Anti-ox40 antibodies and methods of using the same.
Abstract
Human antibodies, preferably recombinant human antibodies, both humanized and chimeric, which specifically bind to human OX40 are disclosed. Preferred antibodies have high affinity for OX40 receptor and activate the receptor in vitro and in vivo. The antibody can be a full-length antibody or an antigen-binding portion thereof. The antibodies, or antibody portions, are useful for modulating receptor activity, e.g., in a human subject suffering from a disorder in which OX40 activity is detrimental. Nucleic acids, vectors and host cells for expressing the recombinant human antibodies are provided, and methods of synthesizing the recombinant human antibodies, are also provided.

Term
4.9 yearsleft in the term
Expires 23 August 2031.
- Priority
- Filed
- Granted
- Today
- Expires
10 claims: 5 independent, 5 dependent
- 1REIVINDICACIONES 5 1,- Un anticuerpo aislado que se une a 0X40, caracterizado porque comprende:(a) una región variable de cadena pesada CDR1 que comprende la secuencia de aminoácidos de la SEQ ID NO: 1;(b) una región variable de cadena pesada CDR2 que comprende la secuencia de aminoácidos de la SEQ ID NO: 2;(c) una región variable de cadena pesada 10 CDR3 que comprende la secuencia de aminoácidos de la SEQ ID NO: 3;(d) una región variable de cadena ligera CDR1 que comprende la secuencia de aminoácidos de la SEQ ID NO: 7;(e) una reglón variable de cadena ligera CDR2 que comprende la secuencia de aminoácidos de la SEQ ID NO: 8;y (f) una región variable de cadena ligera CDR3 que comprende la secuencia de 15 aminoácidos de la SEQ ID NO: 9.
- 22,- Un anticuerpo aislado qu¿ se une a 0X40, caracterizado porque comprende:(a) una región variable de cadena pesada CDR1 que comprende la secuencia de aminoácidos de la SEQ ID NO: 13;(b) una región variable de cadena pesada CDR2 que comprende la secuencia de 20 aminoácidos de la SEQ ID NO: 14;(c) una región variable de cadena pesada CDR3 que comprende la secuencia de aminoácidos de la SEQ ID NO: 15;(d) una región variable de cadena ligera CDR1 que comprende la secuencia de aminoácidos de la SEQ ID NO: 19;(e) una región variable de cadena ligera 123 CDR2 que comprende la secuencia de aminoácidos de la SEQ D€tA «OntDAD MDUSTfUAL una región variable de cadena ligera CDR3 que comprende la secuencia de aminoácidos de la SEQ ID NO: 21.
- 3- El anticuerpo aislado de conformidad con la reivindicación 1, caracterizado además porque comprende una región variable de cadena ligera que tiene una secuencia al menos 90% idéntica a la secuencia de aminoácidos de la SEQ ID NO:10 y una región variable de cadena pesada que tiene una secuencia al menos 90% idéntica a la secuencia de aminoácidos de la SEQ ID NO: 4.
- 4- El anticuerpo aislado de conformidad con la reivindicación 2, caracterizado además porque comprende una región variable de cadena ligera que tiene una secuencia al menos 90% idéntica a la secuencia de aminoácidos de la SEQ ID NO:22 y una región variable de cadena pesada que tiene una secuencia al menos 90% idéntica a la secuencia de aminoácidos de la SEQ ID NO: 16.
- 5- El anticuerpo aislado de conformidad con cualquiera de las reivindicaciones anteriores, caracterizado además porque es un anticuerpo monoclonal.
- 66,- El anticuerpo aislado de conformidad con cualquiera de las 20 reivindicaciones anteriores, caracterizado además porque es un anticuerpo humanizado.
- 77,- El anticuerpo aislado de conformidad con la reivindicación 6, caracterizado además porque comprende una región variable de cadena 124 DE LA PROPIEDAD INDUSTRIAL ligera que tiene una secuencia al menos 90% idéntica a aminoácidos de la SEQ ID NO:1T y una región variable de cadena pesada que tiene una secuencia al menos 90% idéntica a la secuencia de aminoácidos de la SEQ ID NO: 5. 5
- 8- El anticuerpo aislado de conformidad con la reivindicación 6, caracterizado además porque comprende una región variable de cadena ligera que tiene una secuencia al menos 90% idéntica a la secuencia de aminoácidos de la SEQ ID NO:23 y una región variable de cadena pesada que tiene una secuencia al menos 90% idéntica a la secuencia de 10 aminoácidos de la SEQ ID NO: 17. *
- 9- Una porción de unión al antígeno de un anticuerpo como el que se reclama en cualquiera de las reivindicaciones anteriores, caracterizada porque dicha porción de unión al antígeno retiene la habilidad de unirse específicamente a y agonizar al receptor 0X40. 15 10,- Un ácido nucleico aislado, caracterizado porque codifica para un anticuerpo como el que se reclama en cualquiera de las reivindicaciones 1 a 9. 11, - Una célula hospedera, caracterizada porque comprende el ácido nucleico que codifica para un anticuerpo como el que se reclama en 20 cualquiera de las reivindicaciones 1 a 9. 12, - Un método para producir un anticuerpo, caracterizado porque comprende la etapa de cultivar una célula hospedera como la que se reclama en la reivindicación 11. 125 13,- El método de conformidad con la caracterizado además porque comprende adicionalmente ‘T^Sfl^feraréT anticuerpo de la célula hospedera. 14, - El anticuerpo aislado o porción de unión al antígeno del 5 mismo como se reclaman en las reivindicaciones 1 a 9, para usarse como un medicamento. 15, - El anticuerpo aislado o porción de unión al antígeno del mismo como se reclaman en las reivindicaciones 1 a 9, para usarse en el tratamiento de una enfermedad autoinmune.
- 1010 16.- El anticuerpo aislado o porción de unión al antígeno del mismo como se reclaman en las reivindicaciones 1 a 9, para usarse en el tratamiento de cáncer. 17,- El uso del anticuerpo aislado o porción de unión al antígeno del mismo como se reclaman en las reivindicaciones 1 a 9, en la preparación 15 de un medicamento para tratar cáncer o una enfermedad autoinmune. 126
Independent claims10
793 paragraphs in 94 sections, as filed
(54) Title: ANTIBODIES ΑΝΤΙ-ΘΧ40 AND METHODS FOR USING THEM.
(54) Title: ΑΝΤΙ-ΘΧ40 ANTIBODIES AND METHODS OF USING THE SAME.
(57) Summary
The present invention relates to an isolated 0X40-binding antibody, characterized in that it comprises: (a) a CDR1 heavy chain variable region comprising the amino acid sequence of SEO ID NO: 1; (b) a CDR2 heavy chain variable region comprising the amino acid sequence of SEO ID NO: 2, (c) a CDR3 heavy chain variable region comprising the amino acid sequence of SEO ID NO: 3; (d) a CDR1 light chain variable region comprising the amino acid sequence of SEO ID NO: 7; (e) a CDR2 light chain variable region comprising the amino acid sequence of SEO ID NO: 8; and (f) the CDR3 light chain variable region comprising the amino acid sequence of SEO ID NO: 9.
(57) Abstract
Human antibodies, preferably recombinant human antibodies, both humanized and chimeric, which specifically bind to human 0X40 are disclosed. Preferred antibodies have high affinity for 0X40 receptor and activate the receptor in vitro and in vivo. The antibody can be a full-length antibody or an antigen-binding portion thereof. The antibodies, or antibody portions, are useful for modulating receptor activity, eg, in a human subject suffering from a disorder in which 0X40 activity is detrimental. Nucleic acids, vectors and host cells for expressing the recombinant human antibodies are provided, and methods of synthesizing the recombinant human antibodies, are also provided.
I instituted
Icano xican of Pr piety
Industrial
I
PATENT TITLE NO. 339964
Headline (s): BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM
Address: 201 West 7th Street, Austin, Texas, 78701, USA
Name: ANTI-OX40 ANTIBODIES AND METHODS FOR USING THEM
Classification: lnt.CI.8: A61K39 / 395; A61P35 / 00; C07K16 / 28
Inventor (s): YONG-JUN LIU; KUI SHIN VOO; LAURA BOVER; NAOYA TSURUSHITA; J. YUN
TSO; SHANKAR KUMAR
Number:
MX / a'2013 / 002172
Country:
US
US
REQUEST
International filing deadline: August 23, 2011
PRIORITY
Date:
August 20'U September 8, 2010
Number:
61/375,999
61 / 380,827 v | gence: Twenty years
Winning Dateof August 23, 2031 n rae aHiculos 1 “« • nHndftln »
The reference source is organized
Disagreement with article 23 of the Pro Law as of the date of preservation of the request for defects £
2nd fraction V, 6th fraction III, and 59 of the Industrial Property Law ^ this patent has a validity of twenty unexplorable years, φτπφαφΙ and will be subject to the payment of the fee to keep the
Qajen subscribes to the present Industrial Property (Diario 29®1 / 2004, 16/06/2005, 25 naso a), 4 ° and 12 ° fraction of the hd 'flcial of I / 2006, 0 «iy ni d | '/ 2004 and 1 completeness I <on the basis of the provisions of the 6th Fractions Ht and 7th bis 2 of the Toy of the
Federation (DOF) SKKMfteM, amended on 08/02/19 S4. 10/25/1996, 12/26/1997, 5 / 2009,06 / 01/2010, 18 / *> C% ¿afWg3l4 2101 «or 12 and ** ¿/ 04/2012); Articles 1, 3) Regulations of the Mexican Institute of Industrial Property (DOF) 12/14/1999, re and 5th paragraph a) of the Agreement that delegates powers to the Deputy Directors General, Coordinator, Divisional Directors, Heads of Regional 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).
05/1999, action V signed the Organic '7); 1st, 3rd
Issue Date: June 17, 2016
<img file="MX339964B_D0001.tif" />
ANTI-OX4Q ANTIBODIES AND METHODS FOR
43/ ?-/
IMPI ^ J
UftARfciQiBNo ^ - ** »^ **
AD
INDUSTRIAL
FIELD OF THE INVENTION
This invention relates generally to modulation of 0X40 receptor activation, and more particularly, to modulation of 0X40 receptor to inhibit the immunosuppressive function of interleukin 10 (IL10) that produces type 1 regulatory T cells.
CD4<sup>+</sup> ("Tr1 cells") and regulatory T cells expressing 10 Foxp3<sup>+</sup> (sometimes also referred to herein as "T-reg Foxp3 cells<sup>+</sup>”) And the generation of Tr1 cells from CD4 cells<sup>+</sup> or naive cells and IL-10 production.
CROSS REFERENCE TO RELATED APPLICATION <sup>15</sup>
This application claims the benefit and priority of the United States Patent Application Series No. 61 / 375,999 filed on August 23, 2010 and the United States Patent Application Series No. 61 / 380,827 filed on September 08 2010. Both requests are incorporated herein by reference.
DECLARATION REGARDING DEVELOPMENT
O 3l \ MrBoíl
FEDERALLY SPONSORED
MEXICAN INSTITUTE OE THE PROPERTY
INDUSTRIAL
<img file="MX339964B_D0002.tif" />
This invention was made with government support under R01
AI061645-01, R01 AI062888-01 and U19 AI071130-01 granted by the Institutes
National Health. The government has certain rights in the invention.
THE NAMES OF THE CONTRACTING PARTIES TO AN AGREEMENT OF
JOINT INVESTIGATION
None
Sequence list reference
This description includes a sequence listing presented as a text file in accordance with 37 CFR § 1.52 (e) (v) named sequence listing.txt, created on August 23, 2011, with a size of 13,836 bytes, which is incorporated herein by reference. The accompanying sequence descriptions and Sequence Listing comply with the rules governing nucieotide and / or amino acid sequence descriptions in patent applications as described in 37 CFR §§ 1.821-1.825. The sequence listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in accordance with IUPAC3 standards.
<img file="MX339964B_D0003.tif" />
IUBMB described in Nucleic Acids Res. 13: 3021-3030 (11 J. 219 (No. 2): 345-373 (1984). The symbols and format used for nucleotide and amino acid sequence data comply with the rules described at 37 CFR §1.822.
BACKGROUND OF THE INVENTION
Tr1 cells play a critical role in peripheral tolerance. Tr1 cells are particularly important in limiting tissue damage to the host during inflammatory immune responses. The generation of Tr1 cells accompanies both TH1 and TH2 immune responses in vivo and in vitro.
Tr1 cells are generated from CD4 T cells<sup>+</sup> naive during an antigen-driven T-cell immune response. Tr1 cells are allergic in response to signaling through TCR, CD28 and IL-2 receptors and have the ability to suppress naive CD4 + T cell antigen-driven proliferation in vivo and in vitro. Tr1 cells have the ability to inhibit the development of autoimmune diseases and limit the magnitude of immune responses to microbial pathogens.
While the molecular signals that lead to Tr1 cells have been studied, little is known about the molecular signals that negatively regulate the generation of these cells. Although immunosuppressive drugs, cloclines, costimulatory molecules, and DCs have been implicated in the induction of Tr1 cells, the
<img file="MX339964B_D0004.tif" />
negatively generating Tr1 cells remain elusive.
BRIEF DESCRIPTION OF THE INVENTION
Activation of the 0X40 receptor blocks the generation of Tr1 from CD4 T cells<sup>+</sup> memory or naive as well as the production of IL-10 from Tr1 cells and the immunosuppressive function of Tr1 cells. Activation of the 0X40 receptor also blocks the production of IL-10 by Foxp3 T-reg cells.<sup>+</sup> and immunosuppressive function. As such, presented herein are agonist antibodies that bind to the 0X40 receptor, whereby the agonist modulates activation of the 0X40 receptor to block secretion of IL-10 cytokine and / or Tr1 and T-reg Foxp3 cells.<sup>+</sup> in total immunosuppressive function. Essentially, the antibodies can mimic the 0X40 ligand and activate the 0X40 receptor on Tr1 cells and / or natural regulatory T cells ("nTregs"), also referred to as "T-regs. Foxp3<sup>+</sup>”
As shown in co-pending United States Patent Applications Series Nos. 11 / 659,266 and 12 / 861,135, OX40L inhibits the generation and function of Tr1 cells that produce IL-10 from naive and memory CD4 + T cells. that were induced by the immunosuppressive drugs dexamethasone and vitamin D3. OX40L was found to inhibit the generation and function of regulatory T cells that produce IL-10. These findings demonstrate that 0X40 signaling by OX40L supjrr ^ l ^
MEXICAN INSTITUTE OF PROPERTY
Immunosuppressive human T cells that produce deleterious IL-10.
<img file="MX339964B_D0005.tif" />
OX40L's unique function is not shared by other dit niitilllbfóS of the costimulatory TNF family, ligand GITR and ligand 4-1 BB. OX40L strongly also inhibits the generation and function of IL-10 producing Tr1 cells induced by two physiological stimuli provided by immature DCs and inducible co-stimulant ligand. 0X40 receptor signaling in human T cells by monoclonal antibodies, small molecules, or by OX40L, or a protein that has at least 90 percent homology to them, modulates and regulates the generation and function of immunosuppressive T cells that produce IL- 10.
The discovery lends itself to numerous treatment applications. For example, agonistic, small molecule, or OX40L antibodies could be used to suppress the generation and function of immunosuppressive T cells that produce IL-10 and therefore could be used to enhance the immune response to treat cancer and infectious diseases, or as a adjuvant for cancer vaccines. Antagonistic antibodies to 0X40 or OX40L, or small antagonist molecules, could be used to improve the generation and function of immunosuppressive T cells that produce IL-10 and therefore could be used for the development of therapies for autoimmune diseases and graft diseases. versus host. Our discovery also provides high-throughput methods for the selection of antibodies or
IMPI
<img file="MX339964B_D0006.tif" />
small molecules activating either the receptor blocking 0X40 signaling in T cells for the development of cancer therapies, or alternatively autoimmune diseases and graft versus host diseases.
Monoclonal and human antibodies (sometimes referred to herein as an "anti-OX40 antibody" and / or other variations thereof) that bind the human 0X40 receptor are provided herein. These antibodies are useful in the treatment or prevention of acute or chronic diseases or conditions whose pathology involves 0X40.
In one aspect, an isolated human antibody, or an antigen-binding portion thereof, is disclosed that binds to human 0X40 and is effective as a treatment for cancer or treatment against an autoimmune disease. Any of the anti-OX40 antibodies described herein can be used as a medicine. One or more of the anti 15 0X40 antibodies can be used to treat one or more of a variety of cancers or autoimmune diseases described herein.
Isolated humanized antibodies that bind to 0X40 are provided herein. Isolated antibodies as described herein bind to 0X40, and may bind to encoded 0X40 of the following genes; NCBI Access Number NP_003317, Access Number of
Genpept P23510, or genes that have 90 percent homology to these.
The isolated antibody provided herein may additionally bind to the 0X40 receptor having one of the following
GenBank access:
<img file="MX339964B_D0007.tif" />
Like _________ ______, . ,, isolated which binds to 0X40 comprising: (a) a CDR1 heavy chain variable region comprising the amino acid sequence of SEQ
ID NO: 1; (b) a CDR2 heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; (c) a CDR3 heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 3; (d) a CDR1 light chain variable region comprising the amino acid sequence of SEQ ID NO. 7; (e) a CDR2 light chain variable region comprising the amino acid sequence of SEQ ID No. 8; and (f) a CDR3 light chain variable region comprising the amino acid sequence of SEQ ID NO. 9.
Furthermore, another example is an isolated antibody which binds to
0X40 comprising: (a) a CDR1 heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR2 heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14; (c) a CDR3 heavy chain variable region comprising the amino acid sequence of SEQ ID NO. fifteen; (d) a CDR1 light chain variable region comprising the amino acid sequence of SEQ ID NO. 19; (e) a variable light chain region
CDR2 comprising the amino acid sequence of SEQ ID NO. twenty; and (f) a CDR3 light chain variable region comprising the amino acid sequence of SEQ ID NO. twenty-one.
<img file="MX339964B_D0008.tif" />
Alternatively, an isolated antibody can ^^^^^ roaSaK ^ í ^
FROM THE INDUSTRIAL PROPERTY heavy chain variable CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 13; a CDR2 heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 or 14;
and / or a CDR3 heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 or 15, or a CDR heavy chain variable region that has 90 percent homology to it.
Furthermore, an isolated antibody may have a CDR1 light chain variable region comprising the amino acid sequence of SEQ
ID NO: 7 or 19; a CDR2 light chain variable region comprising the amino acid sequence of SEQ ID NO: 8 or 20 and / or a CDR3 light chain variable region comprising the amino acid sequence of SEQ ID NO: 9 or 21, or a variable region heavy chain that has 90 percent homology to it.
The isolated antibody may have a light chain variable region ("VL") comprising the amino acid sequence of SEQ ID NO: 10, 11, 22, or 23, or an amino acid sequence with at least 90 percent identity to the amino acid sequences of SEQ ID NO: 10, 11, 22 or 23. The isolated antibody may have a heavy chain variable region ("VH") comprising the amino acid sequence of SEQ ID NO: 4, 5, 16 and
17, or an amino acid sequence with at least 90 percent identity to the amino acid sequences of SEQ ID NO: 4, 5, 16, and 17. As such, as an example, the isolated antibody may comprise a sequence
<img file="MX339964B_D0009.tif" />
OF INDUSTRIAL PROPERTY heavy variable SEQ ID NO: 5 and a light sequence eg
NO: 11, or a sequence that has 90 percent homology to it. Similarly, the isolated antibody can have a variable heavy sequence of SEQ ID NO: 17 and a variable light sequence of SEQ ID NO: 23 or a sequence that has 90 percent homology to it.
The isolated antibody may have a variable light chain encoded by the nucleic acid sequence of SEQ ID NO: 12, or 24, or a nucleic acid sequence with at least 90 percent identity to the nucleotide sequences of SEQ ID NO: 12 or 24. The isolated antibody may have a variable heavy chain encoded by a nucleic acid sequence of SEQ ID NO: 6 or 18, or a nucleic acid sequence with at least 90 percent identity to the nucleotide sequences of SEQ
ID NO: 6 or 18.
Monoclonal antibodies are also provided herein. Monoclonal antibodies can have a variable light chain comprising the amino acid sequence of SEQ ID NO: 10 or 22, or an amino acid sequence with at least 90 percent identity to the amino acid sequences of SEQ ID NO: 10 or 22 . In addition, monoclonal antibodies are provided that have a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 4 or 16, or an amino acid sequence with at least 90 percent identity to the amino acid sequences of SEQ ID NO: 4 or 16.
An isolated nucleic acid is also provided herein.
IMPI encoding any of the anti-OX40 antibodies present. In addition, host cells are provided herein, each comprising nucleic acid encoding any of the anti0X40 antibodies described herein. Additionally, methods are provided for producing an antibody (such as the host cell comprising nucleic acid encoding any of the anti-OX40 antibodies described herein) comprising culturing the host cell such that the antibody is produced, and / or retrieve the antibody from the host cell.
BRIEF DESCRIPTION OF THE FIGURES
In order that the manner in which the above-cited features, aspects, and advantages of the invention, as well as others that will become apparent, be achieved and understood in detail, the more particular description of the invention briefly outlined above can be take for reference the modalities of the same that are illustrated in the figures that form a part of this specification. It will be noted, however, that the attached figures illustrate some embodiments of the invention and, therefore, will not be considered as limiting the scope of the invention, so that the invention can admit other equally effective embodiments.
FIG. 1 shows that human follicular lymphoma (FL) tissues infiltrated with Tregs FOXP3 * and co-localized with tumor B cells and monocytes. Left: Tregs FOXP3 double immunostaining<sup>+</sup> (red) and cells
<img file="MX339964B_D0010.tif" />
CD20 B lymphoma<sup>+</sup> (green);
IMP i
OF INDUSTRIAL PROPERTY
<img file="MX339964B_D0011.tif" />
Right:
Monocyte / macrophage / DC CD11c tregs<sup>+</sup> (green).
FIGS. 2A and 2B show increased numbers of Tregs CD4 + FOXP3 * in FL patients. Tumor cells and PBMCs were obtained from six FL patients at initial diagnosis prior to therapy. PBMCs were also obtained from six normal donors for comparison. The percentages of regulatory T cells over CD4 T cells<sup>+</sup> Totals were determined by CD4 Tregs flow cytometric analysis<sup>+</sup>CD25<sup>+</sup>CD127<sup>low</sup>FOXP3<sup>+</sup>. FIG. 2A shows representative FACS analysis of Tregs. FL PBMC and FL tumor cells were divided from the same patient. FIG. 2B shows the Tregs percentage of all donors. The horizontal bar indicates means.
FIG. 3 shows the isolation of Tregs ICOS<sup>+</sup>FOXP3 * or ICOS “FOXP3<sup>+</sup> from FL. The single cell suspension was obtained from a spleen specimen before any treatment. The cells were thawed on the day of the assay. CD4 T cells<sup>+</sup>CD8<sup>-</sup>CD14<sup>_</sup>CD16 ~ CD56<sup>_ </sup>CDUc'TCRyó<sup>-</sup> were divided into CD25 subsets<sup>low</sup> and CD25<sup>high</sup>. The Tregs CD4<sup>+</sup>CD25<sup>high</sup>FOXP3<sup>+</sup> additionally they were classified into ICOS subsets<sup>high </sup>and ICOS<sup>baia</sup> based on the superficial expression of ICOS. FOXP3 intracellular expression was determined in all subsets.
FIG. 4 shows intratumoral tregs that inhibit the proliferation of CD4 T cells.<sup>+</sup>CD25<sup>-</sup> infiltrators in FL and inhibition could be partially blocked by neutralizing anti-IL-10 antibodies. The
<img file="MX339964B_D0012.tif" />
CD4 + CD25 'tumor infiltrating T cells labeled with recombinant CD40L preactivated autologous tumor cells in the presence or absence of Tregs ICOS<sup>+</sup>FOXP3<sup>+</sup> or Tregs ICOS FOXP3 +, or antilIL-10 (10 pg / ml). After 72 hours of culture, CD4 cell proliferation<sup>+</sup>CD25<sup>_</sup> was determined by dilution flow cytometric analysis of
CFSE.
FIG. 5A shows intracellular analysis of cytokine production by CD4 T cells.<sup>+</sup> naive as determined by flow cytometry in accordance with an embodiment of a method of the present invention.
FIG. 5B shows the production of cytokine by CD4 T cells<sup>+</sup> naive as determined by ELISA according to an embodiment of a method of the present invention.
FIG. 5C shows the suppressive function by Tr1 cells producing IL-10 as determined by the incorporation of [<sup>3</sup>H] thymidine according to an embodiment of a method of the present invention.
FIG. 6A shows intracellular analysis of cytokine production by CD4 T cells.<sup>+</sup> memory as determined by flow cytometry in accordance with an embodiment of a method of the present invention.
FIG. 6B shows the production of IL-10 by CD4 T cells<sup>+</sup> memory as determined by ELISA according to an embodiment of a method of the present invention.
FIG. 7A shows intracellular analysis of cytokine production by CD4 T cells.<sup>+</sup> naive as determined by flow cytometry according to an embodiment of a method of the present ii
Mexican Institute of Industrial Property
<img file="MX339964B_D0013.tif" />
FIG. 7B shows the production of IL-10 by CD4 T cells<sup>+ </sup>na'ive as determined by ELISA according to an embodiment of a method of the present invention.
FIG. 7C shows the number of viable T cells counted according to an embodiment of a method of the present invention.
FIG. 8A shows intracellular analysis of cytokine production by CD4 T cells.<sup>+</sup> na'ive as determined by flow cytometry in accordance with an embodiment of a method of the present invention.
FIG. 8B shows the production of IL-10 by CD4 T cells<sup>+ </sup>na'ive as determined by ELISA according to an embodiment of a method of the present invention.
FIG. 8C shows intracellular analysis of cytokine production by CD4 T cells.<sup>+</sup> memory as determined by flow cytometry according to a mode of a method of the present invention.
FIG. 8D shows the production of IL-10 by CD4 T cells<sup>+</sup> memory as determined by ELISA according to an embodiment of a method of the present invention.
FIG. 8E shows intracellular analysis of cytokine production by CD4 T cells.<sup>+</sup> na'ive as determined by flow cytometry in accordance with an embodiment of a method of the present invention.
FIG. 8F shows the production of IL-10 by CD4 T cells<sup>+ </sup>na'ive as determined by ELISA according to a modality of a
IMPI »5 method of the present invention. '<sup>Ν8Τ</sup>ΪεΪ?<sub>ρ</sub>Το<sup>χ</sup>Α% αο
INDUSTRIAL
FIG. 9 shows the production of IL-10 by regulatory T cells as determined by ELISA according to an embodiment of a method of the present invention.
FIG. 10 shows the results of the selection of anti-human 0X40 hybridoma supernatants against L-OX40 versus parenteral L cells as determined by ELISA.
FIG. 11 shows the selection of human 0X40 specific monoclonal antibodies as determined by flow cytometric analysis according to an embodiment of a method of the present invention.
FIG. 12 shows confirmation of the specificity of anti-hOX40 monoclonal antibodies using SUPM2 cells expressing 0X40 (SUPM2-OX40) according to an embodiment of a method of the present invention.
FIGS. 13A and 13B show 0X40-specific monoclonal antibodies that can inhibit the generation of IL-10 (Tr1) producing cells from CD4 T cells.<sup>+</sup> stimulated by vit D3 (0.1 pM) / Dex (50 nm), CD32L / ICOSL and anti-CD3 / CD28 (0.2 pg / ml) according to an embodiment of a method of the present invention. The data of the
Classification of Fluorescence Activated Cells (FACS) are shown in FIG. 13A and the percentages of IL-10 producing cells for all 0X40 monoclonal antibody treatments are shown in FIG. 13B.
FIG. 14 shows the results that the antibodies
<img file="MX339964B_D0014.tif" />
monoclonal specific of hOX40 that inhibit the geherasfefbdQHGí • of THE PROPERTY
INDUSTRIAL also stimulate the proliferation of CD4 T cells<sup>+</sup> according to an embodiment of a method of the present invention.
FIGS. 15A and 15B detail the titration of 0X40 monoclonal antibodies for their ability to Inhibit the generation of Tr1 cells from CD4 + T cells according to an embodiment of a method of the present Invention. Representative FACS data is shown in FIG. 15A and the percentage of Tr1 cells after treatment with nine 0X40 monoclonal antibodies are shown in FIG. 15B.
FIGS. 16A-16C show that 0X40-specific monoclonal antibodies that Inhibit the generation of Tr1 cells that produce IL-10 from CD4 T cells<sup>+</sup> They also inhibit the production of IL-10 from Treg ICOS.<sup>+</sup>CD4<sup>+</sup>CD25<sup>high</sup>CD127 'and immunosuppressive function. The ICOS Tregs<sup>+</sup>CD4<sup>+</sup>CD25<sup>high</sup>Recently classified CD127 '(ICOS * Tregs) were stimulated with anti-CD3 (02 pg / ml) in the presence of CD32L / ICOSL cells and CD32L / OX40L cells (FIG. 16A) or 0X40 monoclonal antibodies or control antibody ( FIG. 16B) for five days. The cells were then re-stimulated with anti-CD3 / CD28 for 24 hours and the supernatants were assayed by enzyme linked immunosorbent assay (ELISA). FIG. 16C is a monocyte-based proliferation assay showing that two of the antibodies block ICOS function<sup>+</sup>Treg.
FIGS. 17A and 17B show the Identification of monoclonal antibodies anti-hOX40 that inhibit the generation
MEXICAN INSTITUTE
PROPERTY BLOCKS Treg FOXP3 Function<sup>+</sup>CD4<sup>+</sup>CD25<sup>aita</sup> from acu§fcÍb<sup>TO THE</sup> With another embodiment of a method of the present invention. Representative flow cytometric analyzes are shown in FIG. 17A. The data for six monoclonal antibodies are shown in FIG. 17B.
FIG. 18 demonstrates the identification of anti-hOX40 monoclonal antibodies that do not inhibit Tr1 cell generation but block Treg FOXP3 function.<sup>+</sup>CD4<sup>+</sup>CD25<sup>high</sup> according to an embodiment of a method of the present invention.
FIGS. 19A and 19B show antihOX40 agonist antibodies that block CD4 Treg function.<sup>+</sup>CD25<sup>high</sup> derived from lymphoma according to an embodiment of a method of the present invention. Representative FACS analyzes are shown in FIG. 19A and the data for all experiments are shown in FIG. 19B.
FIG. 20 shows that antihOX40 monoclonal antibodies can bind CD4 T cells<sup>+</sup> of rhesus monkey. As shown, six of the anti-hOX40 mAbs can bind CD4 T cells<sup>+</sup> activated rhesus monkey and will link 0X40 rhesus and activate signaling
0X40.
FIG. 21 shows that each of the Hu106-222 antibodies
Lot I and II of Example I is comprised of a heavy chain with a molecular weight of approximately 50 kD and a light chain with a molecular weight of approximately 25 kD. Purity of Hu10617 Antibodies
222 Lot I and II appear to be over 95%. i- IMPIAS * MEXICAN INSTITUTE
PROPERTY> ¿*** ^ 9 ^
FIG. 22 shows the analysis of cFrTÜoy antibodies
Mouse Hu106-222 (Lot II) for binding to L7OX40 cells (Example l and *
FIG. 23 represents the schematic structure of the expression vector for the Hu106 IgGVkappa antibody (Expression vector). Proceeding clockwise from the Salí site at the top, the plasmid contains the heavy chain transcription unit starting with the primary immediate early promoter of human cytomegalovirus (CMV) and enhancer (CMV promoter) to start transcription of the antibody heavy chain gene. The CMV promoter is followed by the VH exon, a genomic sequence containing the human gamma-1 heavy chain constant region that includes the CH1, hinge, CH2 and CH3 exons with the introns involved, and the polyadenylation site which follows exon CH3. Following the heavy chain gene sequence, the light chain transcription unit begins with the CMV promoter, followed by the VL exon and a genomic sequence containing the human kappa chain constant region (CL) exon with part of the intron that precedes it, and the polyadenylation site that follows the exon CL. The light chain gene is then followed by the SV40 early promoter (SV40 promoter), the xanthine guanine phosphoribosyl transferase gene from E.
coli (gpt), and a segment containing the SV40 polyadenylation site (site!
poly (A) SV40). Finally, the plasmid contains a part of the plasmid pUC19, which comprises the bacterial origin of replication (pUC ori) and the beta-lactamase gene (beta lactamase). The locations of
<img file="MX339964B_D0015.tif" />
restriction enzyme are shown in the figure.
FIG. 24 shows the comparison between Hu 106-222 Lot I and II antibodies for binding to L / OX40 cells (Example I below).
FIG. 25 shows that Hu1 19-122 is comprised of a heavy chain with a molecular weight of approximately 50 kD and a light chain with a molecular weight of approximately 25 kD. The purity of Hu119 appears to be more than 95% (Example II below).
FIG. 26 shows the result of FACS analysis of Ch119-122 and Hu119-122 antibodies described herein (Example II below).
FIG. 27 shows that the humanized anti-human 0X40 mAb clone 119-122 (Hu119), and its mutated FcR-binding antibody (Hu119-AA) enhanced CD4 T-cell proliferation.<sup>+</sup>. Hu119-122 produced better T-cell stimulating activity compared to the parent mouse anti-human 0X40 mAb (Mouse 119-122). However, the chimeric anti-human 0X40 mAb (mouse Ch119, VH, and VL but human gamma-1 and kappa constant regions) did not improve cell proliferation.
T.
FIG. 28 shows that the mutated humanized anti-human 0X40 mAb clone binding to FcR 106-222 (Hu222AA) and chimeric anti-human 0X40 mAb clone 106-222 (Ch222) enhanced CD4 T cell proliferation.<sup>+</sup> naive stimulated by anti-CD3. These antibodies have
MEXICAN INSTITUTE OF PROPERTY el Ab'W<sup>r</sup>B4
<img file="MX339964B_D0016.tif" />
Similar stimulatory activity compared to human parental mouse (Mouse 222). However, fully humanized human, Hu222, does not improve T-cell growth compared to human IgG1.
FIGS. 29A and 29B show that the mouse humanized and anti-human 0X40 mAb clone 119-122 blocks the suppressive function of Treg CD4<sup>+</sup>.
FIGS. 30A-30C provide data showing that anti-human 0X40 antibodies enhance CD4 T cell proliferation.<sup>+</sup> and CD8<sup>+</sup> using antibodies attached to the plate.
FIGS. 31A and 31B show that humanized and mouse anti-human 0X40 antibodies require crosslinking to enhance T cell proliferation.
FIGS. 32A-32C show that anti-human 0X40 antibodies block Tregs CD4 activity<sup>+</sup>FOXP3<sup>+</sup>n using plaque binding antibodies.
FIGS. 33A- 33C show that a high concentration of mouse anti-human 0X40 antibodies preferentially kill Tregs
FOXP3 *.
FIGS. 34A and 34B show that mouse anti-human 0X40 mAbs act directly on either effector T cells or nTregs to block Tregs suppressive function.
FIGS. 35A-35C show the results of tumor treatment of anti-hOX40 mAb in transferred mice
<img file="MX339964B_D0017.tif" />
INDUSTRIAL PROPERTY T cells hOX40<sup>+</sup>CD8<sup>+</sup>. The anti-human 0X40 mAb promotes T cell expansion and survival in vivo. The therapeutic vaccination regimen is shown in FIG. 35A. Representative images of bioluminescence in vivo are shown in FIG. 35B. The results of the antibody tumor treatment are shown in FIG. 35C.
FIG. 36 shows the alignment of the amino acid sequences of humanized 106-222, 106-222 (Hu106), and the VH sequences of human acceptor X61012 are shown (accession number of
GenBank). Amino acid residues are shown in single letter code.
The numbers above the sequences indicate the locations according to
Kabat et al. (Sequences of Proteins of Immunological Interests, Fifth edition, NIH Publication No. 91-3242, US Department of Health and Human Services, 1991). The same sequences as claimed herein are also provided in the Sequence Listing and the position numbers may be different. In Figure 36, the CDR sequences defined by Kabat et al. (1991) are underlined in VH 106-222. X61012 VH CDR residues are omitted in the figure. Human VH sequences homologous to VH scaffolds from 106-222 were searched within the database of
GenBank, and the VH sequence encoded by the human X61012 cDNA (X61012 VH) was chosen as an acceptor for humanization. The 106-222 VH CDR sequences were first transferred to the corresponding X61012 VH positions. Then in
<img file="MX339964B_D0018.tif" />
ta & Aífe<sup>1</sup>
OF INDUSTRIAL PROPERTY
<img file="MX339964B_D0019.tif" />
frame positions where the variable three-dimensional model of 106-222 suggests significant contact with the CDRs, the amino acid residues of mouse 106-222 VH were replaced by the corresponding human residues. These substitutions were made at positions
46 and 94 (underlined in HV of Hu106). Furthermore, a human scaffold residue that was found to be atypical in the corresponding V region subgroup was replaced with the more typical residue to reduce potential immunogenicity. This substitution was made at position 105 (double underlined in Hu106 VH).
FIG. 37 shows the alignment of the amino acid sequences of humanized 106-222, 106-222 (Hu106), and the VL sequences of human accepting AJ388641 are shown (GenBank accession number). Amino acid residues are shown in single letter code.
The numbers above the sequences indicate the locations according to
Kabat et al. (1991). The same sequences as claimed herein are also provided in the Sequence Listing although the position numbers may be different. The CDR sequences defined by Kabat et al. (1) are underlined in VH of 106-222. The CDR residues in VL of AJ388641 are omitted in the figure. Human VL sequences homologous to the 106-222 VL scaffolds were searched within the GenBank database, and the VL sequence encoded by the human AJ388641 cDNA (VL from AJ388641) was chosen as an acceptor for humanization . The CDR sequences of VL from 106-222 were transferred to the corresponding VL positions of AJ388641.
shell substitutions in the humanized form.
<img file="MX339964B_D0020.tif" />
INSTÍTUTOWtfCA '
OF INDUSTRIAL PROPERTY
<img file="MX339964B_D0021.tif" />
FIG. 38 shows the nucleotide sequence of the Hu106 VH gene flanked by the Spel and Hindlll sites (underlined) shown along with the deduced amino acid sequence. Amino acid residues are shown in single letter code. The signal peptide sequence is in italics. The N-terminal (Q) amino acid residue of mature VH is double underlined. CDR sequences according to the definition of Kabat et al. (1991) are underlined. The same sequences as claimed herein are also provided in the Sequence Listing and the position numbers may be different in the Sequence Listing. The intron sequence is in italics. The Spel and Hindlll digested Hu106 VH gene fragment was cloned between the corresponding sites in the Expression Vector shown in Figure 23.
FIG. 39 shows the nucleotide sequence of the VL gene from
Hu 106-222 flanked by Nhel and EcoRI sites (underlined) shown along with the deduced amino acid sequence. Amino acid residues are shown in single letter code. The signal peptide sequence is in italics. The N-terminal (D) amino acid residue of the mature VL is double underlined. CDR sequences according to the definition of Kabat et al. (1991) are underlined. The intron sequence is in italics. The Nhel and EcoRI digested Hu106 VL gene fragment was cloned between the corresponding sites in the Expression Vector shown in FIG. 2. 3.
The same sequences as claimed in the
<img file="MX339964B_D0022.tif" />
OF INDUSTRIAL PROPERTY provided in the Sequence Listing although position numbers may be different in the Sequence Listing.
FIG. 40 shows the alignment of the amino acid sequences of humanized 119-122, 119-122 (Hu119), and the VH sequences of human acceptor Z14189 are shown (GenBank accession number). Amino acid residues are shown in single letter code.
The numbers above the sequences indicate the locations according to
Kabat et al. (Sequences of Proteins of Immunological Interests, Fifth edition,
NIH Publication No. 91-3242, US Department of Health and Human Services, 1991). The CDR sequences defined by Kabat et al. (1991) are underlined in VH 119-122. The CDR residues in VH of Z14189 are omitted in the figure. Human VH sequences homologous to 119-122 VH scaffolds were searched in the GenBank database, and the VH sequence encoded by the human Z14189 cDNA (Z14189 VH) was chosen as an acceptor for humanization. The 119-122 VH CDR sequences were first transferred to the corresponding Z14189 VH positions. Then, at the framework positions where the three-dimensional model of the 119-122 variable regions suggested significant contact with the CDRs, the mouse 119-122 VH amino acid residues were replaced by the corresponding human residues. These substitutions were made at positions 26, 27,
28, 30 and 47 (underlined in the Hu119 VH sequence) as shown
<img file="MX339964B_D0023.tif" />
ΙΐΜΡΙ in the figure. The same sequences as reivftidi ^ g ^ rtol & A & r
INDUSTRIAL • <sup>4</sup> “Are also provided in the Sequence Listing although the position numbers may be different in the Sequence Listing.
FIG. 41 shows the alignment of the amino acid sequences of humanized 119-122, 119-122 (Hu119), and the VL sequences of human acceptor M29469 are shown (GenBank Accession Number). Amino acid residues are shown in single letter code.
The numbers above the sequences indicate the locations according to
Kabat et al. (1991). The CDR sequences defined by Kabat et al. (1) 10 are underlined in VL 119-122. M29469 VL CDR residues are omitted from the sequence. Human VL sequences homologous to 119-122 VL scaffolds were searched within the database of
GenBank, and the VL sequence encoded by the human M29469 cDNA (M29469 VL) was chosen as an acceptor for humanization. The 119-122 VL CDR sequences were transferred to the corresponding VL positions in M29469. Frame substitutions were not necessary in the humanized form. The same sequences as claimed herein are also provided in the Sequence Listing although the position numbers may be different in the
Sequence Listing.
FIG. 42 shows the nucleotide sequence of the Hu119 VH gene flanked by the Spel and Hindlll sites (underlined) shown along with the deduced amino acid sequence. The amino acid residues are • xcde ».!.
IMPI show in single letter code. The sequence of
INDUSTRIAL italic. The N-terminal (E) amino acid residue of mature VH is double underlined. CDR sequences according to the definition of Kabat et al. (1991) are underlined. The intron sequence is in italics. The Spel and Hindlll digested Hu119 VH gene fragment was cloned between the corresponding sites in the Expression Vector shown in FIG. 2. 3. The same sequences as claimed herein are also provided in the Sequence Listing although the position numbers may be different in the Sequence Listing.
FIG. 43 shows the nucleotide sequence of the VL gene from
Hu119 flanked by the Nhel and EcoRI sites (underlined) shown along with the deduced amino acid sequence. Amino acid residues are shown in single letter code. The signal peptide sequence is in italics. The N-terminal (E) amino acid residue of the mature VL is double underlined. CDR sequences according to the definition of Kabat et al. (1991) are underlined. The intron sequence is in italics. The Nhel and EcoRI digested Hu119 VL gene fragment was cloned between the corresponding sites in the Expression Vector shown in FIG. 23. The same sequences as claimed herein are also provided in the Sequence Listing although the position numbers may be different in the Sequence Listing.
<img file="MX339964B_D0024.tif" />
DETAILED DESCRIPTION OF THE INVEN
TMPI MEXICAN talOurO
--ΠΕΤΑ PROPERTY
INDUSTRIAL
<img file="MX339964B_D0025.tif" />
The term "antibody" includes an immunoglobulin molecule comprised of four polypeptide chains, two heavy (H) chains, and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2, and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into hypervariability regions, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amlno-termml to carboxl-term in the following order: FR1, CDR1, FR2,
CDR2, FR3, CDR3, FR4.
The term "antigen binding portion" of an antibody (or "antibody portion") includes fragments of an antibody that retain the ability to specifically bind an antigen (eg, hOX40). It has been shown that the antigen binding function of an antibody can be performed by fragments of a full length antibody. The examples
<img file="MX339964B_D0026.tif" />
Binding fragments encompassed within the term "antigen" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (I) a fragment of F (ab ') 2, a bivalent fragment comprising two fragments
Fabs linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546), consisting of a domain VH; and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked, using recombinant methods, by a synthetic linker that allows them to be made as a single protein chain in which the VL and VH pair to form monovalent molecules (known as single chain Fv (scFv); see eg Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Nati. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies, are also covered. Diabodies are bivalent, biospecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, forcing the domains to mate with domains
IMPIf
IMtrriTi ιτ / -ι »« '
MEXICAN INSTITUTE
<img file="MX339964B_D0027.tif" />
complementary to another chain and creating two sites of enlS¿éí ^ Sf ^ £ itíg <aiT · (see for example, Holliger, P., et al. (1993) Proc. Nati. Acrcd finí VISA flGífiririri „
6448; Poljak, RJ, et al. (1994) Structure 2: 1121-1123). Still further, an antibody or antigen binding portion thereof may be part of large immunoadhesion molecules, formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin core region to produce a tetrameric scFv molecule (Kipriyanov, S. M., et al. (nineteen ninety five)
Human Antibodies and Hybridomas 6: 93-101) and the use of a cysteine residue, a marker peptide, and a C-terminal polyhistidine label to produce biotinylated and bivalent scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31: 1047-1058). Antibody portions, such as Fab and F (ab ') 2 fragments, can be prepared from whole antibodies using standard techniques, such as digestion of papain or pepsin, respectively, of full length antibodies. Furthermore, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein. Preferred antigen binding portions are complete domains or complete domain pairs.
OX40 / OX40-ligand (OX40 Receptor) / (OX40L) are a pair of costimulatory molecules critical for T-cell proliferation, survival, cytokine production, and memory cell generation.
IMPI
<img file="MX339964B_D0028.tif" />
Early in vitro experiments showed that the ^ ws ^^^^ g of 0X40 in CD4 T cells<sup>+</sup> led to TH2, but no development rir »TH1 These results were supported by in vivo studies showing that blocking of OX40 / OX40L interaction prevented the induction and maintenance of TH2-mediated allergic immune responses. However, blocking the OX40 / OX40L interaction alleviates or prevents TH1-mediated disease. Furthermore, administration of soluble OX40L or OX40L gene transfer in tumors was shown to strongly enhance anti-tumor immunity in mice. Recent studies also suggest that 10 OX40 / OX40L may play a role in promoting CD8 T-cell mediated immune responses. As discussed herein, 0X40 signaling blocks the inhibitory function of naturally occurring regulatory CD4 T cells.<sup>+</sup>CD25<sup>+</sup> and the OX40 / OX40L pair plays a critical role in the overall regulation of peripheral immunity versus tolerance. 15 The terms "Kabat numbering", "Kabat definitions" and "Kabat labeling" are used interchangeably herein. These terms, which are recognized in the art, refer to an amino acid residue numbering system which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody. , or an antigen-binding fragment thereof (Kabat et al. (1971) Ann. NY Acad. Sci. 190: 382-391 and, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication
No. 91-3242).
IMPI
MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY
<img file="MX339964B_D0029.tif" />
The phrase "recombinant human antibody includes human antibodies that are prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, recombinant human antibody library, antibodies isolated from an animal (eg, a mouse) that is transgenic for human immunoglobulin genes (see eg, Taylor, LD, et al. (1992) Nucí. Acids Res. 20: 6287-6295) or antibodies prepared, expressed, created or isolated by any other means involving the splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242).
An "isolated antibody" includes an antibody that is substantially free of other antibodies that have different antigenic specificities (eg, an isolated antibody that specifically binds hOX40 is substantially free of antibodies that specifically bind antigens other than hOX40). An isolated antibody that specifically binds hOX40 can bind 0X40 molecules from other species. Furthermore, an isolated antibody may be
<img file="MX339964B_D0030.tif" />
OF INDUSTRIAL PROPERTY substantially free of other cellular material and / or produt
The term "activity" includes activities such as the binding specificity / affinity of an antibody for an antigen, eg, an anti-human 0X40 antibody that binds a 0X40 antigen and / or the activation potency of an antibody, eg, an anti0X40 antibody whose binding to the hOX40 receptor activates the biological activity of hOX40 or activates receptor binding in a human L / OX40 cell assay.
The term "Κ<sub>ο</sub>^ ”, As used herein, is proposed to refer to the dissociation constant for dissociation of an antibody from the antibody / antigen complex. The term "Κ <Λ as used herein, is intended to refer to the dissociation constant of a particular antibody-antigen interaction.
The phrase "surface plasmon resonance" includes an optical phenomenon that allows the analysis of biospecific interactions in real time by detecting alterations in protein concentrations within a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For additional descriptions, see Example 5 and Jonsson, U., et al. (1993) Ann. Biol. Clin. 51: 19-26; Jonsson, U., et al. (1991) Biotechniques 11: 620-627;
Johnsson, B., et al. (1995) J. Mol. Recognit. 8: 125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198: 268-277.
The term "vector" includes a nucleic acid molecule capable of transporting another nucleic acid to which it has been bound. One type of vector is
IMPI a "plasmid", which refers to a DNA loop de'doeteT <^ 0e ^ a), p * INDUSTRIAL
<img file="MX339964B_D0031.tif" />
in which the additional DNA segments can be ligated. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thus replicate along with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors). In general, expression vectors useful in recombinant DNA techniques are frequently in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably since the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors (eg, replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.
The phrase "recombinant host cell" (or simply "host cell") includes a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended • IMPI
<img file="MX339964B_D0032.tif" />
refer not only to the particular object cell, but to lat |
Because certain modifications can occur in generations. to come due to either mutation or environmental influences, such progeny may not, in fact, be identical to the cell of origin, but is still included within the scope of the term "host cell" as used herein.
The term "monoclonal antibody" (monoclonal antibody) refers to an antibody, or population of similar antibodies, obtained from a population of substantially homogeneous antibodies, and will not be construed as requiring the production of the antibody by any particular method, including but not limited to, monoclonal antibodies that can be made by the hybridoma method first described by Kohler and Milstein (Nature, 256: 495-497, 1975), or by recombinant DNA methods.
The term "chimeric antibody" (or "chimeric immunoglobulin") refers to a molecule that comprises a heavy and / or light chain which is identical with or homologous to the corresponding sequences in antibodies derived from or belonging to a particular species. a particular class or subclass of antibody, while the rest of the chain (s) is identical or homologous to the corresponding sequences in the antibodies derived from another species or that belong to another class or subclass of antibody, as well as fragments of such antibodies, as long as they exhibit the activity Biological Desired (Cabilly et al (1984) ,fra; Morrison et al., Proc. Nati. Acad. Sci. USA 81: 6851).
. IMPI @ '* INSTITUTO Mexicano fií' * '
The term "humanized antibody" refers to antibodies containing sequences of »· ηο -human - antibodies (eg, murine) as well as human antibodies. A humanized antibody can include conservative amino acid substitutions or unnatural residues from the same or different species that do not significantly alter their biological and / or binding activity. Such antibodies are chimeric antibodies that contain the minimal sequence derived from non-human immunoglobulins. For the most part, humanized antibodies are human immunoglobulins (receptor antibody) in which the residues of a receptor complementary determining region (CDR) are replaced by the residues of a CDR from a non-human species (donor antibody) such like mouse, rat, camel, bovine, goat, or rabbit that has the desired properties. In addition, humanized antibodies can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize the yield of the antibody. Thus, in general, a humanized antibody will comprise all or substantially all of at least one, and in one aspect two,, variable domains, in which all or substantially all hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of a region
ΪΜΡΙ '$ 3 ^ 5%
INSTITUTO MEXICANO immunoglobulin constant (Fe), or that of an immuno ^ fbbñKSSifiunüBW (see, for example, Cabilly et al., US Patent, No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss et al., US Patent No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, MS et al., WO 86/01533; Neuberger, MS et al., European Patent No. 0,194,276 B1 ; Winter, US Patent No. 5,225,539; Winter, European Patent No. 0.239,400 B1; Padlan, EA et al., European Patent Application No. 0.519,596 A1; Queen et al. (1989) Proc. Nati. Acad. Sci.
USA, Vol 86: 10029-10033).
Each of the antibodies described and claimed herein may be referred to, in the singular or plural, as: "anti-OX40 antibody"; "Anti-hOX40 antibody"; "Anti-hOX40 monoclonal antibody"; "0X40 anti-human antibody"; "Anti-human mAb 0X40"; "Anti-hOX40 mAb"; "HOX40 specific monoclonal antibody"; "Anti-OX40L antibody"; "Anti15 hOX40L antibody"; "OX40L anti-human antibody"; "Human 0X40 specific antibody"; "Human 0X40 specific monoclonal antibody"; "Human 0X40 specific antibody"; "0X40 anti-human specific antibody"; "Anti-human 0X40 specific monoclonal antibody"; "H-OX40 specific antibody"; "H-OX40 specific monoclonal antibody";
"HOX40 agonist antibody"; "HOX40 agonist" and / or other similar variations thereof.
As described in the United States Patent Application
No. 11 / 659,266 entitled “Methods to Treat Disease States by Influencing the
Signaling of ΟΧ-40-Receptors and High Throughput
OF PROPERTY <2 * '
INDUSTRIAL ------ Identifying Substrates Thereof 'which is incorporated herein by reference, it was discovered that a function of OX40L is the down-regulation of Tr1 cell generation induced by immunosuppressive agents Dex and vit D3, ICOSL, or Immature DCs. This discovery demonstrates a general mechanism by which OX40L improves immunity and breaks immune tolerance.
With the use of immunohistological analysis (FIG. 1), intracellular staining (FIGS. 2A and 2B), and cell sorting (FIG. 3), it has been shown that both the Tregs that produce ICOS<sup>+</sup>IL-10 like Tregs that produce ICOS-TGF-β infiltrate human FL tissues. These FL-derived FOXP3 * Tregs can strongly inhibit the proliferation of FOXP3 tumor-infiltrating T cells<sup>-</sup>CD4 * CD25<sup>-</sup> in response to CD40 'ligand pre-activated autologous lymphoma cells (FIG. 4). Suppressive activity of
Tregs ICOS * could be partially blocked by a neutralizing anti-IL-10 antibody, confirming the role of Tregs that produce ICOS * IL-10 in FL (FIG. 4). In the FIGS experiment. 2A and 2B, tumor cells and PBMCS were obtained from 7 patients with an initial diagnosis before therapy. PMBCs were also obtained from 7 healthy donors for comparison. The percentages of regulatory T cells over T cells
Total CD4 * were determined by Tregs flow cytometric analysis
CD4 * CD25 * CD127<sup>baia</sup>FOXP3 *. FIG. 2A provides representative FACS analysis of Tregs, while FIG. 2B shows the percentage of
ΙΜΡΙ
<img file="MX339964B_D0033.tif" />
Tregs from all donors.
MEXICAN INSTITUTE
Dt THE PROPERTY _ ..., .... ....... INDUSTRIAL OX40L was also found to inhibit Tr1 cell generation from CD4 T cells<sup>+</sup> Dex and vit D3 induced. A combination of immunosuppressive drugs Dex and vit D3 is known to consistently induce differentiation of naive CD4 + T cells into Tr1 cells. To investigate whether OX40L can inhibit the generation and function of Tr1 cells, CD4 T cells<sup>+</sup> ships were cultured with monoclonal anti-CD3 plus anti-CD28 antibodies in the presence or absence of L cells transfected with OX40L under four different culture conditions Including: (1) Tr1 (Dex and vit D3); (2) TH1 (IL-12); (3) TH2 (IL-4); or (4) neutral (medium only) for 7 days (FIG. 5A). IL-10 production by primed T cells was analyzed by intracellular clonal cloning and ELISA.
In the FIG. 5A, an intracellular analysis of cytokine production by CD4 T cells<sup>+</sup> naíve was performed using flow cytometry. CD4 T cells<sup>+</sup> Naive were cultured with monoclonal anti-CD3 and anti-CD28 antibodies in the presence of IL-2 in parental L cells or OX40L-L cells with the indicated recombinant cytokines or reagents for 7 days. The percentages of the respective cytokine producing T cells are indicated in each spot blot profile. The results show that OX40L inhibits the generation of Tr1 cells from naive CD4 + T cells induced by different polarization signals. As shown in FIG. 5A, between 2% to 4% of Tr1 cells were generated from CD4 T cells<sup>+</sup> naíve cultivated in neutral conditions or TH1 or TH2.
IMPI
More than 15% of Tr1 cells were generated in culture ¿οη ^ §0 ^ & 5ΰ8 ·; INDUSTRIAL addition of OX40L completely blocked the generation of Tr1 cells. while promoting the generation of T cells that produce TNF-α in all culture conditions.
These data were confirmed by ELISA data (FIG. 5B).
In the FIG. 5B, the production of cytokine by CD4 cells<sup>+ </sup>Naive in supernatants after restimulation with anti-CD3 and anti-CD28 monoclonal antibodies for 24 hours was measured by ELISA. CD4 T cells<sup>+</sup> Naive were cultured with monoclonal anti-CD3 and anti-CD28 antibodies in the presence of IL-2 in parenteral L cells or cells
OX40L-L with the indicated recombinant or reactive cytokines for 7 days. Data are shown as mean ± standard error of the mean (SEM) from four independent experiments. The results show that OX40L inhibits the generation of Tr1 cells from CD4 T cells.<sup>+</sup> naive induced by different polarization signals.
CD4 T cells<sup>+</sup> naive primed with Tr1 condition (Dex plus vit D3) were anergic and had the ability to suppress CD4 T cell proliferation<sup>+</sup> naive in response to anti-CD3 plus anti-CD28 monoclonal antibodies (FIGS. 5A-5C). In the FIGS. 5A-5C, suppressive function in T cells was measured by incorporation of [<sup>3</sup>H] thymidine. The mixtures of the indicated T cell populations were stimulated again by the anti-CD3 and anti-CD28 monoclonal antibodies. The error bars represent the SEM of cavities by
<img file="MX339964B_D0034.tif" />
<img file="MX339964B_D0035.tif" />
triplicate. CD4 T cells were found<sup>+</sup> na'ive condition of Tr1 in the presence of OX40L proliferated vigorously and did not
OF INDUSTRIAL PROPERTY
<img file="MX339964B_D0036.tif" />
inhibited the proliferation of CD4 T cells<sup>+</sup> na'ive in response to anti-CD3 plus anti-CD28 monoclonal antibodies. The data suggests that
OX40L blocks the generation of functional Tr1 cells from CD4 T cells<sup>+</sup> na'ive induced by Dex and vit D3.
Tr1 cells found to be generated from CD4 T cells<sup>+</sup>CD45RA'CD45RO<sup>+</sup> memory, and that OX40L can inhibit the generation of Tr1 cells from CD4 T cells<sup>+</sup> of memory. The cells
T CD4<sup>+</sup>CD45RACD45RO<sup>+</sup> Memory cells were cultured for 7 days with monoclonal anti-CD3 plus anti-CD28 antibodies in the presence or absence of Tr1 condition from L cells transfected with OX40L (Dex plus vit D3). In the FIG. 6A, an intracellular analysis of cytokine production by CD4 T cells<sup>+</sup> Memory was carried out using flow cytometry. CD4 T cells<sup>+</sup>CD45RO<sup>+</sup>Memory CD25s were cultured with the anti-CD3, anti-CD28, and IL-2 monoclonal antibodies in parental L cells or OX40L-L cells in the presence or absence of Dex plus vit D3 for 7 days. The percentages of the respective cytokine producing T cells are indicated in each spot blot profile. The results show that OX40L inhibits the generation of memory CD4 * T cell Tr1 cells under a condition with Dex plus vit D3. FIG.
6A shows that large numbers of Tr1 cells (> 20%) were generated from CD4 T cells.<sup>+</sup> memory in culture with Dex plus vit D3. The adition of
IMPI
OX40L completely blocked the generation of celclaB '^ jí ^^ q ^ n
<img file="MX339964B_D0037.tif" />
INDUSTRIAL generation of cells that produce TNF-α from CD4 T cells<sup>+</sup> of memory.
The ability of Dex plus vit D3 to promote the production of 5 IL-10 from CD4 T cells<sup>+</sup> of memory, and that this capacity can be
Inhibited by OX40L, it was confirmed by ELISA analysis of IL-10 (FIG. 6B). In the FIG. 6B, IL-10 production by CD4 T cells<sup>+</sup> Memory was measured in supernatants after re-formulation with the monoclonal antibodies antl-CD3 and antl-CD28 for 24 h by ELISA. Data is shown as mean ± SEM from four Independent experiments. The results show that OX40L inhibits the generation of Tr1 cells from CD4 T cells.<sup>+</sup> memory under a condition with Dex plus vit D3.
Furthermore it was discovered that OX40L inhibits the generation of cells
Tr1, while other members of the TNF family (GITRL and 4-1BBL) do not. Within the TNF superfamllia, OX40L, glucocortlcolde-induced TNF receptor ligand (GITRL), and 4-1 BB ligand (4-1 BBL) have co-formulating function for T cells. To investigate whether OX40L was the only one in the Inhibition of Tr1 cells, CD4 T cells<sup>+</sup> naives were cultured with monoclonal antibodies antl-CD3 plus antl-CD28 with Dex plus vit D3, with parenteral L cells or L cells transfected with OX40L, GITRL, or 4-1 BBL for 7 days. While OX40L, GITRL, and 4-1 BBL all promoted the generation of cells that produce TNF-α, only OX40L inhibited the
IMPI
MEXICAN INSTITUTE OF PROPERTY
INDUSTRIAL
<img file="MX339964B_D0038.tif" />
generation of Tr1 cells (FIGS. 7A and 7B).
In the FIG. 7A, an intracellular analysis of cytokine production by CD4 T cells<sup>+</sup> naíve was performed by flow cytometry. CD4 T cells<sup>+</sup> ships were cultured with monoclonal antibodies 5-CD3, anti-CD28, and IL-2 in parental L cells, OX40L-L cells, GITRL-L cells, or 4-1BBL-L cells in the presence of Dex plus vit D3 for 7 days. The percentages of the respective cytokine producing T cells are indicated in each spot blot profile. The results show that OX40L but neither GITRL nor 4-1BBL inhibits the generation of Tr1 cells.
In the FIG. 7B, IL-10 by naive CD4 * cells was measured in supernatants after restimulation with anti-CD3 and anti-CD28 monoclonal antibodies for 24 h by ELISA. Data are shown as mean ± SEM from four independent experiments. The results show that OX40L but neither GITRL nor 4-1 BBL inhibits the generation of Tr1 cells.
OX40L, GITRL and 4-1 BBL all promoted the expansion of total numbers of T cells (FIG. 7C). In the FIG. 7C, the number of viable T cells was counted. Data shown as mean ±
SEM of four independent experiments.
As understood by those skilled in the art, the results of FIGS. 7A-7C show that OX40L, but not GITRL or 4-1 BBL, inhibits the generation of Tr1 cells. These data suggest that among the three members
<img file="MX339964B_D0039.tif" />
of the TNF superfamily it is known that they costimulate the célutaegjm ^ g ^ g.
INDUSTRIAL a new and unique role in inhibiting the generation of Tr1 cells.
Furthermore it was discovered that OX40L inhibits the generation of cells
Tr1 induced by ICOSL or immature DCs. ICOS and CD28 represent the two 5 positive costimulatory receptors within the CD28 family expressed in T cells. Signaling through ICOS by agonist antibodies or
ICOSL has been shown to promote CD4 T cells<sup>+</sup> to produce IL-10. To investigate whether OX40L can inhibit the ability of ICOs to induce IL-10 production by CD4 T cells<sup>+</sup>CD4 T cells<sup>+</sup> naive and memory were cultured with anti-CD3 in the presence of ICOSL transfected L cells or ICOSL transfected L cells in the presence of OX40L for 7 days.
In the FIG. 8A, an intracellular analysis of cytokine production by CD4 T cells<sup>+</sup> naíve was performed using flow cytometry. CD4 T cells<sup>+</sup> naive were grown for 7 days in parental L cells, in a mixture of ICOSL-L cells and L cells, or in a mixture of ICOSL-L cells and OX40L-L cells, which were precoated with anti-CD3 monoclonal antibody. The percentages of the respective cytokine producing T cells are indicated in each spot blot profile. The results show that OX40L inhibits the generation of Tr1 cells from CD4 T cells.<sup>+</sup> ICOSL-induced ship.
In the FIG. 8B, IL-10 production by CD4 cells<sup>+</sup> naive was measured in supernatants after restimulation
IMPI with anti-CD3 and anti-CD28 monoclonal antibodies ϊδδϊεΏΟϊ ^ β <sup>r J r</sup> INDUSTRIAL through ELISA. CD4 T cells<sup>+</sup> naive were grown for 7 days in
<img file="MX339964B_D0040.tif" />
parenteral L cells, in a mixture of ICOSL-L cells and L cells, or in a mixture of ICOSL-L cells and OX40L-L cells, which were pre5 coated with anti-CD3 monoclonal antibody. Data are shown as mean ± SEM from three independent experiments. The results show that OX40L inhibits the generation of Tr1 cells from CD4 T cells.<sup>+ </sup>naive induced by ICOSL.
In the FIG. 8C, an intracellular analysis of cytokine production by CD4 T cells<sup>+</sup> Memory was performed using flow cytometry. CD4 T cells<sup>+</sup> Memory cells were cultured for 7 days in parenteral L cells, in a mixture of ICOSL-L cells and L cells, or in a mixture of ICOSL-L cells and OX40L-L cells, which were precoated with anti-CD3 monoclonal antibody. The percentages of the respective cytokine producing T cells are indicated in each spot blot profile. The results show that OX40L inhibits the generation of Tr1 cells from CD4 T cells.<sup>+</sup> memory induced by ICOSL.
In the FIG. 8D, the production of IL-10 by CD4 T cells<sup>+</sup> Memory in supernatants after restimulation with anti-CD3 and anti-CD28 monoclonal antibodies for 24 h was measured by ELISA. CD4 T cells<sup>+</sup> Memory cells were cultured for 7 days in parenteral L cells, in a mixture of ICOSL-L cells and L cells, or in a mixture of ICOSL-L cells and OX40L-L cells, which were pre44 coated with anti-CD3 monoclonal antibody. . Data were nYü'óitfén cBhrcr mean ± SEM from three independent experiments. The results show that OX40L inhibits the generation of Tr1 cells from CD4 T cells.<sup>+</sup> memory induced by ICOSL.
<img file="MX339964B_D0041.tif" />
The results of the experiments in FIGS. 8A-8D show that ICOSL significantly promotes the generation of Tr1 cells from both CD4 T cells<sup>+</sup> na'ive like from memory. The addition of OX40L completely inhibits the generation of Tr1 cells from both CD4 T cells.<sup>+</sup> na'ive as memory, while strongly promoting the generation of cells that produce TNF-a.
Immature DCs or DCs treated with IFN-α or IL10 are known to induce CD4 T cells<sup>+</sup> na'ive to differentiate into Tr1 cells. We investigated whether OX40L could inhibit Tr1 cell generation induced by DCs. As shown in FIG. 8E, immature DCs or DCs treated with
IL-10 or IFN-α all induce the generation of more than 10% of Tr1 cells from CD4 T cells<sup>+</sup> naive. In contrast, CD40L-activated DCs induce a strong TH1 response, accompanied by the generation of approximately 3% Tr1 cells. Addition of Recombinant OX40L in DC-T Cell Cultures Completely Inhibits Tr1 Cell Generation Induced by Immature DCs and DCs Treated with IL-10 and IFN-α. Further,
OX40L also inhibits the generation of residual Tr1 cell number induced by mature CD40L-activated DCs. In the FIG. 8E, an intracellular analysis of cytokine production by T cells
IMPI
CD4<sup>+</sup> na'ive was performed using flow cytometry.
<img file="MX339964B_D0042.tif" />
INDUST RÍAL were co-cultured in the presence or absence of soluble recombinant OX40L for 7 days with immature DCs or DCs cultured with IFN-α, IL-10, and CD40L. The percentages of the respective cytokine producing T cells are indicated in each spot blot profile. The results show that OX40L inhibits the generation of CD4 T cell Tr1 cells.<sup>+</sup> induced by
DCs.
The ability of OX40L to inhibit DCs-induced Tr1 cell generation was confirmed by ELISA data (FIG. 8F). In the FIG. 8F, the production of IL-10 by CD4 cells<sup>+</sup> na'ive was measured in supernatants after restimulation with anti-CD3 and anti-CD28 monoclonal antibodies for 24 h by ELISA. CD4 T cells<sup>+</sup> na'ive were co-cultured in the presence or absence of soluble recombinant OX40L for 7 days with immature DCs or DCs cultured with
IFN-α, IL-10, and CD40L. Data are shown as mean ± SEM from three independent experiments. The results show that OX40L inhibits the generation of Tr1 cells from CD4 T cells.<sup>+</sup> induced by DCs. Therefore, these data demonstrate that OX40L could inhibit Tr1 cell generation induced by further physiological signals provided by ICOSL and
DCs.
It has previously been suggested that regulatory T cells are highly represented in the area of B-cell non-Hodgkin lymphoma and that B cells are involved in the recruitment of T * -ί · -A cells.
IMPI
<img file="MX339964B_D0043.tif" />
regulatory in the lymphoma area. 0X40 receptor signaling was investigated, such as by 0X4QL, it could provide therapy against B-cell lymphoma. Cryopreserved samples from patients with B-cell lymphoma were used to estimate the ability of
OX40L to close Tr1 cells. The samples used were follicular lymphoma obtained from a spleen specimen prior to any treatment. Cells were thawed, with 400x10<sup>6</sup> frozen cells that produced 127χ10<sup>6</sup> living cells and 33.9x10<sup>6</sup> dead cells (79% viability). A sufficient number of CD25 cells<sup>+</sup> were identified by FACS staining. In the FIG. 9, IL-10 secretion from Tregs producing ICOS<sup>+</sup>IL-10 was determined by ELISA. Treg cells were grown under two different conditions. In condition 1, CD25 cells<sup>+</sup>/ ICOS<sup>+</sup> they were cultured with anti-CD3 in the presence of IL-2 (900 µΙ / ml) in parental L cells or OX40L-L cells with anti-ICOS antibody for 3-6 days. In condition 2, CD25 cells<sup>+</sup>/ ICOS<sup>+</sup> they were cultured with anti-CD3 in the presence of IL-2 (900 µΙ / ml) in ICOS-LL cells or a mixture of OX40L-L and ICOS-LL cells for 3-6 days. Cytokine production in supernatants was measured by ELISA. The results show that OX40L greatly inhibits the production of IL-10 by Treg cells.
The findings, that OX40L has the ability to inhibit the generation and function of Tr1 cells induced by immunosuppressive drugs Dex plus vit D3, ICOSL, or DCs, highlight a new mechanism by which OX40L promotes immunity and breaks tolerance.
IMPI
<img file="MX339964B_D0044.tif" />
during different forms of immune responses médiawte ^ $ | g ^ R91 INDUSTRIAL as understood by one skilled in the art. QX40L's ability to inhibit Tr1 cell generation during both IL-2-induced TH1 or IL-4-induced TH2 responses suggests that OX40L may control the magnitude of TH1 or TH2-mediated immune responses. Furthermore, OX40L's ability to inhibit Tr1 cell generation appears to be a unique property of OX40L, because the other two members of the TNF family, GITRL and 4-1BBL do not have this functional property. Furthermore, OX40L's ability to inhibit IL-10 production by Treg cells identifies OX40L as a potent treatment for B-cell lymphoma and other cancers.
Many molecules that promote the generation of Tr1 cells have been identified, including IL-10, IFN-α, ICOSL, and immunosuppressive compounds such as Dex plus vit D3. OX40L represents a powerful inhibitor for the generation of Tr1 cells, not just CD4 T cells.<sup>+</sup> na'ive but also CD4 T cell<sup>+</sup> memory and regulatory T cells. This new property of OX40 / OX40L may explain a recent report showing that 0X40 signaling allows anergic reactive T cells to acquire the functions of effector cells. The address of
OX40 / OX40L accordingly provides treatments for human autoimmune and allergic diseases as well as for the development of treatments for human infectious diseases and cancer including, but not limited to melanoma, brain cancer, bone cancer, leukemia,
IMPI
<img file="MX339964B_D0045.tif" />
lymphoma, neoplasm derived from epithelial cells.<sup>1 </sup>such as basal cell carcinoma, adenocarcinoma, gastrointestinal cancer such as cancer of the lips, cancer of the mouth, cancer of the esophagus, cancer of the small intestine and cancer of the stomach, cancer of the colon, cancer of the liver, cancer of the bladder, cancer of the pancreas , ovarian cancer, cervical cancer, lung cancer, breast cancer, and skin cancer, such as squamous cell and basal cell cancers, prostate cancer, renal cell carcinoma, and other known cancers.
Disorders or conditions that can be prevented or treated by the antibodies and methods described herein include the prevention or treatment of cancer, such as cutaneous T-cell leukemia, head and neck tumors, pancreatic cancer, bladder cancer, gliomas of high grade, brain metastases, melanoma, skin cancer, lung cancer, breast cancer, prostate cancer, colon cancer, leukemia, myelodysplastic syndrome (a pre-leukemia condition), and multiple myeloma. In general, the metastasis of any cancer can be prevented or treated with the methods and compounds described herein. Antibodies can also be used to prevent or treat iterative role angiogenic conditions including telangectasia, venous angiomas, hemangioblastoma. Other disorders, diseases or conditions include viral diseases, some of which can traditionally be considered "intractable". Antibodies, for example, can also be used to classify strains of a single pathogen. Researchers can use antibodies
ΙΜΡΙ Described herein to identify and track the specific industrial laesr ^^^ Jg in the organism.
In general, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. More specifically, cancers that can be treated or prevented using one or more of the antibodies described herein or a variant thereof, include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include, but are not limited to, squamous cell cancer, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or kidney cancer, liver cancer, prostate cancer, vulvar cancer, cancer thyroid, liver carcinoma and various types of head and neck cancer, melanoma, superficial spreading melanoma, lentigo malignant melanoma, acral lentiginous melanoma, nodular melanomas, as well as B-cell lymphoma (including follicular low-grade / non-Hodgkin lymphoma (NHL); small lymphocytic NHL (SL); intermediate-grade / follicular NHL; NHL
<img file="MX339964B_D0046.tif" />
Diffuse intermediate grade IMPI; Immunoblastic NHL from ^ cSÉS * ms jumo p ·
<img file="MX339964B_D0047.tif" />
high-grade lymphoblastic; High-grade Small uncleaved cell NHL; Bulky disease NHL; mantle cell lymphoma; AIDS related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatosis, edema (such as that associated with brain tumors), and Meigs syndrome.
Methods for treating or preventing an immune disorder are also provided herein. These methods comprise administering an effective amount of the antibody to a subject in need of such treatment. In some embodiments, the immune disorder is an immune disorder or an autoimmune disorder. The disorder is asthma, atopic dermatitis, allergic rhinitis, inflammatory bowel disease, multiple sclerosis, GVHD, and / or systemic lupus erythematosus. In some modalities, the disorder is a disease associated with a virus, bacteria, or other infectious agent.
Furthermore, the antibodies and methods described herein can be used to prevent or treat inflammatory conditions and diseases, such as osteoarthritis, rheumatoid arthritis, disease of
Crohn's, ulcerative colitis, and autoimmune diseases such as lupus and mixed autoimmune disease. For example, the antibodies described herein may be useful in the treatment of a variety of autoimmune and inflammatory diseases that administer a therapeutically effective amount of the antibody to a subject in need thereof, wherein the autoimmune disease or inflammatory disease is one or more of the following diseases:
Insulin-dependent diabetes mellitus (IDDM), diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, acute disseminated encephalomyelitis, arthritis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, Hashimoto's disease, primary myxedema, thyrotoxicosis, pernicious anemia, gastritis autoimmune atrophic, Addison's disease, premature menopause, male infertility, juvenile diabetes, Goodpasture syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis, autoimmune hemolyticanemia, idiopathic leukophenia, primary biliary cirrhosis, active chronic hepatitis Hb<sub>s</sub>-<sub>we</sub>, cryptogenic cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, poly / dermatomyositis, discoid LE, systemic lupus erythematosus, Chron's disease, psoriasis, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune hepatitis, autoimmune hepatitis , Graves' disease, Guillain-Barre syndrome (GBS), idiopathic thrombocytopenic purpura, ppsoclono-myoclonus syndrome (WHO), optic neuritis, ORd thyroiditis, pemphigus, polyarthritis, primary biliary cirrhosis, Reiter's syndrome, Takayasu, temporal arteritis, autoimmune hemolytic anemia by hot antibody, Wegener's granulomatosis, universal alopecia, Behcet's disease,
<img file="MX339964B_D0048.tif" />
Chagas disease, oron fatigue syndrome, endometriosis, hidradenitis suppurativa, interstitial cystitis, neuromyotonia, sarcoidosis, scleroderma, ulcerative colitis, vitiligo, vulvodynia, inflammatory skin diseases, allergic contact dermatitis, H. pylori chronic inflammatory disease, arteriosclerosis and graft versus host disease.
More specifically, an "autolnmune disease as referred to herein is a disease or disorder that arises and is directed against an individual's own tissue or organ or a co-segregation or manifestation thereof or condition resulting from this. Autoimmune disease can refer to a condition that results from, or is aggravated by, the production of antibody B cells that are reactive with antigens and normal body tissues. Furthermore, an autolnmune disease is one that may involve the secretion of an autoantibody that is specific for an epitope of a self-antigen (eg, a nuclear antigen).
Autoimmune diseases or disorders that are treatable and / or preventable by one or more of the antibodies described herein include, but are not limited to, arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, arthritis acute gouty, acute immune arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral osteoarthritis, and juvenile rheumatoid arthritis, osteoarthritis, chronic prophylactic arthritis, arthritis re
<img file="MX339964B_D0049.tif" />
INSTITUTE 'ME4CCA
OF THE PROPERTY
INDUSTRIAL
<img file="MX339964B_D0050.tif" />
hyperproliferative inflammatory, deforming, primary chronic polyarthritis, ankylosing), skin diseases psoriasis such as plaque psoriasis, gutatta psoriasis, pustular psoriasis, and nail psoriasis, atopy, including 5 atopic diseases such as hay fever and Job, dermatitis including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, Nummular dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, and atopic dermatitis, hyper-IgM x10 syndrome, allergic infraocular inflammatory diseases, urticaria such as chronic allergic urticaria, and chronic idiopathic urticaria, including chronic autoimmune urticaria, myositis, polymyositis, dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spinous-optic MS, primary progressive MS (PPMS), and recurrent remission MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis, ataxic sclerosis, neuromyelitis optic (NMO), inflammatory bowel disease (IBD) (eg, Crohn's disease, autoimmune-mediated gastrointestinal diseases such as ulcerative colitis, ulcerative colitis, microscopic colitis, collagenous colitis, butterfly colitis, necrotizing enterocolitis, and transmural colitis, and inflammatory autoimmune disease of the intestine), inflammation of the intestine, gangrenous pyoderma, erythema nodosum, sclerosing cholangitis
<img file="MX339964B_D0051.tif" />
primary respiratory distress syndrome, including if ^^ J ^ j ^ ífi
INDUSTRIAL PROPERTY acute or adult respiratory (ARDS), meningitis, inflammation of all or
<img file="MX339964B_D0052.tif" />
part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve degeneration such as meningitis, gestational herpes, gestational pemphigoid, scrotum pruritus, autoimmune premature ovarian failure, loss of ovarian failure sudden due to an autoimmune condition, IgE-mediated diseases such as anaphylaxis, and allergic and atopic rhinitis, encephalitis such as Rasmussen encephalitis and limbic and / or brainstem encephalitis, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, posterior uveitis, and autoimmune (glomerular) uvitis, glomerular uveitis no nephrotic syndrome such as chronic or acute glomerulonephritis such as primary GN, immuno-mediated GN, membranous GN (membranous nephropathy), Idiopathic membranous GN or idiopathic membranous nephropathy, membranous or membranous prolifferative GN (MPGN), including type I and type II, and rapidly progressive GN, prolipherative nephritis, autoimmune polyglandular endocrine failure, balanitis including balanitis plasmacellularis circumscripta, balanoposthitis, erythema antrum dyschromic perstans erythema, erythema multiforme, granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulous, lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis, premalignant keratosis, gangrenous pyoderma, aortic conditions and responses, allergic reaction, eczema including allergic or atopic eczema, asteatotic eczema, PI bronchial eczema, palmoplantar eczema, aSTOTH , and autoimmune asthma, conditions involving T-cell infiltration and chronic inflammatory responses, immune reactions against foreign antigens such as fetal ABO blood groups during pregnancy, chronic inflammatory lung disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, including lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus renal, discoid lupus and discoid lupus erythematosus, alopecia lupus, systemic lupus erythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and disseminated systemic lupus erythematosus, juvenile diabetes (type I), diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM), adult diabetes mellitus (type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, diabetic retinopathy, diabetic nephropathy, diabetic large artery disorder, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, tuberculosis, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitis, including vasculitis, large-vessel vasculitis (including giant cell arteritis (Takayasu), and polymyalgia rheumatica), medium-vessel vasculitis (including disease of
Kawasaki and polyarteritis nodosa / periarteritis nodosa), microscopic polyarteritis, immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as v
IMPI
FOOT
INDUSTRIAL
<img file="MX339964B_D0053.tif" />
Churg-Strauss (CSS) and ANCA-associated small vessel vasculitis, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs-positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), anemia pernicious (pernicious anemia), Addison's disease, pure red blood cell anemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocytic diapedesis, inflammatory CNS disorders, Alzheimer's disease, Parkinson's disease, multiple organ injury syndrome such as those secondary to sepsis, trauma or hemorrhage, diseases mediated by antigen-antibody complexes, basement membrane disease anti-glomerular, anti-phospholipid antibody syndrome, allergic neuritis, Behcet's disease / syndrome, Castleman's syndrome, Goodpasture syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid such as bullous pemphigoid and skin pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus folioseum, mucous membrane pemphigus, and erythematous pemphigus disease), autoimmune polyecrinopathies, Reiter's, thermal injury, pre-eclampsia, an immune complex disorder such as complex immune nephritis, antibody-mediated nephritis, polyneuropathies, chronic neuropathy such as IgM polyneuropathies or IgM-mediated neuropathy, thrombocytopenia (as developed by patients with myocardial infarction, for example), including thrombus purpura ^
<img file="MX339964B_D0054.tif" />
<img file="MX339964B_D0055.tif" />
MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY (TTP), post-transfusion purpura (PTP), heparin-induced thrombocytopenia, and autoimmune or immuno-mediated thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, scleritis such as cerato- idiopathic scleritis, episcleritis, autoimmune disease of the testes and ovaries including orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's, polyglandular autoimmune syndromes (or polyglandular syndromes). ), paraneoplastic syndromes, including neurological paraneoplastic syndromes such as LambertEaton myasthenic syndrome or Eaton-Lambert syndrome, stiff man or stiff person syndrome, encephalomyelitis such as allergic encephalomyelitis or allergic encephalomyelitis, and allergic encephalomyelitis (EAE), myasthenia gravis such as myasthenia gravis associated with , cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus-myoclonus syndrome (WHO), and sensory neuropathy, multifocal motor neuropathy, Sheehan syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or chronic autoimmune active hepatitis, interstitial lymphoid pneumonitis (LIP), bronchiolitis obliterans (without transplantation) vs NSIP, Guillain syndrome -Barre, Berger's disease (IgA nephropathy), z-Jx. -λ-U dlrKíSU ^
INSTITUTO MEXICA m industrial property
<img file="MX339964B_D0056.tif" />
Idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile, subcorneal pustular dermatosis, transient acantholitcal dermatitis, cirrhosis such as primary billiard cirrhosis and pneumonoclrrosis, autologous enteropathy syndrome, celiac or coeliac disease, celiac sprue (celiac sprue) refractory sprue, idlopathic sprue, crloglobullnemia, amllotrophic lateral sclerosis (ALS; Lou Gehrlg's disease), coronary artery disease, autolmune ear disease such as autolmune Inner ear disease (AIED), autolnmune hearing loss, polychondritis such as relapsed or refractory pollchondritis, pulmonary alveolar protelnosls, non-interstitial keratitis slfilítlca / Cogan syndrome, Bell's palsy, Sweet's disease / syndrome, autoimmune rosacea, pain associated with herpes zoster, amlloldosls, a non-cancerous llnfocltosls, a primary lymphocytosis, which includes monoclonal B-cell lymphocytes (eg, benign monoclonal gammopathy 15 and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscle disorders, deafness, blindness, periodic paralysis, and CNS channelopathies, autism, inflammatory mylopathy, focal or segmental or focal segmental glomerulosclerosls (FSGS), twenty endocrine ophthalmopathy, uveorretlnitis, chorioretinitis, autologous liver disease, flbromlalgla, multiple endocrine failure, Schmldt syndrome, adrenalltls, gastric atrophy, presenile dementia, deimlinating diseases such as autoimmune diseases and
IMPIgg
MEXICAN INSTITUTE ^ <»“ 1 chronic inflammatory demyelinating polyneuropathy, alopecia greata syndrome, total alopecia, CREST syndrome (rairJnnsiR fpnómonn Hq
Raynaud's, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility, for example, due to anti5 sperm antibodies, mixed connective tissue disease,
Chagas, rheumatic fever, recurrent miscarriage, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, breeder's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis , interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases such as leishmaniasis, chipanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter syndrome, Caplan syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, pulmonary interstitial fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, fetal erythroblastosis syndrome, eosinophilic phaciitis, , Felty's syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuch cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection,
SCID, acquired immunodeficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-virus infection Barr, mumps,
IMPI Evan syndrome, autoimmune gonadal failure, post-streptococcal nephritis, thromboangiitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, chronic hypersensitivity pneumonitis, keratoconjunctivitis, epidemic nephritic disease, idiopathic nephritic disease, by ischemiareperfusion and familial benign, reperfusion by organ transplantation, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive pulmonary / airway disease, silicosis, thrush, aphthous stomatitis, arteriosclerotic disorders, asperniogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, phacoanaphylactic endophthalmia, idiopathic syndrome, leprosy erythema nodosum, chronic fatigue, rheumatic fevers, Hamman-Rich disease, sensorineural hearing loss, paroxysmal hemoglobinuria, hypogonadism, regional ileitis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrosis, sympathetic ophthalmia, granulomatous orchitis, pancreatitis, acute polyradiculitis, gangrenous pyoderma, Quervain's thyroiditis, non-malignant thymoma, vitiligo, vitilic thymoma, vitiligo food poisoning, conditions involving T-cell infiltration, leukocyte adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen complex mediated diseases-
<img file="MX339964B_D0057.tif" />
<img file="MX339964B_D0058.tif" />
antibody, antiglomerft basement membrane disease ^ jTífefiteüsb
INDUSTRIAL PROPERTY Autoimmune polndocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyndocrine insufficiency syndrome, type I autoimmune hypoparathyroidism (AOIH) cardiomyopathy such as dilated cardiomyopathy, epidermolysis bullosa acquisita (EBD), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or non-purulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, eosinophilic pneumonia chronic, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or eosinophil-containing granulomas, anaphylaxis, seronegative spondyloarthritis, poliendocrina autoimmune disease, sclerosing cholangitis, scleral mucocutaneous candidiasis, episclerotic, chronic, Bruton syndrome, transient hypogammaglobulinemia of childhood, WiskottAldrich syndrome, ataxia telangiectasia syndrome, angiectasis, autoimmune disorders associated with collagen disease, rheumatism , reduced blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and vascularization accompanying the disease, allergic hypersensitivity disorders,
IMPI
INSTITUTO MEXICANO glomerulonephritis, reperfusion injury, reperfusion disorder§lb<sup>TO</sup>i »Di®qu € nTOB ^^ Reperfusion injury of the myocardium or other tissue ^ —tidquuobi'emflitis, inflammatory dermatosis, dermatosis with acute inflammatory components, multiple organ failure, huloid diseases, cortical renal necrosis, acute purulent meningitis or other disorders inflammatory diseases of the central nervous system, inflammatory eye and orbital disorders, syndromes associated with granulocyte transfusion, cytokine-induced toxicity, narcolepsy, acute severe inflammation, chronic intractable inflammation, pyelitis, endarterial hyperplasia, peptic ulcer, valvulitis, and endometriosis.
The antibodies described herein can have a variety of academic, medical, and commercial uses. Antibodies can be used in different types of diagnostic tests, for example, to detect a wide variety of diseases or the presence of drugs (pharmaceuticals), toxins or other proteins including hormones, either in vitro or in vivo. The antibodies described herein may be useful in tests for disease, eg, in serum or blood of patients. The disease may include diseases related to 0X40 or diseases or indications not related to
0X40 including various cancers, inflammatory or autoimmune disease. Antibodies can also be used in radioimmuno-detection and radioimmuno-therapy of cancer and some new test methods can use these described antibodies to target only the
IMPI
INSTITUTE 'EXJjCA **' cell membranes of specific cell types, that is to say. ^ EfliSeríuAt
<img file="MX339964B_D0059.tif" />
The antibodies described herein could be obtained from a kit or another diagnostic package. As such, a diagnostic kit, or article of manufacture is provided herein for use with the treatment method herein. The diagnostic kit may comprise one or more of the following: antagonist / antibody / drug reference material; positive control neutralizing antibody (preferably goat or cynomolgus monkey); Protein A + G column (eg, Protein A / G column); delipidation reagent;
immunoglobulin affinity purification buffers (eg, binding, elution and neutralization buffers): complement serum; cell test diluent; literature or instruction manual; frozen cell vial (eg, WIL2 cells); cell labeling reagent (such as CELL TITER GLO.RTM.), etc. By way of example, the diagnostic kit may include but is not limited to: (a) delipidation reagent; (b) buffers (eg, binding and elution buffers) for affinity purification of immunoglobulins; and (c) an instruction manual that instructs the diagnostic kit user to use the kit to pretreat a biological sample of an autoimmune disease or target cancer before conducting a cell-based bioassay (such as a neutralizing antibody assay) in the sample (for example, to avoid the serum interference problem). The diagnostic kit optionally additionally comprises some or more of: drug reference material,
IMPI
MEXICAN INSTITUTE. PROPERTY Neutralizing Positive Control Antibody, ComplemenW ^ tHhjye Cell Assay Serum, and Cell Labeling Reagent, ele .--—
<img file="MX339964B_D0060.tif" />
The antibodies and other findings described herein also provide high throughput screening methods. More specifically, and as understood by those skilled in the art, high-throughput methods for screening antagonist or agonist monoclonal antibodies or small molecules that bind to 0X40 receptors, and which can inhibit the generation and function of Tr1 cells or promote generation and function of Tr1 cells, are made possible. In such a method, a human T cell line (SU-DHL-1) that has the ability to produce IL-10 was transfected with the human 0X40 gene (SUOX40). 100,000 SUOX40 cells were grown with either 100,000 mouse fibroblast cells (L cells) or 100,000 mouse fibroblast cells expressing the human 0X40 ligand (0X40 ligand L cells) in 96 well plates. After 48 hours of culture, culture supernatants were collected for IL-10 measurement by IL-10 specific ELISA. In a representative experiment, 100,000 SUOX40 cells produced up to 6,000 pg / ml IL-10 cultured in the absence of 0X40 ligand. In the presence of 0X40 ligand, 100,000 SUOX40 cells produced less than 1,000 pg / ml IL-10. This culture method can be used to screen, inter alia, for antagonistic monoclonal antibodies or small molecules that block the 0X40 ligand's ability to inhibit IL-10 production by SUOX40 cells. Alternatively, this method
IMPI
The MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY culture can be modified by replacing L cells expressing the 0X40 ligand with potential monoclonal agonist antibodies or small molecules specific for 0X40 to determine, inter alia, their ability to inhibit IL-10 production by cells from SUOX40.
The anti-OX40 antibodies described herein can be used as an assay or in an assay to test or measure the activity of a drug or other molecule found in an organism or organic sample. They could also be used in a quantitative assay to measure the amount of a substance in a sample. Bioassays and immunoassays are among the many varieties of specialized biochemical assays by which these antibodies can be used. The anti-OX40 antibodies taught herein can be used in other assays to measure processes such as enzyme activity, antigen capture, stem cell activity, and competitive protein binding.
Human GITRL, OX40L, 4-1BBL, ICOSL expressing L cells were generated by retrovirus mediated transduction, as understood by those skilled in the art. Briefly, the full-length coding sequence for human GITRL (Access # NM_005092), OX40L (Access # NM 003326), 4-1 BBL (Access # NM_003811), ICOSL (Access # NM_015259) was amplified by RT-PCR with prepared RNA of HSV-1-stimulated PBMCs. Subsequently, the cDNAs were cloned into an MSCV-based retroviral vector pMIGW2 and the resulting plasmids were verified by restriction enzyme digestion and DNA sequencing.
<img file="MX339964B_D0061.tif" />
, IMPI
To produce the recombinant retrovirus, each vector was pCL-gp (gag / pol) and pHCMV-VSVg (VSV glycoprotein envelope) packaging constructs in HEK293T cells. Two days later, virus-containing culture supernatants were harvested and used to infect CD32 to 100 L cells. Under this condition> 95% cells were productively transduced.
CD14 Monocytes<sup>+</sup> Isolates (purity> 94%) were cultured in the presence of 100 ng / ml of GM-CSF and 50 ng / ml of IL-4 (both from R&D) for 5 days, as understood by those skilled in the art. The resulting immature DCs were washed and cultured for 24 h with IFN-a (1000 U / ml, PBL Biomedical Laboratories), IL-10 (10 ng / ml, R&D), and CD40L-transfected L cells were irradiated (ratio of DC to L cells, 4: 1) to obtain mature DCs, as understood by those skilled in the art.
CD4 T cells<sup>+</sup> nai've and CD4 T cells<sup>+</sup> memory (each purity> 99%) were isolated from PBMCs using CD4 T-cell Isolation Kit II<sup>+</sup> (Miltenyi Biotec) followed by cell sorting (CD4 fraction<sup>+</sup>CD45RA<sup>+</sup>CD45RO'CD25 as naive T cells and CD4 + CD45RA CD45RO * CD25 'fraction as memory T cells), as understood by those skilled in the art. 4x10<sup>4</sup> CD4 T cells<sup>+</sup> Freshly purified allogeneic ships were co-cultivated with immature or cultured DCs (DC to T ratio, 1:10) in the presence or absence of recombinant human OX40L (R&D, 100 ng / ml) in 96-well culture culture dishes. round for 7 days, as understood by experts in the
IMPI
INDUSTRIAL
<img file="MX339964B_D0062.tif" />
technique. CD4 T cells<sup>+</sup> purified were also ¿ng / ml, R&D), IL-4 (25 ng / ml, R&D), or combination of dexamethasone (5x10 '<sup>8</sup>___
M, Life Technologies) and 1alpha, 25-dihydroxyvitamin D3 (10 '<sup>7</sup> M) for 7 days in the presence of soluble anti-CD28 monoclonal antibody (CD28.2, 1 pg / ml) and IL-2 (50 U / ml, R&D) in irradiated CD32 / OX40L-L cells, CD32 / cells. GITRL-L, CD32 / 4-1BBL-L cells, or parental CD32-L cells which have been precoated with antl-CD3 monoclonal antibody (OKT3, 0.2 pg / ml) in 48-well culture dishes (ratio of T cells to L cells,
2.5: 1), as understood by those skilled in the art. In some 10 experiments, CD4 + T cells were cultured for 7 days in CD32-L cells, mix of CD32-L cells and CD32 / ICOSL-L cells (1: 1 ratio), or mix of CD32 / ICOSL-L cells. and CD32 / OX40L-L cells (1: 1 ratio) precoated with antl-CD3 monoclonal antibody (0.2 pg / ml) in 48 well culture dishes, as understood by those skilled in the art. RPMI 1640 was used and supplemented with 10% FCS, 2mM L-glutamlna, 1mM sodium pyruvate, penicillin G, and streptomycin for cultures, as understood by those skilled in the art.
Cultured T cells were collected and washed, and then restimulated with plaque-binding anti-CD3 (5 pg / ml) and soluble anti-CD28 20 (2 pg / ml) at a concentration of 1x10<sup>6</sup> cells / ml for 24 hours, as understood by those skilled in the art. The levels of IL-4, IL-10, TNF-α, and IFN-a in the supernatants were measured by ELISA (all R&D kits), as understood by those skilled in the art. For the production of: IMPI
INSTITUTO MEXICANO intracellular cytokine, cultured T cells are restimulated
<img file="MX339964B_D0063.tif" />
PMA plus 2 pg / ml of yonomycin for 6 h. Brofoldin A (10 pg / nM) ~ ag · added during the last 2 h, as understood by those skilled in the art. Cells were stained with a combination of PE-labeled monoclonal antibodies to FITC-labeled monoclonal antibodies of IL-4 or TNF-α to anti-IL-10 labeled with APC and IFN-α (all from BD) using the FIX kit and PERM (CALTAG), as understood by those skilled in the art.
T cells were collected and resuspended in EDTA-containing medium to dissociate clusters, as understood by those skilled in the art. Viable cells were counted by exclusion of trlpane blue from dead cells, as understood by those skilled in the art. For the suppressive function assay, naive CD4 + T cells (A) and Tr1 cells generated from CD4 T cells<sup>+</sup> naive by anti-CD3 monoclonal antibody, anti-CD28 monoclonal antibody, IL-2, Dex, and vlt D3 in the presence of parental L cells (B) or OX40L-L cells (C), these three cell types and their mixtures at a 1: 1 ratio were then restimulated for 5 days by culture in the presence of 5 pg / ml of anti-CD3 monoclonal antibody and 1 pg / ml of anti-CD28 monoclonal antibody, after this time cell proliferation was evaluated by incorporation of [<sup>3</sup>H], as understood by those skilled in the art.
<img file="MX339964B_D0064.tif" />
INDUSTRIAL
Generation of anti-human monoclonal antibodies
Multiple agonist mouse monoclonal antibodies against human 0X40 were generated. The antigen binding specificity of the antibodies was confirmed by flow cytometry (FIGS. 10-12). The agonist activity of the antibodies was validated through functional assays. Nine of the 20 0X40-specific antibodies were found to be able to block vitamin D3 / dexamethasone-mediated generation of Tr1 cells from CD4 T cells.<sup>+</sup> (FIGS. 13A and 13B), enhance the proliferation of CD4 T cells<sup>+</sup> (FIG. 14), and suppress IL-10 production from Treg ICOS<sup>+</sup>CD4<sup>+</sup>CD25<sup>to</sup>FOXP3<sup>+</sup> (FIGS. 16A-16C). Antibodies were titrated and five were found to have activity in suppressing Tr1 cell generation at concentrations as low as 4 ng / ml (FIGS. 15A and 15B).
0X40 antibodies inhibit Treg function
CD4<sup>+</sup>CD25<sup>there</sup>FOXP3 +
Some of the 0X40 monoclonal antibodies inhibit the suppressive function of Treg F0XP3 + (FIGS. 17A and 17B). Of the five antibodies (119-8B, 119-43, 119-122, 119-173B, and 106-222) that potently inhibit the production of IL-10 from Tr1 and Tregs cells
CD4<sup>+</sup>CD25<sup>high</sup>CD127<sup>+</sup>FOXP3<sup>+</sup>, three (119-43, 119-122, and 106-222) were potent in blocking Treg CD4 function<sup>+</sup>CD25<sup>high</sup>CD127<sup>+</sup>FOXP3<sup>+</sup> (FIGS. 17A and 17B). However, two (119-33 and 120-140A) of the 11
<img file="MX339964B_D0065.tif" />
antibodies that have no activity against the production of CD4 Treg function<sup>+</sup>CD25<sup>to</sup>FOXP3<sup>+</sup> (Fig. 18). _______
Anti-human QX40 monoclonal antibodies
Generation of anti-human 0X40 monoclonal antibodies was performed, for example, by immunizing 6-8 week old BALB / c mice with a mouse cell line transfected with human 0X40 following established protocols. Hybridoma clones that secrete hybridoma monoclonal antibody that specifically stained OX40 cells<sup>+</sup> were established and further analyzed.
A comprehensive screen was designed to detect those clones that activate 0X40 signaling (i.e., agonist antibodies) by inhibiting the generation and function of Tr1 cells. Those clones were further purified. The hOX40 agonist antibodies can be humanized and used in clinical protocols for anti-human tumor therapy, either alone or in combination with anti-tumor vaccination and other adjuvants. Various different tumor types may be the target of these antibodies, including melanoma, lymphoma, and breast cancer.
In another embodiment, female BALB / c mice 6-8 weeks old were used for subcutaneous or foot pad immunization.
Each mouse was injected with 5 million murine L cells transfected with
0X40 human (L-OX40) 6 times at 3-day intervals. Three days after the sixth injection, the mice were sacrificed and the lymph nodes
Popliteal IMPIs (from immunization in planterg pad ^ Mjfl ^
INDUSTRIAL subcutaneous immunization) were removed and cells were fused with
<img file="MX339964B_D0066.tif" />
SP2.0 myeloma or NSO myeloma cells at a 1 to 1 ratio to generate hybridoma clones using established protocols. Hybridoma clones secreting monoclonal antibody were then screened for their L-hOX40 cell binding specificity by ELISA assays. Hybridoma supernatants binding to L-hOX40 cells and non-parental L cells were further confirmed for binding on L-hOX40 and SUPM2-hOX40 cells by flow cytometric analysis.
In the FIG experiment. 10, hOX40 hybridoma supernatants were selected against L-hOX40 cells versus parental L cells by ELISA. Twenty monoclonal antibodies specific for hOX40 were selected. Twenty million L cells or L cells expressing human 0X40 (L-hOX40) were coated on a 96 well plate by mixing the cells with 0.01% magnesium chloride and calcium in PBS and allowed to dry overnight in a laminar hood. The plates were then frozen at 20 ° C for at least one day prior to use. For antibody binding assays, frozen cells were rehydrated with PBS and washed with wash buffer containing PBS plus 0.05% Twen 20, and blocked with 2% BSA in wash buffer. The conditioned cells were then used for binding to antibody supernatants.
0X40. Antibody binding to cells was then detected with a secondary antibody, anti-mouse IgG FC HRP. Hybridoma Supernatants
<img file="MX339964B_D0067.tif" />
Specific hOX40 cells recognize L cells that ex ^ Sreera ^^^ |) parental L cells. _____
In the FIG experiment. 11, hOX40-specific monoclonal antibodies were selected by flow cytometric analysis. 5 Equal numbers (100k) of L and L-hOX40 cells were mixed in FACS buffer (1% FCS / 2mM EDTA / PBS) and incubated with 0.5 pg of purified FPLC antibodies (Protein A HiTrap / Ag elution buffer). / Ab Light). The cells were then washed and stained with a secondary antibody, PE-conjugated anti-mouse IgG. Two peaks indicate positive and negative 10 staining by anti-hOX40 monoclonal antibody. A single peak suggests no binding or no specific binding of antibodies. Twenty hOX40-specific monoclonal antibodies were confirmed by flow cytometric analysis.
In the FIG experiment. 12, the specificity of hOX40 monoclonal antibodies was confirmed using SUPM2 cells expressing hOX40 (SUPM2-hOX40). Equal numbers (100k) of SUPM2 and SUPM2-hOX40 cells were mixed in FACS buffer (1% FACS / 2mM EDTA / PBS) and used for hOX40 monoclonal antibody binding as in FIG. 11. The binding specificity of each antibody was analyzed by flow cytometry. Two peaks indicate positive and negative staining by anti-hOX40 monoclonal antibody, while a single peak suggests no binding or no specific binding by antibodies. Twenty hOX40-specific monoclonal antibodies were confirmed.
IMPI ~
In the FIGS experiment. 13A and 13B, id '
MEXICAN INSTITUTE
<img file="MX339964B_D0068.tif" />
monoclonal antibodies specific for human 0X40 that can be inhibited-! » generation of Tr1 cells from CD4 T cells<sup>+</sup> stimulated by VitD<sub>3</sub> (10 micromoles mM) / Dex (50 nanoM), CD32L / ICOSL and anti-CD3 / CD28 (0.2 micrograms / ml). Anti-hOX40 monoclonal antibodies were added on day 0 "of the cell culture and CD4 * T cells after 7 days of stimulation were subjected to intracellular staining of IL-10 followed by flow cytometric analysis. Representative Fluorescence Activated Cell Classification (FACS) data is shown in A and the percentages of Tr1 cells for all antihOX40 monoclonal antibody treatments are shown in B. Using the cells obtained from this experiment, we sought to identify the antibodies monoclonal specific to hOX40 that stimulate the proliferation of CD4 T cells<sup>+</sup> (FIG. 14, cells were counted on day 7 after stimulation) and inhibit Tr1 generation from CD4<sup>+</sup> (FIGS. 13A and 13B).
To identify such hOX40 monoclonal antibodies for their ability to inhibit the generation of Tr1 cells from CD4 T cells<sup>+</sup>Tr1 cells were generated and cultured as described in the experiments for FIGS. 13A and 13B above. Representative FACS data is shown in A and the percentage of Tr1 cells after treatment with nine anti-hOX40 monoclonal antibodies is shown in
B. Five monoclonal antibodies specific to hOX40 strongly inhibited the generation of Tr1 cells at a concentration of 4 ng / ml (FIGS.
IMPI
IHSTTVUTO MEXICANA DtLAÍROMEOAO induttmal
<img file="MX339964B_D0069.tif" />
FIGS. 16A-16C, classified T cells were stimulated with CD32L / ICOSL cells and cells
15Ay 15Β).
In the ICOS experiment<sup>+</sup>CD4<sup>+</sup>CD127CD25<sup>high</sup> recently anti-CD3 (0.2 pg / ml) in the presence of
CD32 / hOX40L or monoclonal anti-hOX40 antibodies or control antibody for 5 days. Then the cells were counted and 5x10<sup>4</sup> cells were restimulated with anti-CD3 / CD28 for 4 hours and the supernatants were assayed for IL-10 secretion with an Elisa kit. Specific monoclonal antibodies to hOX40 that inhibit Tr1 generation from CD4 T cells were identified<sup>+</sup> which also inhibit the production of IL-10 naturally from ICOS T cells<sup>+</sup>CD4<sup>+</sup>CD2<sup>high</sup>. The ICOS Tregs<sup>+</sup>ICOS'CD4<sup>+</sup>CD127 CD25<sup>high</sup> were grown with CD4 cells<sup>+</sup>CD25<sup>low </sup>CFSE-tagged in the presence of irradiated monocytes and anti-CD3 (0.3 pg / ml) and anti-hOX40 mAbs. After 3.5 days of culture, cell proliferation was evaluated for dilution of CFSE in cells by FACS (FIG. 16C).
FIGS. 17A and 17B show the identification of anti-hOX40 monoclonal antibodies that inhibit the generation of Tr1 cells and block the function of Treg FOXP3.<sup>+</sup>CD4<sup>+</sup>CD25<sup>high</sup>. T cells
FOXP3<sup>+</sup>CD4<sup>+</sup>CD127'CD25<sup>high</sup> recently rated (3.5 x 10<sup>4</sup>) were cultured with CD4 cells<sup>+</sup>CD25<sup>low</sup> tagged with CFSE (7x10<sup>4</sup>) in the presence of irradiated monocytes (7x10<sup>4</sup>, 6000 rad) and 0.3 pg / ml of anti-CD3 and various concentrations of anti-hOX40 monoclonal antibody. After 3
<img file="MX339964B_D0070.tif" />
at 4 days of culture, cell proliferation was evaluated in cells by flow cytometric analysis. The percentage of divided cells was indicated. Representative flow cytometric analyzes are shown in FIG. 17A. The data for 6 monoclonal antibodies are shown in FIG.
17B.
In the FIG experiment. 18, FOXP3 T cells<sup>+</sup>CD4<sup>+</sup>CD127CD25<sup>to</sup> recently rated (3.5 x 10<sup>4</sup>) were cultured with CD4 cells<sup>+</sup>CD25<sup>bath</sup> tagged with CFSE (7x10<sup>4</sup>) in the presence of irradiated monocytes (7x10<sup>4</sup>, 6000 rad) and 0.3 pg / ml anti-CD3 and various concentrations of monoclonal antibody 0X40. After 3-4 days of culture, cell proliferation was evaluated for dilution of CFSE stain in cells by FACS. The data is representative of two experiments. Antl-hOX40 monoclonal antibodies that do not inhibit Tr1 generation but block Treg function were identified
FOXP3<sup>+</sup>CD4<sup>+</sup>CD25<sup>high</sup>.
In the FIGS experiment. 19A and 19B, CD4 T cells<sup>+</sup>CD25<sup>a ,, a</sup> derived from lymphoma were cultured with CD4 cells<sup>+</sup>CD25<sup>low </sup>tagged with CFSE (7x10<sup>4</sup>) isolated from a healthy donor in the presence of irradiated allogeneic monocytes (7x10<sup>4</sup>, 6000 rad) and 0.3 micrograms / ml of anti-CD3 and 25 pg / ml of anti-hOX40 monoclonal antibody. After 3 to days of culture, cell proliferation was evaluated for dilution of CFSE by FACS. Representative FACS analyzes are shown in FIG. 19A and the data for all experiments are shown in FIG. 19B. I know
<img file="MX339964B_D0071.tif" />
discovered that hOX40 agonist antibodies blocked uXI ^ TUffiSKMn
D £ INDUSTRIAL PROPERTY
<img file="MX339964B_D0072.tif" />
CD4<sup>+</sup>CD25<sup>high</sup> derived from lymphoma.
FIG. 20 shows the identification of 0X40 agonist antibodies that specifically bind to 0X40 of rhesus monkey and human. The rhesus monkey peripheral blood mononuclear cells were obtained by ficoll centrifugation. CD4 T cells<sup>+</sup> were obtained by CD4 microbeads. CD4 T cells<sup>+</sup> they were stimulated with 10 pg / ml of phaseolus vulgaris lectin (PHA). Two days after stimulation, cells were stained with anti-hOX40 mAbs followed by goat anti-mouse IgG-APC and CD69-PE.
106-317 served as a negative control. Six anti-hOX40 mAbs shown that strongly activate T cell proliferation could bind CD4 T cells<sup>+</sup> of rhesus monkey activated. These results indicate that the toxicity of these six anti-hOX40 monoclonal antibodies can be tested in monkeys.
Only seven out of 500 anti-human 0X40 positive clones obtained using conventional fusion protocols exhibited the activation properties of 0X40, including but not limited to, the ability to block IL-10 producing Tr1 generation and suppressor function of nTreg as described in Table 1.
TABLE 1
List of 0X40-specific monoclonal antibodies
<td></td><td>Monoclonal antibody clone</td><td>They block IL-10</td><td>Block nTreg</td>
<td> 1</td><td> 106-108</td><td></td><td></td>
<td> 2</td><td> 106-317</td><td></td><td></td>
<td> 3</td><td> 106-107</td><td></td><td></td>
<td> 4</td><td> 106-148</td><td></td><td></td>
<td> 5</td><td>119-204A</td><td></td><td></td>
<td> 6</td><td>119-220C</td><td></td><td> +</td>
<td> 7</td><td>119-33A</td><td></td><td> +</td>
<td> 8</td><td> 119-58</td><td></td><td> +</td>
<td> 9</td><td>119-181A</td><td></td><td> +</td>
<td> 10</td><td>119-157A</td><td></td><td> +</td>
<td> 11</td><td>120-140A</td><td></td><td> +</td>
<td> 12</td><td>119-8B</td><td> +</td><td></td>
<td> 13</td><td>119-173B</td><td> +</td><td></td>
<td> 14</td><td> 106-132</td><td> +</td><td> +</td>
<td> 15</td><td> 106-222</td><td> +</td><td> +</td>
<td> 16</td><td> 119-43</td><td> +</td><td> +</td>
<td> 17</td><td> 119-122</td><td> +</td><td> +</td>
<td> 18</td><td>119-69A</td><td> +</td><td> +</td>
<td> 19</td><td> 120-56</td><td> +</td><td> +</td>
<td> 20</td><td> 120-270</td><td> +</td><td> +</td>
IMPI
MEXICAN INSTITUTE OF PROPERTY
INDUSTRIAL
<img file="MX339964B_D0073.tif" />
Hybridoma clones 106-222 and 119-122 were selected based on three criteria
one. Inhibit Tr1 cell generation from CD4 + T cells (inducible Treg)
2. They reverse the suppressive function of nTreg FOXP3 cells<sup>+</sup>
<img file="MX339964B_D0074.tif" />
3. Exhibit Tr1 dose-dependent inhibition and inversion of Treg FOXP3 function<sup>+</sup> _______—
Chimeric and Humanized Antibodies
Humanization (also called reorganization or insertion of
CDR) is an established technique to reduce the immunogenicity of monoclonal antibodies from xenogeneic sources (including but not limited to rodents) and to enhance their activation of the human immune system. Although the mechanisms of production of genetically modified monoclonal antibody using molecular biology techniques are known, the simple insertion of the complementarity determining regions (CDRs) in human frameworks does not always reconstitute the binding affinity and specificity of the original monoclonal antibody.
To humanize an antibody, the design of the humanized antibody becomes the critical stage in reproducing the function of the original molecule. This design includes several options; CDR extensions, human scaffolds to use and substitution of rodent monoclonal antibody residues in human scaffold regions (retromutations). The positions of these retromutations have been identified primarily by sequence / structural analysis or by analysis of a homology model of the 3D structure of the variable region.
Recently, phage libraries have been used to vary amino acids at chosen positions. Similarly, many
Η ·, h „„, -, IMPI procedures have been used to choose the frames <sup>R r</sup> ° de la ra © rier> AO
INDUSTRIAL
<img file="MX339964B_D0075.tif" />
suitable in which to graft the rodent CDRs. Early experiments used a limited subset of well-characterized human monoclonal antibodies (frequently but not always where the structure was available), without considering sequence identity with the rodent monoclonal antibody (the so-called fixed framework procedure). Some groups use variable regions with high amino acid sequence identity with rodent variable regions (best fit or homology match); others use consensus or germline sequences while still others select fragments of the framework sequences within each light or heavy chain variable region of several different human monoclonal antibodies. There are also procedures for developed humanization which replace surface rodent residues with the most common residues found in human monoclonal antibodies ("recapped" or "plated") and those which use different definitions of the extensions of the CDRs. Humanized antibodies are described below. However, a chimeric antibody comprising the heavy and light variable regions of SEQ ID NOs: 4 and 10, or, SEQ ID NOs: 16 and 22 are also described herein.
IMPI
Humanized monoclonal antibodies Tgerc ^^ yaro ^^ tejF '^ murine anti-OX4Q antibody.
The isolated humanized anti-OX40 antibody may have a CDR1 variable heavy chain comprising the amino acid sequence of SEQ ID NO: 1 or 13. The isolated humanized anti-OX40 antibody may have a CDR2 variable heavy chain comprising the amino acid sequence of SEQ ID NO: 2 or 14. The isolated humanized anti-OX40 antibody may have a CDR3 variable heavy chain comprising the amino acid sequence of SEQ ID NO: 3 or 15.
The isolated humanized anti-OX40 antibody may have a CDR1 variable light chain comprising the amino acid sequence of SEQ ID NO: 7 or 19. The isolated humanized anti-OX40 antibody may have a CDR2 variable light chain comprising the amino acid sequence of SEQ ID NO: 8 or 20. The isolated humanized anti-OX40 antibody may have a variable CDR3 light chain comprising the amino acid sequence of SEQ ID NO: 9 or 21.
The isolated humanized anti-OX40 antibody can have a variable light chain comprising the amino acid sequence of SEQ ID NO: 11 or 23, or an amino acid sequence with at least 90 percent identity to the amino acid sequences of SEQ ID NO : 11 or 23. The isolated humanized anti-OX40 antibody may have a variable heavy chain comprising the amino acid sequence of SEQ ID NO .: 5 or 17, or an amino acid sequence with at least 90 percent identity to the amino acid sequences of SEQ ID NO: 5 or 17.
IMPI
MEXICAN INSTITUTE OF INDUSTRIAL I XOW.DAD
<img file="MX339964B_D0076.tif" />
Isolated humanized anti-OX40 antibody could be tone * · a variable light chain encoded by the nucleic acid sequence of SEQ ID NO: 12 or 24, or a nucleic acid sequence with at least 90 percent identity to the amino acid sequences from SEQ ID NO: 12 or 24. The isolated humanized anti-OX40 antibody may have a variable heavy chain encoded by a nucleic acid sequence of SEQ ID NO: 6 or 18, or a nucleic acid sequence with at least 90 percent identity to the amino acid sequences of SEQ ID NO: 6 or 18.
Expression of humanized anti-OX4Q antibodies
An antibody, or antibody portion, of the invention can be prepared by recombinant expression of immunoglobulin heavy and light chain genes in a host cell. To express an antibody recombinantly, a host cell was transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and, preferably, are secreted into the medium in which the host cells were grown, antibodies can be recovered from the medium. Standard recombinant DNA methodologies are used to obtain the antibody heavy and light chain genes, Incorporate these genes into the recombinant expression vectors and Introduce the vectors into host cells, such
<img file="MX339964B_D0077.tif" />
<img file="MX339964B_D0078.tif" />
such as those described in Sambrook, Fritsch and
OF INDUSTRIAL PROPERTY
Cloning; A Laboratory Manual, Second Edition, Coid Spring Harbor, NY, (1989), Ausubel, FM et al. (eds.) Current Protocols in Molecular Blology, Greene Publishing Associates, (1989) and in US Patent No.
4,816,397 to Boss et al.
Antibodies and antibody fragments and variants can be produced from a variety of animal cells, preferably mammalian cells, with murine and human cells being particularly preferred. Furthermore, recombinant DNA expression systems could include those that use host cells and expression constructs that have been genetically modified to produce high levels of a particular protein. Such host cells and expression constructs can include Escherichia coli; host expression constructs derived from plasmids or viruses (bacteriophage); yeast such as
Saccharomyces cerevisieae or Pichia pastoras harboring episomal or chromosomally integrated expression constructs; insect and virus cells such as Sf9 and baculovirus cells; and mammalian cells harboring episomal or chromosomally integrated expression constructs (including, but not limited to, retroviral) (such methods, for example, can be seen from Verma et al., J. Immunol. Methods 216: 165- 181,
1998). Antibodies can also be produced in plants (such methods, for example, can be seen from US Patent No. 6,046,037;
Ma et al., Science 268: 716-719, 1995) or by phage display technology (such methods, for example, can be seen from
OF INDUSTRIAL PROPERTY
<img file="MX339964B_D0079.tif" />
Immunol. 12: 433-455, 1994).
Human anti-OX40 antibodies that exhibit a level of activity and binding specificity / affinity can be further manipulated by standard recombinant DNA techniques, for example to convert variable region genes to antibody chain length gene. complete, to Fab fragment genes or to a scFv gene. In these manipulations, a DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked", as used in this context, is understood to mean that the two DNA fragments are joined so that the amino acid sequences encoded by the two DNA fragments remain framework.
In another aspect, isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3). Human heavy chain constant region gene sequences are known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242) and the DNA fragments spanning these regions can be obtained by PCR amplification
IMPI standard. The heavy chain constant region 'pwe ^ {j ^ c ^) g<sub>to</sub>
INDUSTRIAL
<img file="MX339964B_D0080.tif" />
constant of IgG-1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD and any allotypic variant herein as described in Kabat (, Kabat, E. A, et al.
(1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US
Department of Health and Human Services, NIH Publication No. 91-3242), but most preferably it is a constant region of lgG1 or lgG4. For a Fab fragment heavy chain gene, VH-encoding DNA can be operably linked to another DNA molecule encoding only the CH1 heavy chain constant region.
Furthermore, a humanized antibody bound to the surface antigen can interact with cells bearing FcR. Such interaction can produce effector function such as ADCC and / or improve signaling due to Fe-mediated crosslinking. The interaction can be beneficial or harmful to therapy. Such harmful side effects include chills, fever, hypotension, and in some cases, dyspnea (Thistlethwaite JR Jr., Cosimi AB, Delmonico FL, et al.).
Certain harmful effects can originate from the protein complex found on the surface of a T cell. In T cell activation, the protein complex becomes involved in the transduction of signals generated via an antigen receptor. In short, activation of T cells initiates a cascade of events which includes enhanced antigen receptor crosslinking. Reticulation of the receptor may contribute to strong mitogenic signaling leading to induction of
IMPISg
INSTITUTO MEXICANO certain cytokines such as tumor necrosis factor alpha (TNF ^ ¿| 0 | g ^ ue¡Q »2 (IL-2) and interferon gamma (IFN-y). These cytokines are» they generate in large quantities.
For example, anti-CD3 mAbs are currently used in the treatment of autoimmune disease including diabetes mellltus type I in which T cells mediate the attack against insulin-producing pancreatic islets (Kaufman A, and Herold K. Anti-CD3 mAbs for treatment of type 1 diabetes Diabetes Metab Res Rev 2009; 25: 302-306). AntiCD3 antibodies are known to inhibit lysis of targets by T cells and improve crosslinking of the antigen receptor CDR. Furthermore, along with its potent mitogenic activity, the anti-CD3 antibody is known to be a potent cytokine inducer, specifically, tumor necrosis factor alpha (TNFa), interleukin-2 (IL-2), and gamma interferon (IFN- and). The enormous release of cytokines, particularly TNF-α from T cells in response to the drug (Chatenoud L.) produces toxic effects. These undesirable side effects have been attributed to the crosslinking of T cells that carry CD3 molecules and cells that carry FcR that bind the Fe portion of the antibodies. Crosslinking activates both T cells and FcR-bearing cells leading to massive cytokine release as previously mentioned.
Similarly, undesirable potential side effects could result from using anti-OX40 antibodies. For example, anti-OX40 antibodies which bind cells expressing 0X40 also
IMPI can bind cells bearing FcR and activate prodCIfíESSíííüSi ^ o
INDUSTRIAL which may be beneficial or harmful to patients treated with the antibody. To overcome this potential problem, methods for mutating the FcR portion of anti-OX40 antibodies have been designed and presented herein to avoid toxic effects and provide mutations to the FcR portion which may be desirable.
The human lgG1 site that interacts with FcR is known (CD16, CD32, and CD64). Map the upper CH2 domain. The most important amino acids are the two Leu residues at positions 234 and 235. By mutating these two residues to two Ala residues, the interactions of IgG1 with all FcRs are abolished. Humanized antl-CD3 Incorporates these mutations (KuOKT3AA), is a much safer drug, and has a mechanism of action that is different from that of HuOKT3. See for example, US Patent No. 6,491,916, incorporated for reference in its entirety.
The positions of the AA mutant are shown as follows:
—A — P — E — L --- L — G — G — P— CH2 superior of wild type lGG1 —A — P — E — A — A — G — G — P — CH2 superior of IGG1 from Muíante AA
Hu222AA and Hu122AA described herein may contain these mutations. If the assay system contains cells that carry FcR, you can see the difference between the wild type and the AA mutant. Otherwise, the two antibodies must behave the same.
Isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a
<img file="MX339964B_D0081.tif" />
IMPI
<img file="MX339964B_D0082.tif" />
Fab light chain) by operably linking the C8®0 £ S8t & A8e DNA
INDUSTRIAL another DNA molecule that encodes the light chain constant region, CL. Human light chain constant region gene sequences are known in the art (see for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242) and the DNA fragments spanning these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.
To create a scFv gene, the DNA fragments that encode
VH and VL are operably linked to another fragment encoding a flexible linker, for example, encoding the amino acid sequence (Gly.sub.4-Ser) .sub.3, so that the VH and VL sequences can be expressed as a contiguous single chain protein, with the VL and VH regions linked by the flexible linker (see eg Bird et al. (1988) Science 242: 423-426; Huston et al. (1988) Proc. Nati. Acad. Sci. USA 85: 5879-5883; McCafferty et al., Nature (1990) 348: 552-554.
Amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody.
Amino acid sequence variants of the antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, or by peptide synthesis. Such modifications include, for
IMPI
<img file="MX339964B_D0083.tif" />
For example, deletions of, and / or insertions in and / or substitute TSSe® within the amino acid sequences of the antibody. Any. combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid alterations can be introduced into the amino acid sequence of the target antibody at the time the sequence is made.
A useful method for identifying certain antibody residues or regions that are preferred locations for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244: 1081-1085. Here, a target residue or group of residues (eg, charged residues such as arg, asp, his, lys, and glu) is identified and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the Interaction of amino acids with antigen. Those amino acid locations that demonstrate functional sensitivity to substitutions are then refined by introducing additional or other variants at, or for, the substitution sites. Accordingly, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, random or wing-scanning mutagenesis is conducted at the target region or codon and the expressed immunoglobulins are selected for the desired activity.
<img file="MX339964B_D0084.tif" />
ΙΜΡΙ i IHSTITUTO MEXICANO
Amino- and / or carboxyl-terminal amino acid sequence inserts that vary in length from spray to.
polypeptides containing hundreds or more residues, as well as intersection inserts of single or multiple amino acid residues.
Examples of terminal inserts include an antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide. Other variants of Insertion of the antibody molecule include fusion to the N- or C-term of the antibody to an enzyme (eg, for ADEPT) or a polypeptide which Increases the serum half-life of the antibody. Another type of amino acid variant of the antibody alters the pattern original glycosylation of the antibody. Such alteration includes deletion of one or more carbohydrate portions found in the antibody, and / or addition of one or more glycation sites that are not present in the antibody.
Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. Sites of greatest interest for substitution mutagenesis include hypervariable strands, but FR alterations are also contemplated. Conservative substitutions are shown in Table 1 of the
United States No. 7,812,133, Col. 43, Is. 55 to Col. 44, I. 49, incorporated herein by reference, and under the heading of "preferred substitutions. If such substitutions result in a change in biological activity, then the most substantial changes, called
IMPI ^^ Mexican Institute Exemplary Substitutions ”in Table 1, or as described below with reference to amino acid classes, products may be introduced and selected.
Furthermore, substantial modifications in the biological properties of the antibody are made by selecting substitutions that differ significantly from their effect in maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a helical conformation or leaf, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. Naturally occurring residues are divided into groups based on common side chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, le; (2) neutral hydrophilic: Cys, Ser, Thr, Asn,
Gln; (3) acids: asp, glu; (4) basic: his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatics: trp, tyr, phe. Non-conservative substitutions will result in the exchange of a member of one of these classes for another class.
To express the antibodies, or antibody portions described herein, the DNAs encoding full or partial length light and heavy chains, obtained as described above, are inserted into expression vectors such that genes are operably linked to sequences transcriptional or translational control. In this context, the term "operably linked" is intended to mean that an antibody gene is ligated into a vector such that the sequences of
<img file="MX339964B_D0085.tif" />
transcriptional and translational control within the 'SWíkafc> SH¡wen <sup>one and</sup> THE PROPERTY
INDUSTRIAL proposed function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into a separate vector or, more typically, both genes are inserted into the same expression vector. Antibody genes are inserted into the expression vector by standard methods (eg, ligation of complementary restriction sites in the vector and antibody gene fragment, or blunt-ended ligation if no restriction sites are present).
As shown in FIG. 23, such a schematic structure of the IgG1 / kappa antibody expression vector Hu106-222. Proceeding clockwise from the Salí site at the top, the plasmid contains the heavy chain transcription unit that starts with the primary immediate early promoter of human cytomegalovirus (CMV) and enhancer (CMV promoter) to initiate transcription of the antibody heavy chain gene. The CMV promoter is followed by the VH exon, a genomic sequence containing the human gamma-1 heavy chain constant region that includes the CH1, hinge, CH2, and CH3 exons with the introns involved, and the polyadenylation site that follow the exon
CH3. Following the heavy chain gene sequence, the light chain transcription unit begins with the CMV promoter, followed by the VL exon and a genomic sequence containing
<img file="MX339964B_D0086.tif" />
human kappa chain constant (CL) with part of the intron that precedes it, and the polyadenylation site that follows the exon CL. The light chain gene is then followed by the SV40 early promoter (SV40 promoter), the E. coli xanthine guanine phosphoribosyl transferase (gpt) gene, and a segment containing the SV40 polyadenylation site (poly site (A) of SV40). Finally, the plasmid contains a part of the plasmid pUC19, which comprises the bacterial origin of replication (pUC ori) and the beta-lactamase gene (beta lactamase). The locations of relevant restriction enzyme sites are shown in the figure.
The recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide binds to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).
As noted above, in addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals) that control the
<img file="MX339964B_D0087.tif" />
INDUSTRIAL PROPERTY transcription or translation of gene regulatory chain sequences are described, for example, in Goeddel; Gene
Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). It will be appreciated that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the desired level of expression of the protein, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that drive high levels of protein expression in mammalian cells, such as cytomegalovirus (CMV) derived promoters and / or enhancers (such as the CMV promoter / enhancer). ), Simian Virus 40 (SV40) (such as the SV40 promoter / enhancer), adenoviruses (eg, Adenovirus Major Late Promoter (AdMLP)) and polyoma. For further description of viral regulatory elements, and sequences thereof, see for example, US Patent No. 5,168,062 to Stinski, US Patent No. 4,510,245 to Bell et al. and US Patent No. 4,968,615 to Schaffner et al., US Patent No. 5,464,758 to Bujard et al. and United States Patent No. 5,654,168 to Bujard et al.
In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate vector replication in host cells (eg, origins of replication) and selectable marker genes.
<img file="MX339964B_D0088.tif" />
iNSTrnr
OF INDUSTRIAL PROPERTY
<img file="MX339964B_D0089.tif" />
Selectable facilitates selection of host cells into which the vector has been introduced (see for example, US Patent Nos. 4,399,216, 4,634,665, and 5,179,017, all from Axel et al). For example, the selectable marker gene typically confers resistance to drugs, such as G418, hlgromycin, or methotrexate, in a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr.sup. Host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).
For expression of the light and heavy chains, the expression vectors encoding the heavy and light chains are transfected into a host cell by standard techniques. The various forms of the term "transfection" are proposed to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, eg, electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and Similar. Although it is theoretically possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells, the expression of antibodies in eukaryotic cells, and most preferably mammalian host cells, is most preferred because such eukaryotic cells, and in particular cells In mammals, prokaryotic cells are more likely to assemble and secrete an immunologically active and appropriately folded antibody. Host cells
<img file="MX339964B_D0090.tif" />
M INDUSTRIAL PROPERTY expression of the recombinant antibodies described herein include Chinese Hamster Ovary cells (CHO cells) (such as dhfrCHO cells, described in Urlaub and Chasin, (1980) Proc. Nati. Acad. Sci. USA
77: 4216-4220, used with a selectable DHFR marker, for example, as described in RJ Kaufman and PA Sharp (1982) Mol. Biol. 159: 601-621), NSO myeloma cells, COS cells, and SP2 cells. When the antibody genes encoding recombinant expression vectors are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells or secretion of the antibody into the host cells. culture medium in which the host cells grow. Antibodies can be recovered from the culture medium using standard protein purification methods.
Host cells can also be used to produce intact antibody portions, such as Fab fragments or scFv molecules. Variations in the above procedure will be understood to be within the scope of the present invention. For example, it may be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an antibody of this invention. Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both light and heavy chains that are not necessary for binding to 0X40. The expressed molecules of such
<img file="MX339964B_D0091.tif" />
truncated DNA molecules are also spanned so
Invention. Furthermore, blfunclonal antibodies can be produced in which one heavy and one light chain are in an antibody of the invention and the other heavy and light chain is specific for an antigen other than
0X40 by crosslinking an antibody of the Invention to a second antibody by standard chemical crosslinking methods.
Pharmaceutical Compositions and Pharmaceutical Administration
The antibodies and antibody portions of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises an antibody or antibody portion of the invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" Includes each and every solvent, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyols such as manltol, sorbltol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may additionally comprise
IMPI minor amounts of auxiliary substances such as ageprfq ^^ jm ^ ota
INDUSTRIAL or emulsifiers, preservatives or buffers, which improve shelf life or effectiveness of the antibody or antibody portion.
The antibodies and antibody portions of the invention can be incorporated into a pharmaceutical composition suitable for parenteral administration (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). The compositions of this invention can be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (eg, injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends on the proposed mode of administration and therapeutic application. Typical compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. The antibody can be administered by intravenous injection or infusion, or intramuscular or subcutaneous injection.
The route and / or mode of administration will vary depending on the desired results. In certain embodiments, the active compound can be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biocompatible polymers can be used
<img file="MX339964B_D0092.tif" />
<img file="MX339964B_D0093.tif" />
biodegradable, such as ethylene vinyl acetate, loiMftiifridb 'INSTITUTO MEXICANO; PROPERTY polyglycolic, collagen, polyorthoesters, and polylactic acid. Mucfios<sup>1</sup> for the preparation of such formulations they are patented or generally known to those skilled in the art. See, for example, Sustained and 5 Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel
Dekker, Inc., New York, 1978.
Supplemental active compounds can also be incorporated into the compositions. In certain embodiments, an antibody or antibody portion of the invention is co-formulated with and / or co-administered with one or more additional therapeutic agents that are useful in treating disorders in which inactivation of 0X40 is deleterious. For example, an anti-OX40 antibody or antibody portion of the invention may be co-formulated and / or co-administered with one or more additional antibodies that bind other targets (eg, antibodies that bind other cytokines or that bind cell surface molecules ). Furthermore, one or more antibodies of the invention can be used in combination with two or more of the above therapeutic agents. Such combination therapies can advantageously use lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies. It will be appreciated by the skilled practitioner that when the antibodies of the invention are used as part of a combination therapy, a lower dosage of antibody may be desirable than when the antibody is only administered to a subject (eg, an effect
IMPI
<img file="MX339964B_D0094.tif" />
therapeutic synergist can be achieved through dej
INDUSTRIAL combination which, in turn, allows the use of a lower dose of the antibody to achieve the desired therapeutic effect.
The antibodies described herein, or antigen binding portions thereof, can be used alone or in combination to treat such diseases. It should be understood that these antibodies or antigen binding portion thereof can be used alone or in combination with an additional agent, eg, a therapeutic agent, the additional agent is selected by the skilled artisan for its intended purpose.
For example, the additional agent may be a therapeutic agent recognized in the art as being useful in treating the disease or condition that is treated by the antibody noted herein. The additional agent may also be an agent which imparts a beneficial attribute to the therapeutic composition eg an agent which governs the viscosity of the composition.
The pharmaceutical compositions described herein can include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antibody portion of the invention. A "therapeutically effective amount" refers to an effective amount, dosages and per time period necessary to achieve the desired therapeutic result. A therapeutically effective amount of the antibody or antibody portion can vary according to factors such as the disease state, age, sex, and weight of the individual; and the
100 capacity of antibody or portion of antibody for pn
<img file="MX339964B_D0095.tif" />
PE INDUSTRIAL RROHEDAD
<img file="MX339964B_D0096.tif" />
desired in the individual. A therapeutically effective amount is also one in which any of the toxic or deleterious effects of the antibody or antibody portion is offset by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an effective amount, at dosages, and for periods of time necessary to achieve the desired prophylactic result.
Dosage regimens can be adjusted to provide the optimal desired response (eg, a therapeutic or prophylactic response). For example, a single bolus may be administered, various divided doses may be administered over time, or the dose may be reduced or increased proportionally as indicated by the demands of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in unit dosage forms for easy administration and uniformity of dosage. The unit dosage form as used herein refers to physically discrete units suitable as unit dosages for mammalian subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification of unit dosage forms is dictated by and is directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the
101
<img file="MX339964B_D0097.tif" />
composition technique of such an active compound for sensitivity in individuals.
EXAMPLE I
The chimeric and humanized IgG1 / kappa 106-222 monoclonal antibodies (Ch222 and Hu222, respectively) were purified from culture supernatants of the corresponding NSO stable transfectants using a Protein A column as described in
Appendices A and B. Hu222 was eluted from the column in two different ways. Briefly, Hu222 Lot I was eluted with a low pH buffer and Lot II with Pierce Light Ag / Ab Elution Buffer. Hu222 performance was better when the low pH buffer was used for elution. Ch222 was eluted from the column with Light Ag / Ab Elution Buffer.
The purified Hu222 Lot I and II antibodies were characterized by SDS-PAGE together with mouse 106-222 according to standard procedures. Five pg of each antibody were analyzed under reducing conditions. As shown in FIG. 21, each of the Hu222 Lot I and II antibodies is comprised of a heavy chain with a molecular weight of approximately 50 kD and a light chain with a molecular weight of approximately 25 kD. The purity of the Hu222 Lot I and II antibodies appears to be more than 95%.
Endotoxin contamination in antibodies
102 humanized was analyzed with Lisad QCL-1000 kit
<img file="MX339964B_D0098.tif" />
<img file="MX339964B_D0099.tif" />
DISTtTU. _. . _
M INDUSTRIAL PROPERTY
Limulus de Lonza (LAL). Endotoxin level was less than 0.5 EU / mg protein for both Hu222 Lot I and Lot II antibodies.
Characterization of Hu106-222 to link L / OX4Q cells
Antibody binding 106-222, Ch 106-222 and mouse Hu 106-222 to 0X40 was examined in a FACS binding assay with L / hOX40 cells essentially according to the protocol provided by Dr. Laura Bover. Antibodies bound to L / hOX40 cells were detected with PE-labeled goat anti-mouse IgG antibody (for mouse 106-222) or PE-labeled goat anti-human IgG antibody (for Ch106 and Hu106).
FIG. 22 shows analysis of mouse 106-222, Ch106 and Hu106-222 (Lot II) antibodies to bind UOX40 cells. The Hu106-222 titration curve (Lot II) was almost identical to that of Ch106-222, indicating that the binding affinity of mouse 106-222 antigen is retained in Hu 106-222. The titration curve of mouse 10-222 was similar to that of Ch106 and Hu106; however, due to the difference in secondary antibodies, the data only indicates that the affinity of mouse 10 620 222 is similar to that of Hu106-222.
FIG. 24 shows the comparison between Hu106222 Lot I and II antibodies to bind L / hOX40 cells. Although further analysis is necessary, the affinity of the two batches of Hu-106-222 appeared to be similar, if not
103 identical to each other. Therefore, the acid elution of Hu106 from protein A does not appear to affect its affinity.
<img file="MX339964B_D0100.tif" />
Ch106-222 purification
The stable NSO C8 transfectant grew in 500 ml of
Invitrogen SFM hybridoma in a rotary culture bottle until exhaustion. The culture was spun in Corning 250 ml Centrifuge Tube (Cat # 430776) in Beckman Coulter Allegra X-12R Centrifuge (2000 RPM for 15 min). The culture supernatant was loaded onto a GE column
Healthcare HiTrap MabSelect SuRe 1 ml (Cat # 11-034-95) using a Pharmacia P1 pump. The column was washed with Tris buffered saline (Pierce, Cat # 28379) and eluted with Pierce Light Ag / Ab Elution Buffer (Cat # 21027). Fractions (approximately 1 ml) were collected and their OD was read at 280 nm.
_
<td>Fraction #</td><td>OD at 280 nm</td>
<td> 3</td><td> 0.12</td>
<td> 4</td><td> 0.30</td>
<td> 5</td><td> 0.18</td>
<td> 6</td><td> 0.11</td>
Fractions 3 to 6 were pooled (volume = 3.0 ml, OD at 280 20 nm = 0.14). The pooled fractions were desalted on a column of 10 ml Sephadex G25 medium in PBS. Fractions of 1 ml were collected.
104
IMPIS
<td>Fraction #</td><td>OD at 280 nm</td><td>Ib</td>
<td> 5</td><td colspan="2"> 0.09</td>
<td> 6</td><td colspan="2"> 0.19 -</td>
<td> 7</td><td colspan="2"> 0.12</td>
<td> 8</td><td colspan="2"> 0.12</td>
<td> 9</td><td colspan="2"> 0.00</td>
MEXICAN INSTITUTE · · -ROPieDAD
<img file="MX339964B_D0101.tif" />
Fractions 6 to 9 were pooled (volume = 3.0 ml, OD at 280 nm = 0.11). The pooled fractions were dialyzed overnight in PBS. After dialysis, the volume was 3.0 ml and the OD at 280 nm was 0.19. This preparation is called Ch106, lot 8/31/09, with a concentration of 0.13 mg / ml
Purification of Hu 106-222
The stable NS0 1-C6 transfectant grew in 500 ml of
Invitrogen SFM hybridoma in a rotary culture bottle until exhaustion. The culture was spun in Corning 250 ml Centrifuge Tube (Cat # 430776) in Beckman Coulter Allegra X-12R Centrifuge (2000 RPM for 15 min).
Lot 1: 150 ml of the culture supernatant was loaded onto a 1 ml GE Healthcare HiTrap MabSelect SuRe column (Cat # 11-03495) using a Pharmacia P1 pump. The column was washed with PBS and the bound antibody was eluted with 0.1 M glycine-HCI, 0.1 M NaCI (pH 3.0). The eluted fractions (1 ml each) were collected in tubes containing 50 pl of 1MTris-HCI (pH 8.0).
105
5je<sup>-</sup>»- .*·
<img file="MX339964B_D0102.tif" />
flMPÍ! »· MÜUCANC INSTITUTE
<td>Fraction #</td><td>OD at 28Chim 'Ί</td><td rowspan="2">TFtAL -</td>
<td> 2</td><td> 0.88</td>
<td> 3</td><td> 2.84</td><td rowspan="4"></td>
<td> 4</td><td> 1.29</td>
<td> 5</td><td> 0.63</td>
<td> 6</td><td> 0.18</td>
Fractions 2 to 5 were pooled (volume = 4.2 ml, OD at 280 nm = 1.59). The pooled fractions were dialyzed overnight in PBS. After dialysis, the volume was 4.2 ml and the OD at 280 nm was 1.54. The antibody solution (lot 9/18/09 I; 1.1 mg / ml) was filter sterilized.
Lot II:
The remaining culture supernatant (350 ml) was loaded onto a 1 ml GE Healthcare HiTrap MabSelect SuRe column using a Pharmacia P1 pump. The column was washed with Tris buffered saline and eluted with Light Ag / Ab Elution Buffer. Fractions (approximately 1 ml) were collected and their OD was read at 280 nm.
<td>Fraction #</td><td>OD at 280 nm</td>
<td> 2</td><td> 0.12</td>
<td> 3</td><td> 0.85</td>
<td> 4</td><td> 2.17</td>
<td> 5</td><td> 1.47</td>
<td> 6</td><td> 1.02</td>
<td> 7</td><td> 0.81</td>
<td> 8</td><td> 0.66</td>
<td> 9</td><td> 0.54</td>
<td> 10</td><td> 0.44</td>
<td> 11</td><td> 0.46</td>
Fractions 3 to 7 were pooled (volume = 4.2 ml, OD at 280 nm = 1.22). The column was washed again with buffered saline with
106
Tris and the antibody were eluted with 0.1M glycine-HCI, 0.1M
<img file="MX339964B_D0103.tif" />
<img file="MX339964B_D0104.tif" />
OF INDUSTRIAL PROPERTY
<img file="MX339964B_D0105.tif" />
examine whether the elution by Light Ag / Ab Elution Buffer was efficient.
<td>Fraction #</td><td>OD at 280 nm</td>
<td> 1</td><td> 0.05</td>
<td> 2</td><td> 0.05</td>
<td> 3</td><td> 1.23</td>
<td> 4</td><td> 0.49</td>
<td> 5</td><td> 0.10</td>
Fractions 3 to 7 eluted with Light Ag / Ab Elution Buffer were pooled and desalted on a column of medium
10 ml Sephadex G25 in PBS. Fractions of 1 ml were collected.
<td>Fraction #</td><td>OD at 280 nm</td>
<td> 4</td><td> 0.38</td>
<td> 5</td><td> 0.96</td>
<td> 6</td><td> 1.38</td>
<td> 7</td><td> 1,33</td>
<td> 8</td><td> 1.10</td>
<td> 9</td><td> 0.12</td>
Fractions 5 to 8 were pooled (volume = 4.0 ml, OD at 280 nm = 1.12). The pooled fractions were dialyzed overnight in PBS. After dialysis, the volume was 4.0 ml and the OD at 280 nm was 1.12. The antibody solution (lot 9/18/09 II; 0.8 mg / ml) was filter sterilized.
The High Salt Elution Method with Elution Buffer
Pierce's Light Ag / Ab was not as efficient as the low pH method to elute bound human lgG1 antibody from protein A column.
As the antibodies were not eluted in an acute peak with
Light Ag / Ab elution, it was necessary to group many fractions to
107
<img file="MX339964B_D0106.tif" />
IgG Collection Eluted and Desalting Clustered Fractions
OF INOWSTMA HOMEDAD
The poor elution profile with Light Ag / Ab Elution Buffer and the extra purification step affected the performance of the antibody. The high salt elution method was contemplated to be used only if the IgG to be purified is acid unstable.
EXAMPLE II
Purification of Ch119-122 and Hu119-122 antibodies
The chimeric IgG1 / kappa 119-122 monoclonal antibody (Ch119) was purified from the culture supernatant of the corresponding stable NSO transfectant (clone G11) grown in Hybridoma-SFM medium (Invitrogen) using a protein A column. After elution With Pícerce Light Ag / Ab Elution Buffer, the Ch119 buffer was exchanged to PBS by gel filtration and then dialysis. The Ch119 concentration was 0.21 mg / ml.
The humanized IgG1 / kappa 119-122 monoclonal antibody (Hu122) was purified from the culture supernatant of the corresponding stable NSO transfectant (clone 2F5) grown in Hybridoma-SFM medium using a protein A column. Hu106-222 was eluted from the column with low pH buffer was neutralized with 1M Tris-HCI (pH 8.0), and dialyzed in PBS. The Hu122 concentration was 1.6 mg / ml.
The purified Hu106-222 was characterized by SDS-PAGE together with
108
<img file="MX339964B_D0107.tif" />
119-122 mouse according to standard procedures <eBVa; iflcoJ
I each antibody was analyzed under reducing conditions'<sup>ND</sup>Sorno is shown in FIG. 25, Hu119-122 is comprised of a heavy chain with a molecular weight of approximately 50 kD and a light chain with a molecular weight of approximately 25 kD. The purity of Hu119 appeared to be more than 95%.
Characterization of Hu119-122 to bind L / hQX40 cells
Binding of mouse 119-122, Ch119-122 and Hu119-122 antibodies to 0X40 was examined in a FACS binding assay with L / OX40 cells essentially according to the protocol provided by Dr. Laura Bover. Antibodies bound to L / OX40 cells were detected with PE-labeled goat anti-mouse IgG antibody (for mouse 119-122) or PE-labeled goat anti-human IgG antibody (for
Ch119-122 and Hu119-122).
FIG. 26 shows the result of the FACS analysis. The Hu119-122 titration curve was similar to that of Ch119-122, suggesting that the antigen binding affinity of mouse 119-122 is retained in Hu119122. However, MCF values at higher antibody concentrations of Ch109-122 and Hu119-122 do not fall straight on the corresponding curves. After adjusting the experimental conditions, the FACS analysis should be repeated.
109
EXAMPLE III
IMPI
MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY, Χά
<img file="MX339964B_D0108.tif" />
To assess the ability of our humanized anti-human 0X40 antibodies to enhance T cell proliferation, proliferation assays were performed using anti-CD3 coated CD32-L cells and CD4 T cells.<sup>+</sup> na'ive recently classified. FIG. 27 shows that the humanized anti-human 0X40 mAb clone 119-122 (Hu122), and its mutated FcR-binding antibody (Hu122-AA) enhanced CD4 T-cell proliferation.<sup>+</sup> na'ive. Hu122 produced better T cell stimulating activity compared to parental mouse (mouse 122) anti-human mAb 0X40 (FIG. 27).
The mutated FcR binding humanized anti-human 0X40 mAb clone 106-222 (Hu222-AA) and chimeric anti-human 0X40 mAb clone 106-222 (Ch222) enhanced CD4 T cell proliferation.<sup>+</sup> na'ive stimulated with anti-CD3. These antibodies have similar stimulatory activity compared to the parent mouse anti-human mAb 0X40 (mouse 106222). However, fully humanized anti-human Ab 0X40, Hu106, does not enhance T cell proliferation (FIG. 28).
To assess the ability of humanized anti-human 0X40 antibodies to block CD4 regulatory T-cell (Tregs) suppressive function<sup>+</sup>, proliferation assays were performed using CD4 T cells<sup>+</sup> Recently Classified Naive and CD4 Tregs<sup>+</sup>CD25<sup>high</sup>CD127<sup>low</sup>. Ch122 chimeric antibody and mutated humanized antibody of
110 Fe binding (Hu122-AA) exhibited better potency than g [dML
INSTITUTO MEXICANO 1% ^ D £ LA PK0fl £ 3A0 human parental mouse (122 mouse) in blockade *<sup>!</sup>T £ FTuncioTreg CD4 suppressor<sup>+</sup> (FIGs. 29A and 29B).
In the FIG experiment. 27, naive T cells
CD4<sup>+</sup>CD25<sup>bath</sup>CD127<sup>+</sup>CD45ROCD45RA<sup>+</sup> Recently classified, they were stimulated with CD32-expressing L cells (CD32-L) coated with 4 concentrations of anti-CD3 antibodies plus 2 pg / ml of anti-human Ab 0X40 clone 119 antibodies or control antibodies. Three days after challenge, tritium radioisotope was added and cultured for an additional 16-18 hours prior to cell harvest. The data is representative of two donor experiments. The hOX40 ligand expressing CD32-L cells (CD32-L / hOX40L) serves as a positive control, whereas human and mouse lgG1 serve as negative controls.
In the FIG experiment. 28, CD4 T cells<sup>+</sup> Newly classified naves were stimulated with CD32-L cells coated with anti-CD3 antibody concentrations plus 2 pg / ml of anti-human mAb 0X40 clone 106222 antibodies (Hu222) or control antibodies. Three days after stimulation, the tritium radioisotope was added and cultured for an additional 16-18 hours prior to cell harvest. The data is representative of two donor experiments. CD32-L / hOX40L serves as a positive control, whereas human and mouse lgG1 serve as negative controls.
In the FIGS experiment. 29A and 29B, CD4 T cells
111 recently classified naíve were cultivated in the pr <
MEXICAN INSTITUTE Df THE INDUSTRIAL FRWPIEDAD
<img file="MX339964B_D0109.tif" />
CD4<sup>+</sup>CD25<sup>to</sup>CD127<sup>baia</sup> at three effector Tregs: T ratios and stimulated with CD32-L cells coated with 0.2 pg / ml anti-CD3 antibody plus 10 pg / ml anti-human mAb 0X40 clone 119-122 antibody or control antibody. Three days after stimulation, the tritium radioisotope was added and cultured for an additional 16-18 hours prior to cell harvest. The data is representative of three experiments. CD32-L / hOX40L serves as a positive control, whereas human and mouse lgG1 serve as negative controls.
EXAMPLE IV
Since the antibodies will find total peripheral blood mononuclear cells (PBMCs) when administered to patients via intravenous injection, the ability of our anti-human 0X40 antibodies to stimulate T cell proliferation was tested using PBMCs as antigen presenting cells (APCs) ) our proliferation tests. However, highly variable data was obtained with our mouse anti-human 0X40 mAbs when using PBMCs as
APCs that are not seen when monocytes are used as APCs, suggesting that our antibodies require some form of crosslinking for activity.
To test this possibility, the plates were coated with our mAbs
0X40 anti-human and anti-CD3, were washed, and used to stimulate
112
IMPI CD4 T cell proliferation<sup>+</sup> or CD8<sup>+</sup> in the absence of céted ^ g><sub>?</sub>^ g ^ j ^ gri
INDUSTRIAL
<img file="MX339964B_D0110.tif" />
FIGS. 30A-30C show results that 0X40 anti-human antibodies enhance CD4 T-cell proliferation<sup>+</sup> and CD8 +.
1x10<sup>5</sup> CD4 naive T cells<sup>+</sup>CD25<sup>low</sup>CD45ROCD45RA<sup>+ </sup>recently classified (FIG. 30A) or CD3 T cells<sup>+</sup>CD8<sup>+</sup> (FIG. 30B) were stimulated with plaque-binding anti-CD3 (3 pg / ml) and mouse anti-human 0X40 mAb (2 pg / ml). Tritiated thymidine was added on the third day of culture and the cells were harvested after another 15 hours of incubation. T cell proliferation was evaluated by thymidine incorporation. Anti-human 0X40 mAbs were derived from three hybridoma fusions. The numbers after the fusion number denote a specific antibody. Mouse IgG1 and 119-42 served as negative controls. Each treatment was performed in triplicate. Representative data from 4 T-cell donors is shown (FIG. 30C) All three versions of humanized anti-human 0X40 mAbs [Hu106-222 and Hu119-122; Hu106-222AA and Hu119122AA (AA denotes that two of the Fe binding residues were mutated to the amino acid alanine); and Ch 119-122 (similar to humanized 119-122 except that the "paratope" mouse variable region was maintained)] stimulated proliferation of CD4 T cells<sup>+</sup> naive. Anti-CD28 served as a positive control.
FIGS. 30A and 30B show that plaque-binding mouse anti-human 0X40 mAbs potently stimulate proliferation of CD4 T cells<sup>+</sup> naive and CD8 T cells<sup>+</sup> for a range of 10 to
113 times. Studies were extended to our mAbs
<img file="MX339964B_D0111.tif" />
DELA INDUSTRIAL PROPERTY humanized and it was found that the three versions of our humanized antibodies, if they are completely humanized, chimeric or have AA mutants in which the residues responsible for binding to the receptor of
Fe were altered to alanine, they were potent stimulants of CD4 T cell proliferation<sup>+</sup> naíve (FIG. 30C).
FIGS. 31A and 31B show that humanized and mouse anti-human 0X40 antibodies require crosslinking to enhance T cell proliferation. CD4 T cells<sup>+</sup> Newly classified navi were stimulated with plaque-binding anti-CD3 (3 pg / ml) plus soluble or plaque-binding humanized anti-human 0X40 mAbs (2 pg / ml) in the absence of helper cells. Tritiated thymidine was added on the third day of the culture and the cells were harvested after another 15 hours of incubation. T cell proliferation was evaluated by thymidine incorporation. Mouse IgG1 and anti-CD28 served as negative and positive controls, respectively. Representative data from two donors are shown. CD4 T cells<sup>+</sup> naive were stimulated with anti-CD3-binding plaque in the absence of helper cells. The following day, anti-human mAb 0X40 119-122 (2 pg / ml) was added alone or in combination with an equal amount of a secondary FC antibody. Cell proliferation was evaluated as described in FIG. 31A.
The potency of our humanized anti-human 0X40 mAbs Hu106-222 and Hu119-122 was comparable to that of anti-CD28. In
114 »Α>». ·
<img file="MX339964B_D0112.tif" />
<img file="MX339964B_D0113.tif" />
instituto mexícaí * DE la FSOni.PAI> industrial contrast, when the anti-human 0X40 antibody r
<img file="MX339964B_D0114.tif" />
T cell culture, the stimulatory effect was abolished. (FIG. 31 A). However, when soluble anti-human mAb 0X40 119-122 was added together with a F (ab ') fragment goat anti-mouse IgG<sub>2</sub>, the Fe fragment specific secondary antibody, the stimulatory effect was restored (FIG. 31B). These results demonstrate that anti-human 0X40 mAbs require crosslinking for their biological activities.
To evaluate the ability of our 0X40 anti-human agonist mAbs to block Tregs suppressive function <sub>+</sub> high
CD4 CD25 CD12T, proliferation assays were performed in the presence of CD4 effector T cells<sup>+</sup>CD25<sup>ba) a</sup>CD127 + CD45RO + (Teff) and nTregs CD4<sup>+</sup>. Using our plate binding system in which anti-human 0X40 mAbs along with anti-CD3 were plated on a plate and in the absence of helper cells, twelve (222, 132, 8B, 33A, 43, 58B, 122, 157A , 173B, 220C,
140A, 270) of our mouse anti-human 0X40 mAbs potently inhibited nTreg suppression (FIGS. 32A and 32B). Although the ratio of nTregs to effector T cells used in these assays was 1: 1, these antibodies were able to stimulate effector T cells to proliferate 10 to 35 percent above the percentage achieved by effector T cells in the absence of nTregs. Humanized anti-human 0X40 mAbs also reversed the suppressive function of nTregs to similar levels (FIG. 32C). These results taken together suggest that mouse anti-human 0X40 mAbs are potent 0X40 stimulants, resulting in
115 Significant improvement in proliferative suppressive function of nTreg. Furthermore, humanized maintained potent parental mouse antibodies.
cell »T
<img file="MX339964B_D0115.tif" />
OF THE INDUSTRIAL FROFIEDAD
<img file="MX339964B_D0116.tif" />
mAbs 0X40 anti-human biological activities of their
FIGS. 32A-32C show that anti-human 0X40 mAb blocks Tregs CD4 activity<sup>+</sup>FOXP3<sup>+</sup>n. CD4 effector T cells<sup>+</sup>CD25'CD45RO<sup>+</sup> tagged with CFSE and Tregs CD4<sup>+</sup>FOXP3<sup>+</sup> they were derived from the same healthy donor. T cells were stimulated with soluble antiCD28 (0.5 pg / ml) and anti-plaque-binding CD3 (3 pg / ml) and anti-human 10X40 mAbs (2 pg / ml). The proliferation of effector T cells was evaluated by flow cytometry for dilution of CFSE. The ratio of nTregs to effector T cells was 1: 1. Mouse IgG1 served as a negative control. CD4 T cells<sup>+</sup> naive served as control T cells to demonstrate specific inhibition of effector T cell proliferation by nTregs. The
FIG. 32A is representative FACS data showing proliferation of effector T cells in the presence of CD4 T cells<sup>+</sup> naíve, nTregs or nTregs plus mAb 0X40 anti-human 119-33A. FIG. 32B shows the percentage of effector T cell proliferation in the presence of nTregs after treatment with a mouse anti-human 0X40 mAb (20 tested). FIG. 32C shows that all three versions of humanized anti-human 0X40 mAbs restored effector T-cell proliferation.
A recent report suggests that 0X40 activation may induce apoptosis of a human T cell line that expresses 0X40.
116
<img file="MX339964B_D0117.tif" />
INSTITUTO MEXICANO D £ LA PROPIEDAD (Yoshiaki Takahashi et al., 200B, Aids Research and hu / n
Therefore the effect of irfcremental concentrations on the mAb was tested.
0X40 anti-human 106-222 plus a low fixed dose of anti-CD3 in the survival of three subsets of T cells in the presence of monocytes.
FIG. 33A shows that high concentrations of anti-human 0X40 mAb 106-222 (20-30 pg / ml) preferentially kill activated FOXP3 * nTregs while CD4 T cells<sup>+</sup> Naí've and memory triggered were either resistant or less susceptible to this effort. To test whether anti-human mAb 0X40 acts directly on Tregs to induce cell death, further experiments were performed in the absence of helper cells. FIG. 33B shows that strong 0X40 signaling in combination with anti-CD3 specifically kills nTregs in the absence of helper cells. To confirm whether anti-human mAb 0X40 mediated annihilation effects mimic 0X40 activation by natural 0X40 ligand, a mouse fibroblast L cell line overexpressing hOX40L was used and used to stimulate nTregs in the presence of a low dose anti-CD3 and similar annihilation effects were obtained in Tregs (FIG. 33C). These results suggest that strong activation of 0X40 kills Tregs cells expressing 0X40.
Specifically, FIGS. 33A-33C show that the high concentration of anti-human 0X40 mAb preferentially kills Tregs F0XP3<sup>+</sup>. In FIG. 33A, T cell subsets (naí've,
CD4<sup>+</sup>CD25<sup>baia</sup>CD127<sup>+</sup>CD45R0CD45RA<sup>+</sup>;
memory,
117
CD4<sup>+</sup>CD25<sup>low</sup>CD127<sup>+</sup>CD45RACD45RO<sup>+</sup>; and nTregs, QD-
<img file="MX339964B_D0118.tif" />
OF INDUSTRIAL PROPERTY were grown with an equal ratio of CD14 monocytes<sup>+</sup> in the presence of soluble anti-CD3 (0.3 pg / ml) and increased concentrations of mouse anti-human mAb 0X40 106-222. Cell viability was determined after 3 days of culture by flow cytometric analysis, synchronizing viable lymphocytes. Data from two T cell donors are shown. FIGS. 33B and 33C show that strong activation of 0X40 kills Tregs CD4<sup>+</sup>FOXP3<sup>+</sup>. FIG. 33B shows that CD4 Tregs<sup>+</sup>FOXP3<sup>+</sup> they were stimulated with plaque-binding anti-CD3 (2 pg / ml) plus soluble mAb 119-122 (30 pg per million cells) or mouse lgG1 control antibody. Live trypan blue negative cells after one day of culture were counted with a hemacytometer. FIG. 33C shows that CD4 Tregs<sup>+</sup>FOXP3<sup>+</sup> additional L cells or L cells expressing the hOX40 ligand (L / hOX40L) were stimulated with soluble anti-CD3 (0.2 pg / ml). Live cells were counted after one day of stimulation.
The objective is to determine if the anti-human mAb 0X40 acts directly on T cells to block the suppressive function of nTreg. The nTregs or CD4 effector T cells<sup>+</sup> Recently classified, they were preactivated overnight with anti-CD3 and then pulsed with anti-human 0X40 mAbs for 4 hours. Effector T cells were then washed, CFSE-tagged, and co-cultured with nTregs in the presence of an equal number of CD14 monocytes<sup>+</sup> and anti-CD3. Similarly, the pre-stimulated nTregs were washed and cultured with labeled effector T cells
118
<img file="MX339964B_D0119.tif" />
FIGS. 34A and 34B show that the QX40 mAbs were untreated with CFSE.
IMPI
<img file="MX339964B_D0120.tif" />
Humans act directly on T cells to block the suppressive function of Tregs. FIG. 34A shows that the anti-human mAb 0X40 acts directly on memory effector T cells to confer resistance to suppression by nTregs. CD4 memory T cells<sup>+ </sup>CD25<sup>low</sup>CD127<sup>+</sup>CD45RA CD45RO<sup>+</sup> they were stimulated with anti-CD3 plate binding (0.8 pg / ml) in culture medium (RPMI / 10% FCS / P / S plus IL-2 at 30 lU / ml) for 12 hours, then pulsed with mAb 0X40 anti-human (119-122,
22 pg per 0.5 million cells) in culture medium, for 4 hours, washed times, and 8x10<sup>4</sup> CFSE-labeled effector T cells were cultured with decreased nTregs ratios. The proliferation of effector T cells was evaluated by flow cytometry for dilution of CFSE. The anti-human mAb 0X40 acts on Tregs rendering them unable to suppress the proliferation of effector T cells (FIG. 34B). CD4 nTregs<sup>+</sup>CD25<sup>high</sup>CD127<sup>ba</sup>’<sup>to</sup> they were pre-stimulated with plate-binding anti-CD3 (2 pg / ml) in culture medium for 12 hours, then pulsed with an anti-human 0X40 mAb, 119-122 or 106222, or a control antibody, anti- Mouse ICOS or IgG1, as described in FIG. 34A, washed and cultured with CFSE-tagged memory effector T cells. The proliferation of effector T cells was evaluated by flow cytometry for dilution of CFSE.
Effector T cells treated with anti-human mAb 0X40 became resistant to suppression by nTreg cells. (FIG. 34A) In
119
<img file="MX339964B_D0121.tif" />
In contrast, proliferation of mouse effector T cells treated from mouse lgG1 remained susceptible to suppression by nTregs. FIG. 34B shows that nTregs treated with anti-human 0X40 mAbs were unable to suppress the proliferation of effector T cells. In contrast, nTregs treated with control antibodies, such as mouse antiICOS or IgG1, remained s uppressive. These results suggest that anti-human 0X40 mAbs act directly on both effector T cells and nTregs to restore proliferation of effector T cells.
EXAMPLE V
Preliminary preliminary data in vivo shows that the anti-human 0X40 antibody that works in mice improves T-cell expansion and tumor rejection in mice. It was previously shown that anti-human 0X40 mAb can specifically activate the NF-kB cascade in CD8 T cells<sup>+</sup> mouse transduced with human 0X40. To determine if the anti-hOX40 mAb can improve tumor rejection by promoting the survival of CD8 effector T cells<sup>+</sup> and clonal expansion in vivo, Pmel CD8 T cells<sup>+</sup> transgenes transduced with the luciferase gene and hOX40 were adaptively transferred into C57BL / 6 albino mice bearing un pigmented MC38 tumors. After adoptive transfer of the transduced T cells, the mice were treated with Abs. OX40 T cells were found to be significantly<sup>+</sup> luciferase * Pmel human migrate
120
<img file="MX339964B_D0122.tif" />
35B),
IMPie in the lung on day 4 in mice treated with fNDUSTWAL compared to mouse treated with lgG1 control antibody (FIG. Indicating that activation of hOX40 in mice promoted expansion of CD8 T cells<sup>+</sup>. On day 8 (data not shown) and day 12 after treatment, the same group of mice treated with anti-hOX40 mAb was found to retain significantly more luciferase T cells<sup>+</sup>Pmel at the tumor site compared to the control group of lgG1-treated mice (FIG. 35B), again indicating that activation of hOX40 in mice promoted CD8 T-cell survival<sup>+</sup>. Finally, the tumor sizes of 10 mice that received hOX40 T cells<sup>+</sup>Pmel CD8<sup>+</sup> and subsequently treated with anti-hOX40 mAb were significantly lower compared to those from mice receiving untransduced Pmel T cells and treated with anti-hOX40 mAb or hOX40 T cells.<sup>+</sup>Pmel followed by treatment with control mouse lgG1-matching antibody. These results show that activation of human 0X40 in mice results in biological effects similar to those of mouse 0X40 (Gough MJ et, 2008). Therefore, the data demonstrate the ability of anti-human mAb 0X40 to stimulate CD T cell expansion.<sup>+</sup> and in vivo survival and improve tumor rejection.
121
IMPI
The anti-human 0X40 mAb promotes
<img file="MX339964B_D0123.tif" />
tNOUSTRIAL in vivo survival.
The therapeutic vaccination regimen is shown in FIG. 35A. C57BL / 6 albino mice in groups of 5 were implanted subcutaneously (SC) with 5x10<sup>5</sup> non-pigmented MC38 / gp100 tumor cells (day 0). On day 6, lymphopenia was induced by the administration of a radiation dose of 350 cGy. On day 7, 1 x 10<sup>6</sup> Luciferase Pmel-1 T cells transduced with or without expression of human 0X40 were adoptively transferred into tumor-bearing mice (n = 5 per group), followed by intravenous injection of 5x10<sup>5</sup> Gp100 Peptide Pulsed DCs. Recombinant human IL-2 was administered intraperitoneally for 3 days after T cell transfer. Antibodies were administered on days 7, 9, and 11 with 100, 50, and 50 pg, respectively, by injection by mouse (FIG. 35B ). Bioiuminiscence images in vivo showed accumulation of CD8 T cells<sup>+</sup> luciferase-expressing pmel-1 at the lung and tumor sites on days 4 and 12. Two out of five mice per group are shown on day 4 and day 12 (FIG. 35C). Tumors responded to treatments using anti-hOX40 mAb. Tumor size was measured every 3 days. Pmel-1 and Pmel-1 plus mouse lgG1 served as controls.
122
NOVELTY OF THE IMPI INVENTION
MEXICAN INSTITUTE OF PROPERTY
INDUSTRIAL
Contents94
134 sheets
Sheet 1 Sheet 2 Sheet 3 Sheet 4 Sheet 5 Sheet 6 Sheet 7 Sheet 8 Sheet 9 Sheet 10 Sheet 11 Sheet 12 Sheet 13 Sheet 14 Sheet 15 Sheet 16 Sheet 17 Sheet 18 Sheet 19 Sheet 20 Sheet 21 Sheet 22 Sheet 23 Sheet 24 Sheet 25 Sheet 26 Sheet 27 Sheet 28 Sheet 29 Sheet 30 Sheet 31 Sheet 32 Sheet 33 Sheet 34 Sheet 35 Sheet 36 Sheet 37 Sheet 38 Sheet 39 Sheet 40 Sheet 41 Sheet 42 Sheet 43 Sheet 44 Sheet 45 Sheet 46 Sheet 47 Sheet 48 Sheet 49 Sheet 50 Sheet 51 Sheet 52 Sheet 53 Sheet 54 Sheet 55 Sheet 56 Sheet 57 Sheet 58 Sheet 59 Sheet 60 Sheet 61 Sheet 62 Sheet 63 Sheet 64 Sheet 65 Sheet 66 Sheet 67 Sheet 68 Sheet 69 Sheet 70 Sheet 71 Sheet 72 Sheet 73 Sheet 74 Sheet 75 Sheet 76 Sheet 77 Sheet 78 Sheet 79 Sheet 80 Sheet 81 Sheet 82 Sheet 83 Sheet 84 Sheet 85 Sheet 86 Sheet 87 Sheet 88 Sheet 89 Sheet 90 Sheet 91 Sheet 92 Sheet 93 Sheet 94 Sheet 95 Sheet 96 Sheet 97 Sheet 98 Sheet 99 Sheet 100 Sheet 101 Sheet 102 Sheet 103 Sheet 104 Sheet 105 Sheet 106 Sheet 107 Sheet 108 Sheet 109 Sheet 110 Sheet 111 Sheet 112 Sheet 113 Sheet 114 Sheet 115 Sheet 116 Sheet 117 Sheet 118 Sheet 119 Sheet 120 Sheet 121 Sheet 122 Sheet 123 Sheet 124 Sheet 125 Sheet 126 Sheet 127 Sheet 128 Sheet 129 Sheet 130 Sheet 131 Sheet 132 Sheet 133 Sheet 134
98 members in 39 offices
Priority claims14
| Document | Office | Kind | Date |
|---|---|---|---|
| 37599910 | United States of America | P | |
| 37599910 | United States of America | P | |
| 61375999 | United States of America | – | |
| 38082710 | United States of America | P | |
| 38082710 | United States of America | P | |
| 61380827 | United States of America | – | |
| 2011048752 | United States of America | W | |
| 2011048752 | United States of America | W | |
| 61375999 | – | – | – |
| 61380827 | – | – | – |
| PCTUS2011048752 | – | – | – |
| US20100375999P | – | – | – |
| US20100380827P | – | – | – |
| WO2011US48752 | – | – | – |
Members98
| Document | Office | Kind | |
|---|---|---|---|
| CA2809089A1 | Canada | A1 | |
| WO2012027328A2 | World Intellectual Property Organization (WIPO) | A2 | |
| WO2012027328A3 | World Intellectual Property Organization (WIPO) | A3 | |
| CA2845810A1 | Canada | A1 | |
| WO2013028231A1 | World Intellectual Property Organization (WIPO) | A1 | |
| AU2011293558A1 | Australia | A1 | |
| SG187945A1 | Singapore | A1 | |
| AP2013006771A0 | African Regional Intellectual Property Organization (ARIPO) | A0 | |
| CO6680693A2 | Colombia | A2 | |
| CR20130126A | Costa Rica | A | |
| MX2013002172A | Mexico | A | |
| EP2609118A2 | European Patent Office (EPO) | A2 | |
| CN103221427A | China | A | |
| EA201390278A1 | Eurasian Patent Organization (EAPO) | A1 | |
| JP2013538057A | Japan | A | |
| US2013280275A1 | United States of America | A1 | |
| CU20130025A7 | Cuba | A7 | |
| PE20131403A1 | Peru | A1 | |
| DOP2013000045A | Dominican Republic | A | |
| AU2012299421A1 | Australia | A1 | |
| EP2609118A4 | European Patent Office (EPO) | A4 | |
| TN2013000076A1 | Tunisia | A1 | |
| EP2748199A1 | European Patent Office (EPO) | A1 | |
| KR20140090976A | Republic of Korea | A | |
| CN103946238A | China | A | |
| KR20140093600A | Republic of Korea | A | |
| ZA201301442B | South Africa | B | |
| AU2011293558B2 | Australia | B2 | |
| CL2013000545A1 | Chile | A1 | |
| NZ608033A | New Zealand | A | |
| JP2014526898A | Japan | A | |
| US2014308276A1 | United States of America | A1 | |
| MA35686B1 | Morocco | B1 | |
| UA107851C2 | Ukraine | C2 | |
| US9006399B2 | United States of America | B2 | |
| EP2748199A4 | European Patent Office (EPO) | A4 | |
| RU2562874C1 | Russian Federation | C1 | |
| US9163085B2 | United States of America | B2 | |
| EP2933268A1 | European Patent Office (EPO) | A1 | |
| US2015315281A1 | United States of America | A1 | |
| EA201500204A1 | Eurasian Patent Organization (EAPO) | A1 | |
| NZ629913A | New Zealand | A | |
| AU2012299421B2 | Australia | B2 | |
| AU2016200435A1 | Australia | A1 | |
| US2016068604A1 | United States of America | A1 | |
| CL2015002526A1 | Chile | A1 | |
| MX339964BThis record | Mexico | B | |
| HK1213272A | Hong Kong, China | A | |
| HK1213272A1 | Hong Kong, China | A1 | |
| BR112013004266A2 | Brazil | A2 | |
| CN103221427B | China | B | |
| JP5984810B2 | Japan | B2 | |
| CN103946238B | China | B | |
| JP6038920B2 | Japan | B2 | |
| KR101685262B1 | Republic of Korea | B1 | |
| KR20160149322A | Republic of Korea | A | |
| US9527917B2 | United States of America | B2 | |
| EP2609118B1 | European Patent Office (EPO) | B1 | |
| JP2017018110A | Japan | A | |
| KR101704865B1 | Republic of Korea | B1 | |
| AU2016200435B2 | Australia | B2 | |
| KR101719118B1 | Republic of Korea | B1 | |
| PT2609118T | Portugal | T | |
| CA2845810C | Canada | C | |
| DK2609118T3 | Denmark | T3 | |
| CN106554417A | China | A | |
| LT2609118T | Lithuania | T | |
| IL250691A0 | Israel | A0 | |
| BR112014003982A2 | Brazil | A2 | |
| NZ716369A | New Zealand | A | |
| SI2609118T1 | Slovenia | T1 | |
| ME02678B | Montenegro | B | |
| HRP20170568T1 | Croatia | T1 | |
| US9695246B2 | United States of America | B2 | |
| EP2933268B1 | European Patent Office (EPO) | B1 | |
| HUE031371T2 | Hungary | T2 | |
| PL2609118T3 | Poland | T3 | |
| CL2016003052A1 | Chile | A1 | |
| ES2630328T3 | Spain | T3 | |
| US2017267773A1 | United States of America | A1 | |
| RS55952B1 | Serbia | B1 | |
| IL224886A | Israel | A | |
| BR112013004266A8 | Brazil | A8 | |
| PE20180042A1 | Peru | A1 | |
| CY1118928T1 | Cyprus | T1 | |
| ES2649155T3 | Spain | T3 | |
| JP6317788B2 | Japan | B2 | |
| EA029793B1 | Eurasian Patent Organization (EAPO) | B1 | |
| CA2809089C | Canada | C | |
| US10196450B2 | United States of America | B2 | |
| EA031849B1 | Eurasian Patent Organization (EAPO) | B1 | |
| US2019100596A1 | United States of America | A1 | |
| IL250691A | Israel | A | |
| IL250691B | Israel | B | |
| EP2748199B1 | European Patent Office (EPO) | B1 | |
| MY171312A | Malaysia | A | |
| CN106554417B | China | B | |
| US10851173B2 | United States of America | B2 |
1 legal event, as the office reported them to INPADOC
Events
| Event | Code | |
|---|---|---|
| Grant or registrationFG | FG |
Numbers
- Publication
- 339964
- Publication, DOCDB
- 339964
- Publication, EPODOC
- MX339964
- Application
- 2013002172
- Application, DOCDB
- 2013002172
- Application, EPODOC
- MX20130002172
Titles2
- Spanish
- ANTICUERPOS ANTI-OX40 Y METODOS PARA USARLOS.
- English
- ANTI-OX40 ANTIBODIES AND METHODS OF USING THE SAME.
Classification
- CPC, 13
- C07K16/2878
- A61K39/395
- C07K16/28
- A61K2039/505
- C07K2317/24
- C07K2317/75
- C07K2317/74
- A61P35/00
- A61P37/00
- A61P37/02
- C07K2317/56
- C07K2317/565
- C07K7/06
- IPC, 3
- C07K16 28
- A61K39 395
- A61P35 00