 of the selected antibody with human sequences could be minimized by replacing those residues that differ with the residues of human sequences, for example, by site-directed mutagenesis of individual residues, or by full-cycle CDR grafting . However, antibodies with entirely human sequences can also be created. Printed selection of epitopes therefore offers a method of making partially human or entirely human antibodies that bind to the same epitope as the animal or to the same epitope as the animal antibodies or partially. human, respectively. In the SIE, repertoires of antibody fragments can be displayed on the surface of the phase filaments and genes encoding fragments with binding activities of antigens selected by binding of phage to antigen.

194 ύ

Additional methods pS ¥ S¿ ?, '_ humanize ι: ώΛι' ;; ',. ϊ _ antibodies contemplated for use in the present invention are described in US Patent Nos. 5,750,078; 5,502,167;

5,705,154; 5,770,403; 5,698,417; 5,693,493; 5,558,864;

4,935,496 and 4,816,567, each incorporated in, 1a present by reference.

B10. Polymerase Chain Reaction Mutagenesis

Site-specific mutagenesis is a useful technique in preparing individual antibodies through specific mutagenesis of the underlying DNA. The technique further provides a ready-to-prepare ability and test sequence variants, which incorporate one or more of the following considerations, whether humanizing or not, by introducing one or more nucleotide sequence changes into the DNA.

Although many methods are suitable for use in mutagenesis, the use of the polymerase chain reaction (PCR^mr) is generally preferred. This technology offers a fast and efficient method to introduce the desired mutations into a given sequence. The following text particularly

IMPKí®

IKSTI-ÜT. '. * &lt;? «:. ·;

Introduce

195

X describes the use of PCR for point in a sequence, as you can .. use to change the encoded amino acid by the given sequence.

Adaptations of this method are also convenient for introducing restriction enzyme sites into a molecule.

In this method, the oligonucleotides

<td>synthetic</td><td>I know</td><td>they design</td><td>for</td><td>to incorporate</td><td>a mutation</td>

<td>punctual in</td><td>a</td><td>extreme</td><td colspan="2">of a segment</td><td>amplified.</td>

<td>10 After</td><td>the</td><td>reaction</td><td colspan="3">polymerase chain, the</td>

<td>fragments</td><td colspan="2">amplified</td><td>I know</td><td>flattens them</td><td>the extreme</td>

<td>treating them</td><td>with</td><td colspan="2">fragments of</td><td colspan="2">Klenow, and the shards</td>

<td>with extreme</td><td colspan="2">flattened</td><td>link</td><td>and subclone</td><td>in a vector</td>

to facilitate analysis

To prepare you want to mutagenize the DNA high copy number, such as the sequence.

template DNA that is subcloned into a vector such as pUC19, using restriction sites flanking the areas to be mutated. Template DNA is prepared using a plasmid miniprep. Suitable oligonucleotide primers that are based on the parent sequence, but that contain the desired point mutation and that are flanked at the 5 'end by a restriction enzyme site, are synthesized using an automated synthesizer. It is generally required that

196

INSTITUTO mexicana Ι.Λ η r * the initiator is homologous to the DNA in pla'ft-t'tll ^^^ F approximately 15 bases or something like that. LoF ^ TnTigiáttOTTffi¹· Or „can be purified by denaturing polyacrylamide, absolutely necessary for its end 5¹ of the oligonucleotides

<td>□ foresis</td><td>of</td><td>gel</td><td>of</td>

<td>even if</td><td>this</td><td>not</td><td>is</td>

<td>use in</td><td>the</td><td>PCR.</td><td>The</td>

<td>they should</td><td colspan="2">so</td><td>to be</td>

phosphorylated.

Template DNA must be amplified by PCR, using the oligonucleotide primers containing the desired point mutations. MgCl concentration₂ in the amplifier regulator it will generally be approximately 15 mM. Generally approximately 20-25 cycles of PCR should be carried out as follows: denaturation, 35 seconds at 95EC; hybridization, 2 min at 50EC; and extension, 2 minutes at 72EC. The PCR will generally include a last extension cycle of approximately 10 minutes at 72EC. After the final extension step, approximately 5 units of Klenow fragments should be added to the Xa reaction mixture and incubated for another 15 minutes at approximately 30EC. The exonuclease activity of the Klenow fragments is required to make the ends wash and convenient for blunt end cloning.

197

The reaction mixture

L should generally be assayed by non-denaturing acrylamide or agarose gel electrophoresis to verify that amplification has produced the predicted product. The reaction mixture would then be processed by removing most of the mineral oils, extracting with chloroform to remove the remaining oil, extracting with regulated phenol then concentrating by precipitation with 100 percent ethanol. Next, approximately half of the fragments amplified would be digested with a restriction enzyme that cuts at the flanking sequences used in the oligonucleotides. The digested fragments are purified on a low gelation / fusion agarose gel.

To subclone the fragments and verify the point mutation, the two amplified fragments would be subcloned into a properly digested vector by blunt end ligation. This would be used to transform E. coli, from which the plasmid DNA could subsequently be prepared using a mini-preparation. The amplified portion of the plasmid DNA would then be analyzed by DNA sequencing to confirm that the correct point mutation was generated. This is important, since

198

IMP^{V &lt;} 'Taq DNA polymerase can introdusi ^ To ^ utacionespy additional DNA fragments.

The introduction of a point mutation can also be done using sequential PCR steps. In this procedure, the two fragments encompassing the mutation are strengthened with each other and spread by mutual insertion synthesis. This fragment is then amplified by a second PCR step, thereby avoiding the blunt-ended ligation required in the previous protocol. In this method, template DNA preparation, generation of the oligonucleotide primers, and the first PCR amplification are performed as described above. However, in this process the chosen oligonucleotides should be homologous to the template DNA for a span of approximately 15 to 20 bases, and should also overlap each other by approximately 10 bases or more.

In the second PCR amplification, each amplified fragment and each flanking sequence primer would be used and would carry PCR for approximately 20 and approximately 25 cycles, using the conditions described above. Again the fragments would be subcloned and verified that the point mutation was correct using the steps outlined above.

199

<img file="MX337052B_D0188.tif" />

When using any of the above, mutation is generally preferred by amplifying a fragment as small as possible. Of course, parameters such as the melting temperature of the oligonucleotide, as it will generally be influenced by the GC content and the length of the oligo, should be carefully considered. The execution of these methods, and their optimization, if necessary, will be known to those skilled in the art, and will be further described in various publications, such as Current Protocole in Molecular Biology, 1995, incorporated herein by reference.

When site specific mutagenesis is performed, Table A can be used as a reference.

TABLE A

Amino acids

Codons

<td>To the girl</td><td>To</td><td>TO</td><td>GCA</td><td>GCC</td><td>GCG</td><td>GCU</td>

<td>Cysteine</td><td>Cys</td><td>C</td><td>UGC</td><td>ÜGU</td><td></td><td></td>

<td>Aspartic acid</td><td>Asp</td><td>D</td><td>GAC</td><td>GAU</td><td></td><td></td>

<td>Glutamic acid</td><td>Glu</td><td>AND</td><td>GAA</td><td>GAG</td><td></td><td></td>

<td>Phenylalanine</td><td>Phe</td><td>F</td><td>ÜUC</td><td>UUU</td><td></td><td></td>

<td>Glycine</td><td>Gly</td><td>G</td><td>GGA</td><td>GGC</td><td>GGG</td><td>GGU</td>

200

<td>Histidine</td><td>His</td><td>H</td><td>CAC</td><td colspan="2">CAU</td><td>IM INSTITUTE&gt;</td><td>FA // Μ ··. '..:' &lt;</td><td></td>

<td>Isoleucine</td><td>lie</td><td>I</td><td>AUA</td><td>AUC</td><td>AUU</td><td></td><td></td><td></td>

<td>Lysine</td><td>Lys</td><td>K</td><td>AAA</td><td>AAG</td><td> -</td><td></td><td></td><td> ----</td>

<td>Leucine</td><td>Leu</td><td>L</td><td>USA</td><td>UUG</td><td>CUA</td><td>cuc</td><td>CUG</td><td>CUU</td>

<td>Methionine</td><td>Met</td><td>M</td><td>AUG</td><td></td><td></td><td></td><td></td><td></td>

<td>Asparagine</td><td>Asn</td><td>N</td><td>AAC</td><td>WOW</td><td></td><td></td><td></td><td></td>

<td>Proline</td><td>Pro</td><td>P</td><td>CCA</td><td>CCC</td><td>CCG</td><td>ccu</td><td></td><td></td>

<td>Glutamine</td><td>Gln</td><td>Q</td><td>CAA</td><td>CAG</td><td></td><td></td><td></td><td></td>

<td>Arginine</td><td>Arg</td><td>R</td><td>AGA</td><td>AGG</td><td>CGA</td><td>CGC</td><td>CGG</td><td>CGU</td>

<td>Serine</td><td>To be</td><td>S</td><td>AGC</td><td>AGU</td><td>UCA</td><td>ucc</td><td>UCG</td><td>UCU</td>

<td>Threonine</td><td>Thr</td><td>T ·</td><td>HERE</td><td>ACC</td><td>ACG</td><td>ACU</td><td></td><td></td>

<td>Valine</td><td>Val</td><td>V</td><td>GÜA</td><td>GUC</td><td>GUG</td><td>GUU</td><td></td><td></td>

<td>Tryptophan</td><td>Trp</td><td>W</td><td>UGG</td><td></td><td></td><td></td><td></td><td></td>

<td>Tyrosine</td><td>Tyr</td><td>AND</td><td>UAC</td><td>UAU</td><td></td><td></td><td></td><td></td>

Ε6. Antibody fragments and derivatives

Regardless of the source of the original antibody against an anionic phospholipid. or aminophospholipid, either the intact antibody, antibody multimers, or any of a variety of functional antigen binding regions of the antibody can be used in the present invention. Exemplary functional regions include scFv, Fv, Fab ', Fab, and F (ab') fragments.₂ of anti-VEGF antibodies. Techniques for preparing these constructs are well known to those skilled in the art and are further exemplified.

201 at the moment.

INSTi TU I ·

<img file="MX337052B_D0189.tif" />

The choice of the antibody construct can be influenced by several factors. For example, prolonged half-life may be the result of active readsorption of intact antibodies into the kidney, a property of the Fe piece of immunoglobulin. IgG-based antibodies, therefore, are expected to exhibit slower clearance in the blood than their Fab 'counterparts. However, Fab 'fragment-based compositions will generally exhibit better tissue penetration ability.

The . Antibody fragments can be obtained by proteolysis of the entire immunoglobulin using the non-specific thiol protease, papain. Papain digestion produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fe fragment.

Papain must first be activated by reducing the sulfhydryl group at the active site with cysteine, 2 mercaptoethanol, or dithiothreitol. The heavy metals in the enzyme material could be removed by chelation with EDTA (2mM) to ensure maximum enzyme activity. The enzyme and the substrate are normally mixed with each other in a ratio of 1: 100 by weight. After incubation,

202 the reaction can be stopped by

<img file="MX337052B_D0190.tif" />

irreversible of the thiol group with iodoacetamide. or simply by dialysis. Completion of digestion should be monitored by SDS-PAGE and separate fractions should be separated by ion exchange or protein A Sepharose chromatography .

The usual procedure for the preparation of F (ab ') 2 fragments from rabbit and human origin IgG is limited to proteolysis by the enzyme pepsin. The conditions, 100-fold excess weight / weight antibody in acetate buffer with pH 4.5, 37EC, suggests that the antibody dissociates on the C-terminal side of the disulfide bond of the weighted chain. Digestion rates of mouse IgG may vary with the subclass and it may be difficult to obtain high yields of the F (ab ') fragments.₂ active with some undigested or completely degraded IgG. In particular, IgG₂b is susceptible to complete degradation. The other subclasses require different incubation conditions to produce optimal results, all of which are known in the art.

Pepsin treatment of intact antibodies produces an F (ab ') fragment₂ it has two antigen combining sites and is still capable of crosslinking 25 antigen. Digestion of rat IgG by pepsin requires

203 terms

<img file="MX337052B_D0191.tif" />

NDU5 acetate, pH 4.5, and then incubation for four hours with 1 weight / weight percent pepsin;

digestion of

IgGi and IgG_2nd is improved if first dialyzed against 0.1 M of

<td>regulator</td><td>Format,</td><td>pH 2.8, a</td><td>4EC,</td><td colspan="2">for 16 hours</td>

<td colspan="2">followed by regulator</td><td>acetate.</td><td>igG₂b</td><td>gives results</td><td>plus</td>

<td>consistent</td><td>with the</td><td>incubation</td><td>in</td><td>protease V8</td><td>of</td>

<td>staphylococci</td><td>(3 for</td><td colspan="3">percent weight / weight) in 0.1 M</td><td>of</td>

<td>regulator</td><td>phosphate</td><td>sodium, pH 7</td><td colspan="3">.8, for four hours</td>

to 37EC.

A Fab fragment also contains the light chain constant domain and the heavy chain first constant domain (CH1). Fab 'fragments differ from Fab fragments by the addition of some residues at the carboxyl terminus of the heavy chain of the CH1 domain including one or more cysteines from the hinge region of the antibody. F (ab ') antibody fragments₂They were originally produced as Fab 'fragment pairs that had joint cysteines between them. Other chemical couplings of antibody fragments are also known.

An Fv fragment is a minimal antibody fragment that contains a complete antigen recognition and binding site. This region consists of a heavy chain dimer and a domain

204

MEXICAN INSTITUTE

OF THE ΡΓΤ.

light chain variable in association cbri / Covatente narrow. It is in this configuration quer- ^ ars ^ reá regicrfé'S 'hypervariables of each variable domain interact to define an antigen binding site on the surface of the V dimer_H-V_L. Collectively, the six hypervariable regions confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv fragment comprising only three antigen-specific hypervariable regions) has the ability to recognize and bind antigen, albeit at a lower affinity than the entire binding site.

Single chain Fv or sFv antibody fragments (also known as single chains comprise V domains_H and V_L of the antibody, where these domains are present on a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the V domains._H and V_L which allows sFv to form the desired structure for antigen binding.

The following patents are specifically incorporated herein by reference for the purposes of further supplementing the present teachings regarding the preparation and use of antibody antigen binding regions, including scFv, Fv, Fab ', Fab and the

205

L ^Ar F (ab ') 2 fragments of anti-V $ GF antibodies: Patents

5,877,289; 5,965,132; 6,093,399; 6,261,535 and 6,004,555. The

WO 98/45331 is also incorporated by reference herein for purposes of further including and describing and teaching the preparation of variable, hypervariable, and complementary antibody (CDR) regions. Furthermore, the successful production of scFv constructs within the scope of the present invention is detailed in the example.

XIV.

Diabodies are small fragments of antibodies with two sites these fragments comprise heavy (V_H) connected with a of a slight

Using matching variable domain binding variable domain (V_L) on the same polypeptide chain a linker as between the two antigen, chain-chain (V_H - V_L) .

It is too short to allow the domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites.

are described in the patent

European EP 404,097 Publication

International

WO. 93/11161, each specifically incorporated herein by reference.

Linear antibodies, which can be bispecific or monospecific, comprise a pair of c

Fd segments online.

206

<img file="MX337052B_D0192.tif" />

described in Zapata and (Vh-C_h1-V_h-Ch1) that form an antigen binding, as a partner pair, (1995), specifically incorporated herein by reference.

By using a Fab 'or antigen binding fragment in the antibody, with the expected benefits of tissue penetration, additional benefits can be derived from modifying the fragment to increase its half-life. A variety of techniques can be employed, such as manipulation or modification of the antibody molecule itself, and also conjugation with inert carriers. Any conjugation for the sole purpose of increasing the half-life, rather than administering a substance to a target, should be carried out carefully because Fab 'and other fragments are chosen to penetrate tissues. However, conjugation with non-protein polymers, such as PEG and the like, is contemplated.

Modifications other than conjugation are therefore based on modifying the structure of the antibody fragment to make it more stable, and / or reducing the rate of catabolism in the body.

<td colspan="3">A mechanism for these</td><td>modifications is</td><td>the</td><td>use</td><td>d</td>

<td>amino acids</td><td>D instead</td><td>of</td><td>amino acids L.</td><td colspan="2">people</td><td>with</td>

<td>experience</td><td>ordinary</td><td>in</td><td colspan="2">the technique will understand</td><td>than</td><td>the</td>

introduction of these modifications needs to be followed

207 desirable. Other stabilization modifications include the use of fractions at the N-terminus or at the C-terminus or both, which is generally used to extend the half-life of biological molecules. By way of example only, one may wish to modify the terms by acylation or amination.

Moderate conjugation modifications for use in the present invention include incorporating an antibody fragment epitope. Techniques to accomplish this include mutating the appropriate region of the antibody fragment or incorporating the epitope as a peptide marker that binds to the antibody fragment.

The international publication

WO 96/32478 is specifically incorporated herein by reference for the purposes of further exemplifying this technology.

Receptor receptor epitopes typically are regions of three or more amino acids of one or two cycles of the Fe domain that are transferred to the analogous position of the antibody fragment. The epitope-binding recovery receptor of WO 98/45331 is incorporated herein by reference for use.

208 with the present invention.

E i /. · '· Insmuyo mexicana VCS

<img file="MX337052B_D0193.tif" />

F. Binding of Immunoconjugates to Anionic Phospholipids and Aminophospholipids

The inventors herein have previously developed a range of aminophospholipid-binding immunoconjugates for use in targeting the vasculature of tumors (US Pat.

United of North America

No.

6,312,694, specifically incorporated herein, use proteins as reference). These bind to aminophospholipids, such as annexins and kininogens, and antibodies to aminophospholipids, such as PS and PE, to deliver bound therapeutic agents to the tumor and intratumoral vasculature. The present invention now provides selected anti-PS antibodies, with improved properties, such as 3G4 (ATCC 4545) and 9D2, and these antibodies as well as competing antibodies can now be used as the antibody moieties of immunoconjugates.

<td></td><td>Besides of</td><td colspan="2">use of agents</td><td>for</td><td>the</td>

<td>location</td><td colspan="2">directed vascular which</td><td>I know</td><td>link</td><td>to</td>

<td colspan="2">aminophospholipids (patent</td><td colspan="2">of the states</td><td>United</td><td>of</td>

<td>North America</td><td>No. 6,312,694),</td><td>the present</td><td colspan="2">discovery</td><td>of</td>

<td>that the</td><td>phospholipids</td><td>anionic,</td><td>So</td><td>how</td><td>the</td>

209

<img file="MX337052B_D0194.tif" />

aminophospholipids, they are stable entities

<img file="MX337052B_D0195.tif" />

INSTiTUTw;

it can come with targeted localization, within the tumor vasculature, allows the use of a range of new vascular targeted localization agents for tumors. The new compounds, not suggested in previous work, targeting aminophospholipids, use antibodies directed against anionic phospholipids to deliver toxins, cytokines, coagulants, and other therapeutic agents to up-regulated anionic phospholipids in the tumor and intratumoral vasculature.

As detailed above with respect to naked antibodies, the development of these aspects of the invention required the generation of biological tools, particularly antibodies, with exquisite specificity for different phospholipids, anionic phospholipids, and aminophospholipids.

<td></td><td>As the</td><td>present invention shows</td><td>than</td><td>the</td>

<td>phospholipids</td><td>anionic</td><td>and aminophospholipids, such</td><td>how</td><td>,</td>

<td>PE, Pl, PA</td><td>and PG, and</td><td>more particularly PS and</td><td>PE,</td><td>are</td>

Safe and effective targets for antiviral therapy, the antibodies and peptides that bind to these components, particularly PS and PE, can now be advantageously linked to a range of known antiviral agents. These antiviral conjugates include both peptide-based and antibody-based conjugates. The

210 last of which can be ^stdK denominated Λ 7

¡. • DUSi.-G / '. L ___jS immunoconjugates antiviral or immunoviroeidas.

In these aspects of the invention, any antibody against an anionic phospholipid can be used to prepare an immunoconjugate or coaguligand, with antibodies such as second generation antibodies, particularly type 9D2 antibodies and type 3G4 antibodies, their advantageous binding profiles are preferred. anionic phospholipids. Agents for use in these immunoconjugates preferably include anti-cellular or cytotoxic agents, coagulants (clotting factors), cytokines, radiation therapy agents, anti-angiogenic agents, apoptosis-inducing agents, anti-tubulin drugs, and antiviral agents (and PE-binding peptides, such as duramycin derivatives, as described in detail herein.) In antiviral immunoconjugates there is no requirement to use a second generation antibody as described here, although these can be used. Any antibody to aminophospholipids or anionic phospholipids can then be linked to an antiviral agent to form antiviral or immunovirocidal immunoconjugates, in accordance with the present invention.

211

<img file="MX337052B_D0196.tif" />

Pl. Agent s Toxic and Antic luíLA ™_wltUAU · * IMni KTüi λ i

INDUSTRIAL

For certain applications the therapeutic agents will be cytotoxic or pharmacological agents, particularly cytotoxic, cytostatic, or otherwise anti-cellular agents that have the ability to kill or suppress the growth of cell division of cells, particularly tumor or endothelial cells. tumor cells. In general, these aspects of the invention contemplate the use of any pharmacological substance that can be conjugated with an antibody against an anionic phospholipid, preferably an antibody based on 9D2 or an antibody based on 3G4 and actively administered to the target endothelium.

Exemplary anti-cellular agents include chemotherapeutic agents, as well as cytotoxins.

Chemotherapeutic agents that can be used include: hormones, such as spheroids; antimetabolites, such as cytokine arabinoside, fluorouracil, methotrexate, or aminopterin; anthraeclines; mitomycin C; vinca alkaloids; demecolcin; etoposide; mithramycin; anti-tumor alkylating substances, such as chlorambucil or melphalan. Other modalities can include substances such as cytokines. Basically, any anti-cellular agent can be used, as long as it can be successfully conjugated to, or associated with, an antibody in a way that

212

<img file="MX337052B_D0197.tif" />

will allow its addressing, internal i zacjl ^^^ íbér

D £ L / a PRC? Íí-OAD INDUSTRi / XL and / or presentation to blood components at the site of the target endothelial cells.

There may be circumstances, such as when the target antigen is not internalized via a route consistent with efficient poisoning by 1 toxic compound, where it will be desired to target chemotherapeutic substances, such as antitumor drugs, cytokines, antimetabolites, alkylating agents, hormones, and the like. A variety of chemotherapeutic agents and other pharmacological agents have now been successfully conjugated to antibodies if they are shown to work pharmacologically, including doxorubicin, daunomycin, methotrexate, vinblastine, neocarcinostatin, macromycin, trenimon, and V-amanitin.

In other circumstances, any potential side effects of cytotoxin-based therapy can be eliminated - through the use of DNA synthesis inhibitors, such as daunorubicin, doxorubicin, adriamycin, and the like. These substances are therefore preferred examples of anti-cellular substances for use in the present invention. In terms of cytostatic agents, these compounds generally interrupt the natural cycle of cells in a target cell, preferably so that the cell breaks out of the cycle.

213

A wide variety of cytotoxic agent is released from the cell.

<img file="MX337052B_D0198.tif" />

knows that it can be conjugated with an antibody against an anionic phospholipid, preferably an antibody based on 9D2 or based on 3G4. Examples include numerous toxins derived from plants, fungi, or bacteria, which, by way of example, include various A chain toxins, particularly ricin A chain; ribosome inactivating protein, such as saporin or. gelonin;

V-sarcin; aspergillin; restrictocin; ribonucleases, such as placental ribonuclease; diphtheria toxin;

and pseudomonas exotoxin, just to name a few.

<td></td><td>Of the toxins, use is preferred</td><td>of</td><td>the</td>

<td>chains</td><td>of gelonin and ricin A. The use of gelonin</td><td>how</td><td>the</td>

<td>15 portion</td><td>effector or toxin of immunoconjugates</td><td>than</td><td>I know</td>

<td>link</td><td colspan="2">to the expressed bookmarks, which have access</td><td>to the</td>

binding, were absorbed or located in intratumoral blood vessels of a vascularized tumor, described in the United States patent of

North America No. 6,051,230, specifically incorporated herein by reference and in the United States of America patent

No.

6,451,312, which particularly concerns VEGF-bound gelonin as a targeted targeting agent.

<img file="MX337052B_D0199.tif" />

As for the ricin A chains, a

214 called chain A toxin A that has been treated for mo &lt;

Carbohydrate residues, deglycosylated prolonged is preferred, manufacture at (dgA).

due to and due

Its extremely potent deglycosylated ricin A chain, life span that is economically grade and clinical scale.

It may be desirable from a more pharmacologically feasible midpoint to employ the smallest possible molecule that nevertheless provides an adequate biological response.

that will provide an adequate anti-cellular response. To this end, terminals can be truncated for desired activity, it has been discovered that the ricin A chain is removed by removing 30 amino acids NNagarase (Sigma), a suitable toxin. It is truncated A chain and still retain a proposes that when it can be used in conjugates according to the invention.

Alternatively, it can be found that applying recombinant DNA technology to the toxin A chain moiety will provide additional benefits in accordance with the invention. In order for biologically active ricin A chain cloning and expression to have been achieved, it is now possible to identify and prepare smaller or otherwise peptides.

215

<img file="MX337052B_D0200.tif" />

IM PT variants that nevertheless exhibit adequate activity.

Furthermore, the fact that ricin A has now been cloned allows the application of site-directed mutagenesis, through which peptides derived from the A chain can be easily prepared and selected and additional useful fractions can be obtained for use in in relation to the present invention.

F2. Cytokines

Cytokines and chemokines are particular examples of agents for binding to the antibodies of the present invention. A certain range of cytokines can be used, including IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-11, IL-13, TGF-β, M-CSF, G -CSF, ΤΝΕβ, LAF, TCGF, ​​BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, IFN-oí, IFN-β. The most preferred cytokines include IL-la, IL-Ιβ, IL-2, IL-6, IL-10, GM-CSF, IFNy, monocyte chemoattractant protein-1 (MCP-1), derived growth factor BB platelet (PDGF-BB) and C-reactive protein (CRP) and the like. Particularly preferred examples are TNFa, TNFa inducers, and IL-12.

TNFa increases vascular permeability.

This agent is contemplated for binding to an antibody of the invention, particularly where the resulting immunoconjugate is used in combination therapy to

<img file="MX337052B_D0201.tif" />

216 cancer treatment.

<img file="MX337052B_D0202.tif" />

Institute

The antibody will deliver bound to the tumor environment, and the increased permeability of the tumor will facilitate the penetration of a second anticancer agent into the tumor, thereby amplifying the overall antitumor effect. The scFv constructs are particularly contemplated for use in those modalities. This is partly because TNFa functions as a trimer and the scFv constructs can easily be trimerized.

IL-12, for example, can be bound to an antibody and used to redirect host defenses to attacks on tumor vessels. When using IL-12, a form of antigen-binding region scFv may be preferred. LEC chemokine (liver expressed chemokine, also known as NCC-4, HCC-4, or LMC) is another preferred component (Giovarelli et al., 2000). LEC is chemotactic for dendritic cells, monocytes, T cells, NK cells, and neutrophils and can then enhance host-mediated anti-tumor responses.

F3. Coagulation factors

An antibody against an anionic phospholipid, or a second generation antibody based on the preferred 9D2 and 3G4 antibodies (ATCC 4545) of the

217

<img file="MX337052B_D0203.tif" />

to stimulate coagulation directly or indirectly, to form a coaguligando. United States of America Patents Numbers 6,093,399, 6,004,555,

5,877,289 and 6,036,955 are specifically incorporated herein by reference, for purposes of further describing the functional association of coagulants with antibodies to form coaguligands.

The antibodies of the invention can be linked directly to the coagulant or to the coagulation factor, or they can be linked to a second linked region that binds and then releases the coagulant or the coagulation factor. As used herein, the terms coagulant and coagulation factor each are used to refer to a component that is capable of directly or indirectly stimulating coagulation under suitable conditions, preferably when provided in a specific environment in vivo, such as the vasculature of the tumor.

Preferred clotting factors are Tissue Factor compositions, such as truncated tissue factor (tTF), dimeric, multimeric, and mutant tissue factor molecules. Truncated Tissue Factor (tTF) refers to tissue factor constructs that become deficient in binding to the

218 membrane by removing enough

<img file="MX337052B_D0204.tif" />

amino acids to effect this change in ^^ or ^ age ^ - ^ - eH® · sufficient amount in this context is an amount of transmembrane amino acid sequence originally sufficient to introduce the tissue factor molecule into the membrane, or otherwise mediate the functional membrane binding of the tissue factor protein. Removal of this sufficient amount of the transmembrane extension sequence therefore creates a tissue factor protein or polypeptide deficient in phospholipid membrane binding capacity, such that the protein is substantially a soluble protein that does not bind significantly to phospholipid membranes. Tissue factor thus truncated substantially fails to convert Factor VII to Factor viia in a standard tissue factor assay, and still retains so-called catalytic activity including activation of Factor X in the presence of Factor VHa.

United States Patent Nos. 5,504,067, 6,156,321, 6,156,729 and 6,132,730 are specifically incorporated herein by reference for the purposes of further describing these truncated Tissue Factor proteins. Preferably, Tissue Factors for use in these aspects of the present invention generally

219 They lack the transmembrane and --ci'tósóliíja ^ úÑu ^ (amino acids 220-263) regions of the protein. flin Amba-rgo. np there is a need for truncated tissue factor molecules to be limited to molecules of the exact length of

219 amino acids.

or

Tissue Factor compositions can also be useful as dimers. Any of the truncated, mutated or other tissue factor constructs can be prepared in a dimeric form for use in the present invention. Cortio will be known to those of skill in the art, these Tissue Factor dimers can be prepared using the standard techniques of molecular biology and recombinant expression, in which two coding regions are prepared within the framework and expressed from of an expression vector.

Similarly, various chemical conjugation technologies may be employed in connection with the preparation of Tissue Factor dimers. The individual Tissue Factor monomers can be derived from the conjugation. All of these techniques would be readily known to those skilled in the art.

If desired, the dimers or multimers of Tissue Factor can be linked via a biologically releasable bond, such as a selectively cleavable linker or amino acid sequence.

220 a tumor environment it

For example, cleavage site linkers for a localized or active within are contemplated.

Exemplary forms of these peptide linkers are those that dissociate via urokinase, plasmin, thrombin, Factor

IXa Factor

Xa, or a metalloproteinase, such as collagenase, stromelysin gelatinase.

In certain modalities, the numbers of the

Tissue factor may further comprise a hindered hydrophobic membrane insertion fraction, to subsequently encourage the functional association of the

Tissue factor with the phospholipid membrane, but only under or certain defined conditions.

As described in 1 context of the Factors of

Truncated tissues, hydrophobic membrane association sequences are generally stretches of amino acids that promote phospholipid association with the environment due to their hydrophobic nature. Similarly, fatty acids can be used to provide the potential membrane insert fraction.

These membrane insertion sequences can be located either at the N-terminus or the C-terminus of the Tissue Factor molecule, or generally append at any other point on the molecule as long as their binding to the

221 i ivi FH fep itself does not impede industrial functional properties

The intent of the Tissue Factor fraction.

Impaired insertion is for the Factor construct to remain non-functional until Tissue is located within the tumor environment, allowing the hydrophobic junction to become accessible and further promoting physical association with the membrane. Again, it is contemplated that biologically releasable linkages and selectively cleavable sequences will be particularly useful in this regard, with the linkage or sequence only being dissociated or otherwise modified after localization within the tumor environment and expression to particular enzymes or other bioactive molecules.

In other embodiments, the tTF constructs can be multimeric or polymeric. In this context a polymeric construct contains 3 or more Tissue Factor constructs. A multimeric or polymeric TF construct is a construct that comprises a first TF molecule or a derivative operably linked to at least a second and a third TF molecule or derivative. Multimers can comprise from about 3 to about 20 of these TF molecules. The individual TF units within the multimers or polymers may also be linked by selectively cleavable peptide linkers or other biologically linked

222

<img file="MX337052B_D0205.tif" />

I jM ΡI

MEXICAN INSTITUTE releasable as desired. Again, as with TF discussed above, the ^ ngj-mrEnRs can easily be made using either recombinant manipulation and expression or using standard synthetic chemistry.

Still other TF constructs useful in the context of the present invention are those mutants deficient in the ability to activate Factor VII. These Factor VII activation mutants are generally defined herein as TF mutants that bind to functional Factor VII / VIIa, proteolytically active Factor X, but are substantially free of the ability of proteolytically active Factor VII. Consistent with the above, these constructs are TF mutants that lack Factor VII activation activity.

The ability of these Factor VII activation mutants to function by promoting tumor-specific coagulation is based on their specific administration to the tumor vasculature, and the presence of Factor Vlla at low levels in plasma. Following administration of this Factor VII activating mutant target substance conjugate, the mutant will locate within the vasculature of a vascularized tumor. Before localization, the Tissue Factor mutant would generally be unable to promote

223

<img file="MX337052B_D0206.tif" />

coagulation anywhere else in the body, XJrMLáÍ3eíÍ INSTITUTO McXiCANO

OF THE PROPERTY . , _ _. industpjal inability to convert Factor VII to Factor

However, after localization and accumulation within the tumor region, the mimic will then find sufficient plasma Factor Vlla to initiate the extrinsic coagulation pathway, leading to tumor specific thrombosis. Exogenous Factor Vlla could also be administered to the patient.

One or more of a variety of Factor VII activation mutants can be. prepare and use in connection with the present invention. There is a significant amount of scientific knowledge concerning the recognition sites of the Tissue Factor molecule for Factor VlI / VIIa. Thus it will be understood that the Factor VII activation region generally lies between about amino acid 157 and about amino acid 167 of the TF molecule. However, it is contemplated that residues outside this region may also be shown to be relevant to the activation activity of Factor VII, and therefore it may be considered to introduce mutations in any or more of the residues generally located between approximately amino acid 106 and approximately amino acid 209 from the TF sequence (International Publication WO 94/07515; WO 94/28017; each incorporated herein as

224

As detailed in the reference patents)

United States of

ΙΜΡΪ

INSTITUTE

OF INDUSTRIAL PRODUCTS

North America Numbers 6,093,399,

6,004,555, 5,877,289 and

6,036,955, a variety of other clotting factors can be used in connection with the present invention, as exemplified by the substances presented below.

Thrombin,

Factor V / Va derivatives,

Factor VIII / VIIIa and derivatives, Factor IX / IXa derivatives,

Factor X / Xa and derivatives, Factor Xl / Xla derivatives,

Factor XII / XIIa and derivatives, Factor XlII / XIIIa derivatives,

Activating factor X and activating factor V can be used in the present invention.

Russell's Viper Venom Factor X activator is contemplated for use in this invention. Monoclonal antibodies specific for Factor X activator present in Russell's viper venom have also been produced, and could be used to specifically administer the substance as part of a bispecific binding ligand.

Thromboxane A₂ It is formed of endoperoxides through the sequential actions of the enzymes cyclo-oxygenase and thromboxane synthetase in platelet microsomes. Thromboxane A₂ it is easily generated by platelets and is a powerful vasoconstrictor, by virtue of its ability to cause platelet aggregation.

<img file="MX337052B_D0207.tif" />

225

INSTi; · '··. ·:

KL · .....,

Both thromboxancT A¿ and active analogues thereof are contemplated for its use.^;Only in ^¡ the present invention.

Thromboxane synthase, and other enzymes that synthesize platelet activating prostaglandins, can also be used as coagulants in the present context. Monoclonal antibodies, and immunoaffinity purification of, thromboxane synthase are known;

as well as the cDNA of human thromboxane synthase.

V2-antiplasmin, or the V2plasmin inhibitor, is a proteinase inhibitor naturally present in human plasma that functions to efficiently inhibit lysis of fibrin clots induced by plasminogen activator. V2antiplasmin is a particularly potent inhibitor, and is contemplated for use in the present invention.

As the cDNA sequence for V2-antiplasmin is available, recombinant expression and / or fusion proteins are preferred. Monoclonal antibodies to / 2-antiplasmin are also available that can be used by the bispecific binding ligand modalities of the invention. These antibodies could be used both to administer endogenous V2-antiplasmin to a target site and to accumulate endogenous V2-antiplasmin and co-concentrate it within

226

Λ 'ΐΝυΤΠ the target region. ^D

F4. Anti-tubulin drugs

A range of drugs exert their effects via interference with tubulin activity. Because tubulin functions are essential for mitosis and cell viability, certain antitubulin drugs are powerful chemotherapeutic substances. Some of the best known and currently preferred antitubulin drugs for use with the present invention are colchicine; taxanes, such as taxol; vinca alkaloids, such as vinblastine, vincristine, and vindescine; and combretastatinas. Other antitubulin drugs are cytochalasins (including B, J, E), dolastatin, auristatin PE, paclitaxel, usiloxin D, rizoxin, 1069C85, colcemid, albendazole, azatoxin, and nocodazole.

As described in US Patent Nos. 5,892,069, 5,504,074, and 5,661,143, each specifically incorporated herein by reference, combretastatins are estradiol derivatives that generally inhibit cell mitosis. Exemplary combretastatins that can be used in conjunction with the invention and include those based on combretastatin A, B, and / or D and those described in US Patent Nos. 5,892,069,

<img file="MX337052B_D0208.tif" />

227

<img file="MX337052B_D0209.tif" />

5,504,074 and 5,661,143.

The .combretastatinas ιΆ ^ Τ, A-2 t

<img file="MX337052B_D0210.tif" />

A-4, A-5, A-6, Bl, B-2, B-3 and B-4 are examples of the • ¿«CLuijt.neKnt» previous types.

The United States Patents of

North America Numbers 5,569,786 and 5,409,953, are incorporated herein by reference for purposes of describing the isolation, structural characterization, and synthesis of each of the combretastatins Al, A-2, A-3, Bl, B-2, B- 3 and B-4 and formulations and methods for using these combretastatins to treat neoplastic growth. Any or more of these combretastatins can be used in conjunction with the present invention.

Combretastatin A-4, as described in US Patent Nos. 2

5,892,069, 5,504,074, 5,661,143 and 4,996,237, each specifically incorporated herein by reference, may also be used with it. United States Patent No. 5,561,122 is further incorporated herein by reference.

<td>to describe</td><td>prodrugs</td><td>of</td><td>combretastatin</td><td>A-4</td>

<td>convenient, that</td><td>are contemplated</td><td>for</td><td>combined use</td><td>with</td>

<td colspan="2">the present invention.</td><td></td><td></td><td></td>

<td>The.</td><td>Patent</td><td>the</td><td>United States</td><td>of</td>

North America number 4,940,726, specifically incorporated herein by reference, particularly describes

228 in combination with the present invention.

North America number herein as the compositions and methods of the

The United States Patent of

5,430,062, specifically incorporated reference, refers to stilbene derivatives and combretastatin analogues with anticancer activity that can be used in combination with the present invention.

F5. Anti-Angiogenic Agents

Anti-angiogenic agents are useful for binding of antibodies and peptides of the invention. Many anticancer agents have an anti-angiogenic effect as part of their mechanism of action. Any or more of those agents described for use in combination therapies, including those in Table E, may also be conjugated to an antibody of the invention, as described herein. Certain other agents have been discovered, designed, or selected to have an antiangiogenic effect as a primary mechanism of action. Examples of those agents are described below, any one of which can be used to prepare an immunoconjugate or used separately in combination therapy with the invention.

Numerous useful tyrosine kinase inhibitors

229 for the treatment of angiogenesis, such as

<img file="MX337052B_D0211.tif" />

various disease states are now known. These include, for example, the 4-aminopyrrolo [2,3-d] pyrimidines in US Patent No.

5,639,757, specifically incorporated herein by reference, which can also be used in combination with the present invention. Other examples of organic molecules capable of modulating tyrosine kinase signal transduction via the VEGFR2 receptor are quinazoline compounds and compositions of the US Patent.

United States of America number 5,792,771, which is specifically incorporated herein by reference for the purpose of describing additional combinations for use with the present invention in the treatment of angiogenic diseases.

Compounds from other chemical classes have also been shown to inhibit angiogenesis and to

<img file="MX337052B_D0212.tif" />

they can be used in combination with the present invention. For example, spheroids such as angiostatic 4.9 (11) -spheroids and C21-oxygenated spheroids, as described in U.S. Patent No. 5,972,922, specifically incorporated herein by reference, can be employed in combined therapy. United States Patent Number North America

5,712,291 and 5,593,990, each specifically incorporated

230

INSTí íw '· * * *.

herein for reference, describe related compounds, precursors, analogs ·! metabnites.

hydrolysis products, which can also be used in combination with the present invention to inhibit angiogenesis. Compounds in United States Patents

United States of America numbers 5,712,291 and 5,593,990, can be administered orally. Other exemplary antiangiogenic substances that are useful in connection with combination therapy are listed in Table B. Each of the 10 agents listed herein are exemplary and in no way limiting.

c

TABLE B

Negative Inhibitors and Regulators _1¿ Angiogenesis

<td>SUBSTANCES</td><td>REFERENCES</td>

<td>Angiostatin</td><td>O'Reilly et al., 1994.</td>

<td>Endostatin</td><td>O'Reilley et al., 1997.</td>

<td>16kDa Prolactin Fragment</td><td>Ferrara et al., 1991; Clapp et al., 1993; one D'Angelo et al., 1995; Lee et al., 1998.</td>

<td>Laminin peptides</td><td>Kleinman et al., 1993; one Yamamura et al., 1993; | Iwamoto et al., 1996; j Tryggvason, 1993. |</td>

231

<img file="MX337052B_D0213.tif" />

<td>Fibronectin peptides</td><td>D £ uk: c. .í r 4 Grant et al .; Sheu et al., 1997.</td>

<td>Tissue metalloproteinase inhibitors (TIMP 1, 2, 3, 4).</td><td>Sang, 1998</td>

<td>Plasminogen activator inhibitors (PAI-1, -2).</td><td>Soff et al., 1995</td>

<td>Tumor necrosis factor &lt;* □ (high dose, in vitro).</td><td>Crater-Schroder and collaborators, 1987.</td>

<td>TGF-Sl</td><td>RayChadhury and D'Amore, 1991; Tada et al., 1994.</td>

<td>Interferon (IFN-t, -S, d)</td><td>Moore et al., 1998; Lingen et al., 1998.</td>

<td>ELR-CXC chemokines; IL-12; SDF-1; MIG; Platelet factor 4 (PF-4); IP-10.</td><td>Moore et al., 1998; Hisco and Jiang, 1997; Coughlin et al., 1998; Tanaka et al., 1997.</td>

<td>Thrombospondin (TSP)</td><td>Good et al., 1990; Frazier, 1991; Bornstein, 1992; Tolsma et al., 1993; Sheibani and Frazier, 1995; Volpert et al., 1998.</td>

<td>SPARC</td><td>'Hasselaar and Sage, 1992; Lañe et al., 1992; Jendraschak and Sage, 1996. H</td>

<td>2-Methoxiestradiol</td><td>Fotsis et al., 1994 1</td>

<td>Protein related proliferin</td><td>Jackson et al., 1994. 1</td>

<td>Suramin</td><td>Gagliardi et al., 1992; Takano et al., 1994; Waltenberger et al., 1996; Gagliardi et al., 1998, Manetti et al., 1998.</td>

<img file="MX337052B_D0214.tif" />

<td>Thalidomide</td><td>D'Amato and collaborators Kenyon et al., 1997; Wells, 1998. ................</td>

<td>Cortisone</td><td>Thorpe et al., 1993; Folkman et al., 1983; Sakamoto et al., 1986.</td>

<td>Linomide</td><td>Vukanovic et al., 1993; Ziche et al., 1998; Nagler et al., 1998.</td>

<td>Fumagiline (AGM-1470, TNP-470).</td><td>Sipos. And collaborators, 1994; Yoshida et al., 1998.</td>

<td>Tamoxifen</td><td>Gagliardi and Collins, 1993; Linder and Borden, 1997; Haran et al., 1994.</td>

<td>Korean mistletoe extract Viscum album coloratum).</td><td>Yoon et al., 1995.</td>

<td>Retinoids</td><td>Oikawa et al., 1989; Lingen et al., 1996; Majewski et al., 1996.</td>

<td>CM101</td><td>Hellerqvist et al., 1993; Quinn et al., nineteen ninety five; Wamil et al., 1997; DeVore et al., | 1997. I</td>

<td>Dexamethasone</td><td>Hori 'et al., 1996; Wolff et al., 1997.</td>

<td>leukemia inhibitory factor (LIF).</td><td>Pepper et al., 1995</td>

233

X. JL 7-L · J1, _, INSTITUTO MEXiQLNO V¿ *

Certain preferred components pKÉ ^ R &lt;^ M¡usqg inhibit angiogenesis are angiostatin, endostatin, vasculostatin, canstatin and maspin. The protein called angiostatin is described in US Patents 5,776,704, 5,639,725, and 5,733,876, each incorporated herein by reference. Angiostatin is a protein that has a molecular weight of between about 38 kD and about 45 kD, determined by reducing polyacrylamide gel electrophoresis, which contains about 1 to 4 Kringle regions of a plasminogen molecule. Angiostatin generally has an amino acid sequence substantially similar to that of a murine plasminogen fragment that begins at amino acid number 98 of an intact murine plasminogen molecule.

The amino acid sequence of angiostatin varies slightly between species. For example, in human angiostatin, the amino acid sequence that is substantially similar to the cleavage of the murine plasminogen fragment described above, although an active human angiostatin sequence can start at any amino acid number 97 or 99 of a plasminogen amino acid sequence intact human. Furthermore, human plasminogen can be used, as it has similar anti-angiogenic activity, as

234

INSTI PUTO MEXIRANO

DELA PKOPÍÉDAO

Industrial.

anti-angiogenic therapies mouse tumor.

regressions in tumors, and have the sample in a model of

Certain shown to cause angiostatin is one of those agents. Endostatin, a 20 kDa COOH-terminal fragment of collagen XVIII, bacterial polysaccharide CM101, and antibody LM609 also have angiostatic activity. However, in view of other properties, these are referred to as anti-vascular therapies or toxins against tumor vessels, since they not only inhibit angiogenesis but also initiate the destruction of tumor vessels through mechanisms in their Undefined majority.

Angiostatin and endostatin have become the focus of intensive studies, as they are the first angiogenesis inhibitors to have demonstrated the ability to not only inhibit tumor growth, but also cause tumor regressions in mice. There are multiple proteases that have been shown to produce angiostatin from plasminogen including elastase, macrophage metalloelastase (MME), matrilysin (MMP-7), and 92 kDA gelatinase B / collagenase (MMP-9).

MME can produce angiostatin from plasminogen in tumors, and granulocyte macrophage colony stimulating factor (GMCSF) increases MME expression by angiostatin-inducing macrophages. The role of MME production generation that MME carcinomas is thought

235

<img file="MX337052B_D0215.tif" />

INSTITUTE M

FROM THE INDUSTRIAL PSPSIDIA of angiostatin is supported by the finding is in fact expressed in clinical samples of hepatocellular patients. Another protease that is capable of stromelysin-1 (MMP-3).

similar fragments

Angiostatin plasminogen in vitro.

currently that it binds to an unidentified or about that mitotic arrest endothelial cell.

Failure to produce angiostatin is the

MMP-3 has been shown to produce angiostatin to the mechanism of the unclear, it becomes a superficial receptor and suffers death.

Endostatin appears to stem from the hypothesized action of the cell-inducing programmed cell, or to be an antiangiogenesis agent and anti-tumor biology causing mice.

even more powerful, although its is less clear.

regressions

Endostatin tumors tumors increase the number of cells in non

The various and, after endostatin is effective, tumor models develop resistance to multiple cycles of treatment, enter a dormant state, during volume. In this latent state, the tumor undergoing apoptosis which did not increase in percentage, same size. Endostatin is believed to bind to a receptor on the surface of endothelial cells, not producing a population that essentially remains

236

<img file="MX337052B_D0216.tif" />

identified, which mediates its effect

The Patent of the

North America number 5,854,205, for Folkman and O'Reill and T specifically incorporated herein by reference, refers to endostatin and its use as an inhibitor of endothelial cell proliferation and angiogenesis. The endostatin protein corresponds to the C Terminal fragment of the collagen XVIII type and the protein can be isolated from a variety of sources. United States Patent No. 5,854,205 also shows that endostatin can have an amino acid sequence or a fragment of type XVIII collagen, a type XV collagen, or BOVMPE 1 of pregastric esterase. Combinations of endostatin with other anti-angiogenic proteins, particularly angiostatin, are also described in US Patent No. 5,854,205, so that the combined compositions are capable of effecting effective regression of the mass of a tumor dependent on the angiogenesis.

CM101 is a bacterial polysaccharide that has been well characterized for its ability to induce neovascular inflammation in tumors. CM101 binds to, and cross-linked receptors expressed in dedifferentiated endothelium that stimulate activation of the complement system. It also initiates an inflammatory response

237 cytokine driven

<img file="MX337052B_D0217.tif" />

tumor. It is a unique antipathoangiogenic substance that downregulates the expression of VEGF and its receptors.

CM101 is currently in clinical trials as an anticancer drug, and can be used in combination with this invention.

Thrombospondin (TSP-1) and platelet factor 4 (PF4) can also be used with the present invention. Both are angiogenesis inhibitors that are associated with heparin and are found in platelet alpha-granules. TSP-1 is a 450 kDa large multiple domain glycoprotein that is constituent of the extracellular matrix. TSP-1 binds to many of the proteoglycan molecules found in the extracellular matrix that include HSPGs, fibronectin, laminin, and different types of collagen. TSP-1 inhibits endothelial cell migration and proliferation in vitro and angiogenesis in vivo. TSP-1 also suppresses the malignant phenotype and tumorigenesis of transformed endothelial cells. The tumor suppressor gene p53 has been shown to directly regulate the expression of TSP1, so that loss of p53 activity causes a dramatic reduction in TSP-1 production and a concomitant increase in angiogenesis initiated in the tumor.

<img file="MX337052B_D0218.tif" />

238

INSTITUTE Μ.'Χ · γ_Λ? ο DELA PRO; d

PF4 is a 70aa protein that is a member of the CXC ELR family of chemokines that is highly potent in vitro endothelial cell proliferation, γ angiogenesis in vivo. PF4 administered intratumorally or administered by adenoviral vector is capable of causing inhibition of tumor growth.

Interferons and metalloproteinase inhibitors are two classes of naturally occurring angiogenic inhibitors that can be administered with the present invention. The antiendothelial activity of interferons has been known since the early 1980s, however, the mechanism of inhibition is still unclear. It is known that it can inhibit endothelial cell migration and that it has some anti-angiogenic activity in vivo that is possibly mediated by an ability to inhibit the production of angiogenic promoters by tumor cells. Vascular tumors in particular are sensitive to interferon, for example, proliferating hemangiomas can be successfully treated with IFNV.

Tissue metalloproteinase inhibitors (TIMPs) are a family of naturally-occurring inhibitors of matrix metalloproteases 25 (MMPs) that can also inhibit angiogenesis and are

239 can use

<img file="MX337052B_D0219.tif" />

protocols

<img file="MX337052B_D0220.tif" />

treatment

<img file="MX337052B_D0221.tif" />

invention. MMPs play a key role in the angiogenic process, since they degrade the matrix through which endothelial cells and fibroblasts migrate when they extend or remodel the vascular network. Indeed, a member of the MMPs, MMP-2 has been shown to associate with the endothelium activated via the VvS3 integrin, presumably for this purpose. If this interaction is interrupted by a fragment of MMP-2, then angiogenesis is decreased and tumor growth is inhibited.

There are various pharmacological agents that inhibit angiogenesis, one or more of which can be used as part of the present invention. These include AGM1470 / TNP-470, thalidomide, and carboxyamidotriazole (CAI). Fumagillin was found to be a potent angiogenesis inhibitor in 1990, and since then the synthetic analogues of fumagillin, AGM1470 and TNP-470 have been developed. These drugs inhibit endothelial cell proliferation in vitro and angiogenesis in vivo. TNP-470 has been extensively studied in human clinical trials with data suggesting that long-term administration is optimal.

Thalidomide was originally used as a sedative, but it was found to be a potent teratogen and was

240 discontinued. In 1994 táílrdoniida was found to be .ψΐ · OE LA - &lt;- ··. '&gt; · I:: ·; v ,. . · 'Ίλι, νΈ' -. Λ ·, angiogenesis inhibitor. Thalidomide is currently in clinical trials as an anticancer agent, as well as a treatment for vascular diseases of the eyes.

CAI is a synthetic low-molecular-weight angiogenesis inhibitor that acts as a calcium channel blocker that prevents actin reorganization, endothelial cell migration, and dispersion over collagen IV. CAI inhibits neovascularization at physiologically achievable concentrations and is highly tolerated orally by cancer patients. Clinical trials with CAI have produced disease stabilization in 49 percent of cancer patients who have progressive disease before treatment.

Cortisone in the presence of heparin or heparin fragments has been shown to inhibit tumor growth in mice by blocking endothelial cell proliferation. The mechanism involved in the additive inhibitory effect of the spheroid and heparin is unclear, although it is thought that heparin may increase the assimilation of the spheroid by endothelial cells. The mixture has been shown to increase the dissolution of the basement membrane below the newly formed capillaries, and this is also a possible

241 jívjL A JL

INSTITUTO MEXICANO explanation of the additive angiostatic effect. The^L', don ^ ga¿Í! ^^^ í' of heparin-cortisol also have potent angiostatic and anti-tumor pfpot-ns for in vivo activity.

Other specific angiogenesis inhibitors can be administered to tumors using the targeted tumor localization methods of the present invention. These include, but are not limited to, Anti-Invasive Factor, retinoic acids, and paclitaxel (US Patent No. 5,716,981; incorporated herein by reference); AGM-14 * 70 (Ingber et al., 1990; incorporated herein by reference); shark cartilage extract (United States Patent No. 5,618,925; incorporated herein by reference); anionic polyamide or polyurea oligomers (United States Patent No. 5,593,664; incorporated herein by reference); oxindole derivatives (United States Patent No. 5,576,330; incorporated herein by reference) estradiol derivatives (United States Patent No. 5,504,074; incorporated herein by reference); and thiazolpyrimidine derivatives (United States Patent No. 5,599,813; incorporated herein by reference) are also contemplated for use as anti-angiogenic compositions

242

I for the combined uses of this invention

<img file="MX337052B_D0222.tif" />

a

Compositions comprising V antagonist_v3₃ Integrin can also be used to inhibit angiogenesis in combination with the present

<img file="MX337052B_D0223.tif" />

invention. As described in the United States Patent

United States of America Number 5,766,591 (incorporated herein by reference), polypeptides containing

RGD and salts thereof, including cyclic polypeptides, are convenient examples of V-integrin antagonists_v3₃.

As angiopoietins are the ligands for Tie2, other therapeutic intervention methods based on altered signaling through the receptor can be used

Tie2, in combination with them. For example, a soluble Tie2 receptor can be used, capable of blocking Tie2 activation (Lin et al., 1998a). The supply of that construct that uses »

Recombinant adenoviral gene therapy has been shown to be effective in the treatment of cancer and in reducing metastasis (Lin et al., 1998a).

Angiopoietins, in common with members of the VEGF family, are very specific growth factors for the vascular endothelium (Davis and Yancopoulos, 1999; Holash et al., 1999;

incorporated herein by reference). The

<img file="MX337052B_D0224.tif" />

243 í _ i J Tyr p _T

I INSTITUTE

L ¿E ° “ir 5 ufe naturally agonist, receptor that is angiopoietins first described were activator receptor that presents angiopoietin-1 (Ang-1), and an antagonist naturally presents, angiopoietin-2 (Ang-2), both of These act by means of an endothelial cell tyrosine kinase receptor, Tie2.

Two new angiopoietins, angiopoietin-3 (mouse) and angiopoietin-4 (human) have also been identified (Valenzuela et al., 1999). Angiopoietin-3 appears to act as an antagonist (similar to Ang-2), while angiopoietin-4 appears to function as an agonist (such as Ang-1) (Valenzuela et al., 1999). A protein called angiopoietin-3 was also cloned from the human heart and was reported to have no mitogenic effects on endothelial cells (Kim et al., 1999).

While VEGF is required for the early stages of vascular development, angiopoietin1 is generally required for the post-vascularization stages. VEGF thus acts to promote endothelial cell differentiation, proliferation, and primitive vessel formation. Angiopoietin-I acts, via the Tie2 receptor, to promote the maintenance and stabilization of mature vessels. Angiopoietin-1 is thus a maturing or stabilizing factor,

244 matures promoting interactions between the endothelial cells and the surrounding support cells (Holash et al., 1999).

F6. Agents that Induce Apoptosis

The present invention can also be used to administer apoptosis-inducing substances into any cell within the tumor, including tumor cells and vascular tumor endothelial cells. Although many anticancer agents may have, as part of their mechanism of action, an effect that induces apoptosis. Any one or more of those agents described for use in combination therapy, including those in Table F, may also be conjugated to an antibody of the invention, as described herein. Certain other agents have been discovered, designed, or selected to have an apoptosis-inducing effect as a primary mechanism. Examples of those agents are described below, any one of which can be used to prepare an immunoconjugate or used separately in combination therapy with the invention.

Many forms of cancer have reports of mutation in tumor suppressor genes, such as

245 ρ53. Inactivation of p53 results in

<img file="MX337052B_D0225.tif" />

to promote apoptosis. With this failure, cancer cells progress in tumorigenesis, rather than being destined for cell death. Thus, the provision of tumor suppressors is also contemplated for use in the present invention to stimulate cell death. Exemplary tumor suppressors include, but are not limited to, p53, the Retinoblastoma (Rb) gene, Wilm's tumor (WT1), bax alpha, interleukin Ib and family converting enzyme, the MEN-1 gene, neurofibromatosis, type 1 (NF1), cdk pl6 inhibitor, colorectal cancer gene (DCC), familial adenmatosis polyposis gene (FAP), in multiple tumor suppressor (MTS-1), BRCA1 and BRCA2.

Preferred for use are p53 (US Patent Nos. 5,747,469; 5,6,771.78 and 5,756,455; each incorporated herein by reference), Retinoblastoma, BRCA1 (US Patent Nos. 5,750,400;

5,654,155; 5,710,001;

5,756,294; 5,709,999; 5,693,473

5,753,441; 5,622,829 and 5,747,282; each incorporated herein by reference), MEN-1 (GenBank accession number U93236), and adenovirus E1A (United States Patent 5,776,743; incorporated herein by reference).

246 which include, nL the apo

1NS7.

although 'they are not (other than bcl-1, cyclin y] x «r ^ν

Other oncogenes programmed cell death, limited to, bcr-abl, bcl-2

GAVE; GenBank accession numbers M14745, X06487; United States Patents Numbers 5,650,491; and 5,539,094; each incorporated herein by reference) and family members including Bcl-xl, Mcl-1, Bak, Al, A20. Overexpression of bcl-2 was first discovered in lymph cells of T cells. Bcl-2 functions as an oncogene by binding and inactivating Bax, a protein in the apoptotic pathway. Inhibition of bcl-2 function prevents Bax inactivation, and allows the apoptotic pathway to proceed. Thus, inhibition of this class of oncogenes, for example, using antisense nucleotide sequences, is contemplated for use in the present invention in aspects where increased apoptosis is desired (US Patent Numbers 5,650,491; 5,539,094; and 5,583,034; each of which is incorporated herein by reference).

Other compositions that can be administered by the antibodies of the present invention include genes encoding ligand-inducing apoptosis-related tumor necrosis factor called TRAIL, and the TRAIL polypeptide (US Patent

247

United States of America Number 5,763,223; ii ^ c ^ r jioradaa ·

INSTITU I '' present for reference); the protease to "associated 24 kD apoptosis of the US Pat. No. fisiders ^ ie-North America 5,605,826 (incorporated herein by reference); associated factor Fas 1, FAF1 (US Patent 5,750,653; incorporated herein by reference). Also contemplated for use in these aspects of the present invention is the provision of the enzyme that converts to interleukin-13 and family members, which are also reported to stimulate apoptosis.

Compounds such as carbostyril derivatives (US Patent Number 5,672,603; and 5,464,833; each incorporated herein by reference); branched apogenic peptides (United States Patent 5,591,717; incorporated herein by reference); non-hydrolyzable phosphotyrosine inhibitors and phosphotyrosine analogs (US Patent Number 5,565,491; and 5,693,627; each incorporated herein by reference); RXR retinoid receptor agonists (United States Patent No. 5,399,586; incorporated herein by reference); and even antioxidants (United States Patent No. 5,571,523; incorporated in the present

248 as a reference), tyrosine kinase, such as genistein, may also be used to bind to the antibodies herein

<img file="MX337052B_D0226.tif" />

too

<img file="MX337052B_D0227.tif" />

invention as supported by the US Patent. United States of America

Number 5,587,459; incorporated herein by reference).

F7. Antiviral Agents

Because anionic phospholipids and aminophospholipids, particularly PS and PE, become exposed in virally infected cells, the antibodies of the invention, such as antibodies 9D2 and 3G4 (ATCC 4545), can also be linked to any one of or more antiviral agents. Additional reasons supporting these aspects of the invention, and the advantages thereof, are described in greater detail below with respect to the PE-binding peptide, antiviral conjugates.

Exemplary antiviral agents that are for binding to antibodies or peptides are also described in greater detail in relation to the PE binding peptide, antiviral conjugates of the invention. Some or more antiviral agents, including those in Table G, can be conjugated to an antibody of the invention, as described herein. Those

249 antiviral agents can

<img file="MX337052B_D0228.tif" />

antiviral therapies

<img file="MX337052B_D0229.tif" />

G. Biologically Functional Equivalents

Equivalents, or even enhancements, of antibodies and effectors can also be made now, generally using the materials provided above as a starting point. Modifications and changes can be made to the structure of such an antibody and still obtain a molecule that has similar or otherwise desirable characteristics. For example, certain amino acids can be replaced by other amino acids in a protein structure without appreciable loss of interactive binding capacity. These considerations also apply to toxins, anti-angiogenic substances, apoptosis-inducing substances, coagulants, and the like.

Since it is the interactive capacity and nature of a protein that define the biological functional activity of the protein, certain amino acid sequence substitutions can be made in a protein sequence (or of course, the underlying DNA sequence) and yet obtain a protein with similar properties (agonist). In this way it is contemplated that several changes can be made in the sequence of

250

<img file="MX337052B_D0230.tif" />

antibodies or therapeutic substances (oin1 ^_to secuénc ^^ í'UA-. '·' * '' Of underlying DNA) without appreciable loss of its usefulness or biological activity. Biologically functional equivalents made from the mutated underlying DNA sequence can be made using the codon information provided herein in Table A, and the supporting technical details in site specific mutagenesis.

It is also well understood by those skilled in the art that, inherent in the definition of a biologically functional equivalent protein or peptide, is the concept that there is a limit to the number of changes that can be made within a defined portion of the molecule and still result in a molecule with an acceptable level of equivalent biological activity. Biologically functionally equivalent proteins and peptides in this way are defined herein as those proteins and peptides in which certain, not most, or not all, of the amino acids can be substituted. Of course, a plurality of different proteins / peptides with different substitutions can easily be made and used in accordance with the invention.

Amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, eg, their hydrophobicity, hydrophilicity, charge, size, and the like.

251

An analysis of size, shape

<img file="MX337052B_D0231.tif" />

Chain substituents that arginine, amino acid side lysine reveals, and histidine are all positively charged residues; that alanine, glycine and serine all have a similar size; and that phenylalanine, tryptophan, and tyrosine are all generally similar in shape. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine and serine; and phenylalanine, tryptophan, and tyrosine;

they are defined herein as biologically functional equivalents.

To make further quantitative changes, the hydropathic index of amino acids can be considered. Each amino acid has been assigned a hydropathic index

<td colspan="5">based on its hydrophobicity and load characteristics,</td>

<td colspan="3">these are: isoleucine (+4.5); valine</td><td> (+4.2);</td><td>leucine</td>

<td> (+3.8);</td><td colspan="3">phenylalanine (+2.8); cysteine ​​/ cystine</td><td> (+2.5);</td>

<td>methionine</td><td> (+1.9);</td><td>alanine (+1.8);</td><td>glycine</td><td> (-0.4) ;</td>

<td>threonine</td><td> (-0.7) ;</td><td>serine (-0.8);</td><td>tryptophan</td><td> (-0.9) ;</td>

<td>tyrosine</td><td> (-1-3) ;</td><td>proline (-1.6);</td><td>histidine</td><td> (-3.2) ;</td>

<td>glutamate</td><td> (-3.5) ;</td><td>glutamine (-3.5);</td><td>aspartate</td><td> (-3.5) ;</td>

asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic index of amino acids to confer interactive biological function on a protein is generally understood in the art (Kyte and

252 and they still retain a

Doolittle, 1982, incorporated in reference).

It is known that certain substitutes for other amino acids have a similar hydropathic index or similar biological activity rating. In making changes based on the hydropathic index, substitution of amino acids whose hydropathic indexes are within V2 is preferred, those which are within Vi are particularly preferred, and those within V0.5 are even more particularly preferred.

Thus, it is understood that an amino acid can be replaced by another that has a similar hydrophilicity value and still obtain a biologically equivalent protein. As detailed in United States Patent No. 4,554,101 (incorporated herein by reference), the following hydrophilicity values ​​have been assigned to the amino acid residues: arginine (+3.0); lysine

<td colspan="2">(+3.0); aspartate (+3.0 V</td><td> 1) ;</td><td>glutamate (3.0</td><td>V 1)</td>

<td>serine (+0.3);</td><td>asparagine</td><td> (+0.2),</td><td>; glutamine</td><td> (+0.2)</td>

<td colspan="2">glycine (0); threonine (-0</td><td> • 4) ;</td><td>proline (-0.5</td><td>v 1)</td>

<td>alanine (-0.5);</td><td>histidine</td><td> (-0.5)</td><td>; cysteine</td><td> (-1.0)</td>

<td>methionine (-1.3);</td><td>valine</td><td> (-1.5)</td><td>; leucine.</td><td> (-1.8)</td>

<td>isoleucine (-1.8);</td><td>tyrosine</td><td> (-2.3) ;</td><td>phenylalanine</td><td> (-2.5)</td>

<td>tryptophan (-3.4).</td><td></td><td></td><td></td><td></td>

253 j!. · 'M ¡u u

i. JzO. X, 1.

based on i.Jjqs .-. value

Dt LA FRL't-&gt; l ·. i -f. D

INDUSTRIAL amino acids whose hydrophilicity values, the substitution

When making changes

<img file="MX337052B_D0232.tif" />

Hydrophilicity are within V2 is preferred, those within Vi are particularly preferred, and those within V0.5 are even more particularly preferred.

H. Conjugation

Antibodies to anionic phospholipids and aminophospholipids, including selected anti-PS antibodies, with improved properties, such as

9D2 and

3G4 (ATCC 4545), can be conjugated or linked to, or functionally associated with, cytotoxic anti-cellular agents to prepare immunotoxins;

coagulants, either directly or indirectly, to prepare coaguligands;

antiviral agents, such as nucleosides, for preparing immuno-viral antiviral immunoconjugates. PE-binding peptides such as duramycin can also be conjugated or linked to, or functionally associated with, inert carriers, targeting agents, or antiviral agents, to prepare a range of derivatives of PE-binding peptides and antiviral peptide conjugates.

Although links are preferred

254 covalent, can also be used ot ^&gt; ^? ^ nédibs de / '&gt; |

MEXICAN INSTITUTE OF THE R'JrlBí.V) functional union. For example, the constructs^{! N}Emphasis ^ OS 'can be generated using arvidliidíMótrna bridges. In addition to the knowledge available to those of ordinary skill in the art, the common property U.S. Patent No. 6,093,399 is specifically incorporated herein by reference for purposes of further describing and enabling the use of avidin. : Biotin in the functional union of antibodies and targeted localization agents, to biological and therapeutic agents.

The two agents can also be linked by a second binding region, preferably a binding region to the antibody or to the antigen thereof. This is exemplified by coaguligands where the target agent binds to the coagulant through a second binding region (US Patent Nos. 6,093,399,

6,004,555, 5,877,289 and 6,036,955, each specifically incorporated herein by reference), which have been successfully produced and used in the treatment of cancer. Wherein the first targeting agent is an antibody or an antigen region, the use of a second binding region

255

<img file="MX337052B_D0233.tif" />

institute .v.cA ··

FROM THE ΡλΟΡϋ '.

RestitTACT which is also an antibody, binding to the antigen, gives bispecific construct of the antibody. repair and use of bispecific antibodies is generally well known in the art, and is further described herein.

Immunoconjugate technology is now generally known in the art. However, certain advantages can be achieved by applying a certain preferred technology, both in preparation and in purification for subsequent clinical administration. For example, although IgG-based constructs will typically exhibit better binding capacity and slower clearance in blood than their Fab 'counterparts, Fab' fragment-based constructs will generally exhibit better tissue penetrability.

Additionally, although numerous types of disulfide-containing linkers are known, which can be successfully employed in the conjugation of antibodies and peptides, certain linkers will generally be preferred over other linkers, based on different pharmacological characteristics and capabilities. For example, preferred

256 linkers that contain a link

<img file="MX337052B_D0234.tif" />

is sterically hindered, due to its greater stability in vivo, thus preventing the release of the coagulant before binding at the site of action.

Each type of crosslinker, as well as how crosslinking is carried out, will tend to vary the pharmacodynamics of the resulting conjugate.

It may be desired to have a conjugate that remains intact under the conditions found in any part of the body except the desired site of action, at which point it is desirable that the conjugate have good release characteristics. Therefore, the particular crosslinking scheme, including in particular the reagent used, and the particular crosslinking structures, which are crosslinked, will be of some significance.

Depending on the specific agents we provide a peptide spacer operably linked to the antibody or to the PE binding peptide and to the second or therapeutic agent. Certain peptide separators can be folded into a disulfide-bonded coil structure. Proteolytic cleavage within the loop will then produce a polypeptide

257 the anticue

<img file="MX337052B_D0235.tif" />

Bound only by one utilize certain compounds can a cleavage be provided, to bind heterodimer where therapeutically single disulfide bond is found. A toxin of .chain Ricin A.

Where different from toxins, peptide spacer is non-functionally to the antibody and the toxin compound of the fusion protein. The toxins that can be used in conjunction with non-cleavable peptide separators are those that can themselves be converted by proteolytic cleavage into a disulfide-bound, cytotoxic form. An example of that toxin compound is the Pseudomonas exotoxin compound.

A variety of chemotherapeutic agents and other pharmacological agents have now been successfully conjugated to antibodies and are shown to function pharmacologically. Exemplary anti-neoplastic agents that have been investigated include doxorubicin, daunomycin, methotrexate, vinblastine, and several others. Furthermore, the binding of other agents such as neocarzinostatin, macromycin, trenimon and aamanitin has also been described. These bonding methods can be adapted for use with the present.

Any covalent bond to the antibody or

258 PE-binding peptide will be

<img file="MX337052B_D0236.tif" />

in a site other than (functional)

The compositions linked in an operational way

<img file="MX337052B_D0237.tif" />

The site (s) are then allowing each region to perform its intended function, without significant harm, in particular such that the resulting construct still binds to the intended antigen or PE and in such a way that the bound agent maintains substantially biological activity 10 and / or regain biological activity when released from the construct.

Hl. Biochemical crosslinkers

<img file="MX337052B_D0238.tif" />

Besides the . General information provided above, PE binding peptide antibodies can be conjugated to therapeutic agents or other agents, using certain preferred biochemical crosslinking crosslinkers. The reagents are used to form molecular bridges that bind functional groups of two different molecules together. To link two different proteins in a staggered manner, hetero-bifunctional crosslinkers can be used that eliminate unwanted homopolymer formation. Exemplary bifunctional hetero25 crosslinkers are presented in Table C.

259

<img file="MX337052B_D0239.tif" />

MWCANO INSTITUTE

OF INDUSTRIAL PROPERTY

<img file="MX337052B_D0240.tif" />

TABLE C

HETERO-BIFUNCTIONAL CROSSLINERS

<td>Linker</td><td>Reactive towards</td><td>Advantages and Applications</td><td>Separation Length after Crosslinking</td>

<td>SMPT</td><td>Sulfhydryl primary amines</td><td>Greater stability</td><td>11.2 A</td>

<td>SPDP</td><td>Sulfhydryl primary amines</td><td>Rape Reticulation scissile</td><td>6.8 A</td>

<td>LC-SPDP</td><td>Sulfhydryl primary amines</td><td>Extended spacer arm</td><td>15.6 A</td>

<td>Sulfo-LC-SPDP</td><td>Sulfhydryl primary amines</td><td>Extended Soluble Water Spread Arm</td><td>15.6 A</td>

<td>SMCC</td><td>Sulfhydryl primary amines</td><td>Stable Maleimide Reagent Group Enzyme Antibody Conjugation Protein Conjugation</td><td>11.6 A</td>

<td></td><td></td><td>Hapten - carrier</td><td></td>

<td>Sulfo-SMCC</td><td>Sulfhydryl primary amines</td><td>Stable maleimide reactive group Soluble in water Enzyme-antibody conjugation</td><td>11.6 A</td>

<td>MBS</td><td>Sulfhydryl primary amines</td><td>Conjugation enzyme antibody Hapten-carrier protein conjugation</td><td>9.9 A</td>

<td>Sulfo-MBS</td><td>Sulfhydryl primary amines</td><td>Soluble in water</td><td>9.9 A</td>

260

<img file="MX337052B_D0241.tif" />

Reactive Linker to Advantage vja »Applications Separation after Crosslinking

<td>SIAB ·</td><td>Sulfhydryl primary amines</td><td>Conjugation enzyme antibody</td><td colspan="2">10.6 A</td>

<td>Sulfo-SIAB</td><td>Primary amines</td><td>Soluble in water</td><td> 10.6</td><td>TO</td>

<td></td><td>sulfhydryls</td><td></td><td></td><td></td>

<td>SMPB</td><td>Primary amines</td><td>Extended separator</td><td> 14.5</td><td>TO</td>

<td></td><td>sulfhydryls</td><td>Conjugation enzyme-</td><td></td><td></td>

<td></td><td></td><td>antibody</td><td></td><td></td>

<td>Sulfo-SMPB</td><td>Primary amines</td><td>Extended separator</td><td> 14.5</td><td>TO</td>

<td></td><td>sulfhydryls</td><td>Soluble in water</td><td></td><td></td>

<td>EDC / Sulfo-NHS</td><td>Primary amines</td><td>Conjugation Hapten-</td><td> 0</td><td></td>

<td></td><td>ca.rboxyl groups</td><td>Carrier</td><td></td><td></td>

<td>ABH</td><td>Carbohydrates not c</td><td>React with groups</td><td> 11.9</td><td>TO</td>

<td></td><td>selective</td><td>sugar</td><td></td><td></td>

Hetero-bifunctional crosslinkers contain two reactive groups: one generally reacts with a primary amino group (eg, N-hydroxy succinimide) and the other generally reacts with a thiol group (eg, pyridyl disulfide, maleimides, halogens, etc.). ). Through the primary amine reagent group, the crosslinker can react with the lysine residue (s) of a protein (eg, the selected PE-binding antibody, fragment, or peptide) and through the thiol reagent group, the crosslinker, already attached to the first protein, it reacts with the cysteine ​​residue (. free sulfhydryl group) of the other

261 ^procein· ΙΜΡΙΓ&gt; ϊ&gt; '

Mexican INSTITUTE

The compositions have, or are derived to have, a fnnr-innai growl available for crosslinking purposes. This requirement is not considered to be limiting because a wide variety of groups can be used in this way. For example, primary or secondary amine groups, hydrazide or hydrazine groups, carboxylic alcohol, carbamate, phosphate, or alkylating groups can be used to link or crosslink.

The separator arm between two reactive groups of crosslinkers can have various lengths and chemical compositions. A longer spacer arm allows for better flexibility of the conjugate components although some particular components in the bridge (eg benzene group) can provide extraordinary stability to the reactive group or increased resistance of the chemical bond to action in several respects (eg. example, reducing substance resistant disulfide bond). The use of peptide separators, such as L-Leu-L-Ala-L-Leu-L-Ala, is also contemplated.

It is preferred that a crosslinker having reasonable blood stability be used. Numerous types of disulfide bonds containing linkers are known that can be successfully employed in conjugation.

262 sterically impaired can stability in vivo, avoiding the before the link on the site

<img file="MX337052B_D0242.tif" />

ITUTO MEXICANODE INDUSTRIAL PROPERTY demonstrate having greater

<img file="MX337052B_D0243.tif" />

release of the action substance. These linkers are thus a preferred group of linking agents.

One of the most preferred crosslinking reagents is SMPT, which is a bifunctional crosslinker containing a disulfide bond that is sterically hindered by an adjacent benzene ring and methyl groups. Spherical disruption of the disulfide bond is believed to serve as a protective function of the bond from attack by thiolated anions such as glutathione which may be present in tissues and blood, and thereby helps to prevent decoupling of the conjugate prior to administration of the substance attached to the tumor site. It is contemplated that the SMPT substance can also be used in conjunction with the conjugates of this invention.

The SMPT crosslinking reagent, as with many other known crosslinking reagents, provides the ability to crosslink functional groups such as the SH of cysteine ​​or primary amines (eg, the amino group epsilon of lysine). Another possible type of crosslinker includes heterobifunctional photoreactive phenylazides that contain a cleavable disulfide bond such as sulfosuccinimidyl ethyl-1,3'-dithiopropionate263

I

IΜ ΡI

2- (p-azido salicylamido). The N-hydrosf group ^^^^^ itp:

INDUSTRIAL

<img file="MX337052B_D0244.tif" />

it reacts with primary amino groups and phenylazide (after photolysis) reacts non-selectively with any amino acid residue.

In addition to hindered crosslinkers, unimpeded linkers can also be employed herein. Other useful crosslinkers, not considered to contain or generate a protected disulfide, include SATA, SPDP, and 2-iminothiolane. The use of these crosslinkers is well understood in the art.

As soon as they are conjugated, the conjugate separates from antibodies or peptides and other agents, and from other contaminants. A large number of purification techniques are available for use to provide conjugates with a sufficient degree of purity to make them clinically useful. Purification methods based on size separation, such as gel filtration, gel permeation, or high performance liquid chromatography, will generally be of greater use. Other chromatographic techniques such as Sepharose Blue separation can also be used.

H2. Biologically Liberable Linkers

Although it is preferred that any binding fraction have reasonable stability in the blood, for

264 avoid substantial release of

<img file="MX337052B_D0245.tif" />

INDUSTRIAL disease, eg, linked before targeting the tumor site, in certain respects, the use of selectively cleavable biologically releasable linkers and / or linkers is contemplated. Biologically releasable bonds and selectively cleaved spacers or linkers still have reasonable stability in circulation.

The PE binding antibodies or peptides according to the invention can thus be linked to one or more second agents. therapeutics via a biologically releasable bond. Any form of antibody or targeting agent can be

<img file="MX337052B_D0246.tif" />

employ, including intact antibodies, although ScFv fragments will be preferred in certain embodiments.

Biologically releasable bonds or selectively hydrolyzable bonds include all bonds that are releasable, cleavable, or hydrolyzable only or preferentially under certain conditions. This includes disulfide and trisulfide bonds and labile acid bonds, as described in United States Patent Nos. 5,474,765 and 5,762,918, each specifically incorporated herein by reference.

Using an acid sensitive separator to

265 binding with a therapeutic agent or drug to

<img file="MX337052B_D0247.tif" />

PE-binding peptide from

OF THE PROPERTY

INDUSTRIAL the invention,

<img file="MX337052B_D0248.tif" />

behold particularly.

In these modalities, the therapeutic agents are released into the acidic compartments within a cell. It is contemplated that acid sensitive release can occur extracellularly, but still after specific targeting, preferably to the site of the virally infected tumor or cell. Certain currently preferred examples include 2C3-like antibodies bound to colchicine or doxorubicin via an acid sensitive separator. Binding via the carbohydrate moieties of the antibodies is also contemplated. In these modalities, therapeutic substances or drugs are released into acidic compartments within a cell.

The antibody or peptide binding to the

<td>PE too</td><td>I know</td><td>can derive</td><td>for</td><td colspan="2">enter groups</td>

<td>functional</td><td>than</td><td>allow the</td><td>Union</td><td>of</td><td>the agents</td>

<td>therapeutic</td><td>to</td><td>through a</td><td colspan="2">link</td><td>biologically</td>

releasable. The PE binding antibody or peptide can thus be derived to introduce side chains ending in hydrazide, hydrazine, primary amine, or secondary amine groups. The therapeutic substances can be conjugated through the Schiff base bond, a hydrazone or acyl hydrazone bond or a

266

MENICA INSTITUTE (Patents of the States - * ^

<img file="MX337052B_D0249.tif" />

linker

North America numbers 5,474,765 and 5.76 2,918, each specifically incorporated herein by reference).

Also described in United States Patent Nos. 5,474,765 and 5,762,918, each specifically incorporated herein by reference, the PE-binding antibody or peptide can be operably linked to therapeutic substances through one or more biologically releasable bonds that are enzyme-sensitive bonds, including peptide, ester, amide, phosphodiester, and glycoside bonds.

Certain preferred aspects of the invention relate to the use of peptide linkers that include at least a first cleavage site for a peptidase and / or proteinase that is preferentially located within the site of the disease, particularly within the tumor environment. Antibody-mediated administration of the bound therapeutic substance results in cleavage specifically within the disease site or tumor environment, resulting in the specific release of the active substance. Certain peptide linkers will include a cleavage site that is recognized by one or more enzymes involved in remodeling.

<img file="MX337052B_D0250.tif" />

267

I institute m: '· DE LA PP^r.!

cleavage site for urokinase, pro-urokinase, plasmin, plasminogen, TGFS, staphylokinase, thrombin,

Factor IXa, Factor Xa or a metalloproteinase, such as interstitial collagenase, stromelysin, a gelatinase one is particularly preferred. The

United States patents of

North America Nos. 6,004,555,

5,877,289, and 6,093,399 are specifically incorporated herein by reference for the purpose of further describing and enabling how to make and use immunoconjugates comprising biologically releasable linkers and selectively cleavable linkers and peptides. United States Patent No. 5,877,289, is particularly incorporated herein by reference for the purpose of further describing and enabling how to use immunoconjugates comprising a selectively cleavable peptide linker that is cleaved by urokinase, plasmin, thrombin, Factor IXa, Factor Xa or a metalloproteinase, such as an interstitial collagenase, a gelatinase, or a stromelysin, within a tumor environment.

Currently preferred selectively cleavable peptide linkers are those that include a plasmin cleavage site or a

<img file="MX337052B_D0251.tif" />

<img file="MX337052B_D0252.tif" />

metalloproteinase (also known as me ^ ESpffioiuBás ^ Industrial matrix or MMP), such as an interstitial collagenase, a gelatinase, or a stromelysin. Additional peptide linkers that may be used advantageously in connection with the present invention include, for example, plasmin cleavage sequences, such as those that can be cleaved by pro-urokinase, TGF3, plasminogen, and staph ylokinase '; sequences cleaving factor Xa; sequences that cleave MMP such as those that can be cleaved by gelatinase A; Collagen sequences are cleaved, such as those that can be cleaved by calf skin collagen (chain al (I)), calf skin collagen (chain o2 (I)), bovine cartilage collagen (chain al ( II)), human liver collagen (chain al (III)), a₂Human M, human PZP, rat αχΜ, a₂Rat M, axI₃(2J) rat, axI₃(27J) rat, and autolytic cleavage sites of human fibroblast collagenase. In addition to the knowledge available to those of ordinary skill in the art, the text and sequences in Table B2 in the co-owned United States of America Patent Nos. 6,342,219, 6,524,583, 6,342,221, and 6,416,758 are specifically incorporated herein by reference. for the purpose of further describing and enabling the use of these cleavable sequences.

269 π

.to.

MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL

<img file="MX337052B_D0253.tif" />

H3. Bispecific Antibodies

Bispecific antibodies in general can be used, as long as one arm binds to an anionic phospholipid or aminophospholipid, and the bispecific antibody binds, at a site other than antigen binding sites, to a therapeutic agent.

In general, the preparation of bispecific antibodies is also well known in the art. One method involves the separate preparation of antibodies having specificity for anionic phospholipid or aminophospholipid, on the one hand, and on the other, a therapeutic agent. The peptic fragments F (ab '()₂ they are prepared from two chosen antibodies, followed by the reduction of each to provide Fab 'fragments (_SH. SH groups in one of the two partners to be coupled are alkylated with a crosslinking reagent such as o-phenylenedimaleimide to provide free maleimide groups on the other partner. This partner can then be conjugated to the other by means of a thioether bond, to give the desired heteroconjugate F (ab '()₂. Other techniques are known where crosslinking with SPDP or protein A is carried out, or a trispecific construct is prepared.

Another method to produce antibodies

270

JlVL r 1 \ bispecific is through the fusion of

INDUSTRIAL form a square. As used herein, the term "quadroma" is used to describe the productive fusion of two B-cell hybridomas. Using standard techniques now, two antibody-producing hybridomas fuse to give daughter cells, and these cells that have maintained expression from both clonotype immunoglobulin gene sets are selected.

A preferred method of generating a quadroma involves the selection of an enzyme deficient mutant from at least one of the parent hybridomas. This first mutant hybridoma cell line fuses with cells from a second hybridoma that has been fatally exposed, for example, to iodoacetamide, preventing its continued survival. Cell fusion allows rescue of the first hybridoma by acquiring its enzyme deficiency gene from the lethally treated hybridoma, and rescue of the second hybridoma through fusion with the first hybridoma. Fusion of 20 immunoglobulins from the same isotope, but from a different subclass, is preferred but not required. A mixed subclass antibody allows the use of an alternative assay for the isolation of a preferred square.

In more detail, a development method d

<img file="MX337052B_D0254.tif" />

grid and selection involves obtaining a line of

271 hybridoma that secretes the first

INSTITUTO MEXICANO chosen antibody and make it deficient of the

<img file="MX337052B_D0255.tif" />

essential, hypoxanthine-guanine phosphoribosyltransferase (HGPRT). To obtain deficient hybridoma mutants, sd * cultured cells in the presence of increasing concentrations of 8-azaguanine (1 x ΙΟ⁷ M up to 1 x ΙΟ '⁵ M). Mutants are subcloned by limiting dilution and testing their sensitivity to hypoxanthine / aminopterin / thymidine (HAT). The culture medium may consist of, for example, MEMD supplemented with 10 percent SBF, 2mM L-Glutamine and 1mM penicillin-streptomycin.

A complementary hybridoma cell line that produces the desired second monoclonal antibody is used to generate the quadromas using standard cell fusion techniques. In short, 4.5 x 10⁷ first HAT-sensitive cells mix with 2.8 x 10⁷ second HAT-resistant cells that have previously been treated with lethal dose of irreversible biochemical inhibitor iodoacetamide (5 mM in phosphate buffered saline) for 30 minutes on ice prior to fusion. Cell fusion is induced using polyethylene glycol (PEG) and cells are plated on 96-well microculture plates. Grids are selected using HAT-containing medium. Cultures containing bispecific antibody are identified using, for example, an ELISA specific for the

272 • Α- Λ »Λ x JEL r &lt;&lt; 2 ^^ INSTITUTO MEXICANO isotype of solid phase, and stained immiíñ ^^ g ^ gesQ ^^^ specific for the isotype. _____

In an identification modality to identify the bispecific antibody, the wells of the microtiter plates (Falcon, Becton Dickinson Labware) are covered with a reagent that specifically interacts with one of the parent hybridoma antibodies and lacks cross-reactivity with both antibodies. Plates are washed, blocked, and supernatants (SN) to be tested are added to each well. Plates are incubated at room temperature for two hours, supernatants are discarded, plates are washed, and alkaline phosphatase-anti-antibody conjugate are added for two hours at room temperature. The plates are washed and a phosphatase substrate, eg P-Nitrophenyl Phosphate (sigma, St. Louis) is added to each well. Plates are incubated, 3N NaOH is added to each well to stop the reaction, and OD values ​​are determined₄₁₀ using an ELISA reader.

In another embodiment, poly-L-lysine pretreated microtiter plates are used to bind one of the target cells to each well, the cells are fixed, for example, using 1 percent glutaraldehyde, and the bispecific antibodies are they test to determine their ability to link with the

273 intact cell, immunofluorescence

Further,

FACS,

<img file="MX337052B_D0256.tif" />

Idiotype specific antibodies, antigen binding competition assays, and other common methods in the antibody characterization technique can be used in conjunction with the present invention to identify preferred quadromas.

After isolation of the square, bispecific antibodies are purified from other cellular products. This can be accomplished by a variety of protein isolation procedures known to those of skill in the art of immunoglobulin purification. Means for preparing and characterizing antibodies are well known in the art (see, for example, Antibodies: A Laboratory Manual, 1988).

For example, selected supernatants from plaques are run on protein A or protein G sepharose G columns to bind IgG (depending on the isotype). Binding antibodies are eluted with, for example, a citrate buffer pH 5.0. The eluted fractions containing the bispecific antibodies are dialyzed against an isotonic regulator. Alternatively, the eluate is also run on an anti-immunoglobulin sepharose column. The bispecific antibody is then eluted with 3.5M magnesium chloride. Bispecific antibodies purified in this way are

274 they test for

IMPT

Mexican INSTINJTO £ Μ THE PROPERTY

OíDUSTIUAL determine its activity of 'liaison me'diante,

<img file="MX337052B_D0257.tif" />

for example, an isotype-specific linked enzyme immunosorbent assay and immunofluorescence staining assay of target cells, as described above.

Antibodies

Purified BsAbs parent antibodies can also be characterized and isolated by SDS-PAGE electrophoresis, followed by silver or Coomassie staining. This is possible when one of the parent antibodies has a higher molecular weight than the other. Where the bispecific antibody band migrates midway between the two parent antibodies, sample reduction verifies the presence of heavy chains with two different apparent molecular weights.

H4. Proteins

Fusion and Expression

Recombinant

Antibodies to aminophospholipids and anion phospholipids, including the 9D2 and

3G4 (ATCC 4545) and other competing antibodies with improved properties, and PE binding peptides, can also be used to create fusion proteins using molecular biology techniques. Any fusion protein can be designed and made using any of the antibodies-, PE-binding peptides and seconds

275

<img file="MX337052B_D0258.tif" />

known in the art. Fusion protein technology is readily adapted to prepare fusion proteins with other modifications, such as optimizations in CDR sequences, binding via a selectively cleavable peptide sequence, and the like.

The use of recombinant DNA techniques to achieve these ends is now standard practice for those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and live recombination / genetic recombination. DNA and RNA synthesis can be performed, additionally, using automated synthesizers (see, for example, the techniques described in Sambrook et al., 1989).

Preparation of this fusion protein generally involves the preparation of a first and second DNA coding region and the functional linking or binding of these regions, in box, to prepare a single coding region encoding the desired fusion protein. In the present context, the antibody sequence will be linked in the box to a DNA sequence encoding a therapeutic substance. It is not generally believed to be relevant which portion of the immunoconjugate is prepared as the N-terminal region or the C-terminal region.

276

As for the coding region n ^ eseadC ^

M LA PRCi-níftg Kí industrial has been produced, an expression vector is created. The 'and expression vectors contain one more promoter upstream of the regions of

Inserted DNA that acts to promote transcription of the

DNA and thus promotes the expression of the encoded recombinant protein. This is the meaning of recombinant expression.

To obtain a so-called recombinant version of the immunoconjugate, the vector is expressed in a recombinant cell. The technical design of DNA segments for expression in a prokaryotic or eukaryotic system can be performed using techniques generally known to those skilled in recombinant expression. Virtually any expression system is believed to be employable in expression.

The immunoconjugates of the invention can be successfully expressed in bacterial expression however, the systems

<td>eukaryotic,</td><td>by</td><td>example,</td><td>cells</td><td>CHO,</td><td>without</td>

<td>is considered</td><td>than</td><td colspan="2">systems</td><td colspan="2">expression</td>

<td>, such as</td><td>the</td><td>pQE-60</td><td>from E.</td><td>col i</td><td>will be</td>

large scale and the preparation a particularly useful for the subsequent purification of the constructs. The cDNA encoded proteins expressed as fusions with Ξ can also be expressed as bacterial systems, with

277 rT-Ae that the

<img file="MX337052B_D0259.tif" />

galactosidase, ubiquitin,

Schistosoma japonicum, and similar glutathione.

Bacterial expression will have advantages over eukaryotic expression in terms of ease and use and quantity of materials obtained by these.

In terms of microbial expression, the

United States of America Patents Numbers. 5,583,013; 5,221,619; 4,785,420; 4,704,362; and 4,366,246 are incorporated herein by reference for the purposes of further supplementing the present disclosure in relation to gene expression in recombinant host cells.

Recombinantly produced immunoconjugates can be purified and formulated for human administration. Alternatively, the nucleic acids encoding the immunoconjugates can be administered via gene therapy.

Although natural recombinant DNA or plasmids can be employed, the use of liposomes or vectors are preferred. The ability of certain viruses to enter cells via receptor-mediated endositosis and to integrate between the host cell genome and stably and efficiently express viral genes has made them attractive candidates for

<img file="MX337052B_D0260.tif" />

transfer of foreign genes in mammalian cells.

Preferred gene therapy vectors for use in

278 gene delivery due to its pure ability --¡ ^ «- og-rar ·

<img file="MX337052B_D0261.tif" />

their genes in the genome of the. host, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types, and packing into special cell lines. Other viruses, such as adenoviruses, herpes simplex viruses (HSV), cytomegalovirus (CMV), and adeno-associated virus (VAA), such as those described in the US Patent

United States of America number 5,139,941 (incorporated herein by reference), can also be technically designed to serve as vectors for gene transfer.

Although some viruses that can accept foreign genetic material are limited in the number of nucleotides they can accommodate and in the range of cells they infect, these viruses have been shown to successfully perform gene expression. However, adenoviruses do not integrate their genetic material into the host genome and therefore do not require host replication for gene expression, making them ideally suited for rapid, efficient, heterologous gene expression. Techniques for preparing replication defective infectious viruses are best known in the art.

<img file="MX337052B_D0262.tif" />

279

Pl

MEXICAN INSTITUTE OF PROPERTY In certain other modalities adióYóft'cL'l

<img file="MX337052B_D0263.tif" />

Gene therapy vector will be HSV. One factor that makes HSV an attractive vector is the size and organization of the genome. Because HSV is large, incorporation of multiple genes or expression cassettes is less problematic than in other smaller viral systems. Furthermore, the availability of different viral control sequences with varying performance (eg, temporal, strength) makes it possible to control expression to a greater degree than in other systems. It is also another advantage that the virus has relatively few divided messages, further facilitating genetic manipulations. HSV is also relatively easy to handle and can develop at high titers.

Of course, when using viral delivery systems, it would be desirable to purify the virion sufficiently to render it essentially free of undesirable contaminants, such as viral flaw or pyrogen interference particles so as not to cause any reaction in the receiving cell, animal or individual the vector construct. A preferred means of purifying the vector involves the use of buoyant density gradients, such as cesium chloride gradient centrifugation.

280

I. Essay of Enlazami nto and Fws ^^ n ^^

DELA ΡΚοίίϊDAD

Although the present invention has significant 'Yiti ^ iSSfl' ^ in ST1 — miimaluu — and _ human treatment regimens, it also has many other specific and plausible uses, including practical uses in many in vitro modalities. Certain of these uses are related to specific binding properties of antibodies, peptides, and immunoconjugates. Since each of the constructs of the invention includes at least one antibody or peptide component that binds to an aminophospholipid and / or an anionic phospholipid, they can be used in a variety of binding modalities, including useful binding assays.

The presence of a bound agent, where irrelevant, while providing advantageous properties, does not negate the usefulness of the first antibody or regions of the peptide in any binding assay.

Appropriately useful binding assays include those commonly employed in the art, such as immunostaining, Western staining, spot staining, RIAs, ELISAS, immunohistochemistry, fluorescence activated cell sorting (FACS), immunoprecipitation, affinity chromatography, and the like, as further described in the present.

281

<img file="MX337052B_D0264.tif" />

MEXICO DE LA PROPIEDAD INDUSTRIA !, is immovi binding assays as a solid support antigen, for example or a combination thereof

Certain are those in a nitrocellulose matrix, nylon such as in immunostaining, Western staining, ELISAs, and related assays. Other important assays are those using cells, where the components of the present invention can be used to assay the cells for aminophospholipids and / or anionic phospholipids on the cell surface. Those assays can be applied in preclinical analysis, for example with reference to drug design, analyzing the mechanism of action and / or selecting therapeutic agents for combined uses.

Additional in vitro assays are useful in diagnosing aberrant cell activation related diseases and / or apoptosis, where analysis of the presence of aminophospholipids and / or anionic phospholipids on the cell surface would be particularly useful. The constructs of the invention can then be used in conjunction with paraffin embedded, formalin fixed and fresh frozen tissue blocks in immunohistochemistry; in fluorescence-activated cell sorting, flow cytometry, or flow microfluorometry.

282 that of the antigen, such I even ate cases

Additional practical constructs in affinity chromatography purification modalities, related to biospecific antibodies, rapid one-step purification of one or more antigens at a time; and in many other binding assays that will be known to those skilled in the art given the information presented here.

Additional practical uses of the constructs herein are as controls in functional assays, including many in vitro and ex vivo assays and systems. As the binding and functional properties of antibodies, conjugated peptides of the invention are particularly specific, as described herein, those control uses are indeed extremely valuable. Assays benefiting from that practical application of the present invention include, for example, assays concerning the detection of aminophospholipids and / or anionic phospholipids on the cell surface. These assay systems can also be developed in in vitro or ex vivo drug selection assays, where the present supply of biological materials with well defined properties is particularly important. For example, in the use of

283

<img file="MX337052B_D0265.tif" />

positive constructs in the present invention

MEXICAN INSTITUTE OF PROPERTY small molecule selection ^ ^ ^ fí:

Improved 7 equi VáléH LU yu similar binding properties, for example in drug selection and development.

J. Pharmaceutical compositions

The therapeutic agents of the present invention will generally be formulated as pharmaceutical compositions. The pharmaceutical compositions will comprise a biologically or therapeutically effective amount of at least one first therapeutic agent of the invention, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

Combined therapeutics are also contemplated, with the same type of underlying pharmaceutical compositions being employed for both single and combined medications.

The phrases "pharmaceutically pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or otherwise reaction when administered to an animal, or a human, as appropriate. Veterinary uses are also included within the invention and pharmaceutically acceptable formulations include formulations for both clinical and veterinary use.

284

<img file="MX337052B_D0266.tif" />

As used in

MEXICAN INSTITUTE - Pharmaceutically Acceptable PROPERTY includes any 'and^ustttáo £ r¿l! S® solvents, dispersion media, coating TélitOS, —cusLaiiciiijua. antibacterial and antifungal, isotonic and absorption retarding substances and the like. The use of these media and agents for pharmaceutical active substances is well known in the art. Unless some conventional medium or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. For human administration, preparations should meet the standards - for sterility, pyrogenicity, general safety, and purity that are required by the FDA Office of Biological Standards. Supplemental active ingredients can also be incorporated into the compositions.

Unit dose formulations are those that contain a dose or subdose of the administered ingredient for a particular time supply.

For example, unit dose formulations are those containing one daily dose or unit or daily subdose or one weekly dose or unit or weekly subdose and so on.

Jl. Injectable Formulations

The therapeutic agents of the invention

285

<img file="MX337052B_D0267.tif" />

<img file="MX337052B_D0268.tif" />

They will often be formulated for administration particularly for the treatment of tumors, for example, formulated for injection via intravenous, intramuscular, subcutaneous, transdermal routes, or other routes, including peristaltic administration and direct instillation into a tumor or disease site. (intracavity administration). The preparation of an aqueous composition containing an antibody, immunoconjugate, or peptide conjugate, as an active ingredient, will be known to those skilled in the art in light of the present disclosure. Typically, these compositions can be prepared as injectables, either as liquid solutions or suspensions; Solid forms suitable for use in preparing solutions or suspensions after the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.

Convenient dosage forms for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form should be sterile and fluid to the extent that it comes out of the syringe. It must be stable under

286 conditions preserve manufacturing against action

<img file="MX337052B_D0269.tif" />

Therapeutic agents can be formulated in a sterile aqueous composition in a neutral or salt form. Therapeutic agent solutions such as free base or pharmacologically acceptable salts can be prepared in water conveniently mixed with a surfactant, such as hydroxypropyl cellulose. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein), and those formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, trifluoroacetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like.

Convenient carriers include solvents and dispersion media containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), convenient mixtures thereof, and vegetable oils. In many cases, it will be preferable to include substances

I<img file="MX337052B_D0270.tif" />Pl

MEXICAN INSTITUTE

OF THE PROPERTY 'de-'Sbdi Chloride ^

287

<img file="MX337052B_D0271.tif" />

by oj o-mpler; —iiieUlcLIlLT-r lecithin. by isotonics, for example, sugars or adequate fluidity can be maintained, the use of a coating, such as maintenance of the required particle size in the case of dispersion and / or by the use of surfactants.

Under ordinary conditions of storage and use, all these preparations should contain a preservative to prevent the growth of microorganisms. Prevention of the action of microorganisms can be carried out by various antibacterial and antifungal substances, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Prolonged absorption of injectable compositions can be accomplished through the use of compositions of substances that retard absorption, for example, aluminum monostearate and gelatin.

Before or after formulation, therapeutic agents should be extensively dialyzed to remove undesirable small molecular weight molecules, and / or lyophilized for easier formulation in the desired vehicle, when appropriate. Sterile injectable solutions are prepared by incorporating the active substances in the required amount in the appropriate solvent with several of the other ingredients listed above, as desired, followed by filtered sterilization. Usually,

<img file="MX337052B_D0272.tif" />

288

<img file="MX337052B_D0273.tif" />

sterilized active ingredients in a line-agt-erii containing the basic dispersion medium and the required other ingredients from those listed above.

In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques that produce an active ingredient powder, plus any additional desired ingredients from a previously sterile filtered solution. Of the same.

Convenient pharmaceutical compositions according to the invention will generally include an amount of the therapeutic agent mixed with a pharmaceutically acceptable diluent or excipient, such as sterile aqueous solution, to give a range of final concentrations, depending on the intended use. Preparation techniques are generally well known in the art as exemplified by Remington Pharmaceutical Sciences, 16th Ed. Mack Publishing Company, 1980, incorporated herein by reference. For human administration, the preparations shall meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biological Standards. After formulation, the agents, rapéutics

289 eat way _ _

MEXICAN INSTITUTE

PROPERTY a quantity | -aí^TR'^TOFormulation of a single dose will be administered in

<img file="MX337052B_D0274.tif" />

<img file="MX337052B_D0275.tif" />

therapeutically effective.

J2. Sustained Release Formulations

<img file="MX337052B_D0276.tif" />

The formulations are easily administered in a variety of dosage forms, such as the

<img file="MX337052B_D0277.tif" />

type solutions

<img file="MX337052B_D0278.tif" />

injectables described above, contemplate other acceptable forms

<img file="MX337052B_D0279.tif" />

<img file="MX337052B_D0280.tif" />

pills, capsules

<img file="MX337052B_D0281.tif" />

others

<img file="MX337052B_D0282.tif" />

nasal sprays, suppositories, pessaries, solutions

<img file="MX337052B_D0283.tif" />

aerosols, inhalants, topical formulations, liposomal forms, and the like. The type of administration form will be coupled to the disease or condition to be treated.

Pharmaceutical compositions or preparations can be used in slow-release or sustained-release capsules. Slow release formulations are generally designed to give a constant drug level over an extended period and can be used to

<td>manage</td><td colspan="2">therapeutic agents of</td><td>according to the</td><td>present</td>

<td>invention.</td><td>The</td><td>formulations</td><td colspan="2">slow release</td>

<td>typically</td><td>I know</td><td>implanted in the</td><td>neighborhood of</td><td>site of</td>

<td>25 disease,</td><td>by</td><td>example in the</td><td>site of a</td><td>tumor or</td>

290 Viral infection. I Μ PI

MEXICAN INSTITUTE

Convenient examples of sustained release include mat-rlf · »ggm-ipavmaaKi aa to solid hydrophobic polymers containing therapeutic agents, the matrices of which are in the form of shaped articles, eg, films or microcapsules. Examples of sustained release matrices include polyesters; hydrogels, for example, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol); polyactides, eg, US Patent Number 3,773,919;

<td colspan="5">copolymers of L-glutamic acid and ethyl-L-glutamate range;</td>

<td>copolymers</td><td>of</td><td>ethylene-acetate</td><td>vinyl no</td><td>degradable;</td>

<td>copolymers</td><td>of</td><td>lactic acid-acid</td><td>glycolic</td><td>degradable,</td>

<td>such as</td><td>the</td><td colspan="2">Lupron Depot® (microspheres</td><td>injectable</td>

composed of copolymer of lactic acid-glycolic acid and leuprolide acetate); and poly-D - (-) - 3-hydroxybutyric acid.

Although polymers such as ethylene vinyl acetate and lactic acid-glycolic acid allow molecules to be released for 100 days, certain hydrogels release proteins for shorter periods of time. When encapsulated antibodies remain in the body for a long time, they can be denatured or added as a result of exposure to moisture at 37 ° C, thereby reducing biological activity and / or

291

<img file="MX337052B_D0284.tif" />

<img file="MX337052B_D0285.tif" />

changing immunogenicity. __ Rational industrial property is available for stabilization depending on the mechanism involved.

involves the

For example, if the aggregation mechanism of intermolecular SS bond formation through thio-disulfide exchange, stabilization is achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using suitable additives, developing compositions of specific polymer matrix, and the like.

J3. Liposomes and Nanocapsules

In certain embodiments, liposomes and / or nanocapsules may also be employed with the therapeutic agents. The formation and use of liposomes is generally known to those of skill in the art, as summarized below. The present invention provides particular combinations of antibodies, liposomes and chemotherapeutic agents, which are described below. Furthermore, a liposomal formulation can be used as a routine component of any of the therapeutic agents of the entire invention.

Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form concentric bilayer vesicles of

292

<img file="MX337052B_D0286.tif" />

multiple sheets (also d nominated vesí &lt;&amp; J &amp; fex £ &amp;_TO!3 »áÍ nt fa Mirwfcnán

FROM THE PROPIW.D

INCUSTíU.AL

<img file="MX337052B_D0287.tif" />

multiple (MLV)). Multiple lamella vesicles generally have diameters from 25 nm to 4 Φπι. Sonification of multiple lamella vesicles results in the formation of small single lamellae vesicles (SUVs) with diameters in the range of 200 to 500A, containing an aqueous solution in the nucleus.

Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. In low proportions the liposome is the preferred structure. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures they undergo a phase transition that remarkably alters their permeability . The phase transition involves a change from a well packed, ordered structure, known as the gel state, to a loosely packed, less ordered structure, known as the fluid state. This occurs at a characteristic phase transition temperature and results in increased permeability to ions, sugars, and drugs.

Liposomes interact with cells via four different mechanisms: cell endocytosis

293 phagocytic macrophages of the system and neutrophils reticuloendothelia I Mil! I

MEXICAN INSTITUTE

OF THE PROPERTY , . - INDUSTRIAR adsorption to the superr

<img file="MX337052B_D0288.tif" />

cellular, either through weak non-specific fluidic or electrostatic forces, or through 5 specific interactions with components of the cell surface; fusion with the plasma cell membrane by insertion of the liposome lipid bilayer into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transferring liposomal lipids to cell or subcellular membranes, or vice versa, without any association of liposome content. By varying the liposome formulation it can be altered which mechanism is operative, although more than one can operate at the same time.

Nanocapsules generally trap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overload, such as ultrafine particles (with sizes around 0.1 microns) should be designed using polymers

<img file="MX337052B_D0289.tif" />

capable of being degraded live. Biodegradable polyalkyl cyanoacrylate nanoparticles that satisfy these requirements are contemplated for use in the present invention and these particles can be easily manufactured.

294

J4. Ophthalmic formulations

<img file="MX337052B_D0290.tif" />

JLQ £ L

Many cold diseases, particularly those that have an angiogenic component, can be treated by the present invention. For example, neovascular ocular disease, age-related macular degeneration, diabetic retinopathy, premature retinopathy, graft rejection, neovascular glaucoma, retrolental fibroplasias, and other diseases associated with neovascularization of the cornea or retinal / choroidal neovascularization, as described hereinafter.

The therapeutic agents of the present invention can be advantageously employed in the preparation of convenient pharmaceutical compositions for use as ophthalmic solutions, including those for intravitreal and / or intracameral administration. For the treatment of any of the above or other conditions the therapeutic agents are administered to the eye or eyes of the subject in need in the form of an ophthalmic preparation prepared in accordance with conventional pharmaceutical practice, see for example Remington's Pharmaceutical Sciences 15ava. Edition, pages 1488 to 1501 (Mack Publishing Co., Easton, PA).

The ophthalmic preparations will contain a

295 therapeutic agent in a concentration

<img file="MX337052B_D0291.tif" />

or

0.01 to about 1 per c preferably about 0.05 to about 0.5 percent in a pharmaceutically acceptable solution, suspension, or ointment. Some variation in concentration will necessarily occur, depending on the compound used, the condition of the subject to be treated, and the like, and the person responsible for the treatment will determine the most convenient concentration for the individual subject. The ophthalmic preparation will preferably be in the form of a sterile aqueous solution containing, if desired, additional ingredients, for example preservatives, buffers, tonicity agents, antioxidants and stabilizers, wetting or clarifying nonionic substances, viscosity increasing substances and the like .

Convenient preservatives for use in such a solution include benzalkonium chloride, benzetonium chloride, chlorobutanol, thimerosal, and the like. Convenient regulators include boric acid, sodium and potassium bicarbonate, sodium and potassium borates, sodium and potassium carbonate, sodium acetate, sodium bisphosphate, and the like, in amounts sufficient to maintain the pH at between about pH 6 and pH 8, preferably between about pH 7 and pH 7.5. The

<img file="MX337052B_D0292.tif" />

dextran substances 7 0

296

<img file="MX337052B_D0293.tif" />

dextrose, glycerin, potassium chloride, propylene glycol, sodium chloride, and the like, so

<td>that he</td><td>chloride</td><td>of</td><td>equivalent sodium</td><td>of the solution</td>

<td>ophthalmic</td><td>It's in</td><td>the</td><td>0.9 more interval</td><td>or less 0.2 by</td>

<td>hundred.</td><td></td><td></td><td></td><td></td>

<td></td><td>The</td><td></td><td>antioxidants and</td><td>stabilizers</td>

convenient include sodium bisulfite, sodium metabisulfite, sodium thiosulfite, thiourea, and the like. Convenient wetting and clarifying substances include polysorbate 80, polysorbate 20, poloxamer 282, and tyloxapol. Suitable viscosity increasing substances include dextran 40, dextran 70, gelatin, glycerin, hydroxyethyl cellulose, hydroxymethylpropyl cellulose, lanolin, methyl cellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethyl cellulose and the like. The ophthalmic preparation will be administered topically to the eye of the subject in need of treatment by conventional methods. For example, in the form of drops or bathing the eye in the ophthalmic solution.

J5. Topical formulations

In the broadest sense, formulations for topical administration include those for

<img file="MX337052B_D0294.tif" />

administration in the mouth (buccal) already

297

IM

INSTITUTO MÉXICa, -. Or

4 ^ .vés íflé '^STITel

<img file="MX337052B_D0295.tif" />

Topical delivery systems “'^ Sffl! 51éll include · transdermal patches containing the ingredient to be administered. Administration through the skin can be further accomplished by iontophoresis or electrotransport, if desired.

Convenient formulations for topical administration to the mouth include dragees comprising the ingredients in a flavored base, usually sucrose and acacia or tragacanth; lozenges comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and rinses or

buccal comprising the ingredient to be administered in a convenient liquid carrier.

Convenient formulations for topical administration to the skin include ointments, creams, gels, and pastes comprising the ingredient to be administered in a pharmaceutically acceptable carrier. The formulation of therapeutic agents for topical use, such as in creams, ointments, and gels include the preparation of oil-soluble or water-soluble ointment bases, as well as is well known in the art in view of the present disclosure. For example, these compositions may include vegetable oils, animal fats, and more preferably, semi-solid hydrocarbons obtained from the

298

<img file="MX337052B_D0296.tif" />

Petroleum. Particular components used ^ sTg ^ g ^ in include '¡1NDUSTÜ1 / .L white ointment, yellow ointment, cetyl wax esters, c ΊΐΓΐ i ~ ii — iTii · ηπ · ιη · ππτ · ~ · ι ~~ -' '' - ^ * ~ ** oleic acid, olive oil, paraffin, petrolatum, white petrolatum, spermaceti, starch glycerite, white wax, yellow wax, lanolin, anhydrous lanolin and glyceryl monostearate. Various water soluble ointment bases can also be used, including glycol esters and derivatives, polyethylene glycols, polyoxyl 40 stearate, and polysorbates.

Formulations for rectal administration can be presented as a suppository with a convenient base comprising, for example, cocoa butter or a salicylate. Convenient formulations for vaginal administration may be presented as suitable pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient vehicles as known in the art.

J6. Nasal Formulations

Local administration via the nasal and respiratory routes is contemplated to treat various conditions, particularly for use in the antiviral treatment methods of the present invention. These routes of administration are also convenient for administering substances into the systemic circulation. The formulations

299

<img file="MX337052B_D0297.tif" />

Nasal administration is therefore included within the invention for example, nasal solutions, sprays, sprays and inhalants. When the vehicle is a solid,

<img file="MX337052B_D0298.tif" />

formulations include a coarse powder having a particle size, for example, in the range of 20 to 500 'microns, which is administered, eg, by rapid inhalation through the nasal passage of a container of the maintained powder close to the nose.

Convenient formulations where the carrier is a liquid are useful in nasal administration. Nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays and are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, aqueous nasal solutions are usually isotonic and slightly regulated to maintain a pH of 5.5 to 6.5. Furthermore, antimicrobial preservatives similar to those used in ophthalmic preparations, and suitable drug stabilizers, if required, can be included in the formulation. Various commercial nasal preparations are known and include, for example, antibiotics and antihistamines and are used for asthma prophylaxis.

Inhalations and inhalants are preparations

300 Pharmaceutical companies designed to administer drug or

one. compound in the respiratory tree of a patient. A mist or mist is administered and reaches the affected area. This route can also be used to deliver substances into the systemic circulation. Inhalations can be administered by the nasal or oral respiratory routes. The administration of inhalation solutions is only effective if the drops are fine enough and uniform in size so that the mist reaches the bronchioles.

Another group of products, also known as inhalations, and sometimes called insufflations, comprises drugs in very fine powder or liquid that are carried into the respiratory passages through the use of special delivery systems, such as pharmaceutical aerosols, that maintain a solution or suspension of the drug in a liquefied gas propellant. When released through a convenient valve and oral adapter, an inhalation doser is propelled into the patient's airway. Particle size is of utmost importance to the administration of this type of preparation. The optimal particle size for penetration into the lung cavity has been reported to be in the range of 0.5 to 7 microns. Fine mists are produced by pressurized aerosols and therefore their use is considered advantageous.

301

<img file="MX337052B_D0299.tif" />

INSTITUTED Me ;;;.?&gt; ;. ·.

K. Diagnostic and Therapeutic Games ____

This invention also provides diagnostic, therapeutic and therapeutic kits comprising at least a first therapeutic agent of the present invention, i.e., an antibody, immunoconjugate or peptide conjugate, which binds to an anionic phospholipid or aminophospholipid, for use in treatment methods, combined treatment methods and / or imaging and treatment modalities. These games will generally contain; in at least a first container (or container element), a pharmaceutically acceptable formulation of at least one therapeutic agent, an antibody, immunoconjugate or peptide conjugate, which binds to an anionic phospholipid or aminophospholipid. The games may include written or electronic instructions for their use, for example in preclinical, clinical and / or veterinary modalities.

The kits may also contain other compositions, pharmaceutically acceptable formulations, and second biological and therapeutic agents, including those for combination therapy and / or for diagnosis and imaging. For example, those kits may contain any or more of a range of chemotherapeutic, radiotherapeutic, or anti-angiogenic agents,

302 antitumor, antiviral immunotoxins or vasculature and / or antitumor components or cells, coaguligand stromal antibodies, diagnostic agents. Written or electronic instructions may also be included for use in combination therapy and / or for diagnosis and imaging.

Kits may have a single container containing the first antibody, immunoconjugate or peptide conjugate, which binds to an aminophospholipid or anionic phospholipid, with or without additional components, or may have different containers for each desired agent. When combination therapeutics are provided, a single solution may be premixed, either in a molar equivalent combination, or with one component in excess of the other. Alternatively, the primary therapeutic agent of the invention and the second biological or therapeutic agent, such as a second anticancer or antiviral agent, may be kept separately in separate containers prior to administration to a patient.

The most frequently used diagnostic components will be kept in at least one second container, other than the other container or the first container comprising the one or more therapeutic agents.

303 I included

<img file="MX337052B_D0300.tif" />

FROM INDUSTRIAL PROPERTY same aminophospholipid

Diagnostic kits may be labeled peptides that bind to the anioriic phospholipid as the primary therapeutic agent, or any other agent appropriate for the diagnosis of the disease to be treated. The kits may include diagnostic agents for in vitro use, for in vivo use, or both of those agents. The kits may include written or electronic instructions for use, for example in preclinical, clinical and / or veterinary diagnostic modalities.

For in vitro immunodetection, the antibodies can be bound to a solid support, as well as to a microtiter plate, although solutions or powders are preferred for antibody reconstitution. The immunodetection kits comprise at least one first immunodetection reagent. The kit immunodetection reagents can take any variety of forms, including those detectable markers that are associated or related to the determined antibody, as used in vivo. Detectable markers that are associated with or linked to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.

304

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, ν '·· v 77 Á

Immunodetec reagents ^. ^ Prpiate,

FROM THE PKCI * ¹ C- INDUSTRIAL ---- for use in the games herein, the antibody includes a binding affinity for the first antibody, along with a third antibody having a binding affinity for 1 second antibody, the third antibody is found linked to a detectable marker.

A number of exemplary markers are known in the art and all of those markers can be employed in connection with the present invention. These sets may contain antibody-marker conjugates either in a fully conjugated form, in the form of intermediates, or as separate portions to be conjugated by the user of the set. The imaging kits will preferably comprise a targeted targeting agent or an antibody that is already attached to an in vivo detectable marker. However, the level and the connecting means could be supplied separately.

Any form of diagnostic kit can further comprise control agents such as biological compositions conveniently provided as aliquots, either labeled or unlabelled, that can be used to prepare a standard curve for an assay of

305 detection. Components packaged in either aqueous media

When the kit components are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, with the sterile aqueous solution being particularly preferred. However, the kit components can be provided as dry powders. When the reagents or components are provided as a dry powder, the powder can be reconstituted by adding a suitable solvent.

The solvent can also be provided in another container within the kit.

Therapeutic and diagnostic kit containers will generally include at least one vial, test tube, ampule, bottle, syringe, or other container item, in which the therapeutic agent and any other desired agents can be placed and conveniently taken into aliquots. Since at least two separate components are preferred, the kits will preferably include at least two of those containers. The kits may also contain a third or fourth container to contain a pharmaceutically acceptable buffer or other diluent.

Kits may also contain an element by which therapeutic agents are administered to

306

l.lvi example, an ^^ jjft ^ for eyes, pipette, an animal or patient, by syringes, or even a dropper these similar devices, from which the formulation can be injected into the animal or applied to a diseased area of ​​the Body.

Kits of the present invention will also typically include a means for containing the vials, or any other closed component containment, for commercial sale, such as, for example, injection molded or blow molded plastic containers in which the jars and other desired appliances are placed and retained.

L. Immunodetection and Imaging

The present invention further provides in vitro and in vivo diagnostic and imaging methods. These methods are applicable for use in generating diagnostic, prognostic, or imaging information, for example, related to angiogenic diseases and viral infections, and preferably related to the treatment and imaging methods, of tumors. The methods of the invention include in vitro diagnostic tests, eg where samples can be obtained non-invasively and tested in high throughput assays and / or when the clinical diagnosis is ambiguous and the

307 confirmation is desired. In in vivo imaging,

F diagnose and

DtLA. γ antibodies, and peptides of the invention are linked to one or more detectable agents and are used to image an angiogenic site or tumor, optionally as a first step before treatment.

L1. Immunodetection Methods and Games

The invention then relates to immunodetection methods for binding, purifying, quantifying, or otherwise generally detecting aminophospholipids and anionic phospholipids, for example, for use in the diagnosis of activated and apoptotic cells and associated diseases. The antibodies of the present invention, such as 9D2 and 3G4 (ATCC 4545) can be used to detect anionic phospholipids and aminophospholipids (see below), in isolated tissue samples, biopsies or smears, and / or in homogenized tissue samples. These immunodetection methods have diagnostic utility

<td>evident,</td><td>but they also have</td><td>Applications</td><td>for samples</td><td>not</td>

<td>clinics</td><td>such as in the</td><td>Title</td><td>of samples</td><td>of</td>

<td>antigens,</td><td>and the like.</td><td></td><td></td><td></td>

<td></td><td>The steps</td><td>several</td><td>methods</td><td>of</td>

Useful immunodetections have been described in the scientific literature, such as, eg, Nakámura et al., 1987, ier immuno-linkage methods containing incorporated aminophospholipids

In general, a sample that is suspected and / or anionic phospholipids, which are suspected to be phospholipids in contact

308 cells preferably have aminophospholipids and / or anionics on the cell surface, and placing the sample with an antibody of the invention, such as 9D2 or to allow

3G4 (ATCC 4545), under effective conditions the formation of immune complexes.

Any immune complexes formed during the binding process are then detected and preferably quantified.

The analyzed sample may be a sample of cells, such as cells exposed to certain test conditions in the laboratory. The sample may also be a biological sample from an animal or patient, for example one that is suspected of having a disease associated with activation or apoptosis of one or more cell types. That sample may be a section or specimen of tissue, a biopsy, a smear or stain left on one site, a homogenized tissue extract, or separate or purified forms thereof.

By contacting the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow

309

7Γ &lt;Τδ ΤΓ

IL .iv.

(Immune questioning and incubation of the mixture during primary complex formation) is generally the antibody to the sample long enough for the antibodies to form immune complexes, such as to bind to, any anionic phospholipid and aminophospholipid present. After this time, the sample antibody composition, such as a tissue section or ELISA plate, will generally be washed to remove any species of non-specifically bound antibody, allowing only specifically bound antibodies within the primary immune complexes to be removed. detect.

Detection of immunocomplex formation is well known in the art and can be accomplished through the application of numerous approaches. These methods are generally based on the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic marker or label known in the art. US Patent Nos. 3,817,837; 3,850,752; 3,939,350;

3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated herein by reference. The use of enzymes that generate a colored product after contact with a chromogenic substrate are preferred³¹⁰ ÍIMHS:⁴usually. The s ^ ufiSShtió binding ligands, such as a second antibody or a biotin / avidin ligand binding arrangement, can also be used, as is known in the art.

Antibodies of the invention, such as 9D2 and 3G4 (ATCC 4545) employed in detection, can themselves bind to a detectable marker, where one could simply detect a marker, thereby allowing for the amount of primary immune complexes in the composition. to be determined.

Preferably, the primary immune complexes are detected by means of a second binding ligand that has binding affinity for the antibodies of the invention. In these cases, the second binding ligand can be bound at a detectable level. The second binding ligand itself is frequently an antibody, and may thus be referred to as a secondary antibody. The primary immune complexes are contacted with the labeled secondary binding ligand or antibody under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. Secondary immune complexes are generally washed to remove any specifically labeled unbound antibody or secondary ligand and the remaining marker in secondary immune complexes is detected

311 so.

complex

INSTITUTE a'L; -: ·····; ·: Ij DE LA? ΊΚ · Λ.-_; · &lt;'·? i '. ·!

Additional methods include detecting primary immune using a two-step approach. A second binding ligand such as an antibody, which has binding affinity for the first antibody, is.

used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody having 10 binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow complex formation. immune (complex

<img file="MX337052B_D0302.tif" />

tertiary immune). The third ligand or antibody binds to a detectable marker, allowing detection of the tertiary immune complexes thus formed. This system can provide signal amplification if desired.

Diagnosis or clinical monitoring can be applied to patients with a variety of diseases, particularly those associated with increased exposure to aminophospholipids and / or anionic phospholipids, on the cell surface. Detection of an anionic aminophospholipid and / or phospholipid, or an increase in the levels of an anionic phospholipid and / or phospholipid, compared to levels in a sample *

corresponding biological

312

<img file="MX337052B_D0303.tif" />

it is a patient with this disease.

However, as those skilled in the art know, this clinical diagnosis is unlikely to be made based on this method in isolation. Those of skill in the art are very familiar with the differentiation between significant expression of a biomarker, which represents positive identification, and low or background expression of a biomarker. Certainly, background expression levels are frequently used to form an enzyme cut that increases staining will be rated as significant or positive.

L2. Live Jn Imaging

The present invention provides a variety of in vivo diagnostic and imaging modalities. Certain aspects of the invention concern novel and surprisingly effective compositions for diagnostic and imaging.

I live. For example, any or more of the panel of novel anti-PS antibodies of the invention, preferably 9D2 or 3G4 antibodies (ATCC 4545) or competing antibodies with similar properties are bound to a detectable agent in vivo to form a

313 . IMPI0-5 conjugate for immunodiagnosis of the invention; Although the antibodies represent an important development in the field, the resulting immunodiagnostics can now be used in any previously described diagnostic or imaging modality related to the detection of an anionophospholipid and / or phospholipid.

In this regard, compounds for immunodiagnostics comprising an antibody of the invention, including antibodies 9D2 and 3G4 (ATCC 4545) or competing antibodies with similar properties, can be used in the imaging of vascular thrombosis, particularly in the heart or near it,

<td>such as</td><td>in deep vein thrombosis, embolism</td>

<td>pulmonary,</td><td>myocardial infarction, atrial fibrillation,</td>

<td>problems</td><td>with prosthetic cardiovascular materials,</td>

embolism and the like. Those compositions of the invention can be used in imaging of activated platelets, for example under conditions such as abscesses, restenosis, joint inflammation, and hemostatic disorders, such as arterial, coronary, venous, and cerebral thrombosis, and Similar. The immunodiagnostic compositions of the invention, preferably those comprising the 9D2 or 3G4 antibodies (ATCC 4545) or competing antibodies with similar properties, can also be used in the

314 detection of apoptotic cells,

<img file="MX337052B_D0304.tif" />

used in the diagnosis and imaging of a variety of diseases in which increased or inappropriate apoptosis occurs.

The invention further provides a range of new methods for in vivo diagnosis and imaging, which is not limited to the use of the antibody panel provided herein. For example, in view of the unexpected discovery that anionic phospholipids such as Pl, PA, and PG are accessible markers and which can stably target tumor vasculature, the invention provides methods for the diagnosis and imaging of tumors, which comprise the administration of an immunodiagnostic agent that binds to Pl, PA or PG, which will be specifically localized to the vasculature of solid tumors. Furthermore, virally infected cells can now be detected, and viral infections can be diagnosed, using an immunodiagnostic conjugate that binds to an aminophospholipid and / or an anionic phospholipid, such as PS, PE, Pl, PA, and PG, and preferably PS and PE. .

The compositions and methods for in vivo imaging of the invention can be used in imaging per se, in prior imaging of a site in the body, to form a

315

<img file="MX337052B_D0305.tif" />

<img file="MX337052B_D0306.tif" />

Imaging is the imaging of a tumor. These compositions and methods can also be applied to imaging and diagnosis of 5 other diseases or conditions associated with aminophospholipids and anionic phospholipids, such as those that

<td>involve</td><td>activation</td><td colspan="2">and / or apoptosis</td><td colspan="2">of cells,</td>

<td>including</td><td colspan="3">angiogenic diseases,</td><td colspan="2">atherosclerosis,</td>

<td>infections</td><td>viral, and others</td><td>of those</td><td colspan="2">conditions of</td><td>the</td>

<td>which ones</td><td>want a picture</td><td>internal</td><td>for</td><td>purposes</td><td>of</td>

<td>diagnosis</td><td>or prognosis,</td><td>or for</td><td>the</td><td>design of</td><td>a</td>

treatment.

In these embodiments, antibodies and peptides, preferably antibodies of the invention, such as 9D2, 3G4 (ATCC 4545), and the like, are linked functionally linked or conjugated to a detectable marker. Detectable markers are compounds or elements that can be detected due to their specific functional properties, or chemical characteristics, the use of which allows the component to which they are attached to be detected, and further quantified if desired. In antibody and peptide conjugates for in vivo diagnostic protocols or imaging methods, the markers can be detected using non-invasive methods.

316

In the art,

<img file="MX337052B_D0307.tif" />

suitable for the formation of pcp magnet; &lt;-nmo are the methods for binding to antibodies and binding ligands (see, eg, US Patent Nos. 5,021,236 and 4,472,509, both incorporated herein by reference). Certain methods of binding involve the use of a metal chelated complex employing, for example, an organic chelating agent such as a DTPA bound to the antibody (United States Patent No. 4,472,509). Monoclonal antibodies can also be reacted with an enzyme in the presence of a coupling agent such as a glutaraldehyde or periodate. Fluorescein marker conjugates are prepared in the presence of these coupling substances or by reaction with an isothiocyanate.

An example of detectable markers are paramagnetic ions. In this case, suitable ions include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium ( III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III), with gadolinium being particularly preferred.

Useful ions in other contexts, such

317 as for X-ray imaging,

<img file="MX337052B_D0308.tif" />

they are not limited to lanthanum (III), gold (III), and especially bismuth (III). Fluorescent levels include rhodamine, fluorescein, and renographin. Rhodamine and fluorescein are frequently linked via an isothiocyanate intermediate.

In the case of radioactive isotopes for diagnostic applications, suitable examples include ¹⁴carbon, ⁵¹chrome, ³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, ⁶⁷copper, ¹⁵²eu, ⁶⁷gallium, ³hydrogen, ¹²³iodine, ¹²⁵iodine, ¹³¹iodine, ^{11: L}Indian, ⁵⁹iron, ³²match, ¹⁸⁶rhenium, ¹⁸⁸rhenium, ⁷⁵selenium, ³⁵sulfur, ^99mtechnetium, ⁹⁰yttrium. The¹²⁵Iodine is often preferred for use in certain modalities, and ^99mtechnetium and the ^{i: L1}Indium are also frequently preferred because of their low energy and convenience for detection over an extended range.

Radioactively labeled antibodies and peptides for use in the present invention can be produced according to methods well known in the art. For example, the intermediate functional groups that are frequently used to link radioisotopic metal ions with antibodies are diethylenetriaminepentacetic acid (DTPA) and ethylene diaminotetracetic acid (EDTA).

Monoclonal antibodies can be

318 chemical oxidizing agent such as

<img file="MX337052B_D0309.tif" />

an enzymatic oxidizing substance such as lactoperoxidase.

Anti-tumor antibodies according to the invention can be labeled with technetium⁹⁹¹ by means of a ligand exchange process, for example, reducing the pertechinate with reduced on stannous solution, chelating the technetium a Sefadex column and applying the antibody to this column; or by direct labeling techniques, for example, by incubating pertechinate, a reducing substance such as SNC1₂, a regulatory solution such as sodium potassium phthalate solution, and the antibody.

Any preceding type of detectably labeled antibody and binding ligand can be used in the imaging aspects of

<td colspan="2">the invention,</td><td>already</td><td>be for</td><td>the formation</td><td>of</td><td>images</td>

<td>only</td><td>or</td><td>for</td><td colspan="2">form an image of</td><td>a</td><td>site of</td>

<td>disease</td><td>or</td><td>tumor</td><td>before the</td><td>treatment.</td><td>In</td><td>any</td>

<td>form the</td><td colspan="2">methods</td><td>understand</td><td>administer to</td><td>a</td><td>animal or</td>

patient a diagnostic effective amount of an antibody or binding ligand that is conjugated to a marker that can be detected by non-invasive methods. The binding antibody-marker ligand marker conjugate is allowed sufficient time to locate and bind cells that

319 express aminophospholipids

<img file="MX337052B_D0310.tif" />

disease site, such as in 1 tumor or tumor vasculature. The patient is then exposed to a detection device to identify the detectable marker, thereby forming an image of the diseased site or tumor.

Nuclear magnetic spin resonance isotopes, such as gadolinium, are detected using a nuclear magnetic imaging device; and radioactive substances such as technetium⁹⁹”¹ or indian¹¹¹, are detected using a gamma scintillation detector or camera. United States Patent for North America No. 5,627,036 is also specifically incorporated herein by reference, for purposes of providing additional guidance regarding the safety and effective introduction of detectably labeled constructs into the blood of an individual, and means for determining the distribution of the detectably labeled agent , extracorporeally, for example, using a gamma scintillation chamber or through magnetic resonance measurement.

Dosages for imaging modalities are generally lower than for therapy, but are also dependent on the age and weight of a patient. A single dose of between about 0.1, 0.5 or about 1 mg and about 9 or 10 mg, and

320 more preferably, about 5-10 mg

<img file="MX337052B_D0311.tif" />

<img file="MX337052B_D0312.tif" />

between approximateSSffiífitóYes ^ ?.

Industrial binding of the antibody-binding ligand conjugate, per patient, is contemplated as useful.

<td></td><td>L3.</td><td>Marker</td><td>Surrogate paw</td><td>Therapy</td>

<td>Cancer</td><td></td><td></td><td></td><td></td>

<td></td><td>With</td><td>respect</td><td>to the formation of</td><td>images and</td>

<td>diagnosis</td><td>in</td><td>alive the</td><td>present invention</td><td>provides</td>

further compositions and methods for use as a surrogate marker for cancer therapy. Those modalities concern the use of an antibody that binds to an aminophospholipid and / or an anionic phospholipid, preferably PS, and most preferably to the use of 9D2 or 3G4 antibodies (ATCC 4545) or competing antibodies, bound to a detectable agent. in vivo.

Many anticancer therapies in use today induce apoptosis and necrosis. Aminophospholipids and anionic phospholipids, particularly PS, are pre-apoptotic and apoptotic cell markers. Therefore, imaging with an appropriate antibody, preferably 9D2 3G4 antibodies (ATCC

4545) or competing antibodies, can be used to identify pre-apoptotic and apoptotic cells and thus provide information regarding the

321

IMPI therapy progress. This is what I know

INDUSTRIAL cancer therapy as it is

<img file="MX337052B_D0313.tif" />

surrogate marker for use herein.

The use of preferably those antibodies of the invention, comprising 9D2 or 3G4 antibodies (ATCC 4545) or competing antibodies with similar properties, provides particular advantages as a surrogate marker for cancer therapy. For example, the · ability to identify pre-apoptotic cells is a particular advantage. The specificity of the antibodies will also provide more meaningful imaging data for the clinician. Also, the safety profile of these antibodies is impressive and provides advantages over annexin, for example, since annexin suffers from the disadvantages associated with coagulation.

Accordingly, any of the in vivo diagnostic and imaging methods described above can be adapted for prognostic use, as a surrogate marker for cancer therapy, simply by use in a patient undergoing anti-cancer therapy.

322

M. Treatment of Tumors

Important aspects of the treatment of malignancies,

<img file="MX337052B_D0314.tif" />

tumors and vascularized tumors. These include tumors in which angiogenesis is more or less important and the tumors have prothrombotic blood vessels. The treatment of benign tumors is included in the invention, such as acoustic neuroma, neurofibroma, trachoma, pyogenic granulomas and BPH. Treatment of birth tumors in the blood, such as 10 leukemias, and various acute or chronic neoplastic diseases of the bone marrow are also covered.

The present invention is broadly applicable to the treatment of any malignant tumor, whether or not it has a vascular component. Tumors for treatment include 15 solid tumors, particularly carcinomas, which require a

<img file="MX337052B_D0315.tif" />

vascular component for the provision of oxygen and nutrients · Exemplary solid tumors that can be treated using the invention include, but are not limited to, are carcinomas of the lung, breast, ovaries, stomach, 20 pancreas, larynx , esophagus, testis, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate, thyroid, squamous cell carcinomas, adenocarcinomas, small cell carcinomas, melanomas, gliomas, '1 glioblastomas, neuroblastomas, and the like.

323 solid.

In general the invention can be used to treat tumors of all sizes including those of 0.3-0.5 cm and larger, tumors larger than 0.5 cm and patients presenting with «

Tumors between about 1.0 and about 2.0 cm, although larger tumors that include the largest tumors found in humans can also be treated.

Although the present invention is not generally intended to serve as a preventive or prophylactic treatment, the use of the invention is certainly not confined to the treatment of patients having tumors of large or only moderate size. There are many reasons that support these aspects of the invention. For example, a patient with a primary tumor of moderate or larger size may also have several other metastatic tumors that are considered small in size or even earlier in the seeding of the metastatic tumor. Since the anti-aminophospholipid or anti-anionic phospholipid antibodies or derivatives of PE-binding peptides, or combinations, of the invention, are generally administered to the systemic circulation of a patient, they will naturally have effects on smaller, secondary tumors and metastatic, although this may not be the primary purpose of treatment. Furthermore, even in situations where the mass of the tumor as a whole is one

324 small tumor, certain beneficial antitumor effects resulted from the use of the treatments herein.

The guidance provided herein regarding the most convenient patients for use with the present invention is intended to teach that certain patient profiles may aid in the selection of patients for the treatment of the present invention. The pre-selection of certain Patients, or category of patients, in no way deny the basic utility of the present invention in relation to the treatment of all patients having cancer. A further consideration is the fact that the attack on the tumor provided by the antibody therapy of the invention may predispose the tumor to another therapeutic treatment, so that subsequent treatment results in an overall synergistic effect or still leads to remission or total cure.

It is not believed that any particular type of tumor should be excluded from treatment using the present invention. However, the type of tumor cells may be relevant to the use of the invention in combination with other tertiary therapeutic substances, particularly chemotherapeutics and anti-tumor cellular immunotoxins. As the present invention includes within its modes of action the targeted localization and destruction of the tumor vasculature, and how vasculature 25 is substantially or entirely the same in all tumors

325

<img file="MX337052B_D0316.tif" />

<img file="MX337052B_D0317.tif" />

MEXICAN INSTITL OF PROPERTY, it will be understood that the present methodology is^NEITHERáf! $ ¡l^LIt is not entirely applicable to the treatment of Lódüü luy-umwcesólidos, regardless of the particular genotype or genotype of the same tumor cells. The data presented here is of utmost importance as it shows impressive results in a wide variety of different tumor models.

Therapeutically effective doses can be readily determined using data from an animal model, as shown in the detailed studies herein, and from clinical data using a variety of therapeutic agents. Experimental animals that have solid tumors are frequently used to optimize appropriate therapeutic doses before transferring them to a clinical setting. These models are known to be very reliable in predicting effective anticancer strategies. For example, solid tumors in mice, such as those used in the examples, were. widely used in preclinical testing. The inventors have used these art-accepted mouse models to determine working ranges of therapeutic agents that produce beneficial anti-tumor effects with minimal toxicity.

In terms of tumor therapy, taking into account the concurrent safety benefits associated with the global invention, reference can be made to the scientific and patent literature on the success of the use of other therapies.

326

<img file="MX337052B_D0318.tif" />

5,965,132; 6,051,230; 6,004,555; 5,776,427, 6,004,554; and

6,036,955; and 6,093,399 are incorporated herein by reference for the purpose of further describing the use of those agents as they may be applied to those of the present invention. Patents of the United States of America

Numbers. 6,312,694 and 6,406,693 are further specifically incorporated herein, by reference, for dosing and treatment guidance using PS and PE unconjugated antibodies and related immunoconjugates.

As is known in the art, these are realistic objectives that can be used as guidelines in relation to preclinical testing before proceeding with clinical treatment. However, due to the safety already demonstrated in the accepted models, the preclinical tests of the present invention will be a matter of optimization, rather than confirming its effectiveness. Thus preclinical tests can be used to select the most advantageous agents, doses or combinations.

Any dose of antibody, combination method, or medication that results in any consistently detectable antitumor effect, including regression of the tumor vasculature, detectable thrombosis and / or destruction and necrosis of the tumor, will further be defined as a useful invention.

The regressive, thrombotic, destructive and necrotic effects

327 Tumor tissues should preferably be observed between and approximately 40-50% of the vessels, up to values ​​approximately

99% of those effects

<img file="MX337052B_D0319.tif" />

of the tumor and of approximately 50% and observed. The present invention may also be effective against vessels that are downstream of the tumor, that is, at least a subset of the drainage vessels, particularly as cytokines released from the tumor, which will act on these vessels, changing their antigenic profile.

It will also be understood that even in circumstances where the antitumor effects of therapy are directed at the low end of this range, doses of anti-VEGF antibody, which blocks VEGFR2, or 2C3-based antibody or immunoconjugate, or therapy, may be that this therapy is still the same or even more effective than other known therapies in the context of the goals of the particular tumor or patient. Unfortunately it is evident to a clinician that certain tumors and conditions do not. they can be effectively treated in the intermediate or long term, but the usefulness of the present therapy is not denied, particularly when it is at least as effective as the other strategies generally proposed.

To design suitable doses of anti-aminophospholipid and anti-anion phospholipid antibodies, derivatives of PE-binding peptides, or combined therapeutics for the treatment of vascularized tumors,

328

ÍMPJíía can easily extrapolate from studies on animals d¡eí = fé5 *! Iítos ^ · the present in order to reach an adequate dose. clinical administration. To achieve this conversion, the mass of the agents administered per unit mass of the experimental animal will be taken into account and, preferably, the differences in body surface area between the experimental animal and the human patient would be accounted for. All of these calculations are well known and routine to those skilled in the art.

For example, to take a successful dose of therapeutic products used in mouse studies, and to apply standard calculations based on mass and surface area, the effective doses of agents for use in human patients range from about 1 milligram to about 500 milligrams. of antibody per patient, or preferably, between about 10 milligrams and about 100 milligrams of antibody per patient.

Accordingly, using this information the inventors contemplate that low doses useful for administration to humans will be between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25 or about 30 mg or similar value per patient; and the high useful doses for human administration will be about 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or about 500 mg or the like per patient. Useful intermediate doses for human administration are contemplated to be approximately 35, 40, 50, 60, 70, 80,

329 similar per patient.

between approximately

5-100 mg, about 10-80 mg, about 20-70 mg, about 25-60 mg, about 30-50 mg per patient will be preferred.

However, any particular range using any of the exemplary doses mentioned above, or any intermediate value between the particular mentioned ranges, is contemplated.

<td></td><td>No</td><td>importing</td><td colspan="3">the mentioned intervals,</td>

<td>will understand</td><td>than,</td><td>given the</td><td>parameters and the</td><td>guide</td><td>detailed</td>

<td>filed</td><td>here,</td><td>variations</td><td>additional in</td><td>the</td><td>intervals</td>

active or optimal will be found within the present invention. Therefore it will be understood that lower doses may be more suitable in combination with certain agents, and that high doses can still be tolerated, particularly given the improved safety of the constructs herein. The use of human or humanized antibodies and human effectors makes the present invention even safer for clinical use, further reducing opportunities for toxicity or side effects in healthy tissues.

The intention of the therapeutic regimens of the present invention is generally to produce significant anti-tumor effects while still maintaining the doses below the levels associated with unacceptable toxicity.

330 i

In addition to varying the dose itself, the regime * OF PROPERTY t&gt;.

INDUSTRIAL can also be adapted to optimize the treatment strategy. A currently preferred treatment strategy is to administer between about 1 and 500 mg, and preferably between about 10 and 100 mg of the antibody, or therapeutic cocktail containing the same, about 3 times within a period of about 7 days. For example, doses will be administered around day 1, day 3 or 4, and day 6 or 7.

To administer the same particular doses, it would be preferred to provide a pharmaceutically acceptable composition (in accordance with FUA standards of sterility, pyrogenicity, purity and general safety) to the patient systemically. Intravenous injection is generally preferred, and the most preferred method is to use a continuous infusion over a period of time of about 1 or 2 hours or so. Although it is not required to determine these parameters prior to treatment using the present invention, it will be appreciated that the studies detailed herein result in at least some thrombosis being observed in the blood vessels of a solid tumor within approximately 12-24 hours of injection. , and that the tumor cells themselves begin to die within approximately 24 to 72 hours. The generalized necrosis of the tumor is generally observed in the next period of 48 to 96 hours, 'including an observed necrosis greater than 60%.

331

Naturally, before using

<img file="MX337052B_D0320.tif" />

will conduct clinical trials. The various elements for conducting a clinical trial, including patient management and monitoring, will be known to those skilled in the art in light of the present disclosure. The following information is presented as a general guideline for use in establishing these trials.

Patients chosen for the first treatment studies did not respond to at least one course of conventional therapy, and will have objectively measurable disease as determined by physical examination, laboratory techniques, and / or radiographic procedures. Any chemotherapy should be stopped at least two weeks before entering the study. When using portions of antibody or murine monoclonal antibodies, patients should have no history of allergy to mouse immunoglobulin.

Several advantages will be found in using an internal central venous catheter with a triple lumen port. Therapeutic compounds should be filtered, for example, using a 0.22 filtro filter, and diluted appropriately, such as by saline, to a final volume of 100 milliliters. Before use, the test sample should also be filtered similarly, and its concentration assessed before and after filtration by determining the A₂g₀. The expected recovery should be within the range of 87 percent to 99 percent, and

332

<img file="MX337052B_D0321.tif" />

can determine adjustments for loss of prot ^ 8S2¡ "or ^ g

I * vT'W ΚΎ r&gt; ·

INDUSTRIAL

Constructs can be administered over a period of approximately 4-24 hours, with each patient receiving 2-4 infusions at 2-7 day intervals. Administration can also be done using a stable infusion rate over a period of 7 days. The infusion given at any dose level should be dependent on any observed toxicity. Therefore, if grade II toxicity is achieved after a single infusion, in a particular period of time for a stable rate infusion, other doses should stop or stop the stable rate infusion unless toxicity improves. Increasing doses should be administered to patient groups until approximately 60 percent of patients show unacceptable grade III or IV toxicity in any category. Doses that are 2/3 of this value are defined as safe doses.

Physical examination, tumor measurements, and laboratory tests should, of course, be performed prior to treatment and at intervals of up to one month thereafter. Laboratory tests should include complete blood counts, serum creatinine, creatinine kinase, electrolytes, urea, nitrogen, SGOT, bilirubin, albumin, and total serum protein. Serum samples taken up to 60 days after treatment should be evaluated by radioimmunoassay to detect the presence of the construct and antibodies against

333

<img file="MX337052B_D0322.tif" />

IMPI INSTITUTE w * PE LA l'fa '.L any portion thereof. Serum immunophysical analyzes, using any standard assay such as, for example, Ull ELISA or RIA will allow to evaluate the pharmacokinetics and the elimination of the therapeutic compound to be evaluated.

To assess anti-tumor responses, patients should be examined 48 hours to 1 week and then 30 days after the last infusion. When manifest disease is present, two perpendicular diameters of all treatment masses should be measured daily within one week after completing therapy, and at 30 days. To measure non-overt disease, serial computerized phonography scans could be performed at 1-centimeter intervals across the chest, abdomen, and pelvis within 48 hours and up to 1 week, and again within 30 days. Tissue samples should also be evaluated histologically, and / or by flow cytometry, using biopsies from the sites of the disease or even blood or fluid samples if appropriate.

Clinical responses can be defined by acceptable measurement. For example, a complete response can be defined by the disappearance of all measurable tumors 1 month after treatment. While a partial response can be defined by a reduction to 50 percent or more of the sum of the products of the perpendicular diameters of all evaluable tumor nodules 1 month after treatment, without

334

JL JLV JL A &lt;

MEXICAN INSTITUTE OF PROPERTY tumor sites showing enlargement. From panera, you can define a mixed response using a useful reduction product of perpendicular diameters of all measurable lesions by 50 percent or more 1 month after treatment, with progress at one or more sites.

In light of the results of clinical trials, such as those described above, a more precise treatment regimen can still be formulated. Still, some variation in dosage may subsequently be necessary depending on the condition of the subject being treated. The physician responsible for administration, in light of the present disclosure, will be able to determine the appropriate dose for the individual subject. This optimization and tuning is routinely carried out in the art and in no way reflects an inappropriate amount of experimentation.

N. Combination Therapies against Tumors

The treatment methods of the present invention may be combined with any other methods generally employed in the treatment of particular tumors, diseases or disorders exhibited by the patient, provided that a particular therapeutic approach is not known to be harmful to the condition itself of the patient, and that does not significantly counteract the treatment based on anti-

<img file="MX337052B_D0323.tif" />

Regarding the treatment of solid tumors, the present invention can be used in combination with classical approaches, such as surgery, chemotherapy, radiotherapy, cytokine therapy, anti-angiogenesis, and the like. The invention then provides combination therapies in which the antibodies, immunoconjugates, or peptide conjugates are used simultaneously with, before, or after surgery or radiation treatment; or they are administered to patients with, before, or after conventional chemotherapeutic or radiotherapeutic agents, cytokines, antiangiogenic agents, apoptosis-inducing agents, objective immunotoxins or coaguligands, or the like. Many examples of appropriate therapeutic agents have been described in relation to the immunoconjugate aspects of the present invention. Any of the agents initially described for use as a part of a therapeutic conjugate, or may be used separately, in the therapies of the invention.

336

<img file="MX337052B_D0324.tif" />

In terms of surgery, any surgical intervention can be practiced in combination with the present invention. In relation to radiotherapy, any mechanism to induce DNA damage locally within tumor cells is contemplated, such as irradiation and, X-rays, UV irradiation, microwaves, and even electronic emissions and the like. Targeted delivery of radioisotopes 10 to tumor cells is also contemplated and can be used in connection with a targeted antibody or other means for targeted localization.

The general use of combinations of substances in the treatment of cancer is well known. For example, United States of America Patent No. 5,710,134 (incorporated herein by reference) describes components that induce necrosis in tumors in combination with non-toxic substances or prodrugs. The enzymes released by necrotic processes cleave the non-toxic prodrug to produce the toxic drug, leading to the death of the tumor cell. Also, United States Patent No. 5,747,469 (incorporated herein by reference) describes the combined use of viral vectors encoding p53 and DNA damaging agents. Any of those similar approaches can be used with the present invention.

337

<img file="MX337052B_D0325.tif" />

when each treatment is carried out separately. Although at least additive effects are generally desired, any increased antitumor effect, over and above individual therapies, would be beneficial. Also, there is no particular requirement for the combined treatment to exhibit synergistic effects, although this is certainly possible and advantageous.

NEITHER. Selection of Second Anticancer Agents

The primary therapeutic agents of the present invention, as used herein, are anti-aminophospholipid or anti-anionic phospholipid antibodies, immunoconjugates or derivatives and conjugates of PE-binding peptides. Secondary therapeutic agents, as used herein, are distinct second therapeutic agents, or anticancer agents, i.e. therapeutic agents or anticancer agents other than the primary therapeutic agent. Any secondary therapeutic agent may be used in combination therapies herein. invention. Also, secondary therapeutic agents or second anticancer agents can be selected with the intention of achieving an effect.

338

<img file="MX337052B_D0326.tif" />

<img file="MX337052B_D0327.tif" />

according to the following guide.

To carry out the combined anti-tumor therapy, an anti-aminophospholipid or anti-anianic phospholipid antibody, immunoconjugate or PE-binding peptide-based therapeutic product of the present invention would simply be administered to an animal or patient in combination with another, that is, a different second anticancer agent, in an effective way so that its combined antitumor actions result in the animal or patient. The agents would then be provided in effective amounts and for effective time periods to result in their combined presence in the tumor or tumor vasculature and their combined actions in the tumor environment. To achieve this goal, the primary therapeutic compounds of the present invention and the second, distinct anticancer agents can be administered to an animal substantially simultaneously, either in a single composition or as two distinct compositions using different routes of administration.

Alternatively, the anti-aminophospholipid or anti-anianic phospholipid antibody, immunoconjugate, or PE-binding peptide-based therapeutic product of the present invention may precede, or follow, the second distinct anticancer agent, eg, by intervals ranging from minutes to weeks. In certain modalities in

339 where the therapeutic compounds of the presei

<img file="MX337052B_D0328.tif" />

Second, different, anti-cancer agents are applied separately to the animal, it would be ensured that a significant period has not elapsed between the time of each administration, so that each agent can still exert an advantageous combined effect on the tumor. In these cases, it is contemplated that the tumor would be contacted with both agents, within approximately 5 minutes to approximately one week from each other and, more preferably, from approximately 12 to 72 hours from each other, with a delay time of only approximately 12 to 48 hours, the latter being the most preferred.

Secondary therapeutic agents for synchronized combination therapies, separately, can be selected based on certain criteria, including those discussed below. However, a preference to select one or more different second anticancer agents before or after administration does not preclude their use in substantially simultaneous administration if so desired.

Second, the various anticancer agents selected for administration prior to the primary therapeutic agents of the present invention, and designed to achieve increased and potentially synergistic effects, include agents that induce the expression of aminophospholipids or anionic phospholipids in the vasculature of the

340 tumor. For example, agents who

<img file="MX337052B_D0329.tif" />

localized calcium, activate membrane transporters that move PS and other phospholipids to the outer surface of the plasma membrane, damage the endothelium of the tumor, cause pre-apkotic changes and / or induce apoptosis in the endothelium of the tumor, will generally result an increased expression of aminophospholipid and anionic phospholipid. Examples of such agents are docetaxel and paclitaxel. The aminophospholipids and anionic phospholipids can be targeted using an antibody of the invention, then amplifying the overall therapeutic effect, and also providing increased attack through ADCC host effectors (complement), antibody-mediated phagocytosis, CDC).

Drugs that have selectivity for activated angiogenic, remodeling, or endothelial cells, such as those present in tumor blood vessels but not in normal resting blood vessels, can also be used to selectively cause exposure of PS and other phospholipids on the surface of tumor endothelial cells. Examples of such agents are combretastatins and docetaxel. This would again lead to increased binding of the antibody and improved initiation of host effector mechanisms.

Second, the various anticancer agents selected for subsequent administration to ra

341 Synergists include agents that benefit from the effects of the primary therapeutic agent. The anti-aminophospholipid or anti-anion phospholipid, immunoconjugate, or therapeutic peptide-based antibody of the present invention will cause tumor destruction. Accordingly, the second, distinct, effective anticancer agents for subsequent administration include anti-angiogenic agents that inhibit metastasis; agents for targeted localization of necrotic tumor cells, such as antibodies specific to intracellular antigens that become accessible to malignant cells in vivo (United States Patent Nos. 5,019,368, 4,861,584 and 5,882,626, each of which is specifically incorporated herein as reference), and chemotherapeutic and immunoconjugated agents of antitumor cells, that attack any anti-tumor cells that may survive in the periphery.

In some situations it may be desirable to prolong the treatment time significantly, where several days (2, 3, 4, 5, 6, or 7), several weeks (1, 2, 3, 4, 5, 6, 7 or 8) or even several months (1, 2, 3, 4, 5, 6, 7 or 8) between the respective administrations this would be advantageous in circumstances where a treatment is intended to destroy substantially

342 i

'J Π the tumor, such as the therapeutic agent íprimárío_d§¡

I the present invention, and another i-Tvai-ami ent is intended to prevent micrometastasis or re-growth of the tumor, such as the administration of an anti-angiogenic agent. Anti-angiogenic agents should be administered in a prudent time after surgery, and nevertheless should allow effective wound healing. Anti-angiogenic agents can then be administered throughout the life of the patient.

It is also contemplated that more than one administration of either the primary therapeutic agent or the second distinct anticancer agent be used. The primary therapeutic agent and the second distinct anticancer agent may be administered interchangeably, on alternate days or weeks; or a sequence of one treatment with one agent may be provided, followed by a sequence of the other treatment. In any case, to achieve tumor regression using combination therapy, all that is required is to deliver both agents in a combined effective amount to exert an antitumor effect, regardless of the times of administration.

Whether administered substantially simultaneously or sequentially, anti-aminophospholipid and anti-anion phospholipid antibodies and

343

<img file="MX337052B_D0330.tif" />

Given in combination with one or .. · mSsag ^ nt-Ag or chemotherapeutic drugs, the chemotherapeutic drugs can kill proliferating tumor cells, enhance necrotic areas created by global treatment. The drugs can thus enhance the thrombotic action of the primary therapeutic agents of the invention.

Most chemotherapy drugs for cancer are selective for dividing oxygen cells. These have advantages in combined therapy since the chemotherapeutic drug acts on different objectives from the primary therapeutic agents of the invention, leading to a more complete antivascular or antitumor effect.

For example, chemotherapeutic drugs are selectively active against rapidly dividing oxygenated tumor cells at the periphery of the tumor while the agents of the invention act primarily on the vessels or tumor cells in the nucleus of the damaged tumor, where reactive, activating oxygen species are abundant.

Anti-angiogenic drugs that are selective for well-oxygenated angiogenic vessels at the periphery of the tumor would also be effective in combination, since the

344

<img file="MX337052B_D0331.tif" />

ID] [agents of the invention act on the vasó ^ '^:'¿I; fiáctÍV | ₉ INü'ui »TidAL relatively hypoxic, in the nucleus of the tumor.

By inducing thrombus formation in tumor vessels, the primary therapeutic agents of the present invention can also enhance the action of chemotherapeutic drugs by retaining or trapping the drugs in the tumor. The chemotherapeutic agents are thus retained within the tumor, while the rest of the drug is eliminated from the body. The tumor cells are then exposed to a higher concentration of the drug for a longer period. Trapping the drug in the tumor makes it possible to reduce the dose of the drug, making the treatment safer and more effective.

Additional drugs for combined use in the present invention are those that act on cells that are sensitized to the drug by the action of the primary therapeutic agent such that reduced doses of the second drug are required to achieve its antitumor effect. For example, this could occur where a major component of the action of the second drug is exerted on the tumor vessels and the antibodies or agents of the invention sensitize the cells to the drug. The same is true where the primary therapeutic agent of the invention - sensitizes tumor cells to a second drug, either

Ι, ΜΡΙ

i. FUTO MKttr · /_F,_or

PROPERTY stimulation^{TO THE} d

<img file="MX337052B_D0332.tif" />

individually or through the

3. 4. 5 cytokine release.

<img file="MX337052B_D0333.tif" />

Other suitable second anticancer agents for combination therapy are those that increase the activity of host effector cells, for example by selectively inhibiting the activity of immunosuppressive components of the immune system. These agents enable primary therapeutic agents, which stimulate attack by effector cells as part of their mechanism, to function more aggressively. An example of such an agent is docetaxel.

Although an understanding of the precise mechanism (s) of action of primary therapeutic agents is not necessary to practice the treatment of the invention, data and inferences regarding those mechanisms may be used to select particular second anticancer agents for the combined use in the present invention. The effectiveness of selected combination therapy, in turn, supports the original data and proposed mechanisms of action, and also leads to preferred categories of second anticancer agents for practicing combination therapy.

Drugs that induce apoptosis are preferred for use in combination walls. The

346 docetaxel, by exposure i Jl ', example, induces apoptos ^ s V'ipor therefore / 7&gt;

to the PS by binding to the microtubules and disrupting cell mitosis (Hotchkiss et al., 2002). Treatment of endothelial cells, which line the tumor's blood vessels, and tumor cells with docetaxel at subclinical concentrations, is presently shown to induce PS expression on the cell surface, which is demonstrated by the strong binding of the 3G4 antibody in vitro.

The inventors herein have determined

<img file="MX337052B_D0334.tif" />

also that the antitumor effects of the effects of the invention include the Fe domain mediated increase in immune effector functions, which is shown by increased antibody mediated phagocytosis. Therefore 15; Antibodies should exert other functions mediated by the Fe domain, such as ADCC, CDC, stimulation of cytokine production, and those mechanisms in combination. This is also relevant for docetaxel, as other studies have shown that treatment of breast cancer patients with docetaxel leads to increases in IFN-y cytokine levels.

IL-2, IL-6 and GM-CSF, in serum, increased antitumor immune responses in these patients, by increasing the

<img file="MX337052B_D0335.tif" />

activity of natural suppressor cells (NK) and

347 ? 1.W.1 the liiaf ^ qina i · ... 1NDU-.

ξ £ * ·.

(IAK).

suppressor cells activated by (Tsavaris et al., 2002).

Therefore, the inventors reasoned that docetaxel will induce both PS expression and binding of the administered antibody, and will also increase the activities of immune effectors, which mediate antitumor effects. Based on the foregoing considerations, the inventors have demonstrated that the combination of the antibodies of the present invention, exemplified by the 3G4 antibody, with docetaxel was significantly superior to that of docetaxel or 3G4 alone in mice having xenografts. of human breast cancer MDA-MB-435,

<img file="MX337052B_D0336.tif" />

orthotopic (example XX).

Accordingly, docetaxel and other apoptosis-inducing chemotherapeutic agents are preferred agents for use in the combination treatments of the present invention. Combinations of antibodies with aminophospholipids and / or anionic phospholipids with apoptosis-inducing chemotherapeutic drugs, such as docetaxel, should synergistically target endothelial cells of the tumor vasculature and significantly improved treatment cell compartments.

<img file="MX337052B_D0337.tif" />

of the tumor, leading not only to an efficacy of

348 less toxicity.

These combinations use in the treatment of

IΜ N were sung, to

OF THE

INDUSTRIAL ^ &lt;1 .. - breast cancer, particularly the combination of metronomic chemotherapy using docetaxel with an antibody of the present invention.

N2. Endotoxin

Endotoxin and detoxified endotoxin derivatives can be used in combination treatment, preferably at low doses (PCT publication

No. WO 03/028840 specifically incorporated herein by reference). Various detoxified endotoxins are available, which are preferred for use in animals and particularly for use in humans. The 15 refined and detoxified endotoxins, and combinations thereof, are described in US Patent Nos. 4,866,034; 4,435,386;

4,505,899; 4,436,727; 4,436,728; 4,505,900, each of which is specifically incorporated herein by reference.

The non-toxic derived monophosphoryl lipid A (MPL) is an example of a detoxified endotoxin that can be used in the present invention. MPL is using MPL as an adjuvant, it has been shown that 100 g / m² known to be safe for humans; clinical trials

<img file="MX337052B_D0338.tif" />

they are safe for human use, per patient.

<img file="MX337052B_D0339.tif" />

349

<img file="MX337052B_D0340.tif" />

<img file="MX337052B_D0341.tif" />

INDUSTRY!

N3. Cytokines □

Cytokine therapy has been shown to be an effective standard for combination therapy regimens. Various cytokines can be used in the combined approaches of the present invention. Examples of cytokines include

IL-la, IL-Ιβ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL10 9, IL-10, IL-11, IL-12 , IL-13, TGF-β, GM-CSF, M-CSF, G-CSF,

TNFa, TNFP,

LAF, TCGF, ​​BCGF,

TRF, BAF, BDG,

MP, LIF, OSM,

TMF, PDGF,

IFN-a, IFN-β,

IFN-y

Cytokines are administered from. according to standard regimes, &lt;

consistent with clinical indications such as the patient's condition and the relative toxicity of the cytokine. Uteroglobins can also be used to prevent or inhibit metastasis (US Patent No. 5,696,092; incorporated herein by reference).

N4. TNFa to TNFa Inductors

TNFa and TNFa inducers can also be used in combination with the present invention. The useful in facilitating the penetration of agents

<img file="MX337052B_D0342.tif" />

TNFa increases vascular permeability and is therefore

350

<img file="MX337052B_D0343.tif" />

<img file="MX337052B_D0344.tif" />

, η ho? ή_ί V 4, ¿i Η • zacjlÓnAid

OF THE PRCHITY; · INDUSTRIAL antibody is not in any way a problem when targeting an aminophospholipid and anionic phospholipids, as in the present invention, the combined use of TNFoí can facilitate the access of other chemotherapeutic and immunoconjugated compounds to the tumor, and increase including the binding of the antibodies of the invention to quite distant tumor cells.

<img file="MX337052B_D0345.tif" />

Low endotoxin levels, antagonists of

Racl, such as engineered or engineered adenoviruses, DMXAA (and FAA), CM101, and thalidomide can also be used. Racl antagonists can be used in the combined treatment of the present invention, since approximately 5000 DNA particles per cell cause an increase in TNF independently of CD14 (Sanlioglu et al., 2001). CM101, thalidomide and DMXAA can also be used in combination with it, in standard or reduced doses.

N5. Chemotherapeutic substances

Regardless of the fundamental mechanism (s), a variety of chemotherapeutic substances can be used in the chemotherapeutic methods contemplated for their combined use.

<img file="MX337052B_D0346.tif" />

combined treatment described herein.

Agents include, ν.gr.,

351 tamoxifen, taxol,

<img file="MX337052B_D0347.tif" />

(VP-16), adriamycin, 5-fluorouracil (5FU) '-' j-ijlLCin ?.

actinomycin-D, mitomycin, combretastatin (s), more

<img file="MX337052B_D0348.tif" />

particularly docetaxel, (taxoter), cisplastin - (CDDP), 5 cyclophosphamide, doxorubicin, methotrexate, paclitaxel and vincristine and derivatives and prodrugs thereof.

As will be understood by those skilled in the art, the appropriate doses of chemotherapeutic substances will generally be around those already employed in clinical therapies, where the chemotherapeutic substances are administered alone or in combination with other chemotherapeutic substances. However, lower doses are now possible due to the advantages provided by the present invention. By way of

<img file="MX337052B_D0349.tif" />

example only, substances such as cisplatin, and other DNA alkylating agents can be used. Cisplatin has been widely used to treat cancer, with effective doses used in clinical applications of 20 milligrams / m.² for 5 days every three weeks for a total of 20 three courses. Cisplatin is not absorbed orally, and therefore must be administered intravenously, subcutaneously, intratumorally, or intraperitoneally.

Other useful substances include compounds that interfere with DNA replication, mitosis, chromosomal segregation, and / or tubulin activity. These chemotherapeutic compounds include adriamycin, also known as τ

like doxorubicin,

352

INDUSTIU / j.

podophyllotoxin (s), combrestatin (s)

Pi. »· And the like. Widely used in the clinical setting for the treatment of neoplasms, these compounds are administered through

<img file="MX337052B_D0350.tif" />

Bolus injections intravenously at doses ranging from 25-75 milligrams / m² at 21-day intervals for adriamycin, up to 35-50 milligrams / m² intravenously for etoposide or twice the intravenous dose orally.

Agents that interrupt synthesis and fidelity of polynucleotide precursors can also be used. Particularly useful are substances that have undergone extensive testing and are readily available.

<img file="MX337052B_D0351.tif" />

As such, substances such as 5-fluorouracil (5-FU) are preferentially used for neoplastic tissue, making this substance particularly useful for targeting neoplastic cells. Although somewhat toxic, 5-FU is applicable in a wide variety of vehicles, including topicals, yet

<img file="MX337052B_D0352.tif" />

intravenous administration with

<img file="MX337052B_D0353.tif" />

commonly use.

Exemplary Chemotherapeutic Agents

<img file="MX337052B_D0354.tif" />

are

<img file="MX337052B_D0355.tif" />

therapy

<img file="MX337052B_D0356.tif" />

<img file="MX337052B_D0357.tif" />

substances listed

<img file="MX337052B_D0358.tif" />

<img file="MX337052B_D0359.tif" />

limiting ways.

<img file="MX337052B_D0360.tif" />

<img file="MX337052B_D0361.tif" />

<img file="MX337052B_D0362.tif" />

Expert technician can consult Remington's Pharmaceutical

353

Sciences 15^topages 624-652

Edition, chapter 33,. Some variation in

<img file="MX337052B_D0363.tif" />

dose will likely occur depending on the condition being treated. The physician administering the treatment will be able to determine the appropriate dose for the subject.

<img file="MX337052B_D0364.tif" />

individual.

354

Qulmlot therapeutic substances Useful s in

TABLE D

Neoplastic disease

<img file="MX337052B_D0365.tif" />

<img file="MX337052B_D0366.tif" />

♦·

<td>| CLASS</td><td>KIND OF SUBSTANCE</td><td>NAMES NO COMMERCIAL (OTHER NAMES)</td><td>DISEASE 1</td>

<td rowspan="7">Substances Alkylating agents</td><td rowspan="4">Mustards Nitrogen</td><td>Mechloroethamine (HN₂)</td><td>Hodgkin's disease, non-Hodgkin's lymphines.</td>

<td>Famida Cyclophs Zphosphamide</td><td>Acute and Chronic Lymphocytic Leukemias, | Hodgkin's disease, I Non-Hodgkin lymphomas, 1 multiple myeloma, 1 neuroblastoma, chest, I ovary, lung, tumor of | Wilms, cervix, 1 testicles, sarcomas of 1 soft tissue. ||</td>

<td>Melfalan (1sarcolysin)</td><td>Multiple myeloma, chest, 1 ovary. I</td>

<td>Chlorambucil</td><td>Lymphocytic leukemia | chronic, macroglobu- | primary linemia, 1 Hodgkin's disease, | not Hodgkin's lymph. |</td>

<td rowspan="2">Ethyleneimenos and Methylmelamines</td><td>Hexamethylmelamine</td><td>Ovary 1</td>

<td>Thiotepa</td><td>Bladder, chest, ovary. one</td>

<td>Alkylsulfonates</td><td>Busulfan</td><td>Chronic granulocytic leukemia 1. |</td>

<img file="MX337052B_D0367.tif" />

355

<img file="MX337052B_D0368.tif" />

Antimeta-

<img file="MX337052B_D0369.tif" />

Small triazines

Analogues of

Folic acid

Carmustine (BCNU)

Lomustine (CCNU)

Semustine (methyl-CCNU)

Streptozocin (streptozotocin)

Dacarbazine (DTIC; dimethyltriazenoimidazole ecarboxamide).

Methotrexate (ametopterin).

<img file="MX337052B_D0370.tif" />

<img file="MX337052B_D0371.tif" />

—I

MEXICAN INSTITUTE

Disease &amp;^TO4 ^ r $ j $ b non-Hodgkin lymphomas, turne l¹ cu du LUlUblü ~ 'primaries, multiple myeloma, malignant melanoma.

Hodgkin's disease, non-Hodgkin's lymphomas, primary brain tumors, small cell lung.

Primary brain, stomach, colon tumors.

Malignant pancreatic insulinoma, malignant carcinoid.

Malignant melanoma, Hodgkin's disease, soft tissue sarcomas.

Acute lymphocytic leukemia, choriocarcinoma, mycosis fungoides, chest, head and neck, lung, osteogenic sarcoma.

Antime taFluouracil (5fluoro-uracil; 5-FU). Floxuridine bolitos

Pyrimidine analogues (fluorodeoxyuridine; FUdR).

Chest, colon, stomach, pancreas, ovary, head and neck, urinary bladder, premalignant (topical) skin lesions.

Cytarabine (cytosine arabinoside).

Acute lymphocytic leukemias and acute granulocytic leukemias.

356

P *

<img file="MX337052B_D0372.tif" />

<img file="MX337052B_D0373.tif" />

Purine analogs and related inhibitors

Alkaloids

Vinca

products

Natural

Epipodophyllotoxins

Antibiotics

Mercaptopurine (6mercaptopurine 6-MP) ,.

Thioguanine (6thioguanine; TG).

Pentostatin deoxycoformycin).

Vinblastine (VLB).

Vincristine

Etopcfeido Tertiposide (2Dactinomycin (actinomycin D)

Daunorubicin (daunomycin; rubidomycin).

<img file="MX337052B_D0374.tif" />

Leukemias acute lififbciftica, acute granulocytic and chronic granulocytic.

Acute granulocytic, acute lymphocytic, and chronic granulocytic leukemias.

OR

Hairy cell leukemia, mycosis fungoides, chronic lymphocytic leukemia.

Hodgkin's disease, non-Hodgkin lymphomas, chest, testicles.

Acute lymphocytic leukemia, neuroblastoma, Wilms tumor, rhabdomyosarcoma, Hodgkin's disease, 0 non-Hodgkin lymphomas, small cell lung.

Testes, small cell lung and others ^ lungs, chest, Hodgkin's disease, non-Hodgkin's lymphomas, acute granulocytic leukemia, Kaposi's sarcoma.

Choriocarcinoma, Wilms tumor, rhabdomyosarcoma, · testicles, Kaposi's sarcoma.

Acute granulocytic and acute lymphocytic leukemias.

357

<td></td><td></td><td></td><td>Doxorubicin</td><td>Uní JJ 1 U l MtAlUAJMU OF PROPERTY V Sarcoma ^ of tej £ ^^{STR1 AL}soft, osteogenic and</td><td></td>

<td> 5</td><td></td><td></td><td></td><td>other sarcomas; Hodgkin's disease, non-Hodgkin's lymphomas, acute leukemias, chest, genitourinary, thyroid, lung, stomach, neuroblastoma.</td><td></td>

<td> 10</td><td></td><td></td><td>Bleomycin</td><td>-Testicles, head and neck, skin, esophagus, lung and genitourinary tract; Hodgkin's disease, non-Hodgkin's lymphomas.</td><td></td>

<td> .</td><td></td><td></td><td>Plicamycin (Mithramycin).</td><td>Testicles, malignant hypercalcemia.</td><td></td>

<td></td><td></td><td></td><td>Mitomycin (mitomycin C)</td><td>Stomach, cervix, colon, chest, pancreas, bladder, head, and neck.</td><td></td>

<td> 15</td><td></td><td>Enzymes</td><td>L-Asparaginase</td><td>Acute lymphocytic leukemia.</td><td></td>

<td> 20</td><td></td><td>Biological Response Modifiers</td><td>Alpha interferon</td><td>Hairy cell leukemia, Kaposi's sarcoma, melanoma, carcinoid, renal cell, ovary, bladder, non-Hodgkin lymphomas, mycosis fungoides, multiple myeloma, chronic granulocytic leukemia. |</td><td></td>

I

<img file="MX337052B_D0375.tif" />

<td></td><td></td><td></td><td> 358</td><td>IMPIg MEXICAN INSTITUTE</td><td></td>

<td></td><td></td><td>Platinum Coordination Ripples</td><td>Cisplatin (cis- DDP Carboplatin)</td><td>--------'---------- DL LÁ 'PRUWSXS-- INDUSTRIAL. Testicles, ovary, lung, thyroid, cervix, endometrium, neuroblastoma, osteogenic sarcoma.</td><td></td>

<td> 5</td><td></td><td>Anthracenodione</td><td>Mitoxantrone</td><td>Acute granulocytic leukemia, chest.</td><td></td>

<td rowspan="2"> 10</td><td>Miscellaneous substances</td><td>Urea replaced</td><td>Hydroxyurea</td><td>Chronic granulocytic leukemia, polycythemia vera, essential thrombocytosis, malignant melanoma.</td><td></td>

<td></td><td>Methylhydrazine derivative</td><td>Procarbazine (Nmethylhydrazina, MIH).</td><td>Hodgkin's disease.</td><td></td>

<td></td><td></td><td>Adreno Suppressor-</td><td>Mitotane (o, p'- DDD).</td><td>Adrenal cortex '</td><td></td>

<td> 15</td><td></td><td>cortical</td><td>Aminoglutethimide</td><td>Chest</td><td></td>

<td></td><td>Hormones and Antagonists</td><td>Adrenocorsti costeroides</td><td>Prednisone (various other preparations available)</td><td>Acute and chronic lymphocytic leukemias, non-Hodgkin lymphomas, Hodgkin disease, chest</td><td></td>

<td rowspan="2"> 20 25</td><td></td><td>Progestins</td><td>Hydroxyprogesterone caproate Acetate ' Medroxyprogesterone, Acetate Megestrol</td><td>Endometrium, chest</td><td></td>

<td></td><td>Estrogens</td><td>Diethylstil-</td><td>Chest, prostate</td><td></td>

359

<img file="MX337052B_D0376.tif" />

Ν6. Anti-Angiogenic

The term angiogenesis refers to the generation of new blood vessels, generally in a tissue or organ. Under normal physiological conditions, humans or animals undergo angiogenesis only in specific restricted situations. For example, angiogenesis is typically seen in wound healing, in fetal and embryonic development, and in the formation of the corpus luteum, endometrium, and placenta. However, new evidence shows that angiogenesis is important in certain

360 normal situations such as in the

<img file="MX337052B_D0377.tif" />

INSTITUTO MEXIO N'O

DS.U WWTIEL'AD te j xáosTRiad

<img file="MX337052B_D0378.tif" />

prostate and ovaries. Agents i¹* ?? of ?.?.

Invention, in which anti-angiogenesis is not the only mode of action, then have advantages over prominent anti-angiogenic therapies, such as antibody A4.6.1 (Brem, 1998; Baca et al., 1997; Presta et al., 1997), because desirable or physiological angiogenesis will not be inhibited when using the present invention.

Uncontrolled angiogenesis (persistent and / or unregulated) is related to various disease states, and occurs during the development and metastasis of tumors. Both controlled and uncontrolled angiogenesis is believed to proceed in a similar manner. Endothelial cells and pericytes, surrounded by a basement membrane, form capillary blood vessels. Angiogenesis begins with erosion of the basement membrane by enzymes, released by endothelial cells and leukocytes. The endothelial cells, which line the lumen of the blood vessels, then protrude through the basement membrane. Angiogenic stimulants induce endothelial cells to migrate through the eroded basement membrane. Migrant cells form an outbreak outside the related blood vessel, where endothelial cells undergo mitosis and proliferate. The

361 endothelial outbreaks come together

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hair curls creating the blood vessel. -

Despite new evidence that angiogenesis is required in some normal tissues, anti-angiogenic therapies are still important in the treatment of tumors and other diseases. Anti-angiogenic therapies then serve for use in the combination treatments of the present invention. The combination of a relatively frequent low dose of a therapeutic agent of the present invention, in combination with an agent that inhibits angiogenesis, is particularly contemplated. Exemplary anti-angiogenic agents that are useful in relation to a combination therapy are listed above (in relation to the 15 immunoconjugates). Any one or more of those agents, including those in Table B, can be used in combination therapy with the invention. Angiostatin, endostatin, vasculostatin, canstatin and maspin are currently preferred.

Many known anticancer agents also have an anti-angiogenic effect as part of their mechanism of action. These agents, exemplified by those in Table E, are particularly contemplated for use in combination therapy aspects of the present invention (they may also be conjugated to a

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362 antibody of the invention, above).

TABLE E

Agent »Anticancer with Antiangiogenic Activity

<td>Agent class or type</td><td>Examples</td>

<td>Alkylating agents</td><td>Cyclophosphamide, edelfosine, estramustine, melphalan</td>

<td>Antimetabolites</td><td>Fluorouracil, methotrexate, mercaptopurine, UFT, tegafur, uracil, cytarabine</td>

<td>Antitumor antibiotics</td><td>Bleomycin, daunorubicin, doxorubicin, epirubicin, mitomycin, mitoxantrone</td>

<td>Topoisomerase inhibitors</td><td>Camptothecin, irinotecan, etoposide, topotecano</td>

<td>Taxanes</td><td>Docetaxel, paclitaxel</td>

<td>Alkaloids Vinca</td><td>Vinblastine, vincristine</td>

<td>Various ·</td><td>Cisplatin, octreotide</td>

In addition, the LM609 antibody against the integrin α_νβ3 also induces tumor regressions and can be used in combination therapies. Antagonists of the integrin α_νβ₃, such as LM609, induce apoptosis of

363 angiogenic s endothelial cells j ^ / d ^ athdó; inactive blood. LM609 u involvement of vessels other α antagonists_νβ3 may also work by inhibiting the interaction of α_νβ₃ and MMP-2, a proteolytic enzyme, which are believed to play an important role in the migration of endothelial cells and fibroblasts.

Apoptosis of the angiogenic endothelium by

LM609 may have a cascading effect on the rest of the vascular network. Inhibition of the tumor vascular network to respond fully to the tumor signal to expand may, in effect, initiate partial or total collapse of the network, resulting in the death of tumor cells and loss of volume of the tumor. tumor. Endostatin and angiostatin may work in a similar way. The fact that LM609 does not affect inactive vessels, but that it can cause regressions in the tumor, strongly suggests that not all blood vessels in a tumor need to be reached by treatment in order to obtain an antitumor effect.

for the States

Antibodies can also be used as described in the angiogenin patent,

United States of America specifically here as you find related to 25 can use inhibitors of the

<td>NOT.</td><td> 5,520,914,</td><td colspan="2">incorporated</td>

<td colspan="2">reference. How</td><td>the FGF</td><td>I know</td>

<td>the</td><td>angiogenesis,</td><td>too</td><td>I know</td>

<td>FGF</td><td colspan="2">. Certain examples are</td><td>the</td>

364 compounds that sequence with

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2-O-sulfated uronic acid as its main repeating units, including glycosaminoglycans, such as arcaran sulfate. Those compounds are described in United States Patent No. 6,028,061, specifically incorporated herein by reference, and may be used in combination therewith.

N7. VEGF inhibitors

VEGF is a multifunctional cytokine that is induced by hypoxia and oncogenic mutations. VEGF is a primary stimulant of the development and maintenance of a vascular network in embryogenesis. It functions as a potent permeability inducing agent, an endothelial cell chemotactic agent, an endothelial survival factor, and an endothelial cell proliferation factor. Its activity is required for normal embryonic development, as directed disruption of one or both of the VEGF alleles results in embryonic death.

The use of one or more VEGF inhibition methods is a preferred aspect of the combination therapies of the present invention. Recognition of VEGF as a primary stimulus of angiogenesis under zZ conditions

365 Pathologies have led to various methods of blocking the activity of the VEGF. Any of the inhibitors ^^^^^ '

Developed VEGFs can now be used advantageously herewith. Accordingly, any one or more of the following anti-VEGF antibodies, neutralizers, soluble receptor construct, antisense strategies, RNA aptamers, and tyrosine kinase inhibitors, designed to interfere with VEGF signaling, can then be used.

Appropriate agents include neutralizing antibodies (Kim et al.,

1992; Presta et al., 1997;

Sioussat et al., 1993; Kondo et al., 1993;

Asano et al., 1995), soluble receptor constructs (Kendall and Thomas, 1993; Aiello et al., 1995; Lin et al., 1998; Millauer et al., 1996), tyrosine kinase inhibitors (Siemeister et al. ., 1998), antisense strategies, to RNA aptamers and ribozymes against VEGF or VEGF receptors (Saleh et al., 1996; Cheng et al., 1996). Variants of VEGF with antagonistic properties can also be used, as described in WO 98/16551. Each of the foregoing references is specifically incorporated herein by reference.

Blocking of antibodies against VEGF will be preferred in certain embodiments, particularly for simplicity. Monoclonal Antibodies Against VEGF

366 in human tumors and the formation of (Kim et al.,

1993; Mesiano et al.,