Humanized anti-cxcr5 antibodies, derivatives thereof and their uses.

(54) Title: ANTIBODIES ΑΝΤΙ - CXCR5 HUMANIZED, DERIVED FROM THEM AND THEIR USE.

(54) Title: HUMANIZED ANTI-CXCR5 ANTIBODIES, DERIVATIVES THEREOF AND THEIR USES.

(57) Summary

The present invention relates to an isolated or fragmented monoclonal antibody thereof that competitively inhibits the binding of an anti-CXCR5 antibody OR fragment thereof comprises: (a) a light chain variable domain comprising the amino acid sequence of SEQ ID NO. : 11 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 12; (b) the amino acid sequences of (SEQ ID NO: 58), (SEQ ID NO: 59), (SEQ ID NO: 60), (SEQ ID NO: 61), (SEQ ID NO: 62), (SEQ ID NO: 63); (c) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 and a heavy chain variable domain comprising the amino acid sequence of SEQ 15 ID NO: 16; (d) the amino acid sequences of (SEQ ID NO: 58), (SEQ ID NO: 64), (SEQ ID NO: 60), (SEQ ID NO: 61); (SEQ ID NO: 62) AND (SEQ ID NO: 63); (e) the amino acid sequences (SEQ ID NO: 58), (SEQ ID NO: 65), (SEQ ID NO: 60), (SEQ ID NO: 61) (SEQ ID NO: 62), and (SEQ ID NO: 63); (f) a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO: 21, and a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 23; (g) a variable light chain comprising the amino acid sequence of SEQ ID NO. : 30, SEQ ID NO: 31 or SEQ ID NO: 32 and a variable chain weight comprising the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34; (h) the amino acid sequences (SEQ ID NO: 58), (SEQ ID NO: 66), (SEQ ID NO: 60), (SEQ ID NO: 61), (SEQ ID NO: 62) and (SEQ ID No. 63); (i) the amino acid sequences (SEQ ID NO: 58), (SEQ ID NO: 69) (SEQ ID NO: 60), (SEQ ID NO: 61), (SEQ ID NO: 62) and (SEQ ID NO: .63); (j) amino acid sequences (SEQ ID NO; 58), (SEQ ID NO: 68), (SEQ ID NO: 60), (SEQ ID NO: 51), (SEQ ID NO: 62) and (SEQ ID NO: 63); (k) a variable light chain comprising the amino acid sequence of SEQ ID NO: 35 and a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 37; (I) a variable light chain comprising the amino acid sequence of SEQ ID NO: 39, SEQ ID NO: 41, or SEQ ID NO: 43 and a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 45 or SEQ ID NO: 47, (m) a variable light chain comprising the amino acid sequence of SEQ ID NO: 55 and a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 57; or (n) the amino acid sequences (SEQ ID NO: 69), (SEQ ID NO: 66), (SEQ ID NO: 60), (SEQ ID NO: 61), (SEQ ID NO: 62) and (SEQ ID NO: 63).

(57) Abstract

The present ¡nventlon relates to humanized antlbodles that speclflcally blnd to CXCR5 and can, for example, inhibit CXCR5 f unctlon. The ¡nventlon also ¡ncludes uses of the antlbodles to treat or prevent CXCR5 related dlseases or dlsorders.

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of the ___SE___ ^zft. r ''

Mexican Institute of Industrial Property

<td></td><td>PATENT TITLE NO. 338474</td>

<td>Headlines):</td><td>SANOFI - AVENTIS '' X '</td>

<td>Home:</td><td>174 Avenue de France, 75013, Paris, FRANCE</td>

<td>Denomination:</td><td>ANTIBODIES ΑΝΤΙ - CXCR5 HUMANIZED, DERIVED FROM THEM AND THEIR USE</td>

<td>Classification:</td><td>lnt.CI.8: A61K39 / 00; A61K39 / 395; C07K16 / 28; G06F19 / 12</td>

Inventors):

RENATA LEE; VINCENT MIKOL; ELIZABETH ALLEN; NORMAN RUETSCH; BEATRICE CAMERON; THOMAS OLIGINO; NICOLAS BAURIN

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MX / a / 2013/014466 inramagfoiNgi presentation of August 2008

Divisional Patent Number: 338395

PRIORITY go w

Country:

Date:

US August 2007 'li

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V

Number;

60/968,792

Validity: Twenty years

Expiration Date: £ 7 August 2028 and

The reference patent is granted based on toe articles 1 2nd fraction V, 6th fraction III, and 59 of the IndfestriaI Property Law.

In accordance with article? 3 of the Industrial Property Law, this patent has a validity of twenty improrfflgabies years, counted from the date &lt;fe presentation of the application and will be subject to the payment of the fee to keep watch over the rights.

Whoever signs this title does so based on articles 6 fractions lll and 7a bis 2 of the Lw of the

Pro ^ dad Industrial (Diario O &amp; ial de la Federación (DOF) 1996, 12/26/1997, 17 / B5 / 1999,

26 / Of2004, 06/16/2005, 25 / 0W006, 06 / (1 / 2009,06 / 01/2010, 1 12); items 1^or, 3^or frixion V incised a), 4th and 12th fractions! and III of the Regulations of the Institute (DOF 14/12/1999, retaliated on

01/0> 2002, 07/15/2004, 07/28 ^ 004 and 7 / c | / 2007); Articles 1 », 3», 4, 5 »section V subsection a), 16 sections I and lll and 30 of the French Statute of the IfflHWWKHIMWVM ^ ​​iedad 1, 3 and 5 subsection a) of the Agreement that delegates powers to the Deputy General Directors, Coordinator, Divisional Directors, Regional Office Holders, Divisional Deputy Directors, Departmental Coordinators and other subordinates of the Mexican Institute of Industrial Property. (DOF 12/15/1999, amended on 02/04/2000, 07/29/2004, 08/04/2004 and 09/13/2007).

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Issue Date: April 18, 2016

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THE DIVISIONAL DIRECTOR OF PATENTS

NAHANNY CANAL REYES

Arenal No. 550, Floor 1,

Col. Pueblo Santa María Tepepan.

Xochimílco, CP 16020. Mexico City Tel (55) 53 34 07 00 www irnpi gob nn

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338ΉΗ

HUMANIZED ANTI-CXCR5 ANTIBODIES, DERIVED FROM THEM AND THEIR USE

FIELD OF THE INVENTION

MEXICAN INSTITUTE

OF PROPERTY V? INDUSTRIAL

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The present invention relates to anti-CXCR5 antibodies and their use in the improvement, treatment or prevention of diseases or disorders in mammals, including humans, due to improper activity or metabolism of CXCR5 activity, or to improper or unusual use thereof, for example, by a pathogen. An antibody of interest can block the coupling of a ligand, such as CXCL13, with CXCR5. Prophylactic, immunotherapeutic and diagnostic compositions comprising the antibodies and derivatives thereof of interest and their use in methods to prevent or treat diseases in mammals, including humans, caused by metabolism and / or improper activity of CXCR5 cells are also described.⁺, such as B lymphocytes. These diseases include autoimmune deficiencies and diseases produced or characterized by inflammation, such as rheumatoid arthritis (RA), where CXCR5 is up-regulated.

BACKGROUND

CXCR5, also known as the Burkitt's lymphoma receptor (BLR1), CD185, MDR15, and MGC117347, is a G protein-coupled receptor that is a member of the CXC family of chemokine receptors. A ligand is BLC, also known as CXCL13, which is a B cell chemoattractant.

The unprocessed CXCR5 precursor is 372 amino acids in length with a molecular weight of 42 kD.

CXCR5 has a role in the migration and localization of B cells within particular anatomical compartments. Knockout mice lack peripheral lymph nodes, have fewer Peyer's patches, and have lower levels of B cells than normal mice.

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KA \) MEXICAN

F.cOHLDAD

INDUSTRIAL

-2INSTITUTC

OF THE

COMPENDIUM

The present invention provides novel human and human anthrcnc ^ pos "fragments and derivatives thereof, which specifically bind CXCR5. Some of the antibodies and CXCR5-binding fragments thereof can be altered to prevent the formation of intra-chain disulfide bonds resulting in a molecule that is stable throughout preparation and use in vivo. Other antibodies of interest can be altered to minimize binding to F_CA. Some antibodies to CXCR5 of interest compete with CXCL13 for binding to CXCR5. Other antibodies reduce CXCR5 activity.

The invention includes the amino acid sequences of the variable heavy and light chain of the antibodies and their corresponding nucleic acid sequences.

Another embodiment of the invention includes the sequences of the complementarity determining regions (CDR) of the antibodies to obtain binding molecules comprising one or more CDR regions, or regions derived from CDR, that retain the CXCR5 binding ability of the molecule. parental from which the CDR was obtained.

An antibody of interest may be one that prevents binding of CXCL13, or another ligand, to CXCR5 cells.⁺, such as B cells.

Another embodiment of the present invention includes the cell lines and vectors that carry the antibody sequences of the present invention.

Another embodiment of the present invention is the use of antibodies for the preparation of a medicament or composition for the treatment of diseases and disorders associated with the function and metabolism of CXCR5.

Another embodiment of the present invention is the use of these antibodies in the treatment of disorders associated with atypical or abnormal biology and function of CXCR5.

Other features and advantages are described herein, and will be apparent from the following Detailed Description and figures.

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MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL

DETAILED DESCRIPTION

This invention is not limited to the particular methodology, protocols, cell lines, vectors, or reagents described herein, because they may vary without departing from the spirit and scope of the invention. Furthermore, the terminology used herein is intended to exemplify only particular embodiments and should not be construed as limiting the scope of the present invention. Unless otherwise defined, all technical and scientific terms and any acronyms used herein have the same meanings commonly understood by one of ordinary skill in the art in the field of the invention. In the practice of the present invention any method and material similar or equivalent to those described herein can be used and only illustrative methods, devices and materials are described.

All patents and publications mentioned herein are incorporated by reference in their entirety in order to describe and analyze the proteins, enzymes, vectors, host cells, and methodologies presented herein that could be used with and in the present invention. However, nothing herein is to be construed as an admission that the invention has no right to precede such description due to the prior invention.

Before teaching how to prepare and use the methods and products of interest related to CXCR5, the following non-limiting definitions of some terms and phrases are provided to guide the specialist in the art.

"CXCR5" refers to the naturally known molecule found in lymphocytes, particularly B cells and particularly virgin B cells; to said molecule isolated from said cells; to said molecule manufactured recombinantly using known materials and media, and to the use of a nucleic acid encoding a CXCR5; as well as parts of CXCR5, such as the extracellular domain (EC), which retains the characteristics and properties relevant to the practice of the present invention, such as binding to CXCL13. A soluble CXCR5 molecule can consist essentially of the

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CXCR5 EC domain that generally includes approximately the first sixty amino acids of the molecule, ie, the amino terminal part of CXCR5.

CXCR5 is a non-promiscuous receptor. CXCL13 is a ligand of CXCR5 and is constitutively expressed in stromal cells such as follicular dendritic cells and in lymphoid tissues. CXCL13 specifically attracts B cells and a small subset of T cells called B helper follicular T cells, TFH. Infiltration of lymphocytes into tertiary ectopic germ centers (GC) has been found to correlate well with increased disease severity and loss of tolerance in certain disorders with these atypical lymph node-like structures. Using in vivo murine models, such as CXCR5 - / - and CXCL13 - / - mice, the absence of the receptor or the ligand produces an altered fine GC architecture due to altered localization of T and B cells. These mice are also protected against the development of severe collagen-induced arthritis (CIA). Because CXCR5 is selectively expressed in mature B cells, which are associated with RA pathogenesis, blocking of this receptor will modulate the arthritic response in affected individuals. Treatment of rheumatoid arthritis with biological agents (ie, anti-TNFD and anti-CD20 antibodies, Rituximab) has been found to be clinically effective; in particular, patients undergoing B-cell targeted therapy have demonstrated long-lasting improvements in clinical symptoms and signs. Selective targeting of CXCR5, which is expressed only in mature B cells and B helper T cells, will not affect the development of B cells or immunocompromise the patient. Unlike Rituximab, the antibody herein is a neutralizing antibody that does not mediate cellular cytotoxicity.

A "CXCR5 disease" is a malaise, disorder, disease, condition, abnormality, and the like, characterized or produced by overexpression or a higher level of CXCL13 or other CXCR5 ligand, a higher level of B cells, higher levels B-cell activity, increased levels of CXCR5, or improper metabolism and activity of CXCR5.

By "B cell activity" is meant levels of B cells greater than

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B-cell function, such as Bruton's anti-antibody expression, prooonoia or tyrosine kinase activity, CD19 expression or presence, B cell activation factor expression or presence, and the like.

The phrase substantially identical with respect to a polypeptide sequence of an antibody chain can be considered an antibody chain that has a sequence identity of at least 70%, 80%, 90%, 95% or greater with the reference polypeptide sequence. Expression with respect to a nucleic acid sequence can be considered a nucleotide sequence that has a sequence identity of at least about 85%, 90%, 95%, 97%, or greater with the reference nucleic acid sequence.

The expressions identity or homology can mean the percentage of nucleotide bases or amino acid residues in the candidate sequence that are identical to the rest of a corresponding sequence with which it is compared, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of identity for the entire sequence, and without considering any conservative substitution as part of the sequence identity. No N-terminal or C-terminal insertion or extension should be considered to reduce identity or homology. Alignment methods and software are available and are well known in the art. Sequence identity can be measured using sequence analysis software.

The phrases and terms functional fragment, variant, derivative or analog and the like, as well as forms thereof, of an antibody or antigen refer to a compound or molecule that has a qualitative biological activity in common with a full length antibody or antigen of interest. For example, a functional or analog fragment of an anti-CXCR5 antibody is one that can bind to a CXCR5 molecule or one that can impede or substantially reduce the ability of a ligand, such as CXCL13, or of an agonist or antagonist antibody, to join CXCR5. An example is a scF molecule_v.

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As a variant of CXCR5 or rail derivative itself it is a molecule that is not identical to a native CXCR5 but that can be used for a purpose of the present invention although not identical to wild-type CXCR5, for example, as an immunogen for induce antibodies that selectively bind to wild-type CXCR5.

Substitution variants are those in which at least one amino acid residue from a native sequence has been removed and replaced by a different amino acid inserted in place at the same position. The substitutions can be individual, when only one amino acid in the molecule is replaced, or they can be multiple, when two or more amino acids are replaced in the same molecule. Plural substitutions can be at consecutive sites. Furthermore, an amino acid can be replaced by multiple residues, in which case said variant comprises both a substitution and an insert. Insertion variants are those in which one or more amino acids have been inserted immediately adjacent to an amino acid at a particular position in a native sequence. Immediately adjacent to an amino acid means connected to the ü-carboxyl end or a functional Π-amino group of the amino acid. Deletion variants are those in which one or more amino acids have been removed from the native amino acid sequence. Typically, deletion variants will have one or two amino acids deleted in a particular region of the molecule.

The term antibody is used in the broadest sense and specifically includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, blespecific antibodies), antibody fragments, or synthetic polypeptides that carry one or more CDRs or CDR derived sequences as long as the polypeptides exhibit the desired biological activity. Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins that have the same structural characteristics. Antibodies are generally considered Ig with a defined or recognized specificity. Thus, although the antibodies show specificity of binding to a specific target, the

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IMPI

The Mexican Institute of Industrial Property immunoglobulins include both antibodies and other antibody-like molecules that lack specificity for a target. The antibodies of the invention can be of any class (eg, IgG, IgE, IgM, IgD, IgA, and the like), or subclass (eg, IgG-ι, lGG2, lgG2a, lgG3, IgGzj, IgA-i, lgA2, and similar) ("type" and "class", and "subtype" and "subclass" are used interchangeably herein). Antibodies and native or wild-type immunoglobulins, that is, obtained from a member of a population not artificially manipulated, are normally heterotetrameric proteins of approximately 150,000 daltons, composed of two identical light chains (L) and two heavy chains ( H) identical. Each heavy chain has at one end a variable domain (V_H) followed by several constant domains. Each light chain has a variable domain at one end (Vl) and a constant domain at the other end. By "not artificially manipulated" is meant untreated to contain or express a foreign antigen binding molecule. Wild type refers to the most prevalent species or allele found in a population or to the antibody obtained from an unhandled animal, compared to an allele or polymorphism, or a variant or derivative obtained by a form of manipulation, such as mutagenesis , use of recombinant methods and the like, to change an amino acid of the antigen binding molecule.

As used herein, "anti-CXCR5 antibody" refers to an antibody or polypeptide derived therefrom (a derivative) that specifically binds human CXCR5 as defined herein, including, but not limited to, molecules that inhibit or substantially reduce the binding of CXCR5 to its ligands or inhibit CXCR5 activity.

The term variable, in the context of an antibody variable domain, refers to certain parts of the relevant molecule that differ significantly in sequence between and with the antibodies and are used in the recognition and specific binding of a particular antibody to its target particular. However, the variability is not distributed evenly across the variable domains of the antibodies. Variability focuses on

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IMPI

O MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL three segments called Complementarity Determining Regions (CDR; i.e., CDR1, CDR2, and CDR3) screen known as hypervariable regions, in both light chain variable domains and heavy chain variable domains. The most conserved parts of variable domains are called flanking regions or sequences (FR). Each variable domain of each native heavy and light chain comprises four FR regions, which largely adopt a lamina configuration □, connected by three CDRs, which form loops that connect, and in some cases form part of the lamina structure □ . The CDRs of each chain are often held close together by the FR regions and, with the CDRs of the other chain, contribute to the formation of the target binding site (epitope or determinant) of antibodies (see Kabat et al. Sequences of Proteiris of Immunological Interest, National Institute of Health, Bethesda, MD (1987)). As used herein, the numbering of amlnoacid residues of an immunoglobulin is performed according to the immunoglobulin amino acid residue numbering system of Kabat et al., Unless otherwise indicated. A CDR may have the ability to specifically bind to the cognate epitope.

The term "antibody fragment" refers to an intact or full-length part of a chain or to an antibody, generally the target binding region or the variable region. Examples of antibody fragments include, but are not limited to, F fragments._tob> F_tob ·, F (ab ') 2 and F_v. An "functional fragment" or "analog of an anti-CXCR5 antibody" is one that can prevent or substantially reduce the receptor's ability to bind to a ligand or initiate signaling. As used herein, functional fragment is generally synonymous with antibody fragment and with respect to antibodies, may refer to fragments such as F_v, Fab, F (_tob ') 2 and the like in which it can impede or substantially reduce the receptor's ability to bind to a ligand or initiate signaling. A fragment F_v it consists of a dimer of a heavy chain and a light chain variable domain in a non-covalent association (dimer V_H-V_L). In that configuration, the three CDRs of each variable domain interact to define a

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INSTITUTE

Dfc THE PROPERTY

INDUSTRIAL target binding site on the surface of the V-dimer_h-Vl, as in an intact antibody. Collectively, the six CDRs confer target binding specificity on the intact antibody. However, even a single variable domain (or half of an F_v comprising only three specific CDRs per target) may have the ability to recognize and bind to the target.

F antibody fragments_v single chain ”sF_v or "scAb" comprise the V domains_H and V_L of an antibody, where these domains are present on a single polypeptide chain. In general, the F polypeptide_vit also comprises a polypeptide linker, often a flexible molecule, between the V domains_H and V_L, which allows the sFv to form the desired structure for binding to the target.

The term "diabodies" refers to antibody fragments with two antigen binding sites, said fragments may comprise a heavy chain variable domain (V_H) connected to a light chain variable domain (V_L) on the same polypeptide chain. By using a linker that is too short to allow pairing between the two variable domains on the same chain, the domains of the diabody are forced to pair with the binding domains of another chain to create two antigen binding sites. .

Fragment F_ab it contains the variable and constant domains of the light chain and the variable domain and the first constant domain (Cm) of the heavy chain. Fragments F_ab· Differ from F fragments_ab by the addition of a few residues at the carboxy terminus of the Cm domain to include one or more cysteines from the hinge region of the antibody. Fragments F_ab· Can be produced by cleavage of the disulfide bond in the hinge cysteines of the F-pepsin digestion product (_tob-) 2 · Other enzymatic and chemical treatments of antibodies can produce other functional fragments of interest.

The term monoclonal antibody, as used herein, refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, individual antibodies that

IMPI

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-10 make up the population are identical with the exception of possible natural mutations that may be present in minor amounts.

Monoclonal antibodies herein specifically include "chimeric" antibodies in which a part of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a class or subclass of antibody particular (type or subtype), the rest of the chain (s) being identical or homologous to corresponding sequences in antibodies derived from other species or belonging to another class or subclass of antibody, as well as fragments of said antibodies, provided they have the desired biological activity of binding to CXCR5 or of affecting CXCR5 activity or metabolism (US Patent No. 4,816,567; and Morrison et al., Proc Nati Acad Sci USA 81: 6851 (1984)). In this way, CDRs from one class of antibody can be grafted onto the FR of an antibody from a different class or subclass.

Monoclonal antibodies are very specific, targeting a single target, epitope, or determinant site. Furthermore, unlike conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes) of an antigen, each monoclonal antibody is directed against a single determinant on the target. In addition to their specificity, monoclonal antibodies are advantageously synthesized by a host cell, without being contaminated by other immunoglobulins, allowing the cloning of the relevant gene and the mRNA encoding the antibody or its chains. The monoclonal adjective indicates the character of the antibody that is obtained from a substantially homogeneous population of antibodies, and should not be considered to require production of the antibody by any particular method. For example, monoclonal antibodies for use with the present invention can be isolated from phage antibody libraries using well-known techniques, or they can be purified from a polyclonal preparation. The parenteral monoclonal antibodies to be used according to the present invention

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

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they can be obtained by the hybridoma method described by Kohler et al., Nature 256: 495 (1975), or they can be obtained by recombinant methods well known in the art.

Humanized forms of non-human (eg, murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as F_v, F_tob, F_ab·, F (ab) 2 or other antibody target binding subsequences) containing sequences derived from non-human immunoglobulin, compared to a human antibody. In general, the humanized antibody will comprise substantially one, and typically two variable domains, in which all CDR regions correspond to those of a non-human immunoglobulin and all or substantially all FR regions are those of a human immunoglobulin template sequence. The humanized antibody can also comprise at least a part of an immunoglobulin constant region (F_c), typically that of the chosen human immunoglobulin template. In general, the goal is to have an antibody molecule that is minimally immunogenic in a human. In this way, it is possible that one or more amino acids from one or more CDRs can also be changed to one that is less immunogenic for a human host, without substantially minimizing the specific binding function of said one or more CDRs to CXCR5 or CXCL13. Alternatively, FR may be non-human, but the more immunogenic amino acids are replaced by less immunogenic ones. However, CDR grafting, as described above, is not the only way to obtain a humanized antibody. For example, modification of the CDR regions alone may not be sufficient as it is quite common for flanking residues to have a role in determining the three-dimensional structure of CDR loops and the overall affinity of the antibody for its ligand. Therefore, any means can be practiced such that the non-human parental antibody molecule is modified to be less immunogenic to a human, and global sequence identity with a human antibody is not always necessary. In this way, humanization can also

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-12IMPI

MEXICAN INSTITUTE

INDUSTRIAL PROPERTY can be achieved, for example, by the mere substitution of a few residues, particularly those that are exposed in the antibody molecule and not hidden within the molecule and therefore not easily accessible to the host's immune system. Said method is taught herein regarding the substitution of mobile or flexible residues in the antibody molecule, the objective being to reduce or mitigate the immunogenicity of the resulting molecule without compromising the specificity of the antibody for its epitope or determinant. See, eg, Studnicka et al., Prot Eng 7 (6) 805-814, 1994; Mol Imm 44: 1986-1988, 2007; Sims et al., J Immunol 151: 2296 (1993); Chothia et al., J Mol Biol 196: 901 (1987); Carter et al., Proc Nati Acad Sci USA 89: 4285 (1992); Presta et al., J Immunol 151: 2623 (1993), WO 2006/042333 and US Patent No. 5,869,619.

The adaptive immune response has two main arms: the cellular immune response of T lymphocytes and the humoral immune response of antibody-secreting B lymphocytes. B cell epitopes can be linear, contiguous amino acids, or they can be conformational (Protein Science (2005) 14, 246). In contrast, T-cell epitopes are short linear peptides that cleave from antigenic proteins that arise in the context of major histocompatibility complex (MHC) proteins, or, in humans, human leukocyte antigen molecules (HLA) class I or class II. Epitope presentation depends both on binding to the MHC peptide and on interactions with the T-cell receptor (TCR). MHC proteins are highly polymorphic and each binds to a limited series of peptides. Thus, the particular combination of MHC alleles present in a host limits the range of possible epitopes recognized during an infection.

Two fundamental types of T cells are distinguished by the expression of CD8 and CD4 proteins, which dictate whether a T cell will recognize epitopes presented by class I or class II molecules, respectively. CD4 T epitopes⁺ are processed after encapsulation by antigen presenting cells in membrane-bound vesicles, where the antigen is degraded by proteases

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ZL MEXICAN INSTITUTE. 1 - OF THE PROPERTY

INDUSTRIAL to give peptide fragments that bind to MHC class II proteins. In contrast, CD8 T cells⁺ they generally recognize viral antigens or autoantigens expressed from within a cell, proteins that are cleaved into short peptides in the cytosol by the immunoproteasome. After cleavage, the peptides are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum to load HLA I antigens. CD4 T epitopes⁺ (Auxiliaries) are critical for directing T-cell dependent immune responses against protein antigens.

One humanization method of interest is based on the impact of the antibody's molecular flexibility during and immune recognition. The flexibility of a protein is related to the molecular movement of the protein molecule. The flexibility of a protein is the ability of an entire protein, a part of a protein, or a single amino acid residue to adopt a set of conformations that differ significantly from each other. Information on the flexibility of the protein can be obtained by performing X-ray crystallography experiments (see, for example, Kundu et al. 2002, Biophys J 83: 723-732.), Nuclear magnetic resonance experiments (see, for example, Freedberg et al., J Am Chem Soc 1998, 120 (31): 7916-7923) or by performing molecular dynamics (MD) simulations. An MD simulation of a protein is performed on a computer and allows the movement of all the atoms in the protein to be determined over a period of time by calculating the physical interactions of the atoms with each other. The result of an MD simulation is the path of the protein studied during the simulation time period. The path is a set of protein conformations, also called photos, that are periodically sampled during the simulation period, for example, every 1 picosecond (ps). By analyzing the set of photos, the flexibility of the amino acid residues of the protein can be quantified. In this way, a flexible residue is one that adopts a set of different conformations in the context of the polypeptide within which said residue resides. MD methods are known in the art, see, eg, Brooks et al. Proteins: A Theoretical Perspective of Dynamics, Structure and

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Thermodynamics ”(Wiley, New York, 1988). Various software programs allow MD simulations, such as Amber (see Case et al. (2005) J Comp Chem 26: 1668-1688), Charmm (see Brooks et al. (1983) J Comp Chem 4: 187-217; and MacKerell et al. (1998) in "The Encyclopedia of Computational Chemistry" vol. 1: 271-177, Schleyer et al., eds. Chichester: John Wiley &amp; Sons) or Impact (see Rizzo et al. J Am Chem Soc ; 2000; 122 (51): 12898-12900.)

Most protein complexes share a relatively large and flat hidden surface and the flexibility of the binding partners has been shown to provide the origin of their plasticity, allowing them to conform conformationally to each other (Structure (2000) 8, R137 -R142). As such, certain examples of "induced fit" have been shown to play a dominant role at protein-protein contact surfaces. Furthermore, there is increasing data demonstrating that proteins actually bind ligands of various shapes, sizes, and composition (Protein Science (2002) 11: 184-187) and that conformational diversity appears to be an essential component of capacity. of recognizing different patterns (Science (2003) 299, 1362-1367). Flexible moieties are involved in the binding of protein-protein patterns (Structure (2006) 14, 683-693).

Flexible moieties can adopt a variety of conformations that provide a set of areas of interaction that are likely to be recognized by memory B cells and trigger an immunogenic response. In this way, an antibody can be humanized by modifying various residues of the flanking region so that the set of conformations and recognition areas presented by the modified antibody are as similar as possible to those adopted by a human antibody.

This can be accomplished by modifying a limited number of residues: (1) by constructing a homology model of the parent mAb and performing an MD simulation; (2) analyzing flexible residues and identifying the most flexible residues of a non-human antibody molecule, as well as identifying residues or motifs that are likely to be the source of heterogeneity or a reaction

IMPI

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-15 degradation; (3) identifying a human antibody that presents the set of recognition areas most simulating that of the parental antibody; (4) determining the flexible residues to mutate, where also residues or motifs with probability of being a source of heterogeneity and degradation; and (5) checking for the presence of known T or B cell epitopes. Flexible moieties can be found using an MD calculation as taught herein using an implicit solvent model, which explains the interaction of the aqueous solvent with the protein atoms during the time period of the simulation. Once the series of flexible residues within the light and heavy variable chains has been identified, a series of human heavy and light chain variable region flanking regions are identified that closely resemble those of the antibody of interest. This can be done, for example, using a blast search in the series of flexible residues against a database of human germline antibody sequences. This can also be done by comparing the dynamics of the parent mAb with the dynamics of a library of germ canonical structures. CDR residues and neighboring residues are excluded from the search to ensure that a high affinity for the antigen is preserved.

The flexible debris is then replaced. When several human residues show similar homologies, selection is also driven by the nature of the residues that are likely to affect the solution behavior of the humanized antibody. For example, in exposed flexible loops, polar residues will be preferred over hydrophobic residues. Remains that are a potential source of instability and heterogeneity also mutate even though they are found in CDRs. This will include exposed methionines, as sulfoxide can be formed from oxygen radicals, proteolytic cleavage of acid labile bonds such as those of the Asp-Pro dipeptide (Drug Dev Res (2004) 61: 137-154), sites deamidation found with an exposed asparagine residue followed by a small amino acid such as Gly, Ser, Ala, His, Asn or Cys (J Chromatog (2006) 837: 35-43) and N-glycosylation sites such as the Asn site -X-Ser / Thr. Typically, the exposed methionines will be replaced

<img file="MX338474B_D0026.tif" />

<img file="MX338474B_D0027.tif" />

-16 for a Leu, the exposed asparagines will be replaced by a glutamine or an aspartate, or the next rest will be changed. For the glycosylation site (AsnX-Ser / Thr), either the Asn residue or the Ser / Thr residue will be changed.

In the resulting composite sequence, the presence of known linear T-cell epitopes or known B-cell epitopes is checked. A search is performed, for example, with publicly available IEDB. If a known epitope is found within the compound sequence, another series of human sequences is retrieved and replaced

Unlike the resurfacing (surface modification) method of US Patent No. 5,639,641, this method treats both B cell mediated and T cell mediated immunogenic responses. The method also prevents activity loss problem sometimes seen with CDR graft (US Patent No. 5,530,101). In addition, stability and solubility issues are also considered in the genetic engineering and selection process, resulting in an antibody that is optimized for low immunogenicity, high antigen affinity, and better biophysical properties.

Strategies and methods for modifying the surface of antibodies, and other methods for reducing the immunogenicity of antibodies within a different host, are described, for example, in US Patent No. 5,639,641. In summary, in a preferred method, (1) positional alignments of a group of antibody heavy and light chain variable strands are generated to produce exposed positions on the surface in flanking areas of the heavy and light chain variable region, where alignment positions for all variable regions have an identity of at least about 98%; (2) a series of surface exposed amino acid residues are defined in a flanking region of the heavy and light chain variable region for a non-human antibody, such as a rodent antibody (or a fragment thereof); (3) a series of exposed amino acid residues are identified on the surface of flanking areas of the heavy and light chain variable region that is most similar to the series of

<img file="MX338474B_D0028.tif" />

IMPI

MEXICAN INSTITUTE

1 ^ 7 OF PROPERTY

- 1 / - INDUSTRIAL amino acid residues exposed on the surface of the rodent; and (4) the series of surface exposed amino acid residues in flanking areas of the heavy and light chain variable region defined in step (2) is replaced by the series of surface exposed amino acid residues in flanking areas of the region. heavy and light chain variable identified in step (3), except for amino acid residues that are within 5A distance from any atom of any residue of a CDR of the mouse antibody, to produce a humanized antibody, such as a mouse antibody that retains binding specificity.

Antibodies can be humanized by a variety of different techniques including CDR grafting (EPO 0 239 400; WO 91/09967; and US Patent Nos. 5,530,101 and 5,585,089), veneering or resurfacing (EPO 0 documents 592 106; EPO 0 519 596; Padlan, 1991, Molec Imm 28 (4/5): 489-498; Studnicka et al., 1994, Prot Eng 7 (6): 805-814; and Roguska et al., 1994 , PNAS 91: 969-973) and chain exchange (US Patent No. 5,565,332). Human antibodies can be obtained by a variety of methods known in the art including, but not limited to, phage display methods, see US Patent Nos. 4,444,887, 4,716,111, 5,545,806 and 5,814,318; and WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741, using transgenic animals such as rodents, using chimeric cells and the like. .

"Antibody homolog" or "homolog" refers to any molecule that specifically binds CXCR5 as taught herein. Thus, an antibody homolog includes a native or recombinant antibody, modified or not, parts of antibodies that retain the biological properties of interest, such as binding to CXCR5, such as an F molecule._tobo F_v, a single-chain antibody, a polypeptide bearing one or more CDR regions, and the like. The amino acid sequence of the homolog does not need to be identical to that of the natural antibody, but may have been altered or modified to carry substitute amino acids, inserted amino acids, amino acids

<img file="MX338474B_D0029.tif" />

IMPI io INSTITUTO MEXICANO “10“ D £ LA PROPIEDAD

INDUSTRIAL deleted, amino acids different from the twenty normally found in proteins and the like, to obtain a polypeptide with better properties or other beneficial properties.

Antibodies with homologous sequences are antibodies with amino acid sequences that have sequence homology to the amino acid sequence of an antibody to CXCR5 of the present invention. Preferably, homology is with the amino acid sequence of the variable regions of an antibody of the present invention. "Sequence homology", as applied to an amino acid sequence herein, is defined as a sequence with a sequence homology of at least about 90%, 91%, 92%, 93%, 94% or greater, and more preferably a sequence homology of at least about 95%, 96%, 97%, 98%, or 99% with another amino acid sequence, as determined, for example, by the FASTA search method according to Pearson &amp; Lipman , Proc Nati Acad Sci USA 85, 2444-2448 (1988).

A chimeric antibody is one with different parts of an antibody derived from different sources, such as different antibodies, different classes of antibody, different animal species, for example, an antibody that has a variable region derived from a murine monoclonal antibody paired with a region. human immunoglobulin constant and the like. In this way, a humanized antibody is a kind of chimeric antibody. Methods for producing chimeric antibodies are known in the art, see, eg, Morrison, 1985, Science 229: 1202; Oi et al., 1986, BioTechniques 4: 214; Gillies et al., 1989, J Immunol Methods 125: 191-202; and United States Patent Nos. 5,807,715, 4,816,567, and 4,816,397.

Artificial antibodies include scFv fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, and mru (see reviews by Winter &amp; Milstein, 1991, Nature 349: 293-299; and Hudson, 1999, Curr Opin Imm 11: 548-557), each with antigen binding or epitope binding capabilities. In the single-chain fragment Fv (scFv), the domains V_H and Vl of an antibody are connected by a flexible peptide. Typically the

<img file="MX338474B_D0030.tif" />

-19IMPI

MEXICAN INSTITUTE

D £ LA KD.HEDAD

INDUSTRIAL linker is a peptide of approximately 15 amino acids. If the linker is much smaller, for example 5 amino acids, '80 fuiman dleiiwipos that are d (mere bivalent scFv. If the linker is reduced to less than three amino acid residues, trimeric and tetrameric structures are formed which are called triabodies and tetrabodies, respectively. The smallest binding unit of an antibody is a CDR, typically the heavy chain CDR2 that has sufficient specific recognition and binding capacity. This fragment is called a molecular recognition unit or mru. Several of these mrus can bind with short linker peptides, thereby forming an artificial binding protein with greater avidity than a single mru.

Functional equivalents of an antibody of interest are also included within the scope of the invention. The term "functional equivalents" includes antibodies with homologous sequences, antibody homologs, chimeric antibodies, artificial antibodies, and modified antibodies, for example, where each functional equivalent is defined by the ability to bind CXCR5, inhibit signaling ability, or function CXCR5, or inhibit the binding of CXCL13 and other ligands to CXCR5. The person skilled in the art will understand that there is an overlap in the group of molecules called "antibody fragments" and the group called "functional equivalents." Methods for producing functional equivalents that retain the binding ability of CXCR5 are known to the person skilled in the art and are described, for example, in WO 93/21319, EPO Ser. No. 239,400, WO 89/09622. , EPO Ser. No. 338,745 and EPO Ser. No. 332,424.

Functional equivalents of the present application also include modified antibodies, eg, antibodies modified by covalently binding any type of molecule to the antibody. For example, modified antibodies include antibodies that have been modified, for example, by glycosylation, acetylation, pegylation, deamidation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, binding to a cellular ligand, binding to a toxin or to a cytotoxic residue or other protein, etc. Not necessarily the covalent union produces an antibody that

<img file="MX338474B_D0031.tif" />

IMPI

MEXICAN INSTITUTE

- 20 - D £ THE PROPERTY

INDUSTRIAL is immune from the generation of an anti-idiotypic response. Modifications can be accomplished by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis, etc. Furthermore, the modified antibodies can contain one or more non-classical amino acids.

Many techniques are available to those skilled in the art that allow for optimization of binding affinity. Typically, the techniques include substitution of various amino acid residues at the site of interest, followed by a binding affinity selection analysis of the mutant polypeptide for the epitope or cognate antigen.

Once the antibody has been identified and isolated, it is often useful to generate a variant or mutant antibody, or mutein, in which one or more amino acid residues have been altered, for example, in one or more of the hypervariable regions of the antibody. Alternatively, or in addition, one or more alterations (eg, substitutions) of flanking moieties may be introduced into the antibody that result in an improvement in the binding affinity of the antibody mutant to CXCR5. Examples of flanking region residues that can be modified include those that do not covalently bind to the antigen directly (Amit et al., Science 233: 747-753 (1986)); interact with / affect the conformation of a CDR (Chothia et al., J Mol Biol 196: 901-917 (1987)); and / or participate in the contact surface V_L-V_H (EP 239 400). In certain embodiments, modification of one or more of these residues in the flanking region produces an improvement in the binding affinity of the antibody for the cognate antigen. For example, in this embodiment of the invention, from about one to about five flanking moieties can be altered. Sometimes this may be sufficient to produce a mutant antibody suitable for use in preclinical assays, even though none of the residues in the hypervariable region have been altered. Typically, however, the antibody mutant may comprise one or more alterations of the hypervariable region. The constant regions can also be altered to obtain desirable or more desirable effector properties.

<img file="MX338474B_D0032.tif" />

IMPI

-21 The altered hypervariable region residues can be changed randomly, especially when the starting binding affinity of the. Parental antibody is such that randomly produced antibody mutants can be easily screened for those with altered binding in an assay such as that taught herein.

One method of obtaining antibody mutants, such as CDR mutants, is alanine mutagenesis (Cunningham &amp; Wells, Science 244: 1081-1085 (1989); and Cunningham &amp; Wells, Proc Nat Acad Sci USA 84: 6434-6437 ( 1991)). One or more of the hypervariable region residues are replaced by alanine or polyalanine residues. This or these hypervariable region residues that demonstrate functional sensitivity to substitutions are then refined by introducing additional or distinct mutations at or instead of the substitution sites. Thus, although the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. Similar substitutions with other amino acids can be attempted, depending on the desired property of the explored residues.

A more systematic method of identifying amino acid residues to be modified comprises identifying residues of the hypervariable region involved in binding to CXCR5 and residues of the hypervariable region with little or no involvement in binding to CXCR5. An alanine scan of the remnants of the hypervariable region is performed, testing each wing mutant for better binding to CXCR5. In another embodiment, residues significantly involved in binding to CXCR5 are selected for modification. Modification may involve deletion of a residue or insertion of one or more residues adjacent to a residue of interest. Typically, however, modification involves substitution of the residue for another amino acid. A first substitution can be a conservative substitution. If this substitution produces a change in biological activity (eg, binding affinity), then another conservative substitution can be made to determine if more substantial changes are obtained.

-22IMPI

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Even more substantial modifications can be made to a series of antibodies and the presentation of biological properties by selection of an amino acid that differs more substantially in properties from that normally residing at a site. In this way, this substitution can be performed while maintaining: (a) the structure of the polypeptide skeleton in the area of ​​the substitution, for example, as a sheet or helical conformation; (b) loading or hydrophobicity of the molecule at the target site, or (c) volume of the side chain. .

For example, natural amino acids can be divided into groups based on common side chain properties:

(1) hydrophobic: methionine (M or met), alanine (A or ala), valine (V or val), leuclin (L or leu) and isoleucine (I or ile);

(2) neutral, hydrophilic: cysteine ​​(C or cys), serine (S or ser), threonine (T or thr), asparagine (N or asn), and glutamine (Q or gln);

(3) acids: aspartic acid (D or asp) and glutamic acid (E or glu);

(4) basic: histidine (H or his), lysine (K or lys) and arginine (R or arg);

(5) residues that influence chain orientation: glycine (G or gly) and proline (P or pro), and (6) aromatics: tryptophan (W or trp), tyrosine (Y or tyr) and phenylalanine (F or phe).

Non-conservative substitutions can involve changing an amino acid for an amino acid from another group. Conservative substitutions may involve exchanging one amino acid for another within a group.

Preferred amino acid substitutions include those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter the binding affinity, and (4) confer or modify other physicochemical or functional properties of these analogues. Analogs can include various muteins of a sequence other than the sequence of the natural peptide. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in the natural sequence (preferably in the portion of the polypeptide outside the domain that forms the intermolecular contacts). A substitution of

IMPI

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-23 non-conservative amino acids must not substantially change the structural characteristics of the parental sequence (for example, a replacement amino acid must not tend to break a helix that appears in the parental sequence, or to break other types of secondary structure that characterizes the sequence parental) unless there is a change in the volume or conformation of the R group or side chain, Proteins, Structures and Molecular Principles (Creighton, ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (Branden &amp; Tooze, eds., Garland Publishing, New York, NY (1991)); and Thomton et al. Nature 354: 105 (1991).

Typically, the antibody mutant with the best biological properties will have an amino acid sequence with an identity or sequence similarity of at least 75% with the amino acid sequence of the heavy or light chain variable domain of the parent anti-human CXCR5 antibody, or a identity of at least 80%, at least 85%, at least 90%, and often at least 95%. Identity or similarity to the parental antibody is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., is the same residue) or similar (i.e., are amino acid residues of the same group based in common side chain properties, supra) to the parental antibody residues, after aligning the sequences and introducing gaps, if necessary, to achieve a maximum percentage of sequence identity.

Alternatively, antibody mutants can be generated by a systematic mutation of the FR and CDR regions of the heavy and light chains, or the F region._c of the anti-CXCR5 antibody. Another procedure for generating antibody mutants involves the use of affinity maturation using phage display (Hawkins et al., J Mol Biol 254: 889-896 (1992) and Lowman et al., Biochemistry 30 (45): 10832-10838 (1991)). Fusions with bacteriophage coat proteins are known (Smith, Science 228: 1315 (1985); Scott &amp; Smith, Science 249: 386 (1990); Cwirla et al. Proc Nati Acad Sci USA 8: 309 (1990) ; Devlin et al. Science 249: 404 (1990); Wells &amp; Lowman, Curr Opin Struct Biol 2: 597 (1992); and U.S. Patent No. 5,223,409) are useful for associating the

<img file="MX338474B_D0034.tif" />

phenotype of proteins or peptides presented to the genotype of bacteriophage particles that encode them. F domains have also been featured_ab of phage antibodies (McCafferty et al., Nature 348: 552 (1990); Barbas et al. Proc Nati Acad Sci USA 88: 7978 (1991); and Garrard et al. Biotechnol 9: 1373 (1991)).

Monovalent phage display consists of presenting a series of protein variants as fusions of a bacteriophage coat protein into phage particles (Bass et al., Proteins 8: 309 (1990). Affinity maturation, or improvement in equilibrium affinities of various proteins, has previously been achieved through the successive application of mutagenesis, monovalent phage display, and functional analysis (Lowman &amp; Wells, J Mol Biol 234: 564 578 ( 1993); and US Patent No. 5,534,617), for example, focusing on the CDR regions of antibodies (Barbas et al., Proc Nati Acad Sci USA 91: 3809 (1994); and Yang et al., J Mol Biol 254: 392 (1995)).

Many libraries can be built (for example, 10⁶ or more) protein variants, which differ in defined sequence positions, in bacteriophage particles, each containing a DNA encoding the particular protein variant. After affinity purification cycles, using an immobilized antigen, individual bacteriophage clones are isolated, and the amino acid sequence of the protein presented is deduced from the DNA.

After production of the antibody mutant, the biological activity of that molecule with respect to the parental antibody can be determined as taught herein. As noted above, this may involve determining the binding affinity and / or other biological activities or physical properties of the antibody. In a preferred embodiment of the invention, a panel of antibody mutants is prepared and screened for binding affinity for the antigen. One or more of the antibody mutants selected from the screening are optionally subjected to one or more additional biological activity assays to confirm that the antibody mutant or mutants have new or improved properties. In preferred embodiments, the antibody mutant retains the ability to bind CXCR5 with a binding affinity similar to or better / greater than that of the parent antibody.

<img file="MX338474B_D0035.tif" />

The mutant (s) of antibody selected in this manner can undergo further modifications, often depending on the desired use of the antibody. These modifications may involve further alteration of the amino acid sequence, fusion to heterologous polypeptides, and / or covalent modifications. For example, a cysteine ​​residue not involved in maintaining the proper conformation of the antibody mutant can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, a cysteine ​​can be added to the antibody to improve stability (particularly when the antibody is an antibody fragment such as an F fragment._v).

Another type of antibody mutant has an altered glycosyclone pattern. This can be accomplished by deleting one or more carbohydrate residues found in the antibody and / or adding one or more glycosylation sites that are not present in the antibody. Glycosylation of the antibodies is typically N-linked to Asn or O-linked to Ser or Thr. The tripeptide sequences, asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are common recognition sequences for the enzymatic binding of a carbohydrate moiety to the asparaglin side chain. N-acetylgalactosamine, galactose, fucose or xylose, for example, binds to a hydroxyamino acid, more commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. The addition or substitution of one or more serine or threonine residues to the sequence of the original antibody can improve the probability of O-glycosylation.

It may be desirable to modify the antibody of the invention with respect to the function of the effector, to improve the efficacy of the antibody. For example, cysteine ​​residues can be introduced into the F region_c, thus allowing the formation of interchain disulfide bonds in that region. The homodimeric antibody generated in this way may have a better capacity for internalization and / or greater complement-mediated cell destruction and antibody-dependent cellular cytotoxicity (ADCC), see Carón et al., J Exp Med 176: 1191-1195 ( 1992) and Shopes, Immunol 148: 2918-2922 (1993). How

IMPI

MFYlfAm INSTITUTE

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-26 alternative, an antibody having F regions can be genetically engineered_c double and therefore may have better properties Uu pure lysis complement and ADCC, see Stevenson et al., Anti-Cancer Drug Design 3: 219 230 (1989).

Covalent modifications of the antibody are included within the scope of the invention. These can be obtained by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if applicable. Other types of covalent modifications of the antibody are introduced into the molecule by reacting target amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or with the N-terminal or C-terminal moiety.

The cysteinyl residues can be reacted with D-haloacetates (and the corresponding amines), such as chloroacetic acid or chloroacetamide, to produce carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues can also be derivatized by reaction with bromotrifluoroacetone, □ -bromo-ü- (5-imidozoyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-

2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercura4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1,3-diazole, for example.

Histidyl residues can be derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0. Pbromophenacyl bromide can also be used, the reaction is preferably carried out in 0.1M sodium cacodylate at pH 6.0.

The lysinyl and □ terminal residues can be reacted with succinic acid anhydrides or other carboxylic acids to reverse the charge on the residues. Other suitable reagents for derivatizing □ -amino-containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea and 2,4-pentanedione, and the amino acid can be modified by a reaction with transamine catalyzed glyoxylate .

Arginyl residues can be modified by reaction with one or more conventional reagents, such as phenylglyoxal, 2,3-butanedione, 1,2IMPI fKJCTITT ΙΤΓ · .ΑΓ'Ζ-r ·>

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-27cyclohexanedione and ninhydrin. Derivatization of arginine residues often requires alkaline reaction conditions. Furthermore, the reagents can react with lysine as well as with the ü-amino group of arginine.

Specific modification of tyrosyl residues can be carried out with aromatic diazonium compounds or tetranitromethane. For example, to form O-acetyl tyrosyl and 3-nitro derivatives species, N-acetylimimidazole and tetranitromethane respectively are used. Tyrosyl residues can be iodized using¹²⁵the ¹³¹one to prepare labeled proteins for use in a radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) can be modified by reaction with carbodiimides (RN = C = C-R '), where R and R' can be different alkyl groups such as 1-cyclohexyl-3- (2- morpholyn-4-etl) carbodiimide or 1-ethyl-

3- (4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

The glutaminyl and asparaginyl residues are often deamidated to give the corresponding glutamyl and aspartyl residues, respectively, under neutral or basic conditions. The deamidated form of these residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of serinyl or threonyl residues, methylation of D-amino groups of lysine, arginine, and histidine side chains (Creighton, Proteins: Structure and Molecular Properties, WH Freeman &amp; Co., San Francisco, pages 79-86 (1983)), acetylation of the N-terminal amine and amidation of any C-terminal carboxyl group.

Another type of covalent modification involves the chemical or enzymatic coupling of glycosides to the antibody. These procedures do not require the production of the antibody in a host cell that has glycosylation capabilities for N- or O-glycosylation. Depending on the coupling mode used, the sugar or sugars can bind to: (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups such as those of cysteine; (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline; (e) remains₂₈ ΙΜΡΙ @> ^ ' ²⁸ * MEXICAN INSTITUTE '/> * <<' J

FROM PROPERTY) aromatics such as phenylalanine, tyrosine or tryptophan;^Nor^or(í) 'the glucramide of glutamine. These methods are described in the Ttacui uei iluu in Aplin &amp; Wriston, CRC Crit Rev Biochem, p. 259-306 (1981).

The removal of any carbohydrate residues present in the antibody can be carried out chemically or enzymatically. Chemical deglycosylation, for example, may require exposure of the antibody to the compound, trifluoromethanesulfonic acid, or an equivalent compound, resulting in cleavage of most or all of the sugars except the binding sugar (N-acetylglucosamine or N- acetylgalactosamine), while the antibody remains intact. Chemical deglycosylation is described, for example, in Hakimuddin et al. Arch Biochem Biophys 259: 52 (1987) and in Edge et al., Anal Biochem 118: 131 (1981). Enzymatic cleavage of carbohydrate residues present in antibodies can be accomplished by any of a variety of endoglycosidases and exoglycosidases as described, for example, in Thotakura et al., Meth Enzymol 138: 350 (1987).

Another type of covalent modification of the antibody comprises binding of the antibody to one or a variety of non-protein polymers, for example, polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in US Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;

4,791,192 or 4,179,337.

Another preferred technique for obtaining mutants or muteins is the affinity maturation by phage display (Hawkins et al., J Mol Biol 254: 889-896 (1992); and Lowman et al., Biochemistry 30 (45): 10832- 10838 (1991)). In summary, several hypervariable region sites (eg, 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. Antibody mutants thus generated are presented monovalently on phage particles as fusions with a protein found in the particles. Phage display of the various mutants can be cycled through binding selection rounds, followed by isolation and sequencing of those high affinity mutants.

The selection method for new binding polypeptides can use a

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-29 library of structurally related polypeptides. The library of structurally related polypeptides, for example, fused to a phage coat protein, is produced by mutagenesis, and occurs on the surface of the particle. The particles are then contacted with a target molecule and the particles with the highest affinity for the target are separated from those with the lowest affinity. The high affinity binding agents are then amplified by infection of a suitable bacterial host cell and the competitive binding step is repeated. The process is repeated until polypeptides of the desired affinity are obtained.

Alternatively, multivalent phages can also be used (McCafferty et al. (1990) Nature 348: 552-554; and Clackson et al. (1991) Nature 352: 624-628) to express random point mutations (eg, generated by of using error-prone DNA polymerase) to generate a library of phage antibody fragments that can then be screened for affinity for CXCR5, Hawkins et al., (1992) J Mol Biol 254: 889- 896.

Preferably, during the affinity maturation process, the replicable expression vector is under strict control of a transcriptional regulatory element, and the culture conditions are adjusted so that the number or number of particles that have more than one copy of the fusion protein is less than about 1%. Also preferably, the amount of particles displaying more than one copy of the fusion protein is less than 10% of the number of particles displaying a single copy of the fusion protein. Preferably the amount is less than 20%.

Functional equivalents can be produced by exchanging different CDRs of different antibody chains within a flanking region or a composite FR FR derived from plural antibodies. In this way, for example, different classes of antibody are possible for a given series of CDRs by substitution of different heavy chains, for example, IgG ^, IgM, lgAi.₂ or IgD, to produce different types and isotypes of antibody to CXCR5. Similarly, artificial antibodies within the scope of the invention are

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MEXICAN INSTITUTE OF PROPERTY lNDl'31 R1AL

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they can produce by including a given CDR set in a fully synthetic framework.

Antibody fragments and functional equivalents of the present invention include molecules with a detectable degree of specific binding to CXCR5. A detectable degree of binding includes all values ​​in the range of at least 10-100%, preferably at least 50%, 60%, or 70%, more preferably at least 75%, 80%, 85%, 90%, 95% or 99% of the binding capacity of an antibody of interest. Equivalents with an affinity greater than 100% of that of an antibody of interest are also included.

CDRs are generally important for antibody epitope recognition and binding. However, changes in residues that make up CDRs can be made without interfering with the antibody's ability to recognize and bind to the cognate epitope. For example, changes can be made that do not affect recognition of the epitope, but increase the binding capacity of the antibody by the epitope. Several studies have inspected the effects of introducing one or more amino acid changes at various positions in the sequence of an antibody, based on knowledge of the primary sequence of the antibody, its properties, such as binding and level of expression (Yang et al., 1995, J Mol Biol 254: 392-403; Rader et al., 1998, Proc Nati Acad Sci USA 95: 8910-8915; and Vaughan et al., 1998, Nature Biotechnology 16, 535-539).

In this way, equivalents of an antibody of interest can be generated by changing the sequences of the heavy and light chain genes in CDR1, CDR2 or CDR3, or in flanking regions, using methods such as oligonucleotide-mediated directed mutagenesis, cassette mutagenesis, Error-prone PCR, exchange of DNA fragments, or E. coli mutant strains (Vaughan et al., 1998, Nat Biotech 16: 535-539; and Adey et al., 1996, Chap. 16, p. 277-291, in Phage Display of Peptides and Proteins, eds. Kay et al., Academic Press). Methods for changing the nucleic acid sequence of the primary antibody can produce antibodies with better affinity (Gram et al., 1992, Proc Nati Acad Sci USA 89: 3576-3580; Boder et al., 2000, Proc Nati Acad

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Sci USA 97: 10701-10705; Davies &amp; Riechmann, 1996, Immunotech 2: 169-179; Thompson et al., 1996, J Mol Biol 256: 77-88; Short et al., 2002, J Biol Chem 277: 16365-16370; and Furukawa et al., 2001, J Biol Chem 276: 27622-27628).

Repeated cycles of polypeptide selection "can be used to select for binding of increasing affinity through, for example, the selection of multiple amino acid changes that are selected by multiple cycle selection. After a first round of selection, involving a first amino acid selection region in the ligand or antibody polypeptide, additional rounds of selection are made in other regions or amino acids of the ligand. Selection cycles are repeated until the desired affinity properties are achieved.

Improved antibodies also include those antibodies that have improved characteristics that are prepared by standard animal immunization techniques, hybridoma formation, and selection of antibodies with specific characteristics.

Antagonist refers to a molecule capable of inhibiting one or more biological activities of a target molecule, such as CXCR5 signaling. Antagonists can interfere with binding of a receptor to a ligand, and vice versa, by disabling or destroying ligand-activated cells, and / or by interfering with receptor or ligand activation (eg, tyrosine kinase activation) or signal transduction after binding of a ligand to a receptor. The antagonist can completely block receptor-ligand interactions or can substantially reduce these interactions. All these points of intervention by an antagonist will be considered equivalent for the purposes of the present invention. Thus, within the scope of the invention are antagonists (eg, neutralizing antibodies) that bind CXCR5, CXCL13 or other CXCR5 ligands, or a complex of CXCR5 and its ligand, such as CXCL13; amino acid sequence variants or derivatives of CXCR5 or CXCL13 that antagonize the interaction between CXCR5 and a ligand, such as CXCL13; Soluble CXCR5, optionally fused to a heterologous molecule such as a region

<img file="MX338474B_D0044.tif" />

immunoglobulin (eg, an immunoadhesin); a complex comprising CXCR5 in association with another receptor or biological molecule; synthetic or native sequence peptides that bind to CXCR5; and the like.

"Agonist" refers to a compound, including a protein, a polypeptide, a peptide, an antibody, an antibody fragment, a conjugate, a large molecule, or a small molecule, that activates one or more biological activities of CXCR5. Agonists may interact with the binding of a receptor to a ligand and vice versa, by acting as a ligand-activated cell mitogen, and / or by interfering with cell inactivation or inhibition of signal transduction after binding. from a ligand to a receptor. All of these points of intervention by an agonist will be considered equivalent for the purposes of the present invention. Thus, within the scope of the present invention are included agonists that bind CXCR5, CXCL13, or another CXCR5 ligand, or a complex of CXCR5 and a ligand thereof, such as CXCL13; amino acid sequence variants or derivatives of CXCR5 or CXCL13 that facilitate the interaction between CXCR5 and a ligand, such as CXCL13; Soluble CXCR5, optionally fused to a heterologous molecule such as an immunoglobulin region (eg, an immunoadhesin); a complex comprising CXCR5 in association with another receptor or biological molecule; synthetic or native sequence peptides that bind to CXCR5; and the like. The agonist is generally an entity that directly activates CXCR5, for example for signaling.

The terms cell, cell line and cell culture include their offspring. It is also understood that all offspring may not be exactly identical, such as in DNA content, due to a deliberate or involuntary mutation. Variant offspring that have the same function or biological properties of interest for which the selection was made in the original cell are included. The host cells used in the present invention are generally prokaryotic or eukaryotic hosts, selected according to the choice of design.

The transformation of a cellular organism, cell or cell line with a

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-33IMPI®

MEXICAN INSTITUTE ': /// -

PROPERTY OF INDUSTRIAL PROPERTY Nucleic acid means the introduction of a nucleic acid into the target so that the nucleic acid can be replicated, as an eXliaiiunrosomal element · »by chromosomal integration, and optionally express. Transfection of a cell or organism with a nucleic acid refers to the uptake of the nucleic acid, eg, an expression vector, by the cell or organism, whether or not some coding sequence is expressed. The terms "transfected and transformed host cell" refer to a cell into which a nucleic acid was introduced. Typical prokaryotic host cells include various strains of E. coli. Typical eukaryotic host cells are mammalian cells, such as Chinese hamster ovary cells, or cells of human origin. The introduced nucleic acid sequence can be from the same species as the host cell or from a different species of the host cell, or it can be a hybrid nucleic acid sequence containing some foreign nucleic acids and some homologous nucleic acids. The transformation can also be carried out by transduction or infection with elements derived from viruses.

The term vector means a nucleic acid construct, a vehicle, containing a nucleic acid, the transgene, the foreign sequence, or the gene of interest, which may be operably linked to suitable control sequences for expression of the transgene in a suitable host. . These control sequences include, for example, a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences that control termination of transcription and translation. The vector can be a plasmid, a phage particle, or just a potential genomic insert. Once the appropriate host has been transformed, the vector can replicate and function independently of the host genome, or in some cases, it can be integrated into the genome of the host cell. In the present specification, plasmid and vector are used interchangeably, since the plasmid is a commonly used form of vector. However, the invention is intended to include other forms of vectors that have

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INSTITUTO MEXICANO ΊΛ DE LA PRCHEDAD

INDUSTRIAL equivalent vehicle functions and which are or are being known in the art such as viruses, synthetic molecules carrying nucleic acids, liposomes and the like.

Mammal, for treatment purposes, refers to any animal classified as a mammal, including humans, domestic and farm animals, non-human primates, and zoo, sport or pet animals, such as dogs, horses, cats, cows, etc.

Antibodies of interest can be screened or can be used in an assay such as that described herein or known in the art. Often these assays require that a reagent be detectable, eg, labeled. The word marker, when used herein, refers to a detectable compound or composition that can be conjugated directly or indirectly to a molecule or protein, for example, an antibody. The marker can be detectable on its own (eg, radioisotope markers, particles, or fluorescent labels) or, in the case of an enzyme marker, can catalyze the chemical alteration of a compound or substrate composition that is detectable.

As used herein, "solid phase" means a non-aqueous matrix to which an entity or molecule can adhere, such as the antibody of the present invention. Examples of solid phases included herein include those formed partially or completely of glass (eg, controlled pore glass), polysaccharides (eg, agarose), polyacrylamides, polystyrene, polyvinyl alcohol, and silicones. In certain embodiments, depending on the context, the solid phase may comprise the well of a test plate; in others it can be used in a purification column (for example, in an affinity chromatography column). Thus, the solid phase can be a paper, a bead, a plastic, a chip, and the like, it can be made of a variety of materials such as nitrocellulose, agarose, polystyrene, polypropylene, silicon, and the like, and can be in a diversity of configurations.

Soluble CXCR5 or its fragments, such as the extracellular domain (EC),

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I Μ. Ρ I

MEXICAN INSTITUTE _ Q5 - OF THE PROPERTY

INDUSTRIAL can be used as immunogens to generate antibodies of interest. The immunogen can be obtained or isolated from natural sources or it can be obtained recombinantly. Whole cells such as CXCR5 cells can be used as immunogens to obtain the antibodies of interest.⁺, cells derived from a natural source (eg, B cells, B cell lines, or cancer cell lines) or cells transformed (or transfected) by recombinant techniques to express, and perhaps overexpress CXCR5. Membrane preparations carrying CXCR5 or synthetic peptides or truncated polypeptides corresponding to the EC regions of CXCR5 can also be used, as is known in the art.

EC, which is about 60 amino acids long, or parts thereof, of CXCR5 can be used as an immunogen. Other forms of the immunogen useful for preparing antibodies, such as a conjugate, will be apparent to those skilled in the art. In this way, CXCR5, or parts thereof, can be attached to a carrier molecule, such as albumin or KLH, to be used as an immunogen. Of course, with cells expressing CXCR5, it is the EC domain that is the preferred immunogen or a part of the immunogen.

The gene or cDNA encoding CXCR5, as known in the art, can be cloned into a plasmid or other expression vector and expressed in any of several expression systems according to methods well known to those skilled in the art, and shown later, for example. Due to the degeneracy of the genetic code, a multitude of nucleotide sequences encoding the CXCR5 polypeptide or protein can be used in the practice of expressing recombinant CXCR5 or functional products thereof. The nucleotide sequence can be varied by selecting combinations based on possible codon choices, such as those preferred by the host cell. The combinations are made according to the conventional triplet genetic code applied to the nucleotide sequence encoding wild-type CXCR5 and all these variations can be considered. In this way, the CXCR5 coding sequence can

<img file="MX338474B_D0050.tif" />

-36IMPI

MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL register so that it contains codons that express the amino acid of interest, however, the triplet codon is one favored by the gene expression machinery of the host cell, such as a human cell. Any of these polypeptides can be used in the immunization of an animal, such as a camelid, or other system to generate antibodies that bind to CXCR5.

As mentioned above, when beneficial, the CXCR5 immunogen can be expressed as a fusion protein that has CXCR5 attached to a fusion segment, which is generally a polypeptide with one or more beneficial functions. The fusion segment often aids in protein purification, for example, by allowing the fusion protein to be isolated and purified by affinity chromatography, but it can also be used to increase immunogenicity. Fusion proteins can be produced by culturing a recombinant cell transformed with a fusion nucleic acid sequence encoding a protein bound to the carboxyl and / or amino terminus of the CXCR5 polypeptide. Other fusion segments may include, but are not limited to, F regions_c of immunoglobulin, glutathione-S-transferase, □ galactosidase, a polyhistidine segment capable of binding a divalent metal ion and maltose binding protein.

Nucleic acid molecules encoding amino acid sequence mutants can be prepared by a variety of methods well known in the art. Methods include, but are not limited to, oligonucleotide-mediated (or directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of a previously prepared mutant or a non-mutant version of the molecule of interest (see, eg, Kunkel, Proc Nati Acad Sci USA 82: 488 (1985)).

Recombinant expression of an antibody of the invention, or fragment, derivative, or analog thereof (eg, a heavy or light chain of an antibody of the invention, a single-chain antibody of the invention, or an antibody mutein of the invention) includes constructing an expression vector containing a polynucleotide encoding the antibody or a fragment of the antibody as described herein. Once it has

<img file="MX338474B_D0051.tif" />

-37IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY obtained a polynucleotide encoding an antibody molecule, the vector for antibody production can be produced by recombinant DNA technology as known in the art. An expression vector is constructed such that it contains antibody coding sequences and appropriate transcription and translation control signals. The methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

The expression vector is transferred to a host cell by standard techniques and the transfected cells are then cultured by standard techniques to produce an antibody or fragment of the invention. In one aspect of the invention, vectors encoding heavy and light chains can be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed herein.

A variety of host / expression vector systems can be used to express the antibody molecules of the invention. These expression systems represent vehicles by means of which the coding sequences of interest can be produced and subsequently purified, but also represent cells that, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in if you. For expression of a recombinant antibody molecule, especially for expression of entire recombinant antibody molecules, bacterial cells such as E. coli, and eukaryotic cells are commonly used. For example, mammalian cells such as CHO cells, together with a vector, such as one carrying the promoter element of the human cytomegalovirus major intermediate early gene, are an efficient expression system for antibodies (Foecking et al., Gene 45: 101 (1986); and Cockett et al., Bio / Technology 8: 2 (1990)). To prepare the proteins of interest, as is known in the art, plants and plant cell culture, insect cells and the like can also be used.

In addition, a host cell is chosen that modulates the expression of the inserted sequences, or that modifies and processes the gene product in the manner

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-38 MEXICAN INSTITUTE £} £ LA? RG? 1 íu, ', L> INUCFÍÁLAL specific desired. These modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important to the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure correct modification and processing of the expressed antibody of interest. Therefore, eukaryotic host cells possessing the cellular machinery can be used for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product. These mammalian host cells include, but are not limited to, CHO, COS, 293, 3T3 cells, or myeloma cells.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines can be genetically engineered that stably express the antibody molecule. Instead of using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (eg, promoter, enhancer, transcription terminator sequences, polyadenylation sites etc.) and a selection marker. After the introduction of the foreign DNA, the engineered cells can be allowed to grow for one or two days in an enriched medium, and then are transferred to a selection medium. The recombinant plasmid selection marker confers selection resistance and allows cells to stably integrate the plasmid into a chromosome and expand into a cell line. These genetically engineered cell lines are not only useful for antibody production, but are also useful in the selection and evaluation of compounds that interact directly or indirectly with the antibody molecule.

Various selection systems can be used including, but not limited to, the Herpes simplex virus thymidine kinase genes (Wigler et al., Cell

IMPI

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-3911: 223 (1977)), hypoxanthine-guanine fngfnmhnciitrancfor_g0o (. ^ yhakka pt ai Proc Nati Acad Sci USA 48: 202 (1992)), the selection of glutamate synthase in the presence of methionine sulfoxamide (Adv Drug Del Rev 58, 671, 2006 and see Lonza website or bibliography Group Ltd.) and adenine phosphoribosyltransferase (Lowy et al., Cell 22: 817 (1980)) in tk, hgprt or aprt cells, respectively. In addition, antimetabolite resistance can be used as the basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Proc Nati Acad Sci USA 77: 357 (1980); O'Hare et al., Proc Nati Acad Sci USA 78: 1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan et al., Proc Nati Acad Sci USA 78: 2072 (1981)); neo, which confers resistance to aminoglycoside, G-418 (Wu et al., Biotherapy 3:87 (1991)); and hygro, which confers hygromycin resistance (Santerre et al., Gene 30: 147 (1984)). Methods known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clone, and these methods are described, for example, in Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley &amp; Sons ( 1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press (1990); Dracopoli et al., Eds., Current Protocols in Human Genetics, John Wiley &amp; Sons (1994); and Colberre Garapin et al., J Mol Biol 150: 1 (1981).

Expression levels of an antibody molecule can be increased by vector amplification (eg, see Bebbington et al., In DNA Cloning, Vol. 3. Academic Press (1987)). When a marker present in the vector system expressing the antibody can be amplified, an increase in the level of inhibitor present in the culture will increase the copy number of the marker gene. Since the amplified region is associated with the antibody gene, the production of the antibody will also increase (Crouse et al., Mol Cell Biol 3: 257 (1983)).

The host cell may be co-transfected with two or more expression vectors of the invention, for example, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. Both vectors can contain

<img file="MX338474B_D0054.tif" />

INSTITUTO MfXICÁN D £ LA PROPIEDAD INDUSTRIAL identical selections that allow equal expression of heavy and light chain polypeptides. Alternatively, a single vector can be used that encodes, and is capable of expressing, both heavy and light chain polypeptides. In these situations, the light chain must be placed before the heavy chain to avoid excess toxic heavy free chain (Proudfoot, Nature 322: 52 (1986); and Kohler, Proc Nati Acad Sci USA 77: 2197 (1980) ). The coding sequences for the heavy and light chains can comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it can be purified by any method known in the art for the purification of an immunoglobulin molecule, for example, by chromatography (eg, ion exchange, affinity, particularly affinity for CXCR5 after Protein A chromatography and size exclusion chromatography and the like), centrifugation, differential solubility or any other conventional technique for protein purification. Furthermore, the antibodies of the present invention or their fragments can be fused with heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.

The recombinant CXCR5 protein, exemplified in the examples presented below, was used to immunize mice to generate the monoclonal antibody-producing hybridomas of the present invention. Among the monoclonal antibodies obtained, those with a beneficial therapeutic potential were selected, for example, the prevention of CXCR5 ligand binding. The selected antibodies were then modified to obtain beneficial properties, such as increased stability in vivo.

The antibodies of the present invention can be generated by any suitable method known in the art. The antibodies of the present invention can comprise polyclonal antibodies, although due to the modification of antibodies to optimize the use in humans, as well as to optimize the use of the antibody per se, the antibodies are preferred

<img file="MX338474B_D0055.tif" />

-41 I IvI P1

MEXICAN INSTITUTE

INDUSTRIAL PROPERTY monoclonal due to the ease of production and handling of particular proteins. Methods for preparing polyclonal antibodies are known to the person skilled in the art (Harlow et al., Antibodies: a Laboratory Manual,

Coid Spring Harbor Laboratory Press, 2nd ed. (1988)).

For example, an immunogen, as exemplified herein, can be administered to various host animals including, but not limited to, rabbits, mice, camelids, rats etc., to induce production of serum containing polyclonal antibodies specific for CXCR5. Administration of the immunogen can include one or more injections of an immunizing agent and, if desired, an adjuvant. Various adjuvants can be used to enhance the immune response, depending on the host species, and these include, but are not limited to, Freund (complete and incomplete), mineral oil, gels, alum (aluminum hydroxide), surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins (KLH), dinitrophenol and potentially useful human adjuvants such as BCG (Bacille CalmetteGuerin) and Corynebacterium parvum. Other examples of adjuvants that can be employed include the adjuvant MPL-TDM (monophosphoryl lipid A, synthetic trehalose dichorinomycolate). Immunization protocols are well known in the art and can be performed by any method that induces an immune response in the chosen animal host. In this way, various routes of administration can be used for various periods of time depending on the choice of design.

Typically, the immunogen (with or without an adjuvant) is injected into the mammal by multiple subcutaneous or intraperitoneal injections, or intramuscularly or intravenously. The immunogen can include a CXCR5 polypeptide, a fusion protein, or variants thereof, which can be produced by a cell that produces or overproduces CXCR5, which can be a natural cell, a natural mutant cell, or a genetically engineered cell. In certain circumstances, whole cells expressing CXCR5 can be used. Depending on the nature of the polypeptides (i.e. the percentage of

<img file="MX338474B_D0056.tif" />

-42IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY hydrophobia, hydrophilicity percentage, stability, net charge, isoelectric point etc.), CXCR5 or part of it can be modified or conjugated to make it immunogenic or more immunogenic in the animal, such like a mammal to be immunized. For example, CXCR5 or a part thereof can be conjugated to a vehicle. Conjugation includes chemical conjugation by derivatization of active chemical functional groups such that both the immunogen and the immunogenic protein are conjugated such that a covalent bond is formed, or by a fusion protein-based methodology, or other known methods for specialist in the technique. Examples of these immunogenic vehicles or proteins include, but are not limited to, KLH, ovalbumin, serum albumin, bovine thyroglobulin, soybean trypsin inhibitor, and promiscuous T helper peptides. Various adjuvants can be used to enhance the immune response as described above.

Once a suitable preparation has been obtained, it is possible to isolate particular antibodies from plural antibodies by known separation techniques, such as affinity chromatography, adsorption (panning), absorption and the like. In this way, a single species of antibody can be obtained for further study, for example, by sequencing to obtain the amino acid sequences of one or more CDRs.

The antibodies of the present invention preferably comprise monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma technology, as described by Kohler et al., Nature 256: 495 (1975); United States Patent No. 4,376,110; Harlow et al., Antibodies: A Laboratory Manual, Coid Spring Harbor Laboratory Press, 2nd ed. (1988) and Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas, Elsevier (1981), recombinant DNA methods, eg, preparation and use of transfectomas, or other methods known to one skilled in the art. Other examples of methods that can be employed to produce monoclonal antibodies include, but are not limited to, the human B cell hybridoma technique (Kosbor et al., Immunology Today 4:72 (1983); and Colé et al., Proc Nati

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IMPI _ 43 _w MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL

Acad Sci USA 80: 2026 (1983)), and the EBV hybridoma technique (Colé et al.,

Monoclonal Antíbodies and Cancer Therapy, page “7 / -9b, Aláll R. Llbb (1985)). These antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, and IgD, and any subclass thereof. The hybridoma producing the mAb of the invention can be cultured in vitro or in vivo.

In the hybridoma model, a host such as a mouse, an immunized mouse, a transgenic mouse with genes from the human immune system, hamster, rabbit, rat, camel, or any other appropriate host animal, is immunized to induce lymphocytes that produce or are capable of producing antibodies that specifically bind CXCR5. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, p. 59-103 (1986)).

In general, to prepare antibody-producing hybridomas, peripheral blood lymphocytes (PBL) are used if cells of human origin are desired, or spleen or lymph node cells are used if non-human mammalian sources are desired. Immortalized cell lines are normally transformed mammalian cells, particularly rodent myeloma cells, of bovine or human origin. Typically a mouse or rat myeloma cell line is employed. Hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of immortalized, non-fused cells. For example, if the parent cell lacks the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), substances that prevent the growth of HGPRT-deficient cells. .

Preferred immortalized cell lines are those that fuse efficiently, support stable, high-level production of antibody by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these myeloma cell lines are

<img file="MX338474B_D0058.tif" />

Murine myeloma lines, such as those derived from mouse tumors MOPC-21 and MPC-11 available from the Salk Institute Cell Distribution Center, San Diego, Calif. and SP2 / 0, FO, or X63-Ag8-653 cells available from the American Type Culture Collection (Manassas, VA).

Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J Immunol 133: 3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker , Inc, pp. 51-63 (1987)). The NSO mouse myeloma cell line (European Collection of Cell Cultures, Salisbury, Wilshire, UK) can also be used.

Another alternative is to use electrical fusion instead of chemical fusion to form hybridomas. Instead of by means of fusion, a B cell can be immortalized using, for example, Epstein Barr virus or another transformation gene, see, for example, Zurawaki et al., In Monoclonal Antibodies, ed., Kennett et al., Plenum Press, p. 19-33. (1980). Transgenic mice expressing immunoglobulins and mice with severe combined immunodeficiency (SCID) can also be used in which human B lymphocytes have been transplanted.

The culture medium in which the hybridoma cells grow is assayed for the production of monoclonal antibodies directed against CXCR5. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay, such as a radiolnmunoassay (RIA), fluorocytometric analysis (FACS), or enzyme-linked immunosorbent assay (ELISA). These techniques are known in this field and are within the experience of the specialist. The binding affinity of the monoclonal antibody for CXCR5 can be determined, for example, by a Scatchard analysis (Munson et al., Anal Biochem 107: 220 (1980)).

After identifying hybridoma cells that produce antibodies of the desired specificity, affinity, and / or activity, the clones can be subcloned by limiting dilution procedures and cultured by conventional methods.

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(Goding, Monoclonal Antibodies: Principies and Practice, Academic Press, p. 59-103 (1986)). Suitable culture media include, for example, Dulbecco's Modified Eagle's Medium (D-MEM) or RPMI-1640 medium. Furthermore, hybridoma cells can be cultured in vivo as ascites tumors in an animal.

Monoclonal antibodies secreted by the subclones are conveniently separated or isolated from the culture medium, ascites fluid, or serum by standard immunoglobulin purification procedures such as, for example, protein A-Sepharose, protein G-Sepharose, hydroxylapatite chromatography, chromatography. molecular exclusion, gel electrophoresis, dialysis or affinity chromatography.

There are a variety of methods in the art for the production of monoclonal antibodies, and thus the invention is not limited to its sole production in hybridomas. For example, monoclonal antibodies can be obtained by recombinant DNA methods, such as those described in US Patent No. 4,816,567. In this context, the term monoclonal antibody refers to an antibody derived from a single eukaryotic, phage, or prokaryotic clone.

The DNA encoding the monoclonal antibodies of the invention is easily isolated and sequenced using standard procedures (eg, using oligonucleotide probes that can specifically bind to genes encoding the murine antibody heavy and light chains, or such chains of human origin, humanized or other sources) (Innis et al. in POR Protocols. A Guide to Methods and Applications, Academic (1990), and Sanger et al., Proc Nati Acad Sci 74: 5463 (1977)). Hybridoma cells serve as the source of such DNA. Once isolated, DNA can be placed into expression vectors that are then transfected into host cells such as E. coli cells, NSO cells, COS cells, Chinese hamster ovary (CHO) cells, or non-producing melanoma cells. otherwise immunoglobulin protein, to obtain monoclonal antibody synthesis in recombinant host cells. DNA can also be modified, by

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IMPI _ 46 - MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL example, replacing the coding sequence of heavy chain constant domains and human light chain constant domains ρδι¹ homologous murine sequences (US Patent No. 4,816,567; and Morrison et al., Proc Nati Acad Sci USA 81: 6851 (1984)) or by covalent binding to the immunoglobulin coding sequence of all or part of the coding sequence for a peptide that is not an immunoglobulin. Said non-immunoglobulin polypeptide can substitute for the constant domains of an antibody of the invention, or can substitute for the variable domains of a CXCR5 combination site of an antibody of the invention to create a chimeric bivalent antibody.

Antibodies can be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant light chain and modified heavy chain expression of immunoglobulin. The heavy chain is generally truncated at any point in the F region_c to prevent the formation of crosslinks in the heavy chain. Alternatively, the relevant cysteine ​​residues are replaced by another amino acid residue or deleted to prevent cross-link formation.

Antibody fragments can be generated that recognize specific epitopes by known techniques. Traditionally, these fragments were obtained by proteolytic digestion of intact antibodies (see, eg, Morimoto et al., J Biochem Biophys Methods 24: 107 (1992); and Brennan et al., Science 229: 81 (1985)). For example, F fragments may be produced_ab and F (_tob ') 2 of the invention by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce F fragments_ab) or pepsin (to produce F fragments (_ab· ^. Fragments F (_ab) 2 contain the variable region, the light chain constant region and the Cm domain of the heavy chain. However, these fragments can be produced directly by recombinant host cells. For example, antibody fragments can be isolated from a phage antibody library. Alternatively, fragments F (_ab) 2-SH from E. coli and

<img file="MX338474B_D0061.tif" />

chemically couple to form F (ab ') 2 fragments (Carter et al.,

Bio / Technology 10: 163 (1992). According to fragments F (_tob-) 2 directly from the culture of recombinant host cells. Other techniques for the production of antibody fragments will be apparent to one skilled in the art. In other embodiments, the antibody of choice is an F fragment._v single chain (F_v) (WO document

93/16185).

For some uses, including the in vivo use of antibodies in humans and in vitro detection assays, the use of chimeric, humanized or human antibodies may be preferable. Methods for producing chimeric antibodies are known in the art, see, eg, Morrison,

Science 229: 1202 (1985); Oi et al., BioTechniques 4: 214 (1986); Gillies et al., J

Immunol Methods 125: 191 (1989); and United States Patents No. 5,807,715; 4,816,567; and 4,816,397.

Humanized antibodies come from antibody molecules generated in a non-human species that bind to CXCR5, where one or more CDRs are inserted into the FR regions of a human immunoglobulin molecule. Antibodies can be humanized using a variety of techniques known in this field including, for example, CDR grafting (EPO 239,400; WO 91/09967; and US Patent Nos. 5,225,539; 5,530,101; and 5,585,089). , veneering or resurfacing (EPO 592,106; EPO 519,596; Padlan, Molecular Immunology 28: 489 (1991); Studnicka et al., Protein Engineering 7: 805 (1994); and Roguska et al., Proc Nati Acad Sci USA 91: 969 (1994)), and chain exchange (US Patent No. 5,565,332).

A humanized antibody has one or more amino acid residues from a non-human source. Non-human amino acid residues are often called imported residues, which are typically taken from an imported variable domain. Humanization can be performed essentially by following the methods of Winter et al. (Jones et al., Nature 321: 522 (1986); Riechmann et al., Nature 332: 323 (1988); and Verhoeyen et al., Science 239: 1534 (1988 )), using non-human CDRs or parts of CDR sequences instead of

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX338474B_D0063.tif" />

the corresponding sequences of a human antinuprpn Accordingly, these humanized antibodies are chimeric antibodies (US Patent No. 4,816,567), in which substantially less than an intact human variable domain has been replaced by the corresponding sequence of a non-species human. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced from analogous sites in rodent antibodies. The heavy chain constant region and hinge region can come from any class or subclass to obtain the desired effect, such as a particular effector function.

Often, the flanking residues in the human flanking regions can be replaced by the corresponding residue of the CDR donor antibody to alter, and possibly improve, binding to antigens. Flanking substitutions are identified by methods known in the art, for example, by modeling interactions of CDR and flanking residues to identify flanking residues important for antigen binding and sequence comparison, to identify unusual flanking residues at positions individuals, see, for example, US Patent No. 5,585,089; and Riechmann et al., Nature 332: 323 (1988).

Furthermore, it is preferable that humanized antibodies retain high affinity for CXCR5, and retain or acquire other favorable biological properties. In this way, humanized antibodies are prepared by a process of analyzing parental sequences and various conceptual humanized products using three-dimensional models of the parent and humanized sequences. Three-dimensional models of immunoglobulins are commonly available and those of skill in the art will be familiar with these methods. Computer programs are available that illustrate and present probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of the presentations allows analysis of the likely role of certain residues in the functioning of the candidate immunoglobulin sequence, i.e. analysis

IMPI instítirrn μγυιγάμπ

<img file="MX338474B_D0064.tif" />

-49 residue that influences the ability of the candidate immunoglobulin to bind CXCR5. In this way, FR residues from the receptor can be selected and combined and sequences imported so as to maximize the desired characteristic of the antibody, such as a higher affinity for the target antigen, although it is the CDR residues that directly influence and more substantially at the junction of CXCR5. The CDR regions can also be modified to contain one or more amino acids that vary from that obtained from the parental antibody from which the CDR was obtained, to provide better or different properties of interest, such as higher affinity binding or higher avidity, for example.

Certain parts of the constant regions of an antibody can be manipulated and changed to provide homologs, derivatives, antibody fragments, and the like with properties different or better than those observed in the parent antibody. In this way, for example, many IgG4 antibodies form intra-chain disulfide bonds near the hinge region. The intra-chain bond can destabilize the parent bivalent molecule by forming monovalent molecules comprising a heavy chain with the associated light chain. These molecules can reassociate, but on a random basis.

It was observed that modifying amino acids in the hinge region of lgG4 molecules can reduce the probability of formation of intra-chain bonds, thus stabilizing the lgG4 molecule, which will minimize the probability of forming bispecific molecules. Such a modification will be beneficial if a therapeutic antibody is an lgG4 molecule, since increased stability will minimize the likelihood that the molecule will dissociate during production and manufacture, as well as in vivo. A monovalent antibody may not have the same efficacy as the bivalent parent molecule. For example, when bivalent lgG4 is administered to a patient, the percentage of bivalent lgG4 drops to approximately 30% over a two week period. A substitution of an amino acid at position 228 increases the stability of lgG4. Serine residing at position 228 can be replaced by another amino acid, such as one of the remaining 19 amino acids. This change can

<img file="MX338474B_D0065.tif" />

particularly be made with recombinant antibodies in which the coding nucleic acid sequence can be mutated to produce a replacement amino acid at position 228. For example, S can be replaced with proline.

Another series of amino acids suitable for modification include amino acids in the hinge area that affect the binding of a molecule containing a heavy chain to binding to the F receptor._c and the internalization of the antibody molecule. These amino acids include, in IgG1 molecules, the residues of about 233 to about 237 (Glu-Leu-Leu-Gly-Gly); (SEQ ID NO: 49) from about 252 to about 256 (Met-lle-SerArg-Thr) (SEQ ID NO: 50) and from about 318 (Glu) to about 331 (Pro), including, for example, Lys32o. Lys 322 and Pro329 Fully human antibodies are particularly suitable for the therapeutic treatment of human patients. Human antibodies can be obtained by a variety of methods known in the art, including phage display methods described above using antibody libraries derived from human immunoglobulin sequences, see US Patent Nos. 4,444,887 and 4,716,111; and WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741. The techniques of Colé et al. and Boerder et al. for the preparation of human monoclonal antibodies (Colé et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss (1985); and Boerner et al., J Immunol 147: 86 (1991)).

Human antibodies can also be produced using transgenic mice that cannot express functional endogenous immunoglobulins, but that also express certain human immunoglobulin genes. For example, human immunoglobulin light and heavy chain gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region can be introduced into mouse embryonic stem cells, in addition to light chain genes and

<img file="MX338474B_D0066.tif" />

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-51 human heavy. Mouse immunoglobulin light and heavy chain genes can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, the homozygous deletion of the JH region prevents the production of endogenous antibodies. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. Chimeric mice are then propagated to produce homozygous offspring expressing human antibodies, see, eg, Jakobovitis et al., Proc Nati Acad Sci USA 90: 2551 (1993); Jakobovitis et al., Nature 362: 255 (1993); Bruggermann et al., Year in Immunol 7:33 (1993); and Duchosal et al., Nature 355: 258 (1992)).

Transgenic mice are immunized in the normal manner with a CXCR5, eg, all or part of CXCR5, such as their EC domain. Monoclonal antibodies directed against CXCR5 can be obtained from immunized transgenic mice using standard hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice, during B lymphocyte differentiation are rearranged, and subsequently undergo class change and somatic mutation. In this way, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview, see Lonberg et al., Int Rev Immunol 13: 65-93 (1995). For a description of the production of human antibodies and human monoclonal antibodies and of protocols for producing such antibodies, see, for example, WO 98/24893; WO 92/01047; WO 96/34096; and WO 96/33735; EPO No. 0 598 877; and United States Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598. In addition, companies such as Amgen (Fremont, CA), Genpharm (San Jose, CA) and Medarex, Inc. (Princeton, NJ) can be contracted to provide human antibodies directed against CXCR5 using technology similar to that described above.

In addition, human mAb could be obtained by immunizing mice in which peripheral blood leukocytes, splenocytes, or bone marrow have been transplanted

<img file="MX338474B_D0067.tif" />

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY example, by the XTL tiloma technique

Antibodies can be fully generated from a selected epitope using a technique

In this strategy, an antibody of human origin (by Biopharmaceuticals, Israel), humans that recognize so-called guided selection, selected non-human monoclonal, eg, a mouse antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope (Jespers et al., Bio / technology 12: 899 (1988)).

When using recombinant techniques, the antibody variant can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody variant is produced intracellularly, as a first step, particulate debris, host cells, or lysed fragments can be removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163 (1992) describe a procedure for isolating antibodies that are secreted into the periplasmic space of E. coli. In summary, cell paste is exposed to sodium acetate (pH 3.5) and EDTA. Cell waste can be removed by centrifugation. When the antibody variant is secreted into the medium, the supernatant from these expression systems is generally first concentrated using a commercially available protein concentration filter, eg, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF can be included to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. The suitability of protein A or protein G as an affinity ligand depends on the species and the isotype of any F domain._c of immunoglobulin that is present in the antibody variant. Protein A can be used to purify antibodies that are based on human lgG1, lGG2, or lgG4 heavy chains (Lindmark et al., J Immunol Meth 62: 1 (1983)). G protein can be used for mouse isotypes and for human IgG3 (Guss et al., EMBO J 5: 1567 (1986)). The matrix to which the ligand binds

<img file="MX338474B_D0068.tif" />

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OF INDUSTRIAL PROPERTY of affinity is most often agarose, but other matrices are available. Mechanically stable matrices, such as controlled pore glass or poly (styrene divinyl) benzene, allow for faster flows and shorter processing times than can be achieved with agarose. When the antibody variant comprises a Ch3 domain, the Bakerbond ABX ™ resin (JT Baker; Phillipsburg, NJ) is useful for purification. Other techniques are also available for protein purification, such as fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, silica chromatography, heparin agarose chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromato focus, SDS-PAGE and ammonium sulfate precipitation, depending on the antibody or variant to be recovered.

After any preliminary purification step, the mixture comprising the antibody or variant of interest and the contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of between about 2.5 and 4.5 , preferably performed at low salt concentration (eg, a salt concentration of about 0-0.25 M).

In addition, the antibodies of the invention, in turn, can be used to generate anti-idiotype antibodies that mimic CXCR5 using techniques well known to those of skill in the art (see, eg, Greenspan et al., FASEB J 7: 437 (1989) ; and Nissinoff, J Immunol 147: 2429 (1991)). For example, antibodies that bind and competitively inhibit the multimerization and / or binding of a ligand to CXCR5 can be used to generate anti-idiotypes that mimic CXCR5 and the binding domain and consequently bind and neutralize CXCR5 and / or its ligand, these neutralizing anti-idiotypes or F fragments_ab of these anti-idiotypes can be used in therapeutic regimens, for example, to neutralize CXCL13.

The antibodies of the present invention can be bispecific antibodies. Bispecific antibodies can be monoclonal, preferably human or humanized, antibodies that have

IMPIO * ®

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INDUSTRIAL Iba 2 ^ binding specificities for at least two different antigens. In the present invention, one of the binding specificities is directed towards CXCR5, and the other may be for any other antigen, such as a cell surface protein, receptor, receptor subunit, ligand, antigen with tissue specificity, protein derived from viruses, envelope protein encoded by viruses, protein of bacterial origin, bacterial surface protein etc. In this way, the other specificity could be for CXCL13.

The methods for obtaining bispecific antibodies are well known. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain / light chain pairs, where the two heavy chains have different specificities (Milstein et al., Nature 305: 537 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, hybridomas (quadromas) produce a possible mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is normally done by affinity chromatography steps. Similar procedures are described in WO 93/08829 and in Traunecker et al., EMBO J 10: 3655 (1991). Other methods for obtaining bispecific antibodies are provided, for example, in Kufer et al., Trends Biotech 22: 238-244, 2004.

Antibody variable domains can be fused with the desired binding specificities to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge regions, Ch2 and Ch3- It may be that the first heavy chain constant region (Cm) contains the site necessary for binding of light chain present in at least one of the fusions. The DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into different expression vectors and used to co-transform a suitable host organism. For more details on the generation of bispecific antibodies see, for example, Suresh et al., Meth Enzym 121: 210

<img file="MX338474B_D0069.tif" />

(1986).

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The present invention also contemplates heteroconjugated antibodies.

Heteroconjugate antibodies are made up of two covalently linked antibodies. These antibodies have been proposed, for example, to target cells of the immune system to unwanted cells (US Patent No. 4,676,980). It is contemplated that the antibodies can be prepared in vitro using methods known in protein synthesis chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or through the formation of a thioester bond. Examples of reagents suitable for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate, and those described, for example, in US Patent No. 4,676,980.

Furthermore, single domain antibodies against CXCR5 can be generated. Examples of this technology have been described in WO9425591 for camelid Ig heavy chain derived antibodies, as well as in US20030130496 which describes the isolation of single domain fully human antibodies from phage libraries.

Alternatively, techniques described for the production of single-chain antibodies can be adapted (US Patent No. 4,946,778; Bird, Science 242: 423 (1988); Huston et al., Proc Nati Acad Sci USA 85: 5879 (1988) ; and Ward, et al., Nature 334: 544 (1989)) to produce single-chain antibodies. Single chain antibodies are formed by binding of the heavy and light chain fragments of the F region_v through an amino acid link, resulting in a single chain polypeptide. Techniques for assembling fragments F can also be used._v functional in E. coli (Skerra et al., Science 242: 1038 (1988)).

The present invention includes antibodies recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugates) to a polypeptide. Fused or conjugated antibodies of the present invention can be used to facilitate purification, see, eg, WO 93/21232; EP 439,095;

<img file="MX338474B_D0070.tif" />

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OF INDUSTRIAL PROPERTY

<img file="MX338474B_D0071.tif" />

Naramura et al., Immunol Lett 39:91 (1994); United States Patent No. 5,474,981; Gillies et al., Proc Nati Acad Sci USA 89: 1428 (1992); and Fell et al., J Immunol 146: 2446 (1991). The marker amino acid sequence may be a hexa-histidine peptide, such as the marker provided in a pQE vector (QIAGEN, Inc., Chatsworth, CA), among others, of which many are commercially available, Gentz ​​et al ., Proc Nati Acad Sci USA 86: 821 (1989). Other useful peptide markers for purification include, but are not limited to, the HA marker, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37: 767 (1984)) and the so-called flag marker .

You can also create single peptide chain binding molecules in which the F regions are connected_v heavy and light chain. Single chain antibodies ("scF_v”) And the method of its construction, for example, in US Patent No. 4,946,778. Alternatively, F can be constructed and expressed_tob by similar means. All fully or partially human antibodies may be less immunogenic than fully murine mAbs, and single-stranded fragments or antibodies may also be less immunogenic.

Antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature 348: 552 (1990). Clarkson et al., Nature 352: 624 (1991) and Marks et al., J Mol Biol 222: 581 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity human antibodies (nM interval) by chain exchange (Marks et al., Bio / Technology 10: 779 (1992)), as well as combinatorial infection and recombination in vivo as a strategy to construct very large phage libraries (Waterhouse et al.,

Nucí Acids Res 21: 2265 (1993)). Thus, the techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

Candidate anti-CXCR5 antibodies are tested by

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-57 Enzyme-Linked Immunosorbent (ELISA), faclq, Immunize Western KfarAma, or other immunochemical techniques known in the art. In this way, B cells or cells expressing CXCR5 can be used to detect binding of antibodies using a known technique, or recombinantly expressed CXCR5, or a part thereof, such as the EC domain, can adhere to a solid phase and used as a capturing element in an assay, configured according to design choice.

To determine if a particular antibody homolog binds to human CXCR5, any standard binding assay can be used. Useful CXCR5 binding assays include FACS analyzes, ELISA assays, radioimmunoassays, and the like, which detect binding of an antibody, and the resulting functions thereof, to human CXCR5. Useful in these assays are full-length, soluble forms of human CXCR5 taught herein. Binding of an antibody or homolog to CXCR5, or to soluble fragments thereof, can be conveniently detected through the use of a second antibody specific for immunoglobulins of the species from which the antibody or homolog comes.

To determine whether or not a particular antibody or homolog significantly blocks the binding of CXCL13 or other ligand to human CXCR5, any suitable competitive assay can be used. Useful assays include, for example, ELISA assays, FACS assays, radioimmunoassays, and the like, which quantify the ability of the antibody or homolog to compete with CXCL13 or another ligand for binding to human CXCR5. Preferably, the ability of a ligand to block binding of labeled human CXCR5 to an immobilized antibody or homolog is measured.

The ability of an antibody or homolog to bind to human CXCR5 can be assessed by testing its ability to bind to CXCR5 cells.⁺ human. These are CXCR5 * cells suitable for use in determining whether a particular antibody or homolog binds to human CXCR5. Tissue culture cells transformed with DNA encoding full-length human CXCR5 and expressing CXCR5 on the surface or B cell lines.

<img file="MX338474B_D0073.tif" />

IMPI

-58 Binding of the antibody or homolog to the CXCR5 * cell can be detected by staining the cells with a second fluorescently labeled antibody specific for immunoglobulins of the same species from which the antibody homolog being tested is derived. A fluorescence activated cell sorter (FACS) can be used to detect and quantify any binding, see generally Shapiro, Practical Flow Cytometry, Alan R. Liss, Inc., New York, NY (1985).

Furthermore, the ability of an antibody homolog to block binding of a ligand, such as CXCL13, to human CXCR5 can be determined by preincubating excess ligand with CXCR5 cells.⁺ and quantifying the extent to which the bound ligand blocks binding of the antibody or homolog to cells. Binding of the antibody or homolog to CXCR5 cells⁺ it can be quantified by FACS analysis using a second fluorescently labeled antibody specific for immunoglobulins of the same species from which the antibody homolog being tested is derived. Alternatively, a competitive assay can be configured using a ligand or labeled antibody as known in the art.

A ligand, such as CXCL13, used in the above assays can be provided by cells transformed with the ligand gene, can be an isolated CXCL13, obtained by practicing the methods taught herein, or can be purchased commercially.

To determine if a particular antibody or homolog does not produce a significant reduction in the number of CXCR5 cells⁺ circulating in vivo, the number of CXCR5 cells is quantified⁺ circulating isolates from a mammal within 24 hours of administration of the antibody or homolog to a mammal having normal immune function, and compared to the number before administration or to the number in a control mammal to which A corresponding isotype antibody or homolog of irrelevant specificity has been administered in place of an antibody or homolog of the present invention. Quantification of CXCR5 cells⁺ in animals that have received the dosage of an antibody to CXCR5 or a functional part or derived from

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-59IMPI ¡INSTir / TO MEXICANO Dí LA? ROFiEOAD INDUSTRIAL itself can be performed, for example, by staining the cells obtained with fluorescently labeled antibodies that bind to antiCXCR5 antibodies, as well as labeled antibodies specific for T cells and cells B, followed by FACS analysis.

The antibodies of the present invention can be described or specified in terms of the epitopes or parts of CXCR5 recognized or to which the antibody specifically binds. The epitope or epitopes or the part or parts of polypeptide can be specified as described herein, for example, by the N-terminal and C-terminal positions, by size in contiguous amino acid residues, conformational epitopes, and the like.

The antibodies of the present invention can also be described or specified in terms of cross reactivity. Also included in the present invention are antibodies that bind to CXCR5 polypeptides, having an identity of at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% (calculated using methods known in the art and described herein) with CXCR5.

The antibodies of the present invention can also be described or specified in terms of the binding affinity for a CXCR5 of interest. Anti-CXCR5 antibodies can bind with a K_D less than about 10 '⁷ M, less than about 10 '⁶ M, or less than about 10 '⁵ M. Higher binding affinities may be beneficial in an antibody of interest, such as those having an equilibrium dissociation constant or Kd of approximately 10 '⁸ to about 10¹⁵ M, about 10 '⁸ at about 10 '¹² M, about 10 '⁹ at about 10 '¹¹ M, or about 10 '⁸ at about 10 '¹° M. The invention also provides antibodies that competitively inhibit the binding of an antibody to an epitope of the invention, as determined by any method known in the art to determine competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits epitope binding by at least 95%, at least 90%, by

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-60- industrial minus 85%, at least 80%, at least 75%, at least 70%. at least 60% or at least 50%.

The present invention also includes conjugates comprising an antibody of interest. The conjugates comprise two primary components, an antibody of interest and a second component, which can be a cell binding agent, a cytotoxic agent, and the like.

As used herein, the term "cell-binding agent" refers to an agent that specifically recognizes and binds to a molecule on the surface of the cell. Thus, the cell binding agent may be a CD antigen, a pathogen antigen, such as a virus antigen, a differentiation antigen, a cancer antigen, a cell specific antigen, an antigen with specificity. of tissue, an Ig or Ig-like molecule and the like.

In one embodiment, the cell binding agent specifically recognizes CXCL13 or the CXCR5 complex and a ligand thereof, such as CXCL13. The conjugate may be in contact with the target cell for a period of time sufficient to allow an effector function of the conjugate to act on the cell, and / or to allow the conjugate to be for a sufficient period of time for the cell to internalize it. .

Cell binding agents can be of any type currently known, or being known, and include peptides, non-peptides, saccharides, nucleic acids, ligands, receptors, and the like, or combinations thereof. The cell binding agent can be any compound that can bind to a cell, in a specific or non-specific way. Generally, the agent can be an antibody (especially monoclonal antibodies), lymphokines, hormones, growth factors, vitamins, nutrient transport molecules (such as transferrin), or any other molecule or substance that binds to cells.

Other examples of cell binding agents that can be used include: polyclonal antibodies; monoclonal antibodies; and antibody fragments such as F_tob, F_tob ·, F (_ab') 2 and F_v (Parham, J. Immunol. 131: 2895-2902

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-61 IMPI

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INDUSTRIAL (1983); Spring et al., J. Immunol. 113: 470-478 (1974); and Nisonoff et al., Arch. Biochem. Biophys. 89: 230-244 (1960)).

The second component can also be a cytotoxic agent. The term "cytotoxic agent" as used herein refers to a substance that reduces or blocks the function, or growth, of cells and / or causes destruction of cells. Thus, the cytotoxic agent can be a taxol, a maytansinoid such as DM1 or DM4, CC-1065 or a CC-1065 analog, ricin, mitomycin C, and the like. In some embodiments, the cytotoxic agent, as with any binding agent in a conjugate of the present invention, is covalently bound, directly or via a cleavable or non-cleavable linker, to an antibody of interest.

Examples of suitable maytansinoids include maytansinol and maytansinol analogs. Maytansinoids inhibit microtubule formation and are highly toxic to mammalian cells.

Examples of suitable maytansinol analogs include those that have a modified aromatic ring and those that have modifications at other positions. Such suitable maytansinoids are described in US Patent Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.

Examples of suitable maytansinol analogs having a modified aromatic ring include: (1) C-19-chloro (United States Patent No. 4,256,746) (prepared, for example, by LAH reduction of ansamitocin P2); (2) C-20-hydroxy (or C-20-desmethyl) +/- C-19-chloro (United States Patent Nos. 4,361,650 and 4,307,016) (prepared, for example, by demethylation using Streptomyces or Actinomyces or dechlorination using lithium aluminum hydride (LAH)); and (3) C-20-desmethoxy, C-20-acyloxy (-OCOR), +/- chloro (United States Patent No. 4,294,757) (prepared by acylation using acyl chlorides).

Examples of suitable maytansinol analogs having modifications of other positions include: (1) C-9-SH (US Patent

States No. 4,424,219) (prepared by reaction of maytansinol with H2S or P2S5);

<img file="MX338474B_D0076.tif" />

-62IMPI

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FROM OWNERSHIP

INDUSTRIAL (2) C-14-alkoxymethyl (demethoxy / CH2OR) (US Patent No. 4,331,598); (3) C-14-hydroxymethyl or acyloxymethyl (CH₂OH or Ch ^ OAc) (US Patent No. 4,450,254) (prepared in Nocardia); (4) C-15-hydroxy / acyloxy (US Patent No. 4,364,866) (prepared by conversion of maytansinol by Streptomyces); (5) C-15-methoxy (US Patent Nos. 4,313,946 and 4,315,929) (isolated from Trewia nudiflora); (6) C-18-N-desmethyl (US Patent Nos. 4,362,663 and 4,322,348) (prepared by demethylation of maytansinol by Streptomyces); and (7) 4,5-deoxy (United States Patent No. 4,371,533) (prepared by titanium trichloride / LAH reduction of maytansinol).

Cytotoxic conjugates can be prepared by in vitro methods. To bind a cytotoxic agent, drug, or prodrug to the antibody, a linking group is commonly used. Suitable linking groups are known in the art and include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. For example, conjugates can be constructed using a disulfide exchange reaction or by forming a thioether bond between an antibody of interest and the drug or prodrug.

As described above, the present invention provides isolated nucleic acid sequences encoding an antibody or functional variant thereof as described herein, vector constructs comprising a nucleotide sequence encoding the CXCR5 binding polypeptides of the present invention, host cells comprising said vector, and recombinant techniques for the production of the polypeptide.

The vector normally contains components known in the art and generally includes, but is not limited to, one or more of the following elements: a signal sequence, an origin of replication, one or more marker or selection genes, sequences that facilitate and / or enhance translation, an enhancing element and the like. Thus, the expression vectors include a nucleotide sequence operably linked to said sequences of

<img file="MX338474B_D0077.tif" />

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-63 MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY appropriate transcriptional or translational regulatory nucleotides such as those derived from mammalian, microbial, viral or insect genes. Examples of additional regulatory sequences include operators, mRNA ribosome binding sites, and / or other appropriate sequences that control transcription and translation, such as initiation and termination. Nucleotide sequences are functionally linked when the regulatory sequence is functionally related to the nucleotide sequence for the appropriate polypeptide. In this way, a promoter nucleotide sequence is operably linked to, for example, the antibody heavy chain sequence if the promoter sequence controls the transcription of that nucleotide sequence.

Furthermore, sequences encoding appropriate signal peptides that are not naturally associated with antibody heavy and / or light chain sequences can be incorporated into expression vectors. For example, a nucleotide sequence for a signal peptide (secretion leader) can be fused in phase with the polypeptide sequence so that the antibody is secreted into the periplasmic space or to the medium. A signal peptide that is functional in the desired host cell increases the extracellular secretion of the appropriate antibody or part of it. The signal peptide can be cleaved from the polypeptide upon secretion of the antibody by the cell. Examples of these secretion signals are well known and include, for example, those described in US Patent Nos. 5,698,435; 5,698,417; and 6,204,023.

The vector can be a plasmid, a single-stranded or double-stranded viral vector, a single-stranded or double-stranded RNA or a phage DNA vector, a phagemid, a cosmid or any other vehicle of a transgene of interest. These vectors can be introduced into cells as polynucleotides by well known techniques to introduce DNA or RNA into cells. Vectors, in the case of phage and viral vectors, can also be introduced into cells as packaged or encapsulated viruses by well known techniques for infection and transduction. Viral vectors may be competent for replication or

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MEXICAN INSTITUTE fZA DE LA PROI^;i AGE “INDUSTRIAL defective for replication. In the latter case, viral propagation will generally only occur in host cell supplementation and in the use of plural vectors carrying the various virus components necessary to produce a particle. Cellless translation systems can also be employed to produce the protein using RNA derived from the present DNA constructs (see, eg, WO 86/05807 and WO 89/01036; and US Patent No. 5,122,464. ).

The antibodies of the present invention can be expressed in any suitable host cell. Examples of host cells useful in the present invention include prokaryotic cells, yeast, or higher eukaryotic cells and include, but are not limited to, microorganisms such as bacteria (eg, E. coli, B. subtilis, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, Serratia and Shigella, as well as bacilli, Pseudomonas and Streptomyces) transformed with bacteriophage DNA, plasmid DNA or cosmid DNA vectors containing the antibody coding sequences of interest; yeasts (eg Saccharomyces, Pichia, Actinomycetes, Kluyveromyces, Schizosaccharomyces, Candida, Trichoderma, Neurospora, and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus) transformed with recombinant yeast expression vectors containing coding sequences of antibodies; insect cell systems infected with recombinant virus expression vectors (eg, Baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (eg, cauliflower mosaic virus, CaMV; or tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (eg, the Ti plasmid) containing antibody coding sequences; or mammalian cell systems (eg COS, CHO, BHK, 293 or 3T3 cells) carrying recombinant expression constructs containing promoters derived from the genome of mammalian cells (eg, metallothionein promoter) or from mammalian viruses (for example, the adenovirus late promoter; or the 7.5K promoter of the vaccinia virus).

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-65 MEXICAN INSTITUTE Dt INDUSTRIAL PROPERTY

Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selection marker genes. A phenotypic selection marker gene is, for example, a gene that encodes a protein that confers antibiotic resistance or that provides an autotrophic requirement. Examples of useful expression vectors for prokaryotic host cells include commercially available plasmid derivatives such as pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), pGEM1 (Promega Biotec, Madison, Wl), pET (Novagen, Madison , Wl) and the pRSET vector series (Invitrogen, Carlsbad, CA) (Studier, J Mol Biol 219: 37 (1991); and Schoepfer, Gene 124: 83 (1993)). Commonly used promoter sequences for recombinant prokaryotic host cell expression vectors include the T7 promoter system, (Rosenberg et al., Gene 56: 125 (1987)), n-lactamase (penicillinase), and lactose (Chang et al., Nature 275: 615 (1978); and Goeddel et al., Nature 281: 544 (1979)), the tryptophan (trp) promoter system (Goeddel et al., Nucí Acids Res 8: 4057 (1980)), and the tac promoter (Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Coid Spring Harbor Laboratory (1990)).

Yeast vectors will often contain a sequence of origin of replication, such as that of the 2D yeast plasmid, an autonomous replication sequence (ARS), a promoter region, polyadenylation sequences, sequences for transcription termination, and a gene selection marker. Suitable promoter sequences for yeast vectors include, but are not limited to, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J Biol Chem 255: 2073 (1980)) or other glycolytic enzymes (Holland et al., Biochem 17 : 4900 (1978)) such as enolase, glyceraldehyde-3phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosaphosphate isomerase, phosphoglucose isomerase and glucokinase. Other suitable vectors and promoters for use in expression in yeast are described are described in Fleer et al., Gene 107: 285 (1991). Other promoters and vectors suitable for yeast and for yeast transformation protocols

<img file="MX338474B_D0080.tif" />

they are well known in the art. Yeast transformation protocols are well known. One of these protocols is described by Hinnen et al., Proc Nati Acad Sci 75: 1929 (1978), which selects Trp transformants⁺ in a selective medium.

Any eukaryotic cell culture can be used, be it a vertebrate or invertebrate cell culture. Examples of invertebrate cells include plant and insect cells (Luckow et al., Bio / Technology 6:47 (1988); Miller et al., Genetic Engineering, Setlow et al., Eds., Vol. 8, p. 277-9, Plenum Publishing (1986); and Maeda et al., Nature 315: 592 (1985)). For example, Baculovirus systems can be used for the production of heterologous proteins. In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be cloned under the control of an AcNPV promoter (eg, the polyhedrin promoter). Other hosts that have been identified include Aedes, Drosophila melanogaster and Bombyx morí. Various viral strains for transfection are available to the public, for example, the L-1 variant of AcNPV and the Bm-5 strain of Bombyx died NPV. In addition, plant cell cultures of cotton, corn, potato, soy, petunia, tomato, and tobacco, as known in the art, can also be used as hosts.

Cultures of vertebrate cells and for the propagation of vertebrate cells (tissue culture) can be carried out by means of a routine procedure, although there are bothersome cell lines that require, for example, a specialized medium with unique factors, feeding cells and the like, see Tissue Culture, Kruse et al., eds., Academic Press (1973). Examples of useful mammalian host cell lines are monkey kidney; human embryonic kidney line; hamster pup kidney cells; Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc Nati Acad Sci USA 77: 4216 (1980)); mouse sertoli cells; human cervical carcinoma cells (eg HeLa); dog kidney cells; human lung cells; cells

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-67 MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY human liver; mouse mammary tumor; and NSO cells.

Host cells are transformed with vectors for antibody production and cultured in conventional nutrient medium containing growth factors, vitamins, minerals, and the like, as well as appropriate inducers for the cells and vectors used. The commonly used promoter and enhancer sequences come from polyoma virus, Adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus (CMV). DNA sequences derived from the SV40 viral genome can be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell, eg, SV40 origin, early and late promoter, enhancer, cleavage sites. and splicing and polyadenylation. Viral early and late promoters are particularly useful because they are easily obtained from a viral genome as a fragment that may also contain an origin of viral replication. Exemplary expression vectors are commercially available for use in mammalian host cells.

Commercially available media such as Ham's F10 medium, Minimum Essential Medium (MEM), RPMI-1640 and Dulbecco's Modified Eagle's Medium (DMEM) are suitable for culturing the host cells. Furthermore, any of the media described in Ham et al., Meth Enzymol 58:44 (1979) and Barnes et al., Anal Biochem 102: 255 (1980), and in US Patent Nos. 4,767,704; 4,657,866; 4,560,655; 5,122,469; 5,712,163; or 6,048,728 can be used as a culture medium for host cells. Any of these media can be supplemented when necessary with hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as chlorides, such as sodium, calcium or magnesium chloride; and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics, trace elements (defined as inorganic compounds normally present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplement may be included at appropriate concentrations, depending on design choice. Culture conditions such as temperature, pH, and

<img file="MX338474B_D0082.tif" />

INSTITUTO MEXICANO DE LA PROPIEDAD INDUSTRIAL similar, are those known in the appropriate technique for the cell and those that allow the desired expression of the transgene.

The polynucleotides of interest can be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (eg, as described in Kutmeier et al., Bio / Techniques 17: 242 (1994)) and then amplifying the linked oligonucleotides, for example, by POR.

Alternatively, a polynucleotide encoding an antibody can be generated from nucleic acid in a cell that expresses it. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be obtained from a suitable source, such as a library, which may be specific for antibody-producing cells, such as hybridoma cells selected to express an antibody of the invention. Primers suitable for PCR amplification can be configured. The amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and the corresponding amino acid sequence of the antibody have been determined, the nucleotide sequence of the antibody can be manipulated to obtain the equivalents of interest described herein using methods known in the art for manipulation of sequences of nucleotides, for example, recombinant DNA techniques, targeted mutagenesis, PCR etc. (see, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Coid Spring Harbor Laboratory (1990); and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley &amp; Sons ( 1998) to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions and / or insertions.

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The amino acid sequence of the heavy chain and / or light chain variable domain can be inspected for identification of the ubk by well-known methods, for example, by comparison with known amino acid sequences of other heavy chain variable regions. and light to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs can be inserted into flanking regions, eg, into human flanking regions to humanize a non-human antibody, as described supra. The polynucleotide of interest generated by the combination of the flanking regions and one or more CDRs encodes an antibody that specifically binds CXCR5, or at least the ED domain thereof. For example, these methods can be used to make amino acid substitutions or deletions of one or more cysteine ​​residues of the variable region that participate in an intra-chain disulfide bond to generate antibody molecules that lack one or more intra-chain disulfide bonds.

The antibodies or antibody fragments of the invention can be used to detect CXCR5, and thus CXCR5-expressing cells, in an in vitro or in vivo biological sample. In one embodiment, the anti-CXCR5 antibody of the invention is used to determine the presence and level of CXCR5 in a tissue or in tissue derived cells. Tissue or biopsy levels of CXCR5 can be determined, for example, in an immunoassay with the antibodies or antibody fragments of the invention. Tissue or biopsy of it can be frozen or fixed. These or other methods can be used to determine other properties of CXCR5, such as its level, cell location, mRNA levels, its mutations, and the like.

The method described above can be used, for example, to diagnose cancer in a subject who is known or suspected to have cancer, where the level of CXCR5 measured in that patient is compared to that of a normal reference or standard subject . The assay of interest can also be used to diagnose arthritis or another autoimmune disease characterized by B cell infiltration and concentration, along with the development of lymphoid tissue.

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-70IMPI

MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL differentiated.

The present invention further provides monoclonal antibodies, humanized antibodies, and epitope binding fragments thereof which are further labeled for use in research or diagnostic applications. In some embodiments, the marker is a radiolabel, a fluorophore, a chromophore, an imaging agent, or a metallic ion.

A diagnostic method is also provided in which said labeled antibodies or epitope binding fragments thereof are administered to a subject suspected of having cancer, arthritis, autoimmune diseases or other disease associated with CXCR5, and is measured or measured. controls the distribution of the marker within the subject's body.

The antibody and fragments thereof of the present invention can be used as affinity purification agents. In this process, the antibodies are immobilized on a solid phase, such as a dextran or agarose resin or filter paper, using methods known in the art. The immobilized antibody is contacted with a sample containing CXCR5 or cells carrying it to be purified, and then the support is washed with a suitable solvent that will remove substantially all of the material from the sample except the CXCR5 or the cell to be purify, which are bound to the immobilized antibody of interest. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5.0, which will release the CXCR5 or the cell from the antibody of interest.

For diagnostic applications, the antibody of interest will typically be labeled with a detectable marker. Numerous markers are available that can generally be grouped into the following categories: (a) radioisotopes, such as³⁶Yes, ¹⁴C, ¹²⁵l, ³H and ¹³¹one (The antibody can be labeled with the radioisotope using techniques described in Current Protocols in Immunology, vol. 12, Coligen et al., ed., Wiley-lnterscience, New York (1991), for example, and radioactivity can be measured using cell count. scintillation); (b) fluorescent labels such as rare earth chelates (europium chelates), fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lysamine,

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IMPI τ. MEXICAN INSTITUTE

- / 1- OF THE PROPERTY

INDUSTRIAL phycoerythrin and texas red, the fluorescent labels being able to be conjugated with the antibody using a technique described in Current Protocols in Immunology, supra, for example, where the fluorescence can be quantified using a fluorimeter; and (c) various enzyme substrate markers that are available (US Patent No. 4,275,149 provides a review), the enzyme generally catalysing a chemical alteration of the chromogenic substrate that can be measured using various techniques, eg, the enzyme it can catalyze a color change in a substrate, which can be measured spectrophotometrically, or the enzyme can alter the fluorescence or chemiluminescence of the substrate. Techniques are known to quantify a change in fluorescence, for example, using a luminometer, or the marker donates energy to a fluorescent acceptor. Examples of enzyme labels include luciferases (eg, firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydroftalazinodiones, malate dehydrogenase, urease, peroxidase, such as horseradish peroxidase (HRPO), alkaline phosphatase, □ -galactosidase, glucoamylase, lysozyme, saccharide oxidases ( for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al., Meth Enz, ed. Langone &amp; Van Vunakis, Academic Press, New York, 73 (1981).

When these labels are used, suitable substrates are available, such as: (i) in the case of horseradish peroxidase, hydrogen peroxidase is used as the substrate, where hydrogen peroxidase oxidizes a dye precursor (eg, hydrochloride of orthophenylene diamine (OPD) or 3,3 ', 5,5'tetramethyl benzidine (TMB)); (I) in the case of alkaline phosphatase (AP), p-nitrophenyl phosphate is used as a chromogenic substrate; and (iii) QD-galactosidase (DD-Gal) with a chromogenic substrate (eg, p-nitrophenyl-ü-D-galactosidase) or a fluorogenic substrate such as 4-methylumbelliferyl-DD-galactosidase.

Other enzyme-substrate combinations are available to those skilled in the art. For a general review, see United States Patents.

IMPI

INSTirUTr 'ΜίΧ.'ίΆνη

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-72 No. 4,275,149 and 4,318,980.

Sometimes the marker is indirectly conjugated to the antibody. For example, the antibody can be conjugated to biotin and any of the indicators mentioned above can be conjugated to avidin, or vice versa. Biotin selectively binds to avidin and, in this way, the marker can be conjugated to the antibody in that indirect manner. Alternatively, to achieve indirect conjugation of the marker, the antibody is conjugated to a small hapten (eg, digoxin) and one of the different types of markers or indicators mentioned above is conjugated to an anti-digoxin antibody. In this way, indirect conjugation of the marker to the antibody or mutein can be achieved using a second antibody.

In another embodiment of the invention, the antibody does not need to be labeled, and its presence can be detected using a labeled antibody-binding antibody, another form of second antibody.

The antibodies of the present invention can be used in any known assay method such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques (CRC Press, Inc. 1987).

Competitive binding assays are based on the ability of a labeled standard to compete with the test sample for binding with a limited amount of antibody. The amount of antigen in the test sample is inversely proportional to the amount of standard that binds to the antibodies. To facilitate the determination of the amount of standard that Binds, the antibodies are generally insolubilized before or after competition. As a result, the standard and the test sample that are bound to the antibodies can be conveniently separated from the standard and the test samples that remain unbound.

Sandwich assays involve the use of two antibodies, each being capable of binding to a different immunogenic, determinant or epitope part of the target to be detected. In a sandwich-type assay, the test sample to be analyzed is bound to a first antibody that is directly immobilized or

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INSINVIO

OF THE INDUSTRIAL PROTEIN indirectly on a solid support, and subsequently a second antibody, directly or indirectly labeled, binds to the bound test sample, thereby forming an insoluble three-part complex, see, for example , the

United States Patent No. 4,376,110. The second antibody can itself be labeled with a detectable moiety (direct sandwich assay) or can be measured using an anti-immunoglobulin antibody or another suitable member of the binding pair (antibody / antigen, receptor / ligand, enzyme / substrate, for example) that is marked with a detectable moiety (indirect sandwich assay). For example, one type of sandwich type assay is an ELISA assay, in which case the detectable moiety is an enzyme.

For immunohistochemistry, the cell or tissue sample can be fresh or it can be frozen or it can be embedded in paraffin and fixed with a preservative such as, for example, formalin.

The antibodies can also be used for in vivo diagnostic assays. Generally, the antibody mutant is labeled with a radionucleotide (such as¹¹¹ In, ⁹⁹Tc, ¹⁴C,¹³¹1, ³H ³²P o ³⁵S) so that CXCR5 expressing sites can be located using immunoscintography.

The present invention also includes kits, eg, comprising an antibody, fragment thereof, homologue, derivative thereof, and the like, such as a labeled or cytotoxic conjugate, and instructions for use of the antibody, conjugate for killing particular cell types, and the like. . Instructions may include directions for using the antibody, conjugate, and the like in vitro, in vivo, or ex vivo. The antibody can be in liquid or solid form, usually lyophilized. The kit may contain other suitable reagents such as a buffer, a reconstitution solution and other ingredients necessary for the desired use. A packaged combination of reagents in predetermined amounts is contemplated with instructions for use, such as for therapeutic use or for conducting a diagnostic assay.

When the antibody is labeled, such as with an enzyme, the kit may include substrates and cofactors required by the enzyme (eg, a substrate precursor that provides the detectable chromophore or fluorophore). Furthermore, they can

IMPI

<img file="MX338474B_D0088.tif" />

Other additives such as stabilizers, buffers (eg, a blocking buffer or a lysis buffer), and the like are included in the MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY. LUS uui iliddiliLA utlutivoo of the various reagents can be varied to provide concentrates of a reagent solution that provides user flexibility, space economy, reagent economy and the like. The reagents can be provided as dry powders, usually lyophilized, including excipients, which after dissolution provide a reagent solution having the appropriate concentration.

The antibodies of the present invention can be used to treat a mammal. In one embodiment, the antibody or equivalent of interest is administered to a non-human mammal for the purpose of obtaining preclinical data, for example. Exemplary non-human mammals to be treated include non-human primates, dogs, cats, rodents, and other mammals in which preclinical studies are performed. These mammals can be established animal models for a disease to be treated with the antibody, or they can be used to study the toxicity of the antibody of interest. In each of these embodiments, dose increase studies can be performed in the mammal.

An antibody with or without a second component, such as a therapeutic moiety conjugated thereto, administered alone or in combination with one or more cytotoxic factors can be used as a therapeutic agent. The present invention relates to antibody-based therapies involving administering antibodies of the invention to an animal, a mammal, or a human, to treat a CXCR5-mediated disease, disorder, or condition. The animal or subject can be a mammal that needs a particular treatment, such as a mammal that has been diagnosed with a particular disorder, for example, one related to CXCR5. Antibodies directed against CXCR5 are useful, for example, for the prophylaxis or treatment of arthritis, inflammatory diseases, in general, graft rejection, cancer, and autoimmune disorders. For example, by administering a therapeutically acceptable dose of an anti-CXCR5 antibody of the present invention, or a mixture of a plurality of the antibodies of the present invention or equivalents thereof, or in

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-75 MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY combination with other antibodies of various origins, symptoms of diseases can be improved or prevented in the treated animal, particularly in humans.

Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs, equivalents, and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention as described herein. present specification (including fragments, analogs and derivatives thereof) and anti-idiotypic antibodies as described herein. The antibodies of the invention can be used to treat, inhibit, or prevent diseases, disorders, or conditions associated with aberrant expression and / or activity of CXCR5, including, but not limited to, one or more of the diseases, disorders, or conditions described herein. memory. The treatment and / or prevention of diseases, disorders or conditions associated with the aberrant expression and / or activity of CXCR5 includes, but is not limited to, alleviation of at least one symptom associated with those diseases, disorders or conditions. The antibodies of the invention can be provided in pharmaceutically acceptable compositions known in the art or as described herein. The term "physiologically acceptable," "pharmacologically acceptable," and the like means approved by the regulatory agency of the federal government or a state government or as indicated in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals and more particularly in humans. humans.

The anti-CXCR5 antibody can be administered to a mammal in any acceptable way. Methods of introduction include, but are not limited to, the parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, epidural, inhalation, and oral routes, and if desired for immunosuppressive treatment, intralesional administration. Parenteral infusions include intramuscular, intradermal, intravenous, intra-arterial, or intraperitoneal administration. Antibodies or compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through the coatings.

-76 epithelial or mucocutaneous (eg, through the oral mucosa, rectal and intestinal mucosa etc.) and can be administered together with other biologically active agents, the administration can be systemic or local. Furthermore, it may be desirable to introduce the antibodies or therapeutic compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; Intraventricular injection may be facilitated by means of an intraventricular catheter, eg, attached to a reservoir, such as an Ommaya reservoir. Furthermore, the antibody is conveniently administered by pulsatile infusion, particularly with decreasing doses of the antibody. Preferably, the dosage is administered by injection, preferably intravenous or subcutaneous injections, depending, in part, on whether the administration is brief or chronic.

Other delivery systems are known and can be used to deliver an antibody of the present invention, including, for example, encapsulation in liposomes, microparticles, microcapsules (see Langer, Science 249: 1527 (1990); Treat et al., In Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein et al., eds., p. 353-365 (1989); and LopezBerestein, ibid., p. 317-327) and recombinant cells capable of expressing the compound; receptor mediated endocytosis (see, eg, Wu et al., J Biol Chem 262: 4429 (1987)); construction of a nucleic acid as part of a retroviral vector or other vector etc.

The active ingredients can also be entrapped in microcapsules prepared, for example, by coascervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly (methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems ( for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. These techniques are described in Remington's Pharmaceutical Sciences, 16^to edition, A. Osal, Ed. (1980).

Pulmonary administration may also be employed, for example, through the use of an inhaler or nebulizer, and formulation with a ₇₇ IMPI ^^ ~ ~ MEXICAN INSTITUTE

PROPERTY ¿βτ aerosolization. The antibody can also be administered in^Nfos * pulmoné? nfe a patient in the form of a sooer-vóaoo powder composition, for example, - tai US Patent No. 6,514,496.

In a specific embodiment, it may be desirable to administer the antibodies or therapeutic compositions of the invention locally to the area in need of treatment; which can be achieved, for example, and not by way of limitation, by local infusion, topical application, by injection, by means of a catheter, by means of a suppository or by means of an implant, said implant being of a porous material , non-porous or gelatinous, including membranes such as silastic membranes or fibers. Preferably, when administering an antibody of the invention, care should be taken to use materials that do not absorb or adsorb the protein.

In another embodiment, the antibody can be administered in a controlled release system. In one embodiment, a pump can be used (see Langer, Science 249: 1527 (1990); Sefton, CRC Crit Ref Biomed Eng 14: 201 (1987); Buchwald et al., Surgery 88: 507 (1980); and Saudek et al., N Engl J Med 321: 574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer et al., Eds., CRC Press (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen et al., Eds., Wiley ( 1984); Ranger et al., J Macromol Sci Rev Macromol Chem 23:61 (1983); see also Levy et al., Science 228: 190 (1985); During et al., Ann Neurol 25: 351 (1989); and Howard et al., J Neurosurg 71: 105 (1989)). In another embodiment, a controlled release system can be placed close to the therapeutic target.

Therapeutic formulations of the polypeptide or antibody for storage can be prepared as lyophilized formulations or aqueous solutions by mixing the polypeptide having the desired degree of purity with optional pharmaceutically acceptable carriers, diluents, excipients, or stabilizers typically employed in the art, i.e., buffering agents, stabilizing agents, preservatives, isotonicity agents, nonionic detergents, antioxidants and various other additives, see Remington's Pharmaceutical

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MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL

-78Sciences, 16th ed., Osol, ed. (1980). These additives are generally non-toxic to receptors at the dosages and concentrations employed, therefore, excipients, diluents, vehicles, and the like are pharmaceutically acceptable.

An isolated or purified antibody is substantially lacking in cellular material or other contaminating proteins from the original cell or tissue or the medium from which the protein is derived, or is substantially lacking in chemical precursors or other chemical agents when chemically synthesized. The term "substantially devoid of cellular material" includes preparations of an antibody in which the polypeptide / protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially devoid of cellular material includes antibody preparations that have less than about 30%, 20%, 10%, 5%, 2.5%, or 1% (by dry weight) of the contaminating protein. When the antibody is produced recombinantly, it also preferably lacks culture medium, i.e. the culture medium represents less than about 20%, 10%, 5%, 2.5%, or 1% of the volume of the preparation of protein. When the antibody is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemical agents and reagents, that is, the antibody of interest is separated from chemical precursors or other chemical agents that are involved in protein synthesis. Accordingly, these antibody preparations have less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In a preferred embodiment of the present invention, the antibodies are isolated or purified.

As used herein, the term "low to undetectable levels of aggregation" refers to samples containing not more than 5%, not more than 4%, not more than 3%, not more than 2%, not more than 1% and often not more than 0.5% aggregation, by protein weight, measured, for example, by high-resolution size exclusion chromatography (HPSEC).

As used herein, the term "levels from low to

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-79 undetectable fragmentation refers to samples containing an amount equal to or greater than 80%, 85%, 90%, 95%, 98% or 99%, of the total protein, for example, in a single peak, as determined by HPSEC, or in two (2) peaks (heavy chain and light chain), for example, by reduced capillary gel electrophoresis (rCGE) and that does not contain any other individual peak with more than 5%, more than 4%, more 3%, more than 2%, more than 1% or more than 0.5% of the total protein. RCGE as used herein refers to capillary gel electrophoresis under reducing conditions sufficient to reduce disulfide bonds in an antibody or antibody-like molecule or antibody-derived molecule.

As used herein, the terms stability and stable, in the context of a liquid formulation comprising an antibody to CXCR5 or a binding fragment thereof, refer to the resistance of the antibody or antigen binding fragment of the same in the formulation to the thermal or chemical unfolding, aggregation, degradation or fragmentation in specific conditions of manufacture, preparation, transport and storage. The stable formulations of the invention retain a biological activity equal to or greater than 80%, 85%, 90%, 95%, 98%, 99% or 99.5% under specific conditions of manufacture, preparation, transport and storage. The stability of said antibody preparation can be assessed by degrees of aggregation, degradation or fragmentation by methods known to those skilled in the art, including, but not limited to, rCGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and HPSEC, compared to a reference.

The term vehicle refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. These physiological vehicles can be sterile liquids such as water and oils, including those derived from petroleum or of animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a suitable vehicle when the pharmaceutical composition is administered intravenously. Saline solutions and glycerol solutions can also be used as liquid vehicles, particularly in the case of solutions

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QA MEXICAN INSTITUTE OF PROPERTY ^OV INDUSTRIAL injectables. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, calcium carbonate, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dehydrated skimmed milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, depots, and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include conventional carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin sodium, celluose, magnesium carbonate etc. Examples of suitable vehicles are described in Remington's Pharmaceutical Sciences, Martin. These compositions will contain an effective amount of the antibody, preferably in a purified form, along with a suitable amount of vehicle to provide the form for proper administration to the patient. As known in the art, the formulation will be constructed to suit the mode of administration.

Buffering agents help maintain pH in the range that approximates physiological conditions. Buffers are preferably present at a concentration range of from about 2mM to about 50mM. Buffering agents suitable for use with the present invention include both organic and inorganic acids, and salts thereof, such as citrate buffers (eg, monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, acid mixture citric-monosodium citrate etc.), succinate buffers (for example, succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture etc.), tartrate buffers (for example, tartaric acid-sodium tartrate mixture, tartaric acid potassium tartar mixture, tartaric acid-sodium hydroxide mixture etc.), fumarate buffers (for example, fumaric acid-monosodium fumarate mixture, mixture of

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-81 MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY fumaric acid-disodium fumarate, mixture of monosodium fumarate-disodium fumarate etc.), gluconate buffers (for example, mixture of gluconic gluconic acid sodium, mixture of gluconic acid-sodium hydroxide, mixture of gluconic acid- potassium gluconate etc.), oxalate buffers (eg oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture etc.), lactate buffers (eg, lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture etc.) and acetate buffers (eg, acetic acid-sodium acetate mixture, acid mixture acetic-sodium hydroxide etc.). Phosphate buffers, carbonate buffers, histidine buffers, trimethylamine salts such as Tris, HEPES and other of these known buffers can be used.

Preservatives can be added to delay microbial growth and can be added in amounts ranging from 0.2% -1% (w / v). Preservatives suitable for use with the present invention include phenol, benzyl alcohol, mcresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (eg, chloride, bromide, and iodide), hexamethonium chloride, alkylparabens, such as methyl or propylparaben, catechol, resorcinol, cyclohexanol and 3-pentanol.

Isotonicity agents are present to ensure the physiological isotonicity of the liquid compositions of the present invention and include alcohol of polyhydroxy sugars, preferably alcohols of trihydroxy sugars or higher, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. The polyhydroxy alcohols may be present in an amount of from about 0.1% to about 25%, by weight, preferably from 1% to 5%, taking into account the relative amounts of the other ingredients.

Stabilizers refer to a broad category of excipients that can change depending on an agent to provide volume to an additive that solubilizes the therapeutic agent or helps prevent denaturation or adherence to the container wall. Typical stabilizers can be

IΜ Ρ1 __ MEXICAN INSTITUTE

- 82 - OF THE PROPERTY

INDUSTRIAL Vaa._ polyhydroxy sugar alcohols; amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine etc., organic sugars or sugar alcohols such as lactose, trehalose, stachyose, arabitol, erythritol, mannitol, sorbitol, xylitol, ribitol, myoinositol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, □ -monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (ie &lt;10 residues); proteins, such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, saccharides, monosaccharides such as xylose, mannose, fructose, and glucose; disaccharides such as lactose, maltose, and sucrose; trisaccharides such as raffinose; polysaccharides such as dextran and the like. Stabilizers are present in the range of 0.1 to 10,000 w / w per active protein part.

Various other excipients include bulking agents (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, or vitamin E), and cosolvents.

The formulation herein may also contain more than one active compound when necessary for the particular indication to be treated, preferably those with complementary activities that do not adversely interfere with each other. For example, it may be desirable to further provide an immunosuppressive agent. These molecules are conveniently present in combination in amounts that are effective for the purpose for which they are intended.

As used herein, the term "surfactant" refers to organic substances that have antipathetic structures, particularly they are composed of groups of opposing solubility trends, typically an oil soluble hydrocarbon chain and a water soluble ionic group. Surfactants can be classified, depending on the loading of the surfactant moiety, into anionic, cationic and nonionic surfactants. Surfactants are often

<img file="MX338474B_D0100.tif" />

-83IMPI

IN *: i. UTO MEXICANO I heard THE PROPERTY use as wetting agents, emulsifiers, solubilizers and^{TO THE}I arranged for various pharmaceutical compositions and do-kill-psi preparations.

biological.

Surfactants or nonionic detergents (also known as wetting agents) may be added to help solubilize the therapeutic agent, as well as to protect the therapeutic agent against agitation-induced agitation, which also allows the formulation to be exposed to shear stresses. on the surface without protein denaturation occurring. Suitable nonionic surfactants include polysorbates (20, 80 etc.), poloxamers (184, 188 etc.), Pluronic® polyols, and polyoxyethylene sorbitan monoethers (TWEEN-20®, TWEEN-80® etc.). Nonionic surfactants can be present in a range of from about 0.05 mg / ml to about 1.0 mg / ml, preferably from about 0.07 mg / ml to about 0.2 mg / ml.

As used herein, the term inorganic salt refers to any compound, which does not contain carbon, that results from the replacement of part or all of the acidic hydrogen or an acid by a metal or a group that acts as a metal, since It is often used as a tonicity-adjusting compound in pharmaceutical compositions and preparations of biological materials. The most common inorganic salts are NaCI, KCI, NaH2PO4 etc.

The present invention provides liquid formulations of an anti-CXCR5 binding compound or a fragment thereof, having a pH ranging from about 5.0 to about 7.0, or from about 5.5 to 6.5, or from about 5.8 to about 6.2, or from about 6.0.

The present invention includes liquid formulations that have temperature stability found in a commercial refrigerator and freezer such as that found in a physician's office or laboratory, such as from about -20 ° C to about 5^or C, said stability being evaluated, for example, by high resolution size exclusion chromatography (HPSEC), for storage purposes, such as for approximately 60 days, for approximately 120 days, for approximately 180 days,

-84 for about a year, for about 2 ~ years or more. The liquid formulations of the present invention also exhibit stability, as evaluated, for example, by HSPEC, at room temperature, for at least a few hours, such as one hour, two hours, or approximately three hours before use.

The term "small molecule" and analogous expressions include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heterooganic and / or ganometallic compounds. ) having a molecular weight of less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1.00 gram per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole , and pharmaceutically acceptable salts, esters and other forms of these compounds.

Thus, in the case of cancer, the antibodies of the invention can be administered alone or in combination with other types of cancer treatment, including conventional chemotherapeutic agents (paclitaxel, carboplatin, cisplatin, and doxorubicin), anti-EGFR agents (gefitinib , erlotinib and cetuximab), anti-angiogenesis agents (bevacizumab and sunitinib), as well as immunomodulatory agents such as interferon-Q and thalidomlda.

As used herein, the terms "therapeutic agent and therapeutic agents" refer to any agent that can be used in the treatment, cure, or improvement of a disease, disorder, malaise, and the like associated with aberrant metabolism and activity of CXCR5 and / or or CXCL13. This can manifest at abnormal B-cell levels or abnormal B-cell activity. Also included are known compounds with a pharmacological effect in the treatment of a disorder and the like that is associated with the metabolism and aberrant activity of CXCR5 and / or CXCL13.

Furthermore, the antibodies of the present invention can be conjugated with

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<img file="MX338474B_D0101.tif" />

-85 various effector molecules such as heterologous polypeptides, drugs, radionucleotides or toxins, see, eg, WO 92/08495;

WO 91/14438; WO 89/12624; United States Patent No. 5,314,995; and EPO 396,387. An antibody or fragment thereof can be conjugated to a therapeutic moiety such as a cytotoxin (for example, a cytostatic or cytoclid agent), a therapeutic agent, or a radioactive metal ion (for example, emitters □ such as, for example,²¹³B¡). A cytotoxin or cytotoxic agent includes any agent that is harmful to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracendione, mitoxantrone, actinomycin, actinomicin, , tetracaine, lidocaine, propranolol and puromycin and analogs and homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, and dacarbazine), alkylating agents (eg, mechloretamine, chlorambucil, melphalan, carmustine (BSNU), and lomustine (CCNU), cyclophosphamide, busulfan, dibromomanitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (eg, daunorubicin, daunomycin, and doxorubicin), antibiotics (eg, dactinomycin, actinomycin, bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (eg, vincristine and vinblastine).

Techniques for conjugating this therapeutic moiety to antibodies are well known, see, for example, Arnon et al., In Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. (eds.), p. 243-56 Alan R. Liss (1985); Hellstrom et al., In Controlled Drug Delivery, 2^to ed., Robinson et al., eds., p. 623-53, Marcel Dekker (1987); Thorpe, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al., Eds., P. 475-506 (1985); Monoclonal Antibodies For Cancer Detection and Therapy, Baldwin et al., Eds., P. 303-16, Academic Press (1985); and Thorpe, et al., Immunol Rev 62: 119 (1982). Alternatively, an antibody can be conjugated to a second antibody to form a heteroconjugate of

<img file="MX338474B_D0102.tif" />

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ANTIBODY, such as a bifunctional antibody, see, for example, the Patent of the MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

United States No. 4,676,980.

The conjugates of the invention can be used to modify a given biological response, and the therapeutic agent or drug moiety should not be considered limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide that possesses a desired biological activity. These proteins can include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, interferon □, interferon □, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, an apoptotic agent, eg TNF-D, TNF-D, AIM I (WO 97/33899), AIM II (WO 97/34911), Fas ligand (Takahashi et al., Int Immunol, 6: 1567 (1994)), VEGF (WO 99/23105); a thrombotic agent; an antiangiogenic agent, for example angiostatin or endostatin; or biological response modifiers such as, for example, lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte and macrophage colony stimulating factor ( GM-CSF), granulocyte colony stimulating factor (GCSF) or other growth factors.

The formulations to be used for in vivo administration must be sterile. This can be accomplished, for example, by filtration through sterile filtration membranes. For example, the liquid formulations of the present invention can be filter sterilized using a 0.2 um or 0.22 □ m filter.

Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, said matrices being in the form of shaped articles, eg, films or matrices. Examples of sustained release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethylmethacrylate), poly (vinyl alcohol)), polylactides (US Patent No. 3,773,919), copolymers of glutamic acid and ethyl -L-glutamate, non-degradable ethylene-vinyl acetate, copolymers

<img file="MX338474B_D0103.tif" />

IMPI

MEXICAN INSTITUTE

OF THE PROPERTY

- O / - INDUSTRIAL lactic acid-glycolic acid (such as injectable microspheres composed of copolymer of lactic acid-glycolic acid) and poly-D - (-) - 3-hydroxybutyric acid. Although certain polymers such as ethylene vinyl acetate and lactic acid glycolic acid allow the release of molecules for more than 100 days, certain hydrogels release proteins for shorter periods of time. Rational strategies for stabilization can be devised depending on the mechanism involved. For example, if it is discovered that the aggregation mechanism is the formation of intermolecular SS bonds through thio-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, by lyophilization of acid solutions, controlling the moisture content, using appropriate additives. , by amino acid substitution and creating specific polymeric matrix compositions.

The antibody or variant composition will be formulated, dosed, and administered in a manner consistent with medical practice. Factors to consider in this context include the particular disorder to be treated, the particular mammal to be treated, the clinical status of the individual patient, the cause of the disorder, the site of administration of the agent, the method of administration, the administration schedule, and others. factors known to specialist doctors. The therapeutically effective amount of the antibody or variant to be administered will be dictated by these considerations, and may be the minimum amount necessary to prevent, ameliorate, or treat a CXCR5 mediated disease, condition, or disorder.

The antibody or variant is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of these other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. They are generally used in the same dosages and with the routes of administration previously used herein or from 1 to 99% of the dosages used hitherto.

As used herein, the term "effective amount" refers to the amount of a therapy (eg, a prophylactic agent or

IMPI

<img file="MX338474B_D0104.tif" />

-88 therapeutic), which is sufficient to reduce the severity and / or duration of a CXCR5-mediated disease, improve one or more symptoms of said disease, prevent the progression of a CXCR5-mediated disease or cause the regression of a CXCR5-mediated disease CXCR5, or that is sufficient to prevent the development, recurrence, onset or progression of a CXCR5-mediated disease or one or more symptoms thereof, or enhance or enhance the prophylactic and / or therapeutic effects of another therapy (eg, another therapeutic agent) useful for treating a CXCR5 mediated disease. For example, a treatment of interest can reduce elevated levels of B cells, based on a basal or normal level, at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least

35%, at least 40%, at least 45%, at least 50%, at least 55%, at least

60%, at least 65%, at least 70%, at least 75%, at least 80%, at least

85%, at least 90%, at least 95%, or at least 100%. In another embodiment, an effective amount of a therapeutic or prophylactic agent reduces symptoms of a CXCR5-mediated disease, such as arthritis or graft rejection, by at least 5%, preferably at least 10%, at least 15%, at least 20%. , at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% , at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. Also used herein as the equivalent is the term "therapeutically effective amount."

The amount of therapeutic polypeptide, antibody, or fragment thereof that will be effective in the use or treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. Whenever possible, a dose-response curve should be performed and the pharmaceutical compositions of the invention should first be prepared in vitro. If a suitable animal model is available, a dose response curve can again be obtained and used to extrapolate a suitable human dose by practicing methods known in the art. However, based on common knowledge of the art, a

<img file="MX338474B_D0105.tif" />

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MEXICAN INSTITUTE

- 89 - DELA HIOP'LDAD

INDUSTRIAL pharmaceutical composition effective in promoting a decrease of an inflammatory effect, for example, can provide a local concentration of therapeutic agent comprised between approximately 5 and 20 ng / ml, and preferably, between approximately 10 and 20 ng / ml. In another specific embodiment of the invention, a pharmaceutical composition effective in improving the growth and survival of cells responsible for B-cell dependent autoimmune manifestations or graft rejection can provide a local concentration of therapeutic agent of between about 10 ng / ml and approximately 100 ng / ml.

In a preferred embodiment, an aqueous solution of the therapeutic polypeptide, antibody, or fragment thereof can be administered by subcutaneous injection. Each dose can range from about 0.5 mg to about 50 mg per kilogram of body weight, or more preferably, from about 3 mg to about 30 mg per kilogram of body weight. The dosage can be empirically determined for the particular disease, patient population, mode of administration, and the like, by practicing pharmaceutical methods known in the art.

The dosage schedule for subcutaneous administration can vary from once a week to daily depending on various clinical factors including the type of disease, the severity of the disease, and the subject's sensitivity to the therapeutic agent.

The present invention provides methods for preparing liquid formulations of the antibody or CXCR5-binding fragment thereof, said methods comprising concentrating a fraction of purified antibody to a final concentration of about 15 mg / ml, about 20 mg / ml, about 30 mg / ml, approximately 40 mg / ml, approximately 50 mg / ml, approximately 60 mg / ml, approximately 70 mg / ml, approximately 80 mg / ml, approximately 90 mg / ml, about 100 mg / ml, about 200 mg / ml, about 250 mg / ml, about 300 mg / ml or greater using, for example, a semipermeable membrane with an appropriate molecular weight (pm) limit (for

<img file="MX338474B_D0106.tif" />

IMPI

90 example, a 30 kD limit for fragments F (_tob ') 2Í and a limit of 10 kD for fragments F_ab) and optionally diafiltering the concentrated antibody fraction in the formulation buffer using the same membrane.

Furthermore, the present invention also includes stable liquid formulations of the products of interest that have a better half-life in vivo. Thus, the antibody of interest has a half-life in a subject, preferably human, greater than 3 days, greater than 7 days, greater than 10 days, greater than 15 days, greater than 25 days, greater than 30 days. , older than 35 days, older than 40 days, older than 45 days, older than 2 months, older than 3 months, older than 4 months, older than 5 months or older.

To prolong serum circulation of an antibody in vivo, various techniques can be used. For example, inert polymer molecules, such as high molecular weight polyethylene glycol (PEG), can be attached to an antibody with or without a multifunctional linker by site-specific conjugation of the PEG to the N-terminus or C-terminus of the antibody. or through amino groups □ present in Usina residues. Linear or branched polymer derivatization can be used which causes minimal loss of biological activity. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of the PEG molecules to the antibodies. Unreacted PEG can be separated from the antibody-PEG conjugates by size exclusion chromatography or ion exchange chromatography. PEG-derivatized antibodies can be assayed for binding activity as well as for efficacy in vivo using methods known to those skilled in the art, for example, by immunoassays described herein.

An antibody with a longer half-life can also be generated in vivo by introducing one or more amino acid modifications (i.e., substitutions, insertions or deletions) into an IgG constant domain, or F-binding fragment_CR thereof (such as a fragment of F_and or a fragment of domain F_and hinge), see for example WO 98/23289; WO 97/34631; and United States Patent No. 6,277,375.

IMPI

<img file="MX338474B_D0107.tif" />

-91 Furthermore, an antibody can be conjugated to albumin to make the antibody more stable in vivo or have a longer half-life in vivo. Techniques are known in this field, see for example WO documents

93/15199, WO 93/15200 and WO 01/77137; and EPO 413. 622. The antibody can also be modified, for example, by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, binding to a cellular ligand or other protein, and the like.

In one embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. When necessary, the composition also includes a solubilizing agent and a local anesthetic such as lidocaine or another "caine" anesthetic to prevent pain at the injection site. Generally, the ingredients are supplied separately or mixed together in a unit dosage form, for example, as a dry lyophilized powder or a waterless concentrate in a tightly closed container, such as an ampoule or pouch indicating the amount of agent. active. When the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing saline or sterile pharmaceutical grade water. When the composition is administered by injection, a vial of sterile water for injection or saline may be provided, for example, in a kit, so that the ingredients can be mixed prior to administration.

The invention also provides that a liquid formulation of the present invention is packaged in a hermetically sealed container such as an ampoule or pouch indicating the amount of the product of interest. The liquid formulations of the present invention may be in a hermetically sealed container that indicates the amount and concentration of the antibody or antibody fragment. The liquid formulation of the present invention can be supplied in a sealed container with at least 15 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml,

<img file="MX338474B_D0108.tif" />

100 mg / ml, 150 mg / ml, 200 mg / ml, 250 mg / ml or 300 mg / ml of antibody to CXCR5 in an amount of 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 mi, 8 mi, 9 mi, 10 mi, 15 mi or 20 mi, for example.

An article of manufacture is provided containing materials useful for the treatment of the disorders described above. The article of manufacture comprises a container and a label. Suitable containers include, for example, vials, vials, syringes, and test tubes. Containers can be formed from a variety of materials such as glass or plastic. The container contains a composition that is effective in diagnosing, preventing, or treating a CXCR5-mediated condition or disease and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial that has a plug pierceable by hypodermic injection needle). The label on or associated with the container indicates that the composition is used for the treatment of the particular condition. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. In addition, it may include other desirable materials from a commercial and user point of view, including buffers, diluents, filters, needles, syringes and leaflets with instructions for use.

In another aspect of the invention, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit, or prevent a disease or disorder associated with aberrant expression and / or activity of CXCR5, by means of therapy. gene. Gene therapy refers to therapy performed by administration to a subject of an expressed or expressible nucleic acid of interest. In one embodiment of the invention, nucleic acids produce the protein encoded in and by target host cells that mediate a therapeutic effect. In accordance with the present invention any of the available gene therapy methods can be used.

For a general review of gene therapy methods, see Goldspiel et

<img file="MX338474B_D0109.tif" />

-93IMPI

MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL al., Clinical Pharmacy 12: 488 (1993); Wu et al., Biotherapy 3:87 (1991);

Tolstoshev, Ann Rev Pharmacol Toxicol 32: 573 (1993); Mulligan, Science 260: 926 (1993); Morgan et al., Ann Rev Biochem 62: 191 (1993); and May, TIBTECH 11: 155 (1993).

In one aspect, the compound comprises nucleic acid sequences encoding an antibody, or functional binding fragments thereof, said nucleic acid sequences being part of expression vectors expressing the antibody or chimeric fragments or proteins or heavy or light chains of the same on a suitable host. In particular, said nucleic acid sequences have promoters operably linked to the coding region of the antibody, said promoter being inducible or constitutive, and optionally, tissue-specific, as well as other regulatory sequences.

In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thereby providing intrachromosomal expression of nucleic acids. encoding the antibody (Koller, et al., Proc Nati Acad Sci USA 86: 8932 (1989); Zijlstra et al., Nature 342: 435 (1989)). In specific embodiments, the expressed antibody molecule is a single-chain antibody; alternatively, nucleic acid sequences include sequences that encode both light and heavy chains, or fragments thereof, of the antibody. Other alternative methods of integration include the use of particular transcription factors that recognize specific nucleic acid sequences, zinc fingers, and the like.

The administration of the nucleic acids to a patient can be done directly, in which case the patient is exposed directly to the nucleic acid or to vectors carrying the nucleic acid, or indirectly, in which case cells are first transformed with the nucleic acids. in vitro, and then these cells are transplanted into the patient.

In one embodiment, the nucleic acid sequences are administered directly in vivo and are expected to produce the encoded product. This

<img file="MX338474B_D0110.tif" />

<img file="MX338474B_D0111.tif" />

-94ir i

INSTITUTO MEXICANO DE LA PSGP.rDAD INDUSTRIAL ______ can be achieved by any of a number of methods known in the art, for example, by constructing sequences coding for the Lüiriu palle antibody to an appropriate nucleic acid expression vector and administering such sequences so that the vectors are intracellular, for example, by infection using attenuated retroviral vectors or other viral vectors (see US Patent No. 4,980,286), by indirect injection of naked DNA, through the use of microparticle bombardment (eg, a gene gun; Biolistic, Dupont), using non-viral vectors such as synthetic compositions comprising an antipathetic compound that binds to hydrophilic nucleic acid and has the ability to fuse with cells, generally thus containing a hydrophobic part to combine with membranes, for coating with lipids or cell surface receptors or transfection agents, encapsulation in liposomes, microparticles or microcapsules, by administration of the vector in association with a peptide known to enter the nucleus, by administration of the vector in association with a ligand undergoing receptor-mediated endocytosis (see, eg, Wu et al., J Biol Chem 262: 4429 (1987)) (which can be used to target cell types that specifically express receptors) etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to break endosomes, allowing the nucleic acid to avoid lysosomal degradation. In another embodiment, the nucleic acid can be targeted in vivo for cell-specific uptake and expression, by targeting to a specific receptor (see, eg, WO 92/06180; WO 92/22635; WO92 / 20316; WO93 / 14188 and WO 93/20221).

In connection with vectors, for example, a lentiviral vector can be used as known in the art. Lentiviral vectors contain components for packaging the viral genome and for integration into the DNA of the host cell. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitate the release of the gene in a patient. For example, a lentiviral vector can be used to

<img file="MX338474B_D0112.tif" />

IMPI

-95 administer a transgene to hematopoietic stem cells. References illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J Clin Invest 93: 644 (1994); Kiem et al., Blood 83: 1467 (1994); Salmons et al., Human Gene Therapy 4: 129 (1993); and Grossman et al., Curr Opin Gen and Dev 3: 110 (1993).

Adenoviruses can also be used in the present invention. The targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle, for example. Adenoviruses infect cells that are not dividing, an advantage over previous retroviral vectors. Kozarsky et al., Curr Opin Gen Dev 3: 499 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5: 3 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelium of rhesus monkeys. Other cases of the use of adenovirus in gene therapy can be found in Rosenfeld et al., Science 252: 431 (1991); Rosenfeld et al., Cell 68: 143 (1992); Mastrangeli et al., J Clin Invest 91: 225 (1993); WO94 / 12649; and Wang et al., Gene Therapy 2: 775 (1995).

Aderio-associated virus (AAV) can also be used in gene therapy (Walsh et al., Proc Soc Exp Biol Med 204: 289 (1993); and US Patent Nos. 5,436,146; 6,632,670; and 6,642,051.

Another gene therapy strategy involves transferring a gene to cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate-mediated transfection, or viral infection. Typically, the transfer method includes transferring a selection marker to the cells. The cells are then placed under selection to isolate cells that have been harvested and that express the transferred gene. Those cells are then administered to a patient.

In this way, the nucleic acid can be introduced into a cell prior to in vivo administration of the resulting recombinant cell. Said introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection,

<img file="MX338474B_D0113.tif" />

infection with a viral or bacteriophage vector that contains nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques for introducing foreign genes into cells are known in this field (see, for example, Loeffler et al., Meth Enzymol 217: 599 (1993); Cohén et al., Meth Enzymol 217: 618 (1993); and Cline Pharm Ther 29:69 (1985)) and can be used in accordance with the present invention, provided that the development and physiological functions of the recipient cells are not altered. The technique should allow stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressed by the cell, can be inherited, and is expressed by the cell's offspring.

The resulting recombinant cells can be administered to a patient by various methods known in the art. Recombinant blood cells (eg, hematopoietic stem cells or progenitor cells) are preferably administered intravenously. The number of cells intended for use depends on the desired effect, the condition of the patient etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for gene therapy purposes include any desired available cell type and include, but are not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, and granulocytes; various stem or progenitor cells, in particular progenitor or hematopoietic stem cells, for example, obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver etc.

In one embodiment, the cell used for gene therapy is autologous to the patient. Nucleic acid sequences encoding an antibody of the present invention are introduced into cells such that the transgene is expressed by the cells or their offspring, and the recombinant cells are then administered in vivo to achieve a therapeutic effect. In a specific embodiment stem or progenitor cells are used. According to the realization

<img file="MX338474B_D0114.tif" />

Any stem and / or progenitor cell that can be isolated and maintained in vitro can be used of the present invention (see, eg, WO 94/08598; Stemple et al., Cell 71: 973 (1992); Rheinwald Meth Cell Bio 21A : 229 (1980); and Pittelkow et al., Mayo Clinic Proc 61: 771 (1986)). As CXCR5 is expressed, for example, in B cells, suitable host cells are blood cells and bone marrow cells. However, the scope of the present invention in relation to the use of stem cells as hosts does not contemplate the preparation and use of a transgene to obtain a transgenic organism by means of administration of the transgene of interest to embryos and embryonic stem cells.

The invention provides methods of treatment, prophylaxis, and improvement of CXCR5-mediated diseases or one or more symptoms thereof, by administering to a subject an effective amount of, for example, a liquid formulation of the invention. The subject is preferably a mammal, such as a non-primate (eg, cows, pigs, horses, cats, dogs, rats etc.) and a primate (eg, monkey, such as a cynomolgus monkey and a human). In a preferred embodiment, the subject is a human.

CXCR5 is also expressed in certain cancer cells, such as the pancreas, colon, and bladder, as well as in T-cell leukemias (Qinping et al., Oncogene 24: 573-584, 2005) and B-cell leukemias (Burkel et al., Blood, Jul 2007; doi: 10.1182 / blood-2007-05-089409) and CXCR5 stimulation correlates with carcinoma cell proliferation, Meijer et al., Cañe Res 66: 9576-9582, 2006.

Thus, the antibody or derivative thereof of interest can be used to control the proliferation of CXCR5-expressing cancer cells, those cancers being identified by determining the presence of CXCR5 expression by means of a diagnostic assay taught in present memory. The antibody of interest can reduce malignant cell infiltration, reduce apoptosis resistance, and minimize proliferation. These patients are then administered an amount of cancer cell proliferation inhibiting antibody, or a derivative thereof, of interest such as

<img file="MX338474B_D0115.tif" />

which is provided herein. _______.

Some autoimmune disorders are associated with aberrant and / or elevated expression of CXCL13, such as lupus (Ishikawa et al., J Exp Med 193: 1393-1402, 2001) and Sjoren syndrome (Salomonsson et al., Sean J Imm 55: 336-342, 2002; and Barone et al., Arth Rheum 52 (6) 1773-1784, 2005), or with high CXCR5 expression, such as myasthenia gravis (Sims et al., J Imm 167 : 1935-1944, 2001; Saito et al., J Neuroimm 55: 336-342, 2005; and Tackenberg et al., Eur J Imm 37: 849-863, 2007). Therefore, an antibody of interest is used to minimize the effect of high levels or high activity of CXCL13 or CXCR5 ligand. In autoimmune disorders characterized by high levels of B cells, high levels of CXCR5, or high levels of CXCL13, or another CXCR5 ligand, an inhibitory amount of B cell activity of an antibody of interest is administered as taught in present memory.

Aberrant expression of CXCR5 is observed in multiple sclerosis, Brain 129 (Pt 1) 200-211, 2006.

In colitis, CXCR5 has a role in the formation and function of GALT (Carlseñ et al., Gut, 2002, 51 (3) 364-367). CXCR5-mediated migration and infiltration of B cells in the lamina propria of the intestine, and infiltration in the mucosa in general, (Mazzucchelli et al., J Clin Invest, 1999, 104 (10) R49-R54) and its expression in Ulcerative colitis lesions containing ectopic germ centers, are inhibited by an antibody of interest.

The reduction of B cells has a beneficial therapeutic effect in improving the symptoms of rheumatoid arthritis (Oligino &amp; Dalrymple, Arth Res Ther 5 (Suppl 4) S7-S11, 2003). CXCR5 is expressed at high levels in the synovial tissue of arthritis patients, compared to tissues from individuals without rheumatoid arthritis (Schmutz et al., Arth Res Ther 7: R217-R229, 2005). In this way, the antibody of the present invention can prevent infiltration and interaction of B cells in a joint. Accordingly, treatment includes administering a B-cell level lowering amount of an antibody of interest to a patient who has been diagnosed with arthritis. The antibody can be administered locally to the affected joint.

<img file="MX338474B_D0116.tif" />

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Ectopic lymphoid neogenesis is observed in several conditions, including psoriatic arthritis (Cañete et al., Ann Rheum Dis, Jan 12, 2DÜ7, doi: 10: 1136 / ard.2006.062042), chronic inflammatory diseases, in general (Aloisi &amp; Pujol-Borrell , Nat Rev Imm 6: 205-217, 2006) and in grafts experiencing rejection, both chronic (Baddoura et al., Am J Trans 5: 510-516, 2005) and acute (DiCarlo et al., Am J Trans 7 : 201-210, 2007). CXCL13 and CXCR5 were present in cardiac grafts (DiCarlo et al., Supra); and CXCL13 was present in psoriatic arthritis (Cañete et al., supra). The presence of CXCL13 and / or CXCR5 is associated with the development of ectopic lymphoid follicles with areas of B cells and T cells, such as those found in normal nodes. B-cell alloantigen presentation was also associated with the acute cardiac allograft model (Noorchashm et al., J Imm 177: 7715-7722,2006).

In this way, an antibody of interest can be used to prevent inflammation and graft rejection. The patient is then administered an inhibitory amount of the B cell activity of an antibody to reduce inflammation, to minimize the development of ectopic germ centers, to minimize the recruitment of B cells in a graft, and to minimize the presentation of alloantigens. by B cells before or after a transplant procedure.

The invention will now be exemplified for the assistance of one skilled in the art by means of the following non-limiting examples representing some of the embodiments by means of and in which the present invention may be practiced.

EXAMPLES

EXAMPLE 1: GENERATION OF IMMUNOGEN

Monoclonal anti-CXCR5 antibodies can be induced in CHO cells transformed with DNA encoding full-length human CXCR5 and expressed on the cell surface (r-CXCR5-CHO cells). The CXCR5 sequence used to transform cells can be obtained from bases of

IMPI

<img file="MX338474B_D0117.tif" />

-100 data, such as NM 001716.2, NC000011.8 or NM001716, and synthesized or isolated from a suitable cellular source.

The CXCR5 open reading frame was placed into an expression vector, such as pCDNA3.1neo_DEST, and then this vector was introduced by transfection into 300-19 cells (Immunogen).

Furthermore, the EC domain of CXCR5, with the amino acid sequence

MNYPTLEMDLENLEDLFWELDRLDNYNTSLVENHLC (SEQ ID NO: 1), was conjugated to KLH by the C-terminal cysteine, and used as an immunogen. Cells expressing CXCR5 or the EC domain of CXCR5 were administered IP (5x10⁶ cells in 0.2 ml or 50 Dg of peptide in 100 DI of buffer, optionally mixed with 100 □ I of adjuvant, such as Freund's complete adjuvant). Injections with the antigen were repeated every two weeks until a high antibody titer for CXCR5 was detected in the serum, eg, by FACS using, eg, CXCR5 cells.⁺ and, for example, commercially available MAB190 (R&amp;D Systems) as a positive control.

Cells expressing CXCR5 were maintained at 37 ° C in a 5% CO2 atmosphere in RPMI (Invitrogen, Carlsbad, CA) supplemented with 10% dialyzed fetal bovine serum (FBS) (Invitrogen). Cells were prepared for injection by replacing the above culture medium with phosphate buffered saline (without Ca / Mg) (CMF-PBS) supplemented with 5mM EDTA, and harvesting the cells in that buffer. The collected cells were pelleted by centrifugation at 500 xg for approximately 5 minutes, washed once by resuspension of the pellet in CMF-PBS and centrifugation as indicated above, counted and adjusted to the appropriate volume (such as 5x10⁶ cells in 0.2 ml) for injection by resuspension of the cell pellet in CMF-PBS.

As mentioned, expression of CXCR5 was controlled, for example, by FACS analysis, using commercially available antibodies to CXCR5 such as MAB190 (R&amp;D), clone RF8B2, and 2G8 (2G8 is a rat anti-CXCR5 antibody. mouse, while the other antibodies acquired are anti-human CXCR5) (BD), and 2C1 (Abnova), as well as various

<img file="MX338474B_D0118.tif" />

<img file="MX338474B_D0119.tif" />

-101 * MEXICAN INSTITUTE

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INDUSTRIAL polyclonal antibodies to hCXCR5 obtained by practicing methods known in the art.

To facilitate plasmid construction and enhance CXCR5 expression, oligonucleotides corresponding to the leader peptide sequence comprising the first 135 base pairs of the CXCR5-encoding nucleic acid sequence were generated. The oligonucleotides contained some changes in the fluctuating coding positions to reduce the GC content. All changes in the nucleotide sequence were silent, that is, no changes in the amino acid sequence were obtained. After hybridizing the oligonucleotides to each other, the genetically engineered leader peptide coding sequence was linked to the rest of the coding sequence by SOE-PCR (Ho et al., Gene 77:51 (1989); and Horton et al., BioTéchniques 8: 528 (1990)).

CXCR5 expression was verified prior to use as an immunogen. Cells are grown in RPMI (Invitrogen, Carlsbad, CA) containing 10% FBS, 0.2mM glutamine and 1x non-essential amino acid solution and then seeded approximately 3-5 x 10⁵ cells per well in a T75 flask and grown for approximately 24-48 hours.

Transformed or transfected cells were cultured for approximately two weeks until cells that did not carry the CXCR5 expression plasmid were removed by antibiotic selection. Cells from stable lines can be lysed and proteins can be obtained and subjected to Western blot analysis.

Stable or transient transfected cells were assayed for CXCR5 expression using methods to detect CXCR5 expression on the cell surface, such as FACS analysis. Alternatively, cells can be lysed and proteins studied, for example, by Western blot analysis. Transfected cells collected from the culture plates were washed once with phosphate buffered saline (PBS) and resuspended in deionized water, mixed with an equal volume of 2x protein sample loading buffer (BioRad, Hercules, CA ) and then heated at approximately 100 ° C for 10 minutes. Protein

<img file="MX338474B_D0120.tif" />

<img file="MX338474B_D0121.tif" />

I í

-102 membrane was analyzed using conditioned medium mixed with an equal volume__ of 2x protein sample loading buffer and heated at 100 ° C for minutes. Samples were separated using SDS-PAGE on a gradient of

4-12%. Proteins were transferred from the gel to a nitrocellulose membrane (BioRad, Hercules, CA), which was blocked with 5% dehydrated skimmed milk in PBST (PBS with 0.05% TWEEN-20®) for at least one hour before transferring the protein.

CXCR5 was detected by incubating the membrane with CXCR5-specific primary antibody in blocking buffer for at least one hour at room temperature, with shaking. The membrane was washed at least three times and indicator-conjugated secondary antibody in blocking buffer was added and incubated for at least one hour at room temperature, with shaking. The membrane was washed three times in PBST and developed with, for example, a chemiluminescent substrate.

EXAMPLE 2: GENERATION OF ANTI-CXCR5 mABS

A / J or BALB / cJ mice, approximately 4-6 weeks old (Jackson Labs, Bar Harbor, ME) were immunized with cells transfected with CXCR5-O an EC peptide. A group of mice were primed intraperitoneally on day 0 with a 1: 1 emulsion of KLH-conjugated peptide mixed with adjuvant (CFA), ip boosted on day 20 with the peptides with IFC (Freund's incomplete adjuvant) and / or cells in PBS without adjuvant, and finally boosted intravenously on day 44 with the KLH-peptides mixed in IFC and / or the cells in PBS, without adjuvant. Another group of mice were primed ip on day 0, ip boosted on days 15, 39, 53 and 67, and finally intravenously boosted on day 81 (all injections with cells in PBS, without adjuvant) . In the two groups of mice, each injection contained approximately 3 x 10⁶ a 2 x 10⁷ cells in a volume of about 200 DI. Alternatively, peptide and / or cell immunizations were performed once every two weeks, 3-6 times until a desirable anti-CXCR5 antibody titer was obtained, determined, for example, by analysis.

<img file="MX338474B_D0122.tif" />

<img file="MX338474B_D0123.tif" />

-103FACS or ELISA. _________ _________

Three days after the last injection, the mice were optionally tested for serum anti-CXCR5 antibody titer, sacrificed, and spleens were removed and placed in approximately 10 ml of serum-free DMEM (Bibico) in a Petri dish. The splenocytes were removed from the capsule using forceps and washed twice in 10 ml of IMDM without serum (Cellgro, Hemdon, VA) at 37 ° C. Spleen cell suspensions were transferred to a 15 ml conical bottom tube and the debris was allowed to settle for approximately 2-5 minutes. The supernatant containing the splenocytes was transferred to a 15 ml conical bottom tube and washed three more times with IMDM until fusion. Spleen cells from mice can pool.

Optionally, a 5 ml individual cell suspension of control spleen feed cells was prepared from an immunized mouse essentially as described above for the immunized spleen cells and placed in an incubator (37 ° C, 5 % CO2) until needed.

The fusion partner for immunized spleen cells may be a hypoxanthine / amynopterin / thymidine (HAT) sensitive, non-secreting myeloma cell line, such as P3X63-AG8.653 or SP2 / 0 (ATCC, Manassas, VA) or FO_B lymphoblasts (ATCC, CRL-1646)). Before fusions, lymphoid cells were maintained in 10% IMDM / FBS (37 ° C, 7% CO2) ensuring that the cells are in the logarithmic growth phase on the day of the fusion. An alternative selection mechanism is based on the use of azaserine, which is typically added one day after fusion.

The fusion protocol used is a hybrid of the protocols indicated in Lerner (Yale J Biol Med, 1981, 54 (5) 387-402) and Gefter et al. (Somatic Cell Genet, 1977, 3 (2) 231-236). Before fusion, pooled spleen cells were washed three times with IMDM without serum, and counted. In addition, immediately before fusion, the logarithmic phase myeloma cells were washed three times with serum-free IMDM and counted. Lymphoid cells were resuspended at 1 x 10⁷ cells / ml in IMDM without serum. For each fusion, 1-1.5 x 10 were mixed⁸

<img file="MX338474B_D0124.tif" />

<img file="MX338474B_D0125.tif" />

-104 spleen cells with 1-3 x 10⁷ myeloma cells in a nniipmpíipnn tube h_or50 ml conical bottom, and cells were washed once with IMDM without serum. The ratio of spleen cells to myeloma cells was 5: 1. The tubes were centrifuged at 500 xg for 10 minutes to pellet the cells. After aspirating the supernatants, the pellets were gently resuspended by lightly tapping the bottom of the tubes. The tubes were then placed in a beaker of water at 37 ° C. All subsequent melting steps were performed in that beaker.

Next, 1 ml of preheated 37 ° C polyethylene glycol 1500 (Roche Applied Science, Indianapolis, IN) was slowly added to each cell pellet over the course of about 1 minute, while gently swirling the tube. Cells were incubated in PEG for approximately one minute followed by the addition of one ml of serum-free IMDM to each pellet over the course of 30 seconds, and then 9 ml of serum-free IMDM was added to each pellet during the next minute. The two tubes were centrifuged at 500 xg for 10 minutes at room temperature, and the supernatants were aspirated. The cell pellet was resuspended in 100 ml of filtered complete hybridoma production medium (500 ml of IMDM (Cellgro) mixed with 10% FBS (SeraCare, Millford, MA), 0.2 mM Lglutamine, non-essential amino acid solution 1x, 1 mM sodium pyruvate, 0.01% pen-strep (Invitrogen) and 1X HT supplement (Invitrogen)).

Each 100 ml of cell suspension was placed in ten 96-well flat-bottom microtiter plates with a volume of -100 Ell / well. The plates were kept in an incubator at 37 ° C, 7% CO₂. On day 2 after fusion, cells were selected by adding azaserine 5.7 DM in IMDM to cells fused at 100 DI per well. Supernatants for primary selection, typically days 10-14 after fusion, were removed from the wells containing clones. The fusion efficiency was 75-99% (720-950 out of 960 possible wells developed clones that were screened).

The primary selection may be a radioimmunoassay (RIA) designed to detect antibodies that bind to human CXCR5. To perform the RIA,

-105 MEXICAN INSTITUTE

OF THE PROPERTY ^ íx ^₇* J.Íí «a

INDUSTRIAL adds affinity purified goat anti-mouse IgG (fragment specific

F_c) (Cappell, Cochranville, PA) in PBS to 96 PVC microtiter plates

<img file="MX338474B_D0126.tif" />

wells (50 Dl / well) and incubate overnight at 4 ° C. The goat anti-mouse IgG is removed from the plates and the wells are blocked with 100 Dl / well of

5% FCS / PBS for 1 hour at room temperature. After removing the blocking solution, pure hybridoma culture supernatant is added to the wells (50 Dl / well) and the mixture is incubated 1 hour at room temperature. The plates are washed 3 times with PBS / 0.05% Tween-20. Then 50 DI of¹²⁵I-CXCR5 (-20,000 cpm) in PBS / 5% FCS at each well and mix 10 incubated 1 hour at room temperature. Finally, the wells are washed 3 times with PBS / 0.05% Tween-20. After removing all of the wash buffer, the wells are removed by cutting the plates and analyzed in a gamma counter. Wells to which 5% FCS / PBS is added in place of culture supernatant serve as bottom wells.

The purified CXCR5 is labeled with ¹²⁵l according to the BoltonHunter method, substantially as described by the supplier of the BoltonHunter reagent (New England Nuclear, Boston, MA). The quality of¹²⁵I-CXCR5 is controlled by confirming that the labeling procedure did not destroy the epitopes recognized by commercially available antibodies to CXCR5 (R&amp;D or 20 Becton Dickinson, Mountain View, CA).

Clones are considered positive after primary selection if samples of the supernatant are marked approximately 10 times with respect to the background level in the RIA. Positive clones are removed, expanded, and stored in the frozen state.

A primary hybridoma selection was designed to determine if the antibodies recognized native CXCR5 epitopes. This was accomplished by FACS analysis of CXCR5 from the cell surface presented in CXCR5 cells.⁺ stained with monoclonal antibodies, and visualization of binding with fluorescently labeled second goat anti-mouse antibody. All 30 clones were considered positive after primary selection if samples of the supernatant were labeled approximately 10 times with respect to the level of

<img file="MX338474B_D0127.tif" />

<img file="MX338474B_D0128.tif" />

-106 MEXICAN INSTITUTE OF THE INDUSTRIAL FRGFlcDAD background in the FACS analysis. Furthermore, to locate the CXCR5 epitopes bound to the antibodies, competitive assays with antibodies to CXCR5 were performed. Positive clones were selected, expanded, and stored in the frozen state.

EXAMPLE 3: CELL-BASED JOINT ASSAYS

FOR ANTI-CXCR5 mABS

A cell-based binding assay was used to characterize anti-CXCR5 mAbs. For example, the CXCR5 expressing transfected cells described above, such as hCXCR5 / Hek293, can be used. A full length human CXCR5 open reading frame was cloned into a vector, eg, in pCDNA3.1neo DEST (Invitrogen, Carlsbad, CA). The CXCR5 coding region was synthesized by RT-PCR using human brain and liver RNA (Ambion, Inc., Austin, TX) as a template. The final plasmid construct, CXCR5 / CDNA3.1neo, expressed a full length CXCR5 protein. A stable cell line expressing CXCR5 was generated by transfection of the CXCR5 / pCDNA3.1neo plasmid construct into CHO or Hek293 cells (ATCC # CRL-1573) using a conventional and commercially available Lipofectamine 2000 kit. After transfection, cells were cultured in DMEM overnight, then re-seeded in medium with 200 Og / ml neomycin and cultured for 12-14 days. Isolated single colonies were collected and cultured in separate wells until sufficient clonal cells were amplified. Stable neomycin resistant clones expressing high levels of CXCR5 protein were identified by FACS analysis using polyclonal anti-CXCR5 antibodies (R&amp;D Systems, Minneapolis, MN) or custom generated polyclonal antibodies.

CXCR5 expression in human Sultan HS cells (ATCC No. CRL-1484) expressing CXCR5 naturally was also confirmed by FACS analysis. HS Sultan cells were grown in RPMI 1640 containing 10% fetal bovine serum, 0.2 mM glutamine and 0.1% pen / strep solution (100 □ g / ml penicillin and 10 ug / ml streptomycin).

<img file="MX338474B_D0129.tif" />

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-107 Cell-based antibody binding can be assessed using the FMAT ™ Cell Detection System (High Performance Macroconfocal Selection with Fluorescence) 8100 HTS or 8200 (Applied Biosystems, Foster

City, CA) following the protocol provided by the manufacturer. Cell lines expressing CXCR5 naturally or stably transfected with CXCR5 expression constructs are seeded in 96-well plates. Alternatively, transiently transfected 293T or CHO cells are seeded in the 96-well plate. Cells are seeded at a density of 5,000-30,000 cells per well. After 20-24 hours, anti-CXCR5 mAb and goat anti-mouse IgG antibody conjugated with

FMAT together in the wells and incubate for 1 hr, 2 hr, 4 hr or overnight at room temperature.

Cell-based antibody binding was also assessed by FACS using a stable Hek293 / CXCR5 cell line expressing CXCR5. All 15 cells were incubated with anti-CXCR5 mAb in PBS. After three washes, cells were incubated with secondary antibody conjugated to a fluorescent molecule (BD Sciences, Palo Alto, CA).

The results indicated that several mAbs bind to CXCR5 expressed in recombinant plasmid constructs. For example, 11D6, 20 14C9, 19H5, H28, 54G6, G7, 56H6, 79B7 and 16D7, humanized variants of the last antibody, 16D7-HC1-LC3, 16D7-HC1-LC2, 16D7-HC1-LC1 and 16D7- were tested. HC2LC1, a negative control IL13 antibody, CA13, positive controls, MAB190, 2C1 and RF8B2, three mouse isotype controls for lgG1, lgG2a and lgG2b and a rat lgG2b isotype control (corresponding to RF8B2), with 25 regarding binding to transfected HEK cells to express CXCR5. 2C1 and MAB190 positive control antibodies bound to CXCR5 cells. RF8B2 exhibited intermediate binding levels. Negative control antibodies showed only background binding. All antibodies except CA13 bound to hCXCR5 / HEK cells with binding profiles and titration kinetics similar to that of the parental antibody 16D7 and 79B7.

2993 cells transfected with the immunofluorescence were also stained with

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<img file="MX338474B_D0130.tif" />

transiently containing a CXCR5 / neo plasmid as described above and observed by fluorescence microscopy. Cell-based FACS and FMAT analyzes confirm that mAbs effectively bind to CXCR5 expressed in recombinant plasmid constructs or as a native protein in cultured cells. A positive binding signal is determined based on the FMAT signal reading that is significantly higher than background binding and other negative hybridoma clones (p> 0.01).

The generated CXCR5 mAbs, such as 16D7, 14C9, 19H5, H28, 54G6, G7, 56H6 and 79B7, bind to the EC domain and block the binding of CXCL13 to CXCR5 in the cell.

EXAMPLE 4: BIACORE AFFINITY ANALYSIS

The human and mouse CXCR5 N-terminal EC region (amino acids 1-59) was synthesized with a terminal biotin marker. The synthetic peptides were immobilized on a Biacore chip for approximately 20 response units (RU). The mAbs were then exposed to the chip to collect kinetic measurements and for epitope mapping, following the manufacturer's recommendations (GE Healthcare, Piscataway, NJ).

The anti-hCXCR5 16D7 mouse mAb clone had a calculated kD value of 2.16 '¹² M; the 16D7 mouse / human lgG4 chimeric antibody (16D7 VH and VL regions grafted onto a human lgG4 Fe, optionally codon optimized, using conventional methods such as cloning, amplification of ends, determination of the mass of the regions, and cloning of the parts) had a kD of 1.41 '¹² M; and for the various humanized variants of 16D7, where the structure of the variants and the derivation of their heavy and light chains is denoted by the terms, "HC_" for a particular heavy chain and "LC_" for a particular light chain, where the Chain composition is provided hereinafter, 16D7-HC1-LC1 had a kD of 3.1 Γ¹² M; 16D7-HC1-LC2 had a kD of 1.4Γ¹² M; 16D7-HC2-LC1 had a kD of 2.40 '¹² M; 16D7-HC1-LC3 had a kD of 1.21 '¹² M; 16D7-HC3-LC4 had a kD of 4.92 '¹² M; 16D7-HC3-LC5 had a kD of 1.84¹⁰ M; and 16D7-HC1-LC6

ΙΜ

<img file="MX338474B_D0131.tif" />

-109 MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY had a kD of 9.17 '¹¹ M.

EXAMPLE 5: WESTERN TRANSFER ANALYSIS OF THE MAB ANTI-CXCR5 UNION ACTIVITY

Western blotting was performed to assess the binding activity of anti-CXCR5 mAb to CXCR5 under denaturing conditions, as well as the expression levels of CXCR5 and other CXCR5 related proteins in human cell lines. Protein samples were also prepared from stably transfected cells using the M-PER Mammalian Protein Extraction Reagent Kit (Pierce, Rockland, IL, Cat # 78501) following the manufacturer's instructions, and heated to 70 ° C for 10 minutes after adding an equal volume of 2x protein sample loading buffer. All samples were separated by electrophoresis on a SDS-PAGE gel with a gradient of 4-12%. Proteins were transferred from the gel to a PVDF membrane and anti-CXCR5 mAbs were applied to the Western transfer membrane as the primary detection antibody. For detection, a secondary antibody conjugated with Alexa 680 was used and the membranes were scanned using the Odyssey infrared imaging system (Li-cor, Lincoln, Nebraska) or using electrochemiluminescence (ECL). Positive control antibodies against human CXCR5 were generated as taught herein.

EXAMPLE 6: FACS TEST FOR CONTROL

CXCR5 INTERNALIZATION

Buffy coat cells are obtained from healthy volunteers (Gulf Coast Blood Center, Houston, TX). Human peripheral mononuclear cells (PBMCs) are isolated with a standard Ficoll-Hypaque gradient method. PBMCs are grown (0.5 x 10⁶ cells / well) in a 96-well plate at 4 ° C. Each well contains 0.2 ml RPMI 1640 supplemented with 10% FBS in the presence / absence of monoclonal antibodies (10Dg / ml). After 30 minutes, the medium is replaced by

<img file="MX338474B_D0132.tif" />

RPMI 1640 new cold supplemented with 10% FBS and without antibodies. Cells are transferred to a humidified tissue culture chamber at 37 ° C containing 5% CO₂. Cells treated with monoclonal antibody are harvested immediately, 2 hr or 24 hr after transferring cells at 37 ° C. Cells are washed once with PBS and incubated in cold PBS containing 1% BSA (PBSB) for 30 minutes. The cells are then stained with PE conjugated anti-human CXCR5 antibody (BD Biosciences). After 30 minutes, the cells are washed 3 times with PBSB and fixed in 1% paraformaldehyde solution overnight. The next day, the presence of CXCR5 was analyzed with a BD FACSCalibur ™ System Flow Cytometer (BD Biosciences, San Jose, CA).

EXAMPLE 7: FLIPR TEST

Changes in intracellular calcium were measured by culturing 9000 cells / well and incubating overnight. The cells were from the RBL-2H3 line stably transfected with human CXCR5. The cells were then washed and loaded with 2mM fluo-4 / ΑΜ (Molecular Probes) in a buffer containing 2.5mM probenecid. Cells were exposed to mAb for CXCR5 and then washed with assay buffer. Cells were exposed to 10 nM CXCL13 (R&amp;D). Changes in Ca⁺² Intracellular were recorded using the 384-B FLIPR device (Molecular Devices). Commercially available anti-human CXCR5 mAbs, and mouse IgG1 and IgG2b, were used as controls.

As discussed further herein, several humanized versions of 16D7 mAbs were constructed, such as CHIMERIC 16D7 (the hlgG chimera₄), 16D7-HC1-LC1, 16D7-HC1-LC2, 16D7-HC2-LC1,

16D7-HC1-LC3, 16D7-HC3-LC4, 16D7-HC3-LC5, and 16D7-HC1-LC6, and were assayed for biological activity as demonstrated by calcium flux.

Humanized antibodies, apart from a negative control, CA13, demonstrated a signal neutralizing activity equal to that of the antibody.

Parental 16D7 on transfected cells stably expressing

CXCR5.

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX338474B_D0134.tif" />

EXAMPLE 8: CHEMOTAXIS TEST

HS Sultan CXCR5 cells were added⁺ (ATCC CRL1484) to upper chamber 5 of a transwell board (Millipore) at 0.5 x 10⁶ cells / well in the presence of 100 nM CXCL13 (R&amp;D) or CTX buffer (RPMI without phenol red, containing 1% FBS, 0.5% BSA and 1 nM pyruvate Na) and cell number evaluated that migrated to the lower chamber. The two chambers were assembled and incubated for two hours. Cells from the lower chamber were counted after adding colorimetric reagent (Promega) and reading DO490.

CXCR5 specific migration was determined as the difference between the total number of cells that had migrated and the number of cells that spontaneously migrated. If an anti-CXCR5 antibody is assayed, the cells are incubated with the antibody for 30 minutes prior to addition to the upper chamber. The degree of antibody inhibition is the ratio of the specific migration in the presence of antibody to the amount of migration in the absence of antibody. The ratio can be multiplied by 100% to produce a percentage of inhibition.

The antibodies in Example 7 were compared to the parental 16D7 antibody for the ability to neutralize chemotaxis. All humanized antibodies apart from CA13 negative control mAb neutralized chemotaxis in a profile comparable to that of 16D7 and 79B7, 14C9, 19H5, H28, 54G6, G7, 56H6. R&amp;D MAB190 had intermediate activity whereas H28 and the Abnova 2C1 antibody did not completely neutralize ligand-induced migration of cells.

EXAMPLE 9: PRIMARY HUMAN B CELLS

REACTIVITY TEST

Human PBMC were isolated from whole blood using 30 Accuspin columns (Sigma). PBMC were then resuspended in BD staining buffer (Becton Dickinson) at 20 million cells / ml. An antibody Dg was added

<img file="MX338474B_D0135.tif" />

IMPI _ I 1 7 - INSTITUTO MEXICANO

GIVE THE PROPERTY

INDUSTRIAL monoclonal mouse anti-human CXCR5 at 50 DI of PBMC and allowed to bind for 20 min at 4^or C. Cells were washed several times with tID Stain buffer. Fifty IDs of the second antibody, goat anti-mouse IgG-PE F were added (_ab·) (Beckman Coulter) diluted 1/100, to the PBMC-antibody cocktail and allowed to bind for 20 minutes at 4 ° C. Cells were washed three times with BD Stain buffer. A cocktail containing mouse anti-human CD20 antibody-FITC (BD) and CD4-APC (BD) at 1/50 dilution was added to the cells, and these were then incubated for 20 minutes at 4 ° C to evaluate the specificity for B / T cells. Cells were washed 3 times with BD Stain buffer, resuspended in 250 □! of BD Stain buffer and underwent FACS analysis on a FACStar Plus. As a positive control, mouse anti-human CXCR5 antibody (R&amp;D; mAb190) was used. Titration curves for humanized antibodies were generated and the Mean Fluorescence Intensity (MFI) was plotted against the concentration.

The humanized antibodies of Example 7 were tested for binding to human PBMC.

Antibodies, apart from the CA13 negative control, bind and have the same titration profile on human B cells. The negative control CA13 only showed a background binding. The BD RF8B2 clone binds weakly to human PBMC.

EXAMPLE 10: MONO CYNOMOLGUS B CELLS

REACTIVITY TEST

Whole blood of monkey cynomolgus (cyno) was obtained from Bioreclamation, Inc. (Hicksville, NY). The blood was transported in BD cell preparation tubes (CPT) after centrifugation. The cyno PBMCs contained in the plasma layer were removed from the CPT tube and placed into a 50 ml tube that had the unaltered gradient gel layer. The tube was washed with 5 ml of PBS to completely remove all cells and the wash was added to a clean 50 ml tube. The cyno PBMCs were centrifuged at 1200 RPM for 10 minutes at 4 ° C. The pellet was resuspended in 1 ml of BD FACS buffer

<img file="MX338474B_D0136.tif" />

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-113- OF THE PROPERTY

INDUSTRIAL

Stain (BD). One million cells were used per assay. One Dg of mouse anti-human CXCR5 monoclonal antibody (purified) was added to 50 Di of PBMC and allowed to bind for 20 minutes at 4 ° C. Cells were washed twice with BD Stain buffer. Fifty second antibody IDs were added, goat anti-mouse IgG-PE F_(ab·) (Beckman Coulter) diluted 1/100 to the cells and allowed to bind for 20 minutes at 4 ° C. Cells were washed three times with BD Stain buffer. A cocktail containing mouse anti-human CD20 antibody-FITC (BD) and CD4-APC (BD) was added to the cells at a 1/20 dilution during a 20 minute incubation at 4 ° C to assess specificity for B / T cells. Cells were washed 3 times with BD Stain buffer, resuspended in 250 ID of BD Stain buffer and subjected to FACS analysis on a FACStarPlus. As a positive control, the commercial human anti-CXCR5 mouse mAb (R&amp;D; MAB190) was used.

The monoclonal antibody 11D6 of the present invention reactive with human CXCR5, was compared with an IgG-type control, the humanized versions of 16D7 and MAB190 were tested for reactivity with CXCR5 cynomolgus. 79B7 was also tested.

CD20 and CXCR5 positive cells were found with MAB190 and 11D6. On the other hand, 16D7 and its humanized variants, as well as G7 and RF8B2 from BD and 2C1 from Abnova did not bind to monkey cynomolgus B cells. 14C9, 19H5, H28, 54G6, 56H6 and 79B7 also bound to cynomolgus B cells.

EXAMPLE 11: SEQUENCING OF ANTI-CXCR5 mAb

Mouse monoclonal antibodies were sotyped using a commercially available sotyping kit. Variable sequences of 16D7 and other anti-CXCR5 mAbs were sequenced. Total RNA from approximately 5 million hybridoma cells was isolated using the Qiagen Qianeasy miniprep kit following the kit protocol. First strand cDNA was synthesized using the Invitrogen Superscript Kit (Cat 11904-018), following the kit's protocols.

First, the variable regions of heavy chain and of

<img file="MX338474B_D0137.tif" />

IMPI

MEXICAN INSTITUTE

Λ OWNED 'Τ' INDUSTRIAL light chain using the following degenerate PCR primers and Taq polymerase (Roche) based on methods described in Wang et al. J Immunol Methods. 233: 167-77, 2000.

Heavy Chain: Left Primer:

1: CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC (SEQ ID NO: 2)

2: CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG (SEQ ID NO: 3)

Heavy Chain: Right Primer: GGAGGATCCATAGACAGATGGGGGTGTCGTTTTGGC (SEQ ID NO: 4) Light Chain: Left Primer:

GGAGCTCGAYATTGTGMTSACMCARWCTMCA (SEQ ID NO: 5)

Light Chain: Right Primer:

TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC (SEQ ID NO: 6) where R is A or G; N is A, G, T or C; M is A or C; W is A or T; S is G or C¡ and Y is C or T.

The PCR products were cloned into pCR4-TOPO® using the TOPO TA cloning® Cloning Kit (Cat #: 45-0641) and sequenced using the T3 and T7 primers. The sequences were then compared using the Blast program against the genebank database to deduce the leader sequences for cloning of entire variable regions. Based on the Blast results, the following primers (Chardes et al., FEBS Letters 452: 386-394, 1999) were chosen for a second round of PCR amplification using Pfx polymerase (Invitrogen).

Heavy Chain:

Left primer:

CCAAGCTGTGTCCTRTCC (SEQ ID NO: 7)

Right primer:

CGACAAGTCGACTAGCCCTTGACCAGGCATCC (SEQ ID NO: 8)

Light Chain:

Left primer:

WTCTCTRGAGTCAGTGGG (SEQ ID NO: 9)

<img file="MX338474B_D0138.tif" />

<img file="MX338474B_D0139.tif" />

-115 Right primer: _____

CGACTAGTCGACTGGTGGGAAGATGGATACAG (SEQ ID NO: 10)

The PCR products were cloned into pCR-Blunt ll®-TOPO® using the Invitrogen Zero Blunt® TOPO® PCR Cloning Kit (Cat 45-0245) and sequenced using T7 primers.

Once light and heavy chains are sequenced, nucleic acids can be registered to optimize expression, eg, in human host cells.

EXAMPLE 12: TRANSFECTOMES

NSO-eu cells are grown at a density of 1 x 10⁶ cells / ml. The cells are kept in exponential growth phase and the medium is changed the day before transfection. On the day of transfection, they are washed 40 x 10⁶ cells. Then, 10 Dg of linearized nucleic acid, such as light chain DNA, and 10 Dg of, for example, linearized heavy chain DNA are added to the cell suspension (total DNA volume should be less than 50 OI) and the Culture is incubated on ice for 15 min. The DNA and cell mixture is transferred to a cooled cuvette (0.4 cm) and an electrical pulse (750 V and 25 DF) is applied. The cuvette is placed on ice immediately after the electrical pulse and is kept on ice for 10-15 min. The cells are collected and plated. Cells are incubated in a 5% CO incubator₂ for 12-16 days or until colonies appear. The supernatant from cell colonies or cells grown in suspension culture is tested by ELISA, positive transfectomas are cloned in clean medium. To further select positive transfectomas, ELISA or Biacore assay is performed. The expanded transfectomas are kept in shake flasks and antibody or its derivative is collected from the supernatant.

EXAMPLE 13: IN VIVO TRIALS

Collagen-induced arthritis (CIA), a well-established model for human RA, has been used to demonstrate the efficacy of antibodies against

IΜ ΡI

<img file="MX338474B_D0140.tif" />

-116 MEXICAN INSTITUTE Μ INDUSTRIAL PROPERTY

TNFO (Williams et al., PNAS 1992, 89: 9784-9788), as well as ^ protein CTU-4 and TNFD Usesion (Webb et al., Eur J Immunol. 1990; 20.2320 2328; and Wnnlry et al., J Immunol . 1993, 151: 6602-6607). A rat anti-mouse CXCR5 monoclonal antibody, clone 1038, was profiled in a CIA mouse model in which DBA / 1J mice were immunized and boosted with type II chicken collagen. The severity of the disease (which was visually assessed by measuring the swelling / inflammation of the paw) was monitored twice a week, while changes in inflammation, pannus, cartilage destruction, and changes in the joints collected at the end of the study were evaluated. bone erosion. Clone 1038, when administered in a prophylactic dosing regimen, significantly reduced both disease severity and joint pathology compared to isotype-treated CIA mice (repeated measurement ANOVA, p &lt;0.05).

An acute mouse model was used to evaluate the efficacy of 16D7-HC1-LC3 in chemotaxis in vivo. Briefly, C57 / BI6 mice (8-16 weeks old) selectively expressing huCXCR5 in immunocytes, such as B cells, T cells, and neutrophils, were generated by traditional transgenic methods using a CD11a promoter. The in vivo chemotaxis model is a neutrophil-driven model. Following intraperitoneal administration of 20 Dg of huCXCL13 (R&amp;D) ligand, mouse neutrophils expressing the huCXCR5 receptor migrated to the peritoneal cavity in response to a gradient of huCXCL13. Peritoneal cavity washes were used to recover cells 80 minutes after intraperitoneal administration of huCXCL13, and fluorocytometric analysis was used to quantify the number of neutrophils expressing huCXCR5 in 2 ml samples of peripheral washes that specifically migrated to the peritoneal cavity in response to instillation of huCXCL13. Neutrophils were identified by phenotypic markers, such as Ly6G, CD19, and CD11b. Subcutaneous administration of humanized anti-hCXCR5, 16D7-HC1LC3, at two different doses (7.5 Dg or 15 Dg) 24 hours prior to instillation of huCXCL13 showed efficacy in reducing migration of neutrophils expressing huCXCR5 into the cavity. peritoneal in

<img file="MX338474B_D0141.tif" />

<img file="MX338474B_D0142.tif" />

-117 response to huCXCL13 compared to an isotype-treated control, showing the two samples treated with UXUR5 antibody "level of neutrophils that are essentially not statistically different compared to the level of isotype negative control neutrophils. At 1.5 Dg, the humanized CXCR5 antibody showed a low level of inhibition of neutrophil migration compared to the higher doses of CXCR5 antibody tested.

EXAMPLE 14: RESURFACING

The resurfacing technique of the murine 16D7 clone followed the steps described in in Proc. Nati. Acad. Sci. USA (1994) 91: 969 and in US Patent 5,639,641.

The 16D7 Vl and Vh sequences were compared by the Blast program against the Protein Data Bank (Nucleic Acids Research, 28: 235-242 (2000) or the Protein Data Bank (PDB) can be accessed online, which contains the &lt;3D coordinates of biological macromolecules, and the ten most heavy and light chain amino acid sequences most similar to that of 16D7 were recovered .. PDB identification codes are used to identify the sequences.

The ten most similar homologs for the variable light chain were 20 1MJU (J Mol Biol 332: 423-435, 2003), 1AE6 (Proteins 29: 161-171, 1997), 1QYG (Pozharski et al., "Carving a Binding Site : Structural Study of an Anti-Cocaine Antibody ”in“ Complex with Three Cocaine Analogs ”), 1UZG (J Virol 79: 1223, 2005), 1UB5 (Beuscher et al.,“ Structure and Dynamics of Blue Fluorescent Antibody 19G2 at Blue and Violet Fluorescent Temperatures ”), 1RUR (Proc Nati 25 Acad Sci USA 110: 2247-2252, 2004, 1FPT (Nat Struct Biol 2: 232-243, 1995), 1QFU (Nat Struct Biol 6: 530-534, 1999), 1NAK (Virology 315: 159-173, 2003) and 1CGS (J Mol Biol 236: 247-274, 1994) (redundant sequences removed) and the ten most similar homologs for variable heavy chain are 1FNS (Nat Struct Biol 7: 881-884, 2000), 1OAK (Nat Struct Biol 5: 189-194, 1998), 1VFB (Proc 30 Nati Acad Sci 91: 1089-1093, 1994), 1CIC Natura 348: 254-257, 1990), 1GIG (Acta Crystallogr D Biol Crystallogr 50: 768-777, 1994), 1T4K (J Mol Biol 343: 1269 -1280,

<img file="MX338474B_D0143.tif" />

<img file="MX338474B_D0144.tif" />

-118 FROM INDUSTRIAL PROPERTY

2004), 1A7P (Marks et al.), 1FE8 (J Biol Chem 276: 9985-9991,2001), 1DL7 (J Exp Med 191: 2101-2112, 2000) and 1YY8 (Cancer Cell 7: 301-311, 2005 ). The closest matches for light and heavy chains were 1MJU and 1FNS, respectively. These two sequences were used to build a homology model of the variable domains that was subsequently subjected to energy minimization by a gradient minimization of conjugate atomic coordinate positions with the CHARMM22 force field (J Comput Chem (1983) 4, 187; J Comput Chem (1986) 7, 591) as applied in the MOE series (Chemical Computing Group, Quebec, CA). The model was used to locate CDR regions and flanking debris. Solvent accessibility was calculated for each variable region moiety from the ten closest matches for each antibody variable region and averaged on an Excel spreadsheet as applied in a Scitegic protocol (Hill &amp; Lewicki (2006) Statistics: Methods and Applications, Statsoft, Tulsa, OK). Positions with more than 30% average accessibility were considered remnants of the surface. Positions with average accessibilities between 25% and 30% depending on proximity to CDR loops were also considered.

The surface positions of the murine 16D7 variable region were compared to the corresponding positions in the human antibody sequences. Only those remnants with an accessible surface area greater than 30%, with a few residues presenting an accessible surface area greater than 25% and which were flanking remnants exposed to the solvent, were retained for the search . Some conserved residues were included in all immunoglobulin sequences to improve search convergence. For the correct analysis only germline sequences were retained. The human antibody variable region surface with the closest surface moieties was chosen, giving special consideration to positions that are less than or equal to 5.0 A away from a CDR, to replace the variable region surface moieties. of the murine antibody 16D7.

Neither sequence contains any known B-cell epitopes or

<img file="MX338474B_D0145.tif" />

T cells indicated in the Immune Epitope database (IEDB, Immune Epitope Database and Analysis Resource web site; PLoS Biol. 2005; 3 (3): e91).

The original sequences of the murine 16D7 variable domains are:

the light chain (CDRs are underlined):

DIVMTQAAPSVAVTPRESVSISCRSSKSLLHSSGKTYLYWFLQRPGQSPQ LLIYRMSNLASGVPDRFSGSGSGTAFTLRI SRVEAEDVGV YYCMQHLEYP YTFGGGTKLE IK (SEQ ID NO: 11); and the heavy chain (CDRs are underlined)

QVQLKESGPGLVAPSQSLSITCTVSGFSLIDYGVNWIRQPPGKGLEWLGVl WGDGTTYYNS ALKS RLSIRKDNSQSQVFL KMNSLQTDDTAMYYCARIVYWGQGTLVTVSA (SEQ ID NO: 12).

The retained series of surface debris for the light chain search included D1, V3, A7, P9, P15, R16, E17, S18, P45, G46, Q47, D65, R79, R82, E86, K108, E110, and K112.

The unknown for the light chain contains the series of surface residues defined above and the conserved amino acids C23, W40, Q43, Y91, C93, F103, G104, G106 and T107, which were included for the convergence of the BLAST protocol. All the other amino acids could be any of the 20 natural amino acids. The BLAST search was performed against the human germline antibody sequence database compiled by the IMGT (International Immunogenetics Information Systems website, Molec Immunol, 2004, 40: 647-659). The best match score was found in X72482 (protein_id = CAA51150.1) from which the light chain, LC4, was obtained. LC5 and LC6 are two variants of VL4 (VL stands for variable light) that have been suggested to correct potential troublesome residues in the light chain: 1 exposed methionine (M51) that will mutate to Leu (LC5 &amp; LC6) and 1 possible deamidation site (LC6) in which the N53 asparagine is exchanged for a serine residue. In total, 3 versions are proposed for the variable light chain containing between 4 and 6 mutations compared to the parental murine clone 16D7. Corresponding mutations are provided in Table 1 below. Sequential numbering and Kabat numbering are provided.

<img file="MX338474B_D0146.tif" />

-120 The retained series of surface residues for the variable heavy chain included Q1, Q3, K5, S7, P9, L11, S15, Q16, S20, P41, G42, K43, S61, A62, K64, S65, R70, S74 , Q75, Q86, T87, D88, Q103, L106, A111, A112 and K113 (sequential numbering). The invariant amino acids that were included in BLAST's 5 unknown for search convergence were: C22, W36, I37, Q39, D89, Y93, C95, W101, G102, G104 and T105. The BLAST search was performed against the database of human germline antibody sequences compiled by IMGT. A version for the heavy chain (HC3) was retained. The two Vh domains of AF062266 (protein_id = AAC18304.1) and 10 AY393082 (protein_id = AAS86018.1), which showed the best correspondence score for the series of surface residues, show an equivalent similarity score and show residues of identical surface. Consequently, only a single sequence was retained for the heavy chain with ten mutations. Lower scoring sequences having 15 different surface residues were not retained because they have fewer polar residues, suggesting possible reduced solubility.

TABLE 1

<img file="MX338474B_D0147.tif" />

<td>Ala7</td><td>Ala7</td><td>To be</td><td>To be</td><td>To be</td>

<td>Pro9</td><td>Pro9</td><td>Leu</td><td>Leu</td><td>Leu</td>

<td>Arg16</td><td>Arg16</td><td>Gly</td><td>Gly</td><td>Gly</td>

<td>Met56</td><td>Met51</td><td>Met</td><td>Leu</td><td>Leu</td>

<td>Asn58</td><td>Asn53</td><td>Asn</td><td>Asn</td><td>To be</td>

<td>Arg82</td><td>Arg77</td><td>Lys</td><td>Lys</td><td>Lys</td>

<img file="MX338474B_D0148.tif" />

<img file="MX338474B_D0149.tif" />

-121 -

<img file="MX338474B_D0150.tif" />

<td>Lys5</td><td>Lys5</td><td>Gln</td><td>Gln</td><td>Gln</td>

<td>Gln16</td><td>Gln16</td><td>Glu</td><td>Glu</td><td>Glu</td>

<td>Ser61</td><td>Ser61</td><td>Pro</td><td>Pro</td><td>Pro</td>

<td>Ala62</td><td>Ala62</td><td>To be</td><td>To be</td><td>To be</td>

<td>Arg70</td><td>Arg70</td><td>To be</td><td>To be</td><td>To be</td>

<td>Gln75</td><td>Gln75</td><td>Lys</td><td>Lys</td><td>Lys</td>

<td>Gln86</td><td>Gln83</td><td>Thr</td><td>Thr</td><td>Thr</td>

<td>Thr87</td><td>Thr84</td><td>To</td><td>To</td><td>To</td>

<td>Asp88</td><td>Asp85</td><td>To</td><td>To</td><td>To</td>

<td>Alai 11</td><td>Alai 13</td><td>To be</td><td>To be</td><td>To be</td>

<img file="MX338474B_D0151.tif" />

Three versions are proposed for the light chain (LC4, LC5 and LC6). Individual mutations introduced by the light chain resurfacing technique are indicated by lowercase letters and are underlined and the CDRs are underlined. The sequences subjected to resurfacing of the variable regions are presented below, the constant domain (lgG4) is not included.

LC4:

DIVMTQsAIS VAVTPgESVS ISCRSSKSLL HSSGKTYLYW FLQRPGQSPQ

LLIYRMSNLA SGVPDRFSGS GSGTAFTLkl SRVEAEDVGV YYCMQHLEYP

YTFGGGTKLE IK (SEQ ID NO: 13)

LC5:

DIVMTQsAIS VAVTPgESVS ISCRSSKSLL HSSGKTYLYW FLQRPGQSPQ

LLIYRISNLA SGVPDRFSGS GSGTAFTLkl SRVEAEDVGV YYCMQHLEYP

YTFGGGTKLE IK (SEQ ID NO: 14)

<img file="MX338474B_D0152.tif" />

<img file="MX338474B_D0153.tif" />

-122LC6:

DIVMTQsAIS VAVTPgESVS ISCRSSKSLL HSSGKTYLYW FLQRPGQSPQ LLIYRISsnLA SGVPDRFSGS GSGTAFTLkl SRVEAEDVGV YYCMQHLEYP YTFGGGTKLE IK (SEQ ID NO: 15)

A version for the heavy chain (VH3) was proposed (VH stands for variable weighing). Mutations introduced by the variable chain resurfacing technique are lowercase and underlined, and CDRs are underlined. The constant domain sequence is not included.

HC3:

QVQLgESGPG LVAPSeSLSI TCTVSGFSLI DYGVNWIRQP PGKGLEWLGV IWGDGTTYYN psLKSRLSIs KDNSkSQVFL KMNSLtaaDT AMYYCARIVY s_ (SEQ ID NO: 16)

Nucleotide sequences were generated by OE-PCR and cloned into Nhel / Hindlll sites of the episomal expression vector pXL4214 (Durocher et al., NAR, 2002, 30 (2), E9. The sequences have codons optimized for expression in human cells. V_L merged into IGKC (AAH93097). Vh was fused to IGHG4 (AAH25985), which lacked the C-terminal Lys (IGHG4AK). The sequences were validated by double chain sequencing

LC4:

MGWSCIILFLVATATGVHSDIVMTQSALSVAVTPGESVSISCRSSKSLLHSS

GKTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLKISRVEAEDV

GVYYCMQHLEYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNN

FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

<img file="MX338474B_D0154.tif" />

ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 17) _____———

GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACC GCCACCGGCGTGCACAGCGACATCGTGATGACCCAGAGCGCCCTCAGCGTG GCCGTGACCCCCGGCGAGAGCGTGAGCATCAGCTGCCGCAGCAGCAAGAGC CTGCTGCACAGCAGCGGCAAGACCTACCTGTACTGGTTCCTGCAGCGCCCC GGCCAGAGCCCCCAGCTGCTGATCTACCGCATGAGCAACCTGGCCAGCGGC GTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGCCTTCACCCTGAAG ATCAGCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGCAC CTGGAGTACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGT ACGGTGGCCGCTCCTTCCGTGTTCATCTTCCCTCCCTCCGACGAGCAGCTGA AGTCCGGCACCGCCTCCGTGGTGTGTCTGCTGAACAACTTCTACCCTCGGGA GGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCA GGAGTCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCC ACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTG AGGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGG GCGAGTGCTGAAGCTT (SEQ ID NO: 18)

LC5:

MGWSCIILFLVATATGVHSDIVMTQSALSVAVTPGESVSISCRSSKSLLHSS GKTYLYWFLQRPGQSPQLLIYRLSNLASGVPDRFSGSGSGTAFTLKISRVEAEDV GVYYCMQHLEYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID N^or:19)

GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACC GCCACCGGCGTGCACAGCGACATCGTGATGACCCAGAGCGCCCTCAGCGTG GCCGTGACCCCCGGCGAGAGCGTGAGCATCAGCTGCCGCAGCAGCAAGAGC CTGCTGCACAGCAGCGGCAAGACCTACCTGTACTGGTTCCTGCAGCGCCCC GGCCAGAGCCCCCAGCTGCTGATCTACCGCCTGAGCAACCTGGCCAGCGGC

<img file="MX338474B_D0155.tif" />

<img file="MX338474B_D0156.tif" />

-124GTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGCCTTCACCCTGAAG

ATCAGCCGCGTGGAGGCCGAGGACGTGGGCGTG IAUI Aü I G'OTTOÜAGCAC

CTGGAGTACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGT

ACGGTGGCCGCTCCTTCCGTGTTCATCTTCCCTCCCTCCGACGAGCAGCTGA

AGTCCGGCACCGCCTCCGTGGTGTGTCTGCTGAACAACTTCTACCCTCGGGA

GGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCA

GGAGTCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCC

ACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTG

AGGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGG

GCGAGTGCTGAAGCTT (SEQ ID NO: 20)

LC6:

MGWSCIILFLVATATGVHSDIVMTQSALSVAVTPGESVSISCRSSKSLLHSS

GKTYLYWFLQRPGQSPQLLIYRLSSLASGVPDRFSGSGSGTAFTLKISRVEAEDV GVYYCMQHLEYPYTFGGGTKLEIKRTVAAQSKVKKSQQKKDQQKKSQVKWKSKVQ

GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACC

GCCACCGGCGTGCACAGCGACATCGTGATGACCCAGAGCGCCCTCAGCGTG

GCCGTGACCCCCGGCGAGAGCGTGAGCATCAGCTGCCGCAGCAGCAAGAGC

CTGCTGCACAGCAGCGGCAAGACCTACCTGTACTGGTTCCTGCAGCGCCCC

GGCCAGAGCCCCCAGCTGCTGATCTACCGCCTGAGCAGCCTGGCCAGCGGC

GTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGCCTTCACCCTGAAG

ATCAGCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGCAC

CTGGAGTACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGT

ACGGTGGCCGCTCCTTCCGTGTTCATCTTCCCTCCCTCCGACGAGCAGCTGA

AGTCCGGCACCGCCTCCGTGGTGTGTCTGCTGAACAACTTCTACCCTCGGGA

GGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCA

GGAGTCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCC

<img file="MX338474B_D0157.tif" />

ACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTG AGGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTÜCTTCA'ACCGGG * GCGAGTGCTGAAGCTT (SEQ ID NO: 22)

HC3:

MGWSCIILFLVATATGVHSQVQLQESGPGLVAPSESLSITCTVSGFSLIDYG VNWIRQPPGKGLEWLGVIWGDGTTYYNPSLKSRLSISKDNSKSQVFLKMNSLTA ADTAMYYCARIVYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNV DHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT CVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 23)

GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACC GCCACCGGCGTGCACAGCCAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCT GGTGGCCCCCAGCGAGAGCCTGAGCATCACCTGCACCGTGAGCGGCTTCAG CCTGATCGACTACGGCGTGAACTGGATCCGCCAGCCCCCCGGCAAGGGCCT GGAGTGGCTGGGCGTGATCTGGGGCGACGGCACCACCTACTACAACCCCAG CCTGAAGAGCCGCCTGAGCATCTCCAAGGACAACAGCAAGAGCCAGGTGTTC CTGAAGATGAACAGCCTGACCGCCGCCGACACCGCCATGTACTACTGCGCCC GCATCGTGTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCCAGCA CCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACCTCCGA GTCCACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTG ACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTG CCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCC TTCCTCCTCCCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCCT TCCAACACCAAGGTGGACAAGCGGGTGGAGTCCAAGTACGGCCCTCCTTGC CCTTCCTGCCCTGCCCCTGAGTTCCTGGGCGGACCTAGCGTGTTCCTGTTCC

<img file="MX338474B_D0158.tif" />

<img file="MX338474B_D0159.tif" />

-126 HE.'CCANO UL INSTITUTE THE INDUSTRIAL LAND

CTCCTAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAGGTGACCTG im xmBMcarKriira * ¢.-3 ^ 5, T .iH ^ iy W

TGTGGTGGTGGACGTGTCCCAGGAGGACCCTGAGGTCCAGTTCAACTGGTAC GTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCA GTTCAATTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGAC TGGCTGAACGGCAAAGAATACAAGTGTAAGGTCTCCAACAAGGGCCTGCCCT CCTCCATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAGCCTCA GGTGTACACCCTGCCTCCTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCC CTGACCTGTCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGG AGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGA CTCCGACGGCTCCTTCTTCCTGTACTCCAGGCTGACCGTGGACAAGTCCCGG TGGCAGGAGGGCAACGTCTTTTCCTGCTCCGTGATGCACGAGGCCCTGCACA ACCACTACACCCAGAAGTCCCTGTCCCTGTCTCTGGGCTGAAGCTT (SEQ ID NO: 24)

EXAMPLE 15: HUMANIZATION

V sequences_L and 16D7 Vh were subjected to a Blast search and the closest matches for the variable light chain are 1MH5, 1MJJ and 1MJU (J Mol Biol 332: 423-435, 2003), with equivalent similarity scores. 1MJU was retained as a template due to the high precision of the crystal structure, which had been determined at a resolution of 1.22 A. The closest similar homolog for the heavy chain was found to be 1FNS (Nat Struct Biol 7: 881884, 2000 ). Structures 1 MJU and 1FNS were used to construct a homology model of variable domains that was subsequently subjected to energy minimization using conventional procedures. Subsequently, a molecular dynamics (MD) calculation was performed from a 3D homology model of 16D7 for 1.7 nanoseconds in implicit solvent Generalized Born (see Gallicchio &amp; Levy, J Comput Chem 2004, 25: 479-499).

The MD begins with the initialization of the velocities from a Gaussian distribution at 298.15 ° K, followed by an equilibration period of 300 ps.

During MD, all links are restricted using the SHAKE algorithm (see

Barth. et al., J Comp Chem, 1995, 16: 1192-1209), the time step was 1

<img file="MX338474B_D0160.tif" />

IMPI

-127femtosecond (fs), and the simulation, based on Verlet's integration algorithm, was performed on the canonical NVT set (number of particles, volume and temperature) at a temperature of 298.15 ° K. During the production period, 1,700 photos were stored, one every 1 ps. The 1,700 conformations of the murine antibody constitute the set on which the following analysis was performed to identify the most flexible residues. To encode the following analysis, the Scientific Vector Language (SVL), available within the molecular modeling environment MOE (Molecular Operating Environment (MOE), Chemical Computing Group, Quebec, Canada), was used. First, each photo, N, was optimally superimposed on its predecessor, photo N-1, to control the global rotational and translational movements that occur during MD modeling and computation. The overlap was obtained by minimizing the root mean square distance (RMSD) between all corresponding pairs of atoms in the two photos. In the overlap exercise only the heavy atoms of the antibody main chain were considered. Using the same overlay method, each photo was then superimposed on the medoid photo. The photo medoid is the antibody conformation with the cartesian coordinates closest to the average coordinates of all the conformations.

For each antibody residue i, the RMSDs between the heavy atoms of conformation j and a conformation k of reference medoid were calculated. The

<img file="MX338474B_D0161.tif" />

, defining dj ^ as the

RMSD has the following formula: RMSDj = Euclidean distance expressed in Angstroms (A) between heavy atom I of residue j and its counterpart of the k conformation of reference medoid. For the pairwise association of heavy atoms I, the symmetry of the heavy side chain atoms was also considered for amino acids, Asp, Leu, Val, Glu, Arg, Phe and Tyr. the reference k conformation varies from one remainder to another, and corresponds to the k medoid conformation with the closest Euclidean distance to the mean coordinates of all the conformations of the rest studied i. So for each

-128-

<img file="MX338474B_D0162.tif" />

FROM THE PG-í ->? .- \ 2

INDUSTRY!

remainder i, a distribution of 1,700 RMSD values ​​was obtained, which reflects the variation of coordinates of remainder i in the course of the MD. By means of the aggregation of all the RMSD values ​​of all the residues of the studied antibody, a global distribution of all the RMSD was obtained. The global distribution of all RMSDs was then used as the reference distribution. When the remainder i was very flexible, a statistical test was performed to decide if the observed mean RMSD of the remainder i, m, was significantly higher than the global mean RMSD for all the remains, m_g. Since the sample is large, for example 1,700 for analysis of clone 16D7, a one-sided Z-test was used (see Dorofeev &amp; Grant, "Statistics for real-life sample surveys. Non-simple-random samples and weighted data" 2006. Cambridge University Press) with the null hypothesis Hq that “the observed value of m¡ is less than the value of m_g global ”to calculate the statistical parameter Z¡ according to the formula: Zf = (nti — m_giobai ')

<img file="MX338474B_D0163.tif" />

Ϊ where m¡ is the mean RMSD calculated from the RMSD distribution of the remainder i, m_g is the mean RMSD calculated from the global RMSD distribution, sd¡ is the standard deviation calculated from the RMSD distribution of the remainder, and i is the sample size, i.e. n = 1,700 for the analysis of clone 16D7. The Z, calculated later, was compared with the cumulative probabilities of the standard normal distribution to evaluate a significance level of 99.9% of the alternative hypothesis, that is, that “the value of m, observed is greater than the value of m_g global ”, this corresponded to a Z¡> 3.08. The Zi statistical parameter, which can be considered a flexibility score, is not correlated neither with the molecular weight nor with the number of heavy atoms of the rest of the antibody (¿= 0.014 and 0.0009, respectively, when the MD of the model is analyzed of 16D7 anti-CXCR5).

The series of flexible residues for the light chain Includes the following residues (sequential numbering): D1, T14, P15, R16, E17, Q47, D65, S72 R79,

R82, E86, K108, E110 and K112; and for the heavy chain include the following residues: Q1, V2, Q3, L11, S15, Q16, S61, A62, K64, S65, R70, D72, Q75, K81,

<img file="MX338474B_D0164.tif" />

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M82, N83, Q86, Q103, S110, A111, A112 and K113. The flexible parts of 16D7 were compared to the corresponding positions of the human antibody sequences in the September 2005 version of the ImMunoGeneTics Database website.

Residues showing a significant high flexibility score and a few flanking residues retaining the 3D structures of the flexible residues were retained for search.

The human antibody variable region with the most similar flexible residues was chosen, giving special consideration to positions that are less than or equal to 5.0 A away from a CDR, to replace the variable region flexible residues of the murine antibody 16D7. The resulting humanized sequences were subjected to a Blast search for sequence similarity in the UniProtKB / SwissProt database confirming that reasonable assumptions had been made. All sequences showed a high degree of similarity with various human antibodies. Furthermore, none of the sequences contains any B-cell or T-cell epitopes indicated in the IEDB database.

The best sequence match in the IEDB for the light chain (LC1, LC2 and LC3) was KPGQPPRLLIYDASNRATGIPA (SEQ ID NO: 25), which includes CDR2 but has a significant residue difference typified by a sequence identity of 56 % obtained from a BLAST search within the IEDB database.

The best IEDB match for the heavy chain (HC1 and HC2) was TDDTAMYYCARI (SEQ ID NO: 26) which is located before the start of 25 CDR3. The sequence has a sequence identity of 61% with the SEDSALYYCARD peptide (SEQ ID NO: 27), making it unlikely to be a human T cell epitope (J Exp Med (1995) 181,1540)

The original sequences of the murine 16D7 variable domains are: light chain (underlined CDR):

DIVMTQAAPS VAVTPRESVS ISCRSSKSLL HSSGKTYLYW

FLQRPGQSPQ LLIYRMSNLA SGVPDRFSGS GSGTAFTLRI SRVEAEDVGV

<img file="MX338474B_D0165.tif" />

<img file="MX338474B_D0166.tif" />

-130YYCMQHLEYP YTFGGGTKLE IK (SEQ ID NO: 28); and -------- heavy chain (underlined CDR):

QVQLKESGPG LVAPSQSLSI TCTVSGFSLI DYGVNWIRQP PGKGLEWLGV IWGDGTTYYN SALKSRLSIR KDNSQSQVFL KMNSLQTDDT 5 AMYYCARJVY WGQGTLVTVS A (SEQ ID NO: 29).

Two versions for the heavy chain (HC1 &amp; HC2) and three versions for the light chain (LC1, LC2 and LC3) were suggested. The two versions of the heavy chain come from AF262096 / AAF79987 and AB063657 / BAC01285.1, respectively. The two sequences have a similarity score of 10 equivalent, but were maintained because the series of residues to be mutated appear to be relatively different and have different physicochemical properties. The LC1 sequence comes from BAC01682 / AB064054.1 and there are only two residues to mutate. LC2 and LC3 are variants of LC1 that are suggested to fix potential troublesome residues in the light chain: an exposed methionine (M51) mutated at 15, Leu (versions 3 and 4), and a possible deamidation site (version 4) where the asparagine, N53, is exchanged for a serine residue. Not all combinations were retained, but four cover most of the key points to address. Kabat numbering is used.

Table 2

<img file="MX338474B_D0167.tif" />

<td>Arg 16</td><td>Arg16</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Gly</td>

<td>Glu17</td><td>Glu17</td><td>To</td><td>To</td><td>To</td><td>To</td>

<td>Met56</td><td>Met51</td><td>Met</td><td>Met</td><td>Leu</td><td>Leu</td>

<td>Asn58</td><td>Asn53</td><td>Asn</td><td>Asn</td><td>Asn</td><td>To be</td>

<img file="MX338474B_D0168.tif" />

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<img file="MX338474B_D0169.tif" />

<img file="MX338474B_D0170.tif" />

<td>Gln1</td><td>Gln1</td><td>Gln</td><td>Glu</td><td>Gln</td><td>Gln</td>

<td>Ser15</td><td>Ser15</td><td>To be</td><td>Gly</td><td>To be</td><td>To be</td>

<td>Gln16</td><td>Gln16</td><td>Glu</td><td>Gly</td><td>Glu</td><td>Glu</td>

<td>Ser61</td><td>Ser61</td><td>Pro</td><td>To</td><td>Pro</td><td>Pro</td>

<td>Ala62</td><td>Ala62</td><td>To be</td><td>Pro</td><td>To be</td><td>To be</td>

<td>Ser65</td><td>Ser65</td><td>To be</td><td>Gly</td><td>To be</td><td>To be</td>

<td>Arg70</td><td>Arg70</td><td>To be</td><td>To be</td><td>To be</td><td>To be</td>

<td>Gln75</td><td>Gln75</td><td>Lys</td><td>Lys</td><td>Lys</td><td>Lys</td>

<td>Lys81</td><td>Lys81</td><td>Lys</td><td>Gln</td><td>Lys</td><td>Lys</td>

<td>Met82</td><td>Met82</td><td>Val</td><td>Met</td><td>Val</td><td>Val</td>

<td>Asn83</td><td>Asn82A</td><td>Thr</td><td>Asn</td><td>Thr</td><td>Thr</td>

<td>Gln86</td><td>Gln83</td><td>Thr</td><td>Lys</td><td>Thr</td><td>Thr</td>

<td>Alai 11</td><td>Alai 13</td><td>To</td><td>To be</td><td>To</td><td>To</td>

<img file="MX338474B_D0171.tif" />

Mutations introduced by humanization of variable chains are lowercase and underlined, and CDRs are underlined. Constant domains are not included.

LC1:

DIVMTQAAPS VAVTPgaSVS ISCRSSKSLL HSSGKTYLYW

FLQRPGQSPQ LLIYRMSNLA SGVPDRFSGS GSGTAFTLRI SRVEAEDVGV

YYCMQHLEYP YTFGGGTKLE IK (SEQ ID NO: 30)

<img file="MX338474B_D0172.tif" />

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LC2:

DIVMTQAAPS VAVTPgaSVS ISCRSSKSLL HSSGKTYLYW

FLQRPGQSPQ LLIYRISNLA SGVPDRFSGS GSGTAFTLRI SRVEAEDVGV

YYCMQHLEYP YTFGGGTKLE IK (SEQ ID NO: 31)

LC3:

DIVMTQAAPS VAVTPgaSVS ISCRSSKSLL HSSGKTYLYW

FLQRPGQSPQ LLIYRISsLA SGVPDRFSGS GSGTAFTLRI SRVEAEDVGV

YYCMQHLEYP YTFGGGTKLE IK (SEQ ID NO: 32)

HC1:

QVQLKESGPG LVAPSeSLSI TCTVSGFSLI DYGVNWIRQP

PGKGLEWLGV IWGDGTTYYN psLKSRLSIs KDNSkSQVFL KvtSLtTDDT

AMYYCARIVY

WGQGTLVTVS A (SEQ ID NO: 33)

HC2:

eVQLKESGPG LVAPggSLSI TCTVSGFSLI DYGVNWIRQP

PGKGLEWLGV IWGDGTTYYN apLKgRLSIs KDNSkSQVFL gMNSLkTDDT

AMYYCARIVY

WGQGTLVTVS s_ (SEQ ID NO: 34)

The sequences of the chimeric constructs are as follows:

Chimeric LC Sequence

MGWSCIILFLVATATGVHSDIVMTQAAPSVAVTPRESVSISCRSSKSLLHSS

GKTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDV

GVYYCMQHLEYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNN

- 133 - _____________________

FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 35)

GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACC GCCACCGGCGTGCACAGCGACATCGTGATGACCCAGGCCGCCCCCAGCGTG GCCGTGACCCCCCGCGAGAGCGTGAGCATCAGCTGCCGCAGCAGCAAGAGC CTGCTGCACAGCAGCGGCAAGACCTACCTGTACTGGTTCCTGCAGCGCCCC GGCCAGAGCCCCCAGCTGCTGATCTACCGCATGAGCAACCTGGCCAGCGGC GTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGCCTTCACCCTGCGC ATCAGCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGCAC CTGGAGTACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGT ACGGTGGCCGCTCCTTCCGTGTTCATCTTCCCTCCCTCCGACGAGCAGCTGA AGTCCGGCACCGCCTCCGTGGTGTGTCTGCTGAACAACTTCTACCCTCGGGA GGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCA GGAGTCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCC ACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTG AGGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGG GCGAGTGCTGAAGCTT (SEQ ID NO: 36)

Chimeric HC Sequence

MGWSCIILFLVATATGVHSQVQLKESGPGLVAPSQSLSITCTVSGFSLIDYG VNWIRQPPGKGLEWLGVIWGDGTTYYNSALKSRLSIRKDNSQSQVFLKMNSLQT DDTAMYYCARIVYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNV DHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 37)

GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACC

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-134 GCCACCGGCGTGCACAGCCAGGTGCAGCTGAAGGAGAGCGGCCCCGGCCT GGTGGCCCCCAGCCAGAGCCTGAGCATCACCTGCACCGTGAGCGGCTTCAG CCTGATCGACTACGGCGTGAACTGGATCCGCCAGCCCCCCGGCAAGGGCCT GGAGTGGCTGGGCGTGATCTGGGGCGACGGCACCACCTACTACAACAGCGC CCTGAAGAGCCGCCTGAGCATCCGCAAGGACAACAGCCAGAGCCAGGTGTT CCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATGTACTACTGCGCC CGCATCGTGTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCCGCCAGC ACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACCTCCG AGTCCACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGT GACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCT GCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGC CTTCCTCCTCCCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCC TTCCAACACCAAGGTGGACAAGCGGGTGGAGTCCAAGTACGGCCCTCCTTGC CCTTCCTGCCCTGCCCCTGAGTTCCTGGGCGGACCTAGCGTGTTCCTGTTCC CTCCTAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAGGTGACCTG TGTGGTGGTGGACGTGTCCCAGGAGGACCCTGAGGTCCAGTTCAACTGGTAC GTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCA GTTCAATTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGAC TGGCTGAACGGCAAAGAATACAAGTGTAAGGTCTCCAACAAGGGCCTGCCCT CCTCCATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAGCCTCA GGTGTACACCCTGCCTCCTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCC CTGACCTGTCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGG AGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGA CTCCGACGGCTCCTTCTTCCTGTACTCCAGGCTGACCGTGGACAAGTCCCGG TGGCAGGAGGGCAACGTCTTTTCCTGCTCCGTGATGCACGAGGCCCTGCACA ACCACTACACCCAGAAGTCCCTGTCCCTGTCTCTGGGCTGAAGCTT (SEQ ID NO: 38)

Humanized VL sequences

LC1:

<img file="MX338474B_D0173.tif" />

<img file="MX338474B_D0174.tif" />

-135MGWSCIILFLVATATGVHSDIVMTQAAPSVAVTPGASVSISCRSSKSLLHSS GKTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDV GVYYCMQHLEYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 39)

GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACC GCCACCGGCGTGCACAGCGACATCGTGATGACCCAGGCCGCCCCCAGCGTG GCCGTGACCCCCGGCGCCAGCGTGAGCATCAGCTGCCGCAGCAGCAAGAGC CTGCTGCACAGCAGCGGCAAGACCTACCTGTACTGGTTCCTGCAGCGCCCC GGCCAGAGCCCCCAGCTGCTGATCTACCGCATGAGCAACCTGGCCAGCGGC GTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGCCTTCACCCTGCGC ATCAGCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGCAC CTGGAGTACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGT ACGGTGGCCGCTCCTTCCGTGTTCATCTTCCCTCCCTCCGACGAGCAGCTGA AGTCCGGCACCGCCTCCGTGGTGTGTCTGCTGAACAACTTCTACCCTCGGGA GGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCA GGAGTCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCC ACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTG AGGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGG GCGAGTGCTGAAGCTT (SEQ ID NO: 40)

LC2:

MGWSCIILFLVATATGVHSDIVMTQAAPSVAVTPGASVSISCRSSKSLLHSS GKTYLYWFLQRPGQSPQLLIYRLSNLASGVPDRFSGSGSGTAFTLRISRVEAEDV GVYYCMQHLEYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 41)

<img file="MX338474B_D0175.tif" />

<img file="MX338474B_D0176.tif" />

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GCTAGCACCATGGGCTGGAGCTGCATCATCCT £ H £ XIGGLGaGGAGG GCCACCGGCGTGCACAGCGACATCGTGATGACCCAGGCCGCCCCCAGCGTG GCCGTGACCCCCGGCGCCAGCGTGAGCATCAGCTGCCGCAGCAGCAAGAGC CTGCTGCACAGCAGCGGCAAGACCTACCTGTACTGGTTCCTGCAGCGCCCC GGCCAGAGCCCCCAGCTGCTGATCTACCGCCTGAGCAACCTGGCCAGCGGC GTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGCCTTCACCCTGCGC ATCAGCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGCAC CTGGAGTACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGT ACGGTGGCCGCTCCTTCCGTGTTCATCTTCCCTCCCTCCGACGAGCAGCTGA AGTCCGGCAGCGCCTCCGTGGTGTGTCTGCTGAACAACTTCTACCCT.CGGGA GGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCA GGAGTCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCC ACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTG AGGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGG GCGAGTGCTGAAGCTT (SEQ ID NO: 42)

LC3:

MGWSCIILFLVATATGVHSDIVMTQAAPSVAVTPGASVSISCRSSKSLLHSS GKTYLYWFLQRPGQSPQLLIYRLSSLASGVPDRFSGSGSGTAFTLRISRVEAEDV GVYYCMQHLEYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 43)

GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACC GCCACCGGCGTGCACAGCGACATCGTGATGACCCAGGCCGCCCCCAGCGTG GCCGTGACCCCCGGCGCCAGCGTGAGCATCAGCTGCCGCAGCAGCAAGAGC CTGCTGCACAGCAGCGGCAAGACCTACCTGTACTGGTTCCTGCAGCGCCCC GGCCAGAGCCCCCAGCTGCTGATCTACCGCCTGAGCAGCCTGGCCAGCGGC GTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGCCTTCACCCTGCGC ATCAGCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGCAC

<img file="MX338474B_D0177.tif" />

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-137CTGGAGTACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCÁAGCGT ACGGTGGCCGCTCCTTCCGTGTTCATCTTCCCTCCCTCCGACGAGCAGCTGA AGTCCGGCACCGCCTCCGTGGTGTGTCTGCTGAACAACTTCTACCCTCGGGA GGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCA GGAGTCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCC ACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTG AGGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGG GCGAGTGCTGAAGCTT (SEQ ID NO: 44)

Humanized VH sequences

HC1:

MGWSCIILFLVATATGVHSQVQLKESGPGLVAPSESLSITCTVSGFSLIDYG VNWIRQPPGKGLEWLGVIWGDGTTYYNPSLKSRLSISKDNSKSQVFLKVTSLTTD DTAMYYCARIVYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVD HKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC WVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWL NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 45)

GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACC GCCACCGGCGTGCACAGCCAGGTGCAGCTGAAGGAGAGCGGCCCCGGCCT GGTGGCCCCCAGCGAGAGCCTGAGCATCACCTGCACCGTGAGCGGCTTCAG CCTGATCGACTACGGCGTGAACTGGATCCGCCAGCCCCCCGGCAAGGGCCT GGAGTGGCTGGGCGTGATCTGGGGCGACGGCACCACCTACTACAACCCCAG CCTGAAGAGCCGCCTGAGCATCAGCAAGGACAACAGCAAGAGCCAGGTGTT CCTGAAGGTGACCAGCCTGACCACCGACGACACCGCCATGTACTACTGCGCC CGCATCGTGTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCCGCCAGC

<img file="MX338474B_D0178.tif" />

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-138- ___________

ACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACCTCCG AGTCCACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGT GACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCT GCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGC CTTCCTCCTCCCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCC TTCCAACACCAAGGTGGACAAGCGGGTGGAGTCCAAGTACGGCCCTCCTTGC CCTTCCTGCCCTGCCCCTGAGTTCCTGGGCGGACCTAGCGTGTTCCTGTTCC CTCCTAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAGGTGACCTG TGTGGTGGTGGACGTGTCCCAGGAGGACCCTGAGGTCCAGTTCAACTGGTAC GTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCA GTTCAATTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGAC TGGCTGAACGGCAAAGAATACAAGTGTAAGGTCTCCAACAAGGGCCTGCCCT CCTGCATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAGCCTCA GGTGTACACCCTGCCTCCTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCC CTGACCTGTCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGG AGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGA CTCCGACGGCTCCTTCTTCCTGTACTCCAGGCTGACCGTGGACAAGTCCCGG TGGCAGGAGGGCAACGTCTTTTCCTGCTCCGTGATGCACGAGGCCCTGCACA ACCACTACACCCAGAAGTCCCTGTCCCTGTCTCTGGGCTGAAGCTT (SEQ ID NO: 46)

HC2:

MGWSCIILFLVATATGVHSEVQLKESGPGLVAPGGSLSITCTVSGFSLIDYG VNWIRQPPGKGLEWLGVIWGDGTTYYNAPLKGRLSISKDNSKSQVFLQMNSLKT DDTAMYYCARIVYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNV DHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT CWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV

<img file="MX338474B_D0179.tif" />

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<img file="MX338474B_D0180.tif" />

- 139 FSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 47) --------------- GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACC GCCACCGGCGTGCACAGCGAGGTGCAGCTGAAGGAGAGCGGCCCCGGCCT GGTGGCCCCCGGCGGCAGCCTGAGCATCACCTGCACCGTGAGCGGCTTCAG CCTGATCGACTACGGCGTGAACTGGATCCGCCAGCCCCCCGGCAAGGGCCT GGAGTGGCTGGGCGTGATCTGGGGCGACGGCACCACCTACTACAACGCCCC CCTGAAGGGCCGCCTGAGCATCAGCAAGGACAACAGCAAGAGCCAGGTGTT CCTGCAGATGAACAGCCTGAAGACCGACGACACCGCCATGTACTACTGCGCC CGCATCGTGTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCCAGC ACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACCTCCG AGTCCACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGT GACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCT GCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGC CTTCCTCCTCCCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCC TTCCAACACCAAGGTGGACAAGCGGGTGGAGTCCAAGTACGGCCCTCCTTGC CCTTCCTGCCCTGCCCCTGAGTTCCTGGGCGGACCTAGCGTGTTCCTGTTCC CTCCTAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAGGTGACCTG TGTGGTGGTGGACGTGTCCCAGGAGGACCCTGAGGTCCAGTTCAACTGGTAC GTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCA GTTCAATTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGAC TGGCTGAACGGCAAAGAATACAAGTGTAAGGTCTCCAACAAGGGCCTGCCCT CCTCCATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAGCCTCA GGTGTACACCCTGCCTCCTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCC CTGACCTGTCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGG AGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGA CTCCGACGGCTCCTTCTTCCTGTACTCCAGGCTGACCGTGGACAAGTCCCGG TGGCAGGAGGGCAACGTCTTTTCCTGCTCCGTGATGCACGAGGCCCTGCACA ACCACTACACCCAGAAGTCCCTGTCCCTGTCTCTGGGCTGAAGCTT (SEQ ID NO: 48)

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EXAMPLE 16: PHARMACOKINETICS-

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The study is conducted with an adequate number of animals (eg 4 in one study; 2 for a single dose and 2 for repeated doses, 5 doses per week) healthy, Cynomolgus monkeys reared for this purpose weighing between 2.0 and 5.4 kg and with ages ranging from 2 to 7 years. Animals can be assigned to two treatment groups, one receiving control lgG4 antibody and the other receiving humanized 16D7. Monkeys in each group are given a single bolus intravenous dose, eg, 2.5-10 mg / kg in a dose volume of 2-3 ml / kg, or 5 doses per week, iv Samples are collected of blood at various time points after each dose administration and processed to obtain plasma. Plasma samples are analyzed for the concentration of total lgG4 and CXCR5 mAb using an ELISA.

EXAMPLE 17: COMPARATIVE STUDIES

Some of the antibodies of the present invention were compared with commercially available antibodies in parallel experiments. MAB 190, available from R&amp;D Systems, is a mouse mAb. RF82B is a rat anti-human CXCR5 antibody available from BD. Clone 2C1 is a mouse mAb with a GST marker available from Abnova. The various humanized antibodies described herein were typed using reagents and methods known in the art. For example, those taught herein have a □ light chain, many are lgG1, while 46C9, 68D3, and H28 are lgG2a. Most antibodies bind to the amino terminus of CXCR5, and several of the antibodies compete with each other for binding to the same epitope or region.

The BD antibody binds weakly to human PBMC.

Although the antibodies generally did not bind to cynomolgus monkey cells, the 14C9, 19H5, H28, 54G6, 56H6, and 79B7 antibodies of the present invention did.

16D7 was found to have a higher affinity, at least 10 times more, than commercial antibodies, and has a 100-fold better inactivation rate

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than the other antibodies.

EXAMPLE 18: INCREASE TO SCALE

Each monoclonal antibody variant was produced in HEK293 FS ™ cells grown in suspension by transient transfection of two expression plasmids encoding heavy or light chain complexed with 293fectin ™ (Invitrogen). The secreted proteins were collected eight days after transfection and centrifuged. Proteins were purified by affinity chromatography on Protein A (ProSepvA, Millipore) after elution from the column with 25 mM citrate buffer pH 3, 0.15 M NaCI. Monoclonal antibodies were formulated in PBS and filtered through 0.22 Dm filters. Protein concentration was determined by measuring absorbance at 280 nm. Each batch was analyzed by SDS-PAGE (Nupage Bistris / MES-SDS 10%) under reducing and non-reducing conditions to determine the purity and molecular weight of each subunit and of the monomer. Each batch of protein was also analyzed by molecular exclusion (Tricom 10/300 GL Superdex 200) to determine monomer homogeneity and the presence of high molecular weight species.

From 240 ml cultures, a total of 30 to 40 mg of eight variant 16D7 monoclonal antibodies was produced and of appropriate quality for subsequent in vitro and in vivo assays.

Those skilled in the art will recognize, or be able to discover using only routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

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INDUSTRIAL fragment thereof from