EA043510B1 - ANTIBODIES AND METHODS OF APPLICATION - Google Patents

Description

Field of technology

The invention relates to antibodies capable of binding to the CRTAM protein and their use.

Introduction

Aspects of the present invention include antibodies and other therapeutic proteins against CRTAM, nucleic acids encoding such antibodies and therapeutic proteins, methods for producing antibodies and other therapeutic proteins, and methods for treating diseases, such as cancer, using antibodies and other therapeutic proteins against CRTAM.

State of the art

The target antigen of CRTAM is a type I single-pass membrane protein of the nectin family with V- and C1-like Ig domains (Yeh et al., Cell. 2008 Mar 7; 132(5): 846-5). Its gene was first identified in activated mouse and human NKT cells (Kennedy et al, J Leukoc Biol. 2000 May;67(5):725-34); subsequent studies showed that its expression is tightly regulated by activation and is limited to activated MHC class I-restricted cells, including NKT and CD8 T cells (Patino-Lopez et al., J Neuroimmunol. 2006 Feb;171(1-2):145 -55).

The only known CRTAM ligand is Necl2 (Galibert et al., J Biol Chem. 2005 Jun 10;280(23):21955-64), and interactions between the ligand and receptor are thought to be favored over homophilic interactions of either molecule . (Zhang et al, Structure. 2013 Aug 6;21(8): 1430-9). The CRTAM-Necl2 interaction is proposed to promote the emergence of a mature immune synapse and promote T cell stimulation by nonprofessional APCs. This function has also been found in NK cells, where antibodies to CRTAM stimulated NK cell cytotoxicity (Boles et al., Blood. 2005 Aug 1;106(3):779-86). Application WO 2009/029883 describes that CRTAM is activated on a specific set of T cells and selectively regulates several cytokines, including IFN-γ, IL-22 and IL-17.

Application WO2016/154341 describes that agonistic antibodies against CRTAM can be used to treat cancer by increasing the ADCC potential of immune effector cells, such as NK cells.

Brief description of the invention

Aspects of the present invention include specific anti-CRTAM antibodies, nucleic acids encoding such antibodies of the present invention, host cells containing such nucleic acids encoding an antibody of the present invention, methods for producing anti-CRTAM antibodies, and methods for treating diseases, such as human cancer, including small cell lung cancer, non-small cell lung cancer (including squamous cell cancer and adenocarcinomas), skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, stomach cancer, ovarian cancer, cervical cancer, prostate cancer, cancer kidney, liver cancer, including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other uroepithelial cancers, as well as gastric cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, cancer bone, gallbladder and bile duct cancer, uterine cancer, adrenal cancer, sarcomas, GIST, neuroendocrine tumors and hematological malignancies, but not limited to those.

The present invention provides an antibody that binds to CRTAM (SEQ ID NO:11). The antibody preferentially binds to the extracellular domain of CRTAM (SEQ ID NO:12). Aspects of the present invention include an antibody or antigen binding fragment thereof that binds to, or cross-competes for binding with, an epitope of a CRTAM protein recognized by an antibody described herein, and preferably retains at least about 80, at least about 85, at least about 90, at least about 91, at least about 92, at least about 93, at least about 94, at least about 95, at least about 96, at least about 97, at least about 98 or at least about 99% binding affinity of the antibody described in the present invention to human CRTAM. In some embodiments, the antibody is an isolated antibody.

Aspects of the present invention include an antibody or antigen-binding fragment thereof that binds to CRTAM, wherein said antibody comprises a heavy chain variable region comprising: a CDR-H1 sequence comprising the sequence of SEQ ID NO:5; CDR-H2 sequence containing SEQ ID NO:6; and a CDR-H3 sequence comprising the sequence of SEQ ID NO:7. In some embodiments, the antibody or antigen binding fragment thereof further comprises a light chain variable region comprising at least one CDR sequence selected from the group consisting of: CDR-L1 comprising any of the sequences of SEQ ID NO:8 or SEQ ID NO:15 ; CDR-L2 containing the sequence SEQ ID NO:9; and CDR-L3 containing any of the sequences of SEQ ID NO:10 or SEQ ID NO:16.

In some embodiments, the CRTAM-binding antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region,

- 1 043510 containing one of the 4 combinations of heavy and light chain CDRs shown in table. 1.

Table 1

Heavy chain variable region CDR Light chain variable region CDR CDR-H1 CDR-H2 CDR-H3 CDR-L1 CDR-L2 CDR-L3 1 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO:7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 2 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO:7 SEQ ID NO: 15 SEQ ID NO: 9 SEQ ID NO: 16 4 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO:7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 16 5 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO:7 SEQ ID NO: 15 SEQ ID NO: 9 SEQ ID NO: 10

In some preferred embodiments, the antibody or antigen binding fragment thereof that binds to CRTAM comprises a heavy chain variable region comprising CDRH1 comprising SEQ ID NO:5; CDR-H2 containing SEQ ID NO:6; and CDR-H3 containing SEQ ID NO:7; and a light chain variable region containing CDR-L1 containing SEQ ID NO:8; CDR-L2 containing SEQ ID NO:9; and CDR-L3 containing SEQ ID NO:10.

In yet another preferred embodiment, the antibody or antigen binding fragment thereof that binds to CRTAM comprises a heavy chain variable region comprising CDR-H1 comprising SEQ ID NO:5; CDR-H2 containing SEQ ID NO:6; and CDR-H3 containing SEQ ID NO:7; and a light chain variable region containing CDR-L1 containing SEQ ID NO:15; CDR-L2 containing SEQ ID NO:9; and CDR-L3 containing SEQ ID NO:16.

In a further aspect, the antibodies or antigen binding fragments thereof of the present invention comprise variable chain CDRs mapped to the parent antibody described in the present invention. Thus, the present invention relates to variant antibodies or antigen binding fragments thereof comprising variant variable regions of a parent antibody, wherein the parent antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising a CDR-H1 sequence comprising the sequence of SEQ ID NO:5; a CDR-H2 sequence containing the sequence of SEQ ID NO:6; and a CDR-H3 sequence comprising the sequence of SEQ ID NO:7; and a light chain variable region containing CDR-L1 containing the sequence of SEQ ID NO:15; CDR-L2 containing the sequence SEQ ID NO:9; and CDR-L3 comprising the sequence of SEQ ID NO:16, wherein in one embodiment, the variant antibody or antigen binding fragment thereof contains 1, 2, 3, 4, 5 or 6 amino acid substitutions, additions and/or deletions in any one or more of them; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 amino acid substitutions, additions and/or deletions in the aggregate; a set of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, containing from 1 to 5 or from 1 to 4 or from 1 to 3 substitutions, additions or deletions of a specific purpose, and the specified the antibody or antigen-binding fragment retains the ability to specifically bind to CRTAM. The variations are preferably substitutions, the substitutions being preferably conservative substitutions or substitutions to convert an amino acid in the variable region back to the corresponding human germline amino acid. In a further embodiment, a variant antibody or antigen binding fragment thereof of the present invention comprises: a CDR-H1 sequence comprising the sequence of SEQ ID NO:5; a CDR-H2 sequence containing the sequence of SEQ ID NO:6; and a CDR-H3 sequence comprising the sequence of SEQ ID NO:7; and a light chain variable region containing CDR-L1 containing the sequence of SEQ ID NO:15; CDR-L2 containing the sequence SEQ ID NO:9; and CDR-L3 comprising the sequence of SEQ ID NO:16, wherein one or more of said CDR sequences are changed to be approximately 70, 75, 80, 85, 87, 88, 89, 90, 91, 92, 93 , 94, 95, 96, 97, 98, 99% identical to the corresponding original CDR sequence listed above.

In some embodiments, an antibody or antigen binding fragment thereof is provided comprising a heavy chain variable region described in SEQ ID NO:1 or SEQ ID NO:13, or a sequence at approximately 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to SEQ ID NO:1 or SEQ ID NO:13, and/or the light chain variable region described in SEQ ID NO:2 or SEQ ID NO:14, or a sequence of approximately 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to the sequence of SEQ ID NO:2 or SEQ ID NO:14. Additional embodiments provide an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions, additions, and/or deletions at compared to SEQ ID NO:1 or SEQ ID NO:13, and/or a light chain variable region containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid substitutions compared with SEQ ID NO:2 or SEQ ID NO:14. Preferably, said antibody contains substitutions, more preferably conservative substitutions.

- 2 043510

In addition, it will be appreciated that amino acid substitutions, additions and/or deletions may be in framework regions and/or CDRs.

In one embodiment, an antibody or antigen binding fragment thereof is provided comprising a heavy chain variable region comprising the sequence of SEQ ID NO:1 and a light chain variable region comprising the sequence of SEQ ID NO:2.

In one embodiment, an antibody or antigen binding fragment thereof is provided comprising a heavy chain variable region comprising the sequence of SEQ ID NO:13 and a light chain variable region comprising the sequence of SEQ ID NO:14.

In one embodiment, a full-length antibody is provided comprising a heavy chain sequence comprising SEQ ID NO:17 and a light chain sequence comprising SEQ ID NO:18.

In a further aspect, the present invention provides an antibody or antigen binding fragment thereof that specifically binds to CRTAM, said antibody comprising 3 heavy chain CDRs of SEQ ID NO: 1 or SEQ ID NO:13 and 3 light chain CDRs of SEQ ID NO:2 or SEQ ID NO:14, with CDRs as defined by Kabat or Chothia numbering system. Preferably, said antibody or antigen binding fragment thereof that specifically binds to CRTAM comprises 3 heavy chain CDRs of SEQ ID NO:13 and 3 light chain CDRs of SEQ ID NO:14, as defined by the Kabat or Chothia numbering system.

SEQ ID NOs:13-18 are humanized antibody sequences based on SEQ ID NOs:1 and 2. Those skilled in the art will appreciate that SEQ ID NOs:17-18 are full-length heavy and light chain sequences including the regions , different from the variable regions that are necessary to obtain a full-length functional antibody, i.e. constant regions and Fc regions. One skilled in the art will appreciate that sequences are humanized by replacing amino acids from the variable region of the antibody-producing organism with amino acids from a human germline sequence in order to minimize the immunogenic effects of the antibodies when administered to human subjects. Most amino acid substitutions occur in the framework region, but substitution of a number of amino acids from the CDR located in non-critical positions is possible; such substitutions are preferably conservative in nature or result in the reversal of an amino acid at a particular position to an amino acid present in the corresponding human germline. Here, substituted amino acids from CDRs were identified using structural models that distinguish between paratope-facing and non-paratope-facing residues in the CDR region. This provides a greater degree of humanization of antibodies compared to conventional CDR transplantation.

In the present invention, SEQ ID NO:14 contains 3 amino acid substitutions in CDR 1 (SEQ ID NO:15) compared to CDR1 SEQ ID NO:2 (SEQ ID NO:8). In particular, there is a V-S substitution at position 1 of SEQ ID NO:8; replacing E-V in position 4 SEQ ID NO:8; and replacing V-A at position 10 SEQ ID NO:8. SEQ ID NO:14 additionally contains 1 amino acid substitution in CDR3 (SEQ ID NO:16) compared to CDR3 of SEQ ID NO:2 (SEQ ID NO:10). In particular, there is a substitution S-A at position 5 of SEQ ID NO:10.

It will be clear to one skilled in the art that these humanized sequences do not represent different alternative antibodies compared to the original antibody, but refer to the same antibody having the same characteristics, which is merely modified to be more germline-compatible with the human using structural modeling to minimize immunogenicity.

In some embodiments, the antibody or antigen binding fragment thereof has a binding affinity (KD) of 5 nM, 4 nM, 3 nM, 2 nM, 1 nM or less, in a preferred embodiment the binding affinity is 0.75 nM, 0.5 nM, or less.

In some embodiments, an antibody or antigen binding fragment thereof of the present invention is an antibody or antigen binding fragment that competes for binding to CRTAM with its ligand necl2.

In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody. In some embodiments, the antibody is a chimeric, humanized, or human antibody. In some embodiments, the heavy chain variable region comprises a framework sequence. In some embodiments, at least a portion of the framework sequence comprises a human consensus framework sequence. In some embodiments, the light chain variable region comprises a framework sequence. In some embodiments, at least a portion of the framework sequence comprises a human consensus framework sequence.

In some embodiments, the antibody or antigen binding fragment is an Fc variant designed to enhance binding to FcgRIIa. In one preferred embodiment, the Fc variant includes amino acid substitutions S267E and/or L328F. In one embodiment, the Fc is an IgG1 Fc region. In a further embodiment, the Fc is the Fc region of IgG4. In a preferred embodiment, if the Fc region is an IgG4 Fc region, the Fc region further comprises an S228P substitution. In one embodiment, the heavy chain comprises the sequence comprising SEQ ID NO:17.

In some embodiments, the antibody or antigen binding fragment thereof is an engineered IgG1 antibody or antigen binding fragment produced under Fc silencing conditions and characterized by reduced or absent binding to one or more Fc receptors. In yet another embodiment, the antibody is an IgG4 antibody.

In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody or antigen-binding fragment that mediates T cell cytotoxicity and/or NK cell cytotoxicity.

In some embodiments, the antibody or antigen-binding fragment thereof is capable of inducing and/or enhancing activation of an immune cell. In one embodiment, the immune cell is preferably a T cell. In yet another embodiment, the immune cell is preferably an NK cell. One skilled in the art will understand that the term induction and/or enhancement may refer to the induction and/or enhancement of the release of cytokines by an immune cell and/or the induction and/or enhancement of the proliferation of said immune cells and/or the induction and/or enhancement of cytotoxic activity. It will be apparent to one skilled in the art that the term induce or induce in the present context means activating an immune cell or increasing the activation of an immune cell to a level greater than the level of activation observed in the absence of the antibody or antigen-binding fragment. The term enhancement in the present context refers to increasing the level of activation of an already activated immune cell.

In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific or multispecific antibody or antigen-binding fragment that binds to a CRTAM protein and one or more additional binding targets, wherein said additional binding targets are preferably one or more tumor antigens. In a further embodiment, said one or more additional binding targets are immunomodulatory molecules.

In some embodiments, the antigen binding fragment is selected from the group consisting of: Fab, Fab', F(ab) ₂ , F(ab') ₂ , Fv, scFv, dAb, and single domain antibody.

In another aspect of the present invention, one or more of nucleic acids encoding the heavy chain of an antibody of the present invention and/or the light chain of an antibody of the present invention is provided. It should be understood that the heavy and light chains of an antibody of the present invention may be encoded by a single nucleic acid molecule or by two separate nucleic acid molecules.

In another aspect, vectors are provided containing one or more of the nucleic acids of the present invention.

In another aspect of the present invention, a host cell is provided comprising one or more nucleic acids encoding the heavy and/or light chain of the antibodies of the present invention. In some embodiments, said host cell is grown under conditions in which the nucleic acid(s) are expressed. In other embodiments, a method for isolating an antibody according to the present invention is provided.

Aspects of the present invention include methods for producing an antibody or antigen binding fragment thereof, the methods comprising culturing a host cell under conditions in which the antibody or antigen binding fragment is expressed in the host cell, and optionally isolating the antibody or antigen binding fragment.

Aspects of the present invention include pharmaceutical compositions comprising an antibody or antigen binding fragment described herein and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition or drug further comprises an effective amount of a second therapeutic agent.

In a further aspect, the present invention provides a method of treating a disorder, the method comprising administering to a patient in need thereof an antibody or antigen binding fragment of the present invention that binds to CRTAM (SEQ ID NO:11). In one embodiment, said disorder is cancer.

In a further aspect, there is provided a method of treating cancer, comprising administering an effective amount of an antibody or antigen binding fragment according to the present invention to a subject in need thereof.

In one embodiment, the antibody or antigen binding fragment comprises a heavy chain variable region comprising CDR-H1 comprising SEQ ID NO:5; CDR-H2 containing SEQ ID NO:6, and CDR-H3 containing SEQ ID NO:7, and a light chain variable region containing CDR-1 containing SEQ ID NO:8, or SEQ ID NO:15, CDR- L2 containing SEQ ID NO:9, and CDR-L3,

- 4 043510 containing SEQ ID NO:10 or SEQ ID NO:16. The light chain variable region preferably contains CDR-1 containing SEQ ID NO:15, CDR-L2 containing SEQ ID NO:9, and CDR-L3 containing SEQ ID NO:16.

In some embodiments, a method of treating cancer is provided wherein an antibody or antigen binding fragment of the present invention is administered to a patient in need thereof, and said antibody or antigen binding fragment of the present invention induces and/or enhances an immune response, such as a cytotoxic T cell response and/or or NK cell response.

In some embodiments, the cancer is selected from the group consisting of small cell lung cancer, non-small cell lung cancer (including squamous cell cancer and adenocarcinomas), skin cancer, including melanoma, breast cancer (including TNBC), colorectal cancer, stomach cancer, cancer ovarian, cervical cancer, prostate cancer, kidney cancer, liver cancer, including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other types of uroepithelial cancer, stomach cancer, glioma, glioblastoma , testicular cancer, thyroid cancer, bone cancer, gallbladder and bile duct cancer, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumors and hematological malignancies.

In accordance with a further aspect of the present invention, an antibody or antigen binding fragment of the present invention is provided for use in prophylaxis or therapy.

The antibody or antigen binding fragment thereof is preferably intended for use in the prevention or therapy of cancer.

In accordance with a further aspect of the present invention, there is provided the use of an antibody or antigen binding fragment according to the present invention in the preparation of a medicament for the treatment of cancer.

In some embodiments, the cancer in accordance with the preceding aspects is selected from the group consisting of small cell lung cancer, non-small cell lung cancer (including squamous cell cancer and adenocarcinomas), skin cancer, including melanoma, breast cancer (including TNBC), colorectal cancer , stomach cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer, including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other types of uroepithelial cancer, cancer stomach, glioma, glioblastoma, testicular cancer, thyroid cancer, bone cancer, gallbladder and bile duct cancer, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumors and hematological malignancies.

Brief description of drawings

In fig. 1a shows the amino acid sequence of the heavy chain variable region of the parent antibody 5A11 (SEQ ID NO:1).

In fig. 1b shows the amino acid sequence of the light chain variable region of the parent antibody 5A11 (SEQ ID NO:2).

In fig. 2a shows the amino acid sequence of the heavy chain variable region of the humanized antibody 5A11 (SEQ ID NO: 13).

In fig. 2b shows the amino acid sequence of the light chain variable region of the humanized antibody 5A11 (SEQ ID NO: 14).

In fig. Figure 3 shows the specific binding of antibody 5A11 and the commercially available anti-CRTAM antibody CR24.1 to activated T cells in a dose-dependent manner.

In fig. Figure 4 shows that antibody 5A11 cross-reacts with the cynomolgus CRTAM homologue, but antibody CR24.1 does not.

In fig. Figure 5 shows the increased ability of antibody 5A11 to mediate the production of IFN-γ compared to CR24.1 upon activation of T cells.

In fig. 6a shows an alignment of the heavy chain variable region sequence of the parent 5A11 antibody and the heavy chain variable region sequence of the humanized 5A11 antibody.

In fig. 6b shows an alignment of the light chain variable region sequence of the parent antibody 5A11 and the light chain variable region sequence of the humanized 5A11 antibody.

In fig. 7 shows that antibody 5A11 can activate perforin production in T cells in vitro in a dose-dependent manner.

In fig. 8 shows that antibody 5A11 can activate granzyme B production in T cells in vitro in a dose-dependent manner.

In fig. 9 shows that antibody 5A11 can induce T cell-mediated cytotoxicity against MCF7 tumor cells in vitro. In fig. 10 shows that antibody 5A11 can induce the production of interferon gamma in tumor-infiltrating lymphocytes (TIL),

- 5,043,510 isolated from primary NSCLC tumor samples in ex vivo assays. This assay shows that the 5A11 antibody has increased activity compared to the commercially available antibody

CR24.1.

In fig. 11 shows that antibody 5A11 can induce interferon gamma production in TILs isolated from primary breast cancer specimens in ex vivo assays.

In fig. 12 shows that antibody 5A11 induces interferon-gamma production in TILs in a dose-dependent manner.

In fig. 13 shows that antibody 5A11 can cause NK-mediated lysis of K562 cells.

In fig. 14 shows that antibody 5A11 can significantly reduce tumor growth in mice compared to animals treated with an isotype control antibody.

Detailed Description of the Invention

Aspects of the present invention include anti-CRTAM antibodies, nucleic acids encoding such antibodies, host cells containing such nucleic acids encoding an antibody of the present invention, methods for producing anti-CRTAM antibodies, and methods for treating diseases, such as CRTAM-mediated disorders, such as cancer human, including small cell lung cancer, non-small cell lung cancer (including squamous cell cancer and adenocarcinomas), skin cancer, including melanoma, breast cancer (including TNBC), colorectal cancer, stomach cancer, ovarian cancer, cervical cancer, prostate cancer glands, kidney cancer, liver cancer, including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other types of uroepithelial cancer, stomach cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, Bone cancer, gallbladder and bile duct cancer, uterine cancer, adrenal cancer, sarcomas, GISTs, neuroendocrine tumors and hematological malignancies, but not limited to.

It should be noted that in the present invention and in the accompanying claims, the singular forms include the plural forms unless the context clearly indicates otherwise. In addition, it should be noted that when drawing up the claims, any optional element can be excluded. The implication is that this statement as such is used as the basis of the antecedent for the use of such exclusive terminology as exclusively, only, etc. in connection with the enumeration of the elements of the claims or the use of a negative limitation.

It will be apparent to those skilled in the art upon reading this specification that each of the individual aspects described and illustrated in the present invention contains distinct components and features that can be separated from or combined with features of any of several other aspects without departing from the spirit. of the present invention. Any method outlined can be performed in the order of the listed events or in any other logically possible order.

Definitions

The following definitions are used to interpret this specification, with terms used in the singular also including plural forms as appropriate and vice versa.

The term CRTAM as used herein refers to any native CRTAM protein of any vertebrate, including mammals, such as primates (eg, humans, apes, and rodents (eg, mice and rats)), unless otherwise noted. The CRTAM protein may also be referred to as a CRTAM-like protein. The amino acid sequence of human CRTAM is provided in the present invention in SEQ ID NO: 11.

The term CRTAM covers full-length, unprocessed CRTAM, as well as any form of CRTAM resulting from processing in the cell. The term also covers naturally occurring variants of CRTAM, such as splice variants, allelic variants and isoforms. The term specifically includes naturally occurring truncated or secreted forms of the CRTAM polypeptide (eg, extracellular domain sequence). The CRTAM polypeptides described in the present invention can be isolated from a variety of sources, for example, from various human tissues or other sources, or obtained by recombinant or synthetic methods. The definition of native sequence CRTAM polypeptide includes a polypeptide with the same amino acid sequence as the corresponding CRTAM polypeptide obtained from a natural source. Such native sequence CRTAM polypeptides can be isolated from natural sources or produced by recombinant or synthetic methods. In the present invention, the term CRTAM epitope refers to an epitope bound by an antibody containing at least one or more of the CDR sequences described in the present invention and/or an antibody typified by the binding profile of the anti-CRTAM antibody shown in the Examples section.

The term antibody is used in the broadest sense and specifically covers, for example, single monoclonal antibodies against CRTAM (including agonist, antagonist,

- 6 043510 neutralizing antibodies, full-length or intact monoclonal antibodies), compositions of antibodies against CRTAM with polyepitope specificity, polyclonal antibodies, multivalent antibodies, multispecific antibodies (for example, bispecific antibodies, provided that they exhibit the desired biological activity), formed by at least of two intact antibodies, single chain anti-CRTAM antibodies and antigen binding fragments of anti-CRTAM antibodies, including Fab, Fab', F(ab')2 and Fv fragments, diabodies, single domain antibodies (sdAbs), provided they exhibit the desired biological or immunological activity. The term immunoglobulin (Ig) is used interchangeably with the term antibody in the present invention. The antibody may be a chimeric antibody, a humanized antibody, a human antibody, and/or an affinity matured antibody. Those skilled in the art will appreciate that in some embodiments, minimal antibodies comprise a set of 6 CDRs defined in the present invention; these include traditional antibodies (including both monoclonal and polyclonal antibodies), humanized antibodies, human antibodies and/or chimeric antibodies, antibody fragments, engineered antibodies (for example, with amino acid modifications specified below), multispecific antibodies (including bispecific antibodies), and other analogs known in the art and discussed in the present invention, but are not limited to those.

It should be understood that in other embodiments, the term antibody as used in the present invention refers to structures that do not contain 6 CDRs; including, but not limited to, Nanobody®, Unibody® and scFv fragments.

The term anti-CRTAM antibody, CRTAM antibody, or CRTAM-binding antibody refers to an antibody capable of binding CRTAM with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent to target CRTAM. In some embodiments, an anti-CRTAM antibody binds to a CRTAM epitope that is conserved among CRTAMs from different species of organisms.

An isolated antibody is an antibody that has been identified and separated and/or recovered from a component of its environment. Environmental contaminants are materials that interfere with the therapeutic use of the antibody and may include enzymes, hormones, and other protein or non-protein solutes.

With respect to the binding of an antibody to a target molecule, the term specific binding or specifically binds to or is specific to a particular polypeptide or epitope on a particular target polypeptide means binding that is measurably different from a nonspecific interaction. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is usually a molecule with a similar structure that does not have binding activity.

The term antagonist is used in its broadest sense and includes any molecule that partially or completely blocks, inhibits or neutralizes the biological activity of the native CRTAM polypeptide. Suitable antagonist molecules include, but are not limited to, antagonistic antibodies or antibody fragments, fragments or amino acid sequence variants of native CRTAM polypeptides, peptides, antisense oligonucleotides, small molecule organic compounds, etc. Methods for identifying antagonists of a CRTAM polypeptide may involve contacting the CRTAM polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more of the biological activities typically associated with the CRTAM polypeptide.

The term agonist is used in its broadest sense and includes any molecule that enhances the biological activity of the native CRTAM polypeptide. Suitable agonist molecules include, but are not limited to, agonist antibodies or antibody fragments, fragments or amino acid sequence variants of CRTAM ligand polypeptides, peptides, antisense oligonucleotides, small molecule organic compounds, etc. Methods for identifying agonists of a CRTAM polypeptide may involve contacting the CRTAM polypeptide with a candidate agonist molecule and measuring a detectable change in one or more of the biological activities typically associated with the CRTAM polypeptide.

In the present invention, tumor refers to all growth and proliferation of tumor cells, both malignant and benign, as well as all precancerous and cancerous cells and tissues.

In the present invention, the terms predictive and prognostic are also used interchangeably in the sense that the predictive or predictive methods should allow the practitioner to select patients for whom a response to treatment with an anticancer agent, including an anti-CRTAM antibody, is considered (typically before treatment , but not necessarily) more likely.

CRTAM proteins.

- 7 043510

According to SWISS-PROT, CRTAM is a type I membrane protein of the nectin family. The protein consists of one V-type Ig-like (immunoglobulin-like) domain, one C-type Ig-like (immunoglobulin-like) domain (Yeh et al., Cell. 2008 Mar 7; 132(5): 846-5), one transmembrane region and extracellular tail between amino acids 18 - 287 SEQ ID NO:11.

Its gene was first identified in activated mouse and human NKT cells (Kennedy et al., J Leukoc Biol. 2000 May; 67(5):725-34); subsequent studies showed that its expression is tightly regulated by activation and is limited to activated MHC class I-restricted cells, including NKT and CD8 T cells (Patino-Lopez et al., J Neuroimmunol. 2006 Feb;171(1-2):145 -55).

The only known CRTAM ligand is Necl2 (Galibert et al., J Biol Chem. 2005 Jun 10; 280(23):21955-64), and interactions between the ligand and receptor are thought to be favored over homophilic interactions of either molecule . (Zhang et al., Structure. 2013 Aug 6;21(8): 1430-9). The CRTAM-Necl2 interaction is proposed to promote the emergence of a mature immune synapse and promote T cell stimulation by nonprofessional APCs. This function is also found in NK cells, where antibodies to CRTAM stimulated NK cell cytotoxicity (Boles et al., Blood. 2005 Aug 1;106(3):779-86).

In some embodiments, an antibody of the present invention binds to a human CRTAM. In the present invention, the term human CRTAM or human CRTAM protein refers to the protein with the sequence SEQ ID NO:11 specified in the present invention.

An antibody in accordance with embodiments of the present invention may, in some cases, cross-react with the CRTAM protein of a non-human animal. For example, to facilitate preclinical and toxicological testing, an antibody of the present invention may cross-react with mouse or primate CRTAM proteins. Alternatively, in certain embodiments, the antibody may be specific for the human CRTAM protein and may not exhibit species cross-reactivity or other type of cross-reactivity with non-human proteins.

Antibodies

Aspects of the present invention include anti-CRTAM antibodies, typically therapeutic and/or diagnostic antibodies described in the present invention. Antibodies that find use in the methods of the present invention can be molecules in any of a number of formats described in the present invention, including traditional antibodies, as well as antibody derivatives, antigen binding fragments and mimetics, further described in the present invention. In some embodiments, the antibody contains one or more CDRs selected from the set of 6 CDRs defined in the present invention (including a small number of amino acid substitutions described in the present invention). According to the above review, in the present invention, the term antibody refers to various structures.

In some embodiments of the present invention, IgG isotypes are used. In one embodiment, isotype IgG1 antibodies produced under Fc silencing conditions are used. In another embodiment, isotype IgG4 antibodies are used.

The N-terminal fragment of each antibody chain includes a variable region of approximately 100 to 110 or more amino acids in length primarily responsible for antigen recognition. In the variable region, three loops are formed for each of the V domains of the heavy chain and the light chain, forming an antigen-binding site. Each of these loops is called the complementarity determining region (hereinafter referred to as CDR) whose amino acid sequence change is most significant. The term variable refers to the fact that the sequence of certain segments of the variable region varies greatly between antibodies. Variability within the variable region is not evenly distributed. In contrast, V regions are composed of relatively invariant regions of 15–30 amino acids in length, called framework regions (FR), separated by shorter, highly variable regions, called hypervariable regions, each 9–15 amino acids or more in length.

Each of VH and VL consists of three hypervariable regions (complementarity determining regions, CDRs) and four FRs, located from N-terminus to C-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region typically covers amino acid residues from approximately amino acid residues 24-34 (CDR-L1; L is light chain), 50-56 (CDR-L2) and 89-97 (CDR-L3) in the light chain variable region and approximately 31 -35B (CDR-H1; H is heavy chain), 50-65 (CDR-H2) and 95-102 (CDR-H3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and/or residues forming a hypervariable loop (for example, residues 26-32 (CDR-L1), 50-52 (CDR-L2) and 91-96 (CDR-L3) in the light chain variable region and 26-32 (CDR-H1), 53-55 (CDR-H2) and 96-101 (CDR-H3) in the heavy chain variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917. Specific CDRs according to the present invention are described below.

In the present description, the Kabat numbering system is generally used to indicate the balance in

- 8 043510 variable domain (approximately residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region) (eg, Kabat et al., supra (1991)).

CDRs contribute to the formation of the antigen-binding or, more specifically, epitope-binding site of antibodies. One antigen may contain more than one epitope.

Immunoglobulins of the IgG subclass contain several immunoglobulin domains in the heavy chain. As used herein, the term immunoglobulin (Ig) domain refers to a region of an immunoglobulin having a characteristic tertiary structure. From the point of view of the present invention, heavy chain domains are of interest, including heavy chain constant (CH) domains and hinge domains. In the context of IgG antibodies, each of the IgG isotypes contains three CH regions.

The hinge region is a different type of Ig domain. In the present invention, by hinge or hinge region or antibody hinge region or immunoglobulin hinge region is meant a flexible polypeptide containing amino acids between the first and second constant domains of an antibody.

From the point of view of the present invention, the Fc regions are of particular interest. As used herein, by Fc or Fc region or Fc domain is meant a polypeptide comprising the constant region of an antibody, excluding the first constant region of the immunoglobulin domain and, in some cases, the hinge portion. Thus, Fc refers to the last two immunoglobulin constant region domains of IgA, IgD and IgG, the last three immunoglobulin constant region domains of IgE and IgM, and the flexible hinge located N-terminal to these domains. For IgA and IgM, the Fc may contain a J chain. For IgG, the Fc domain contains the immunoglobulin domains Cy2 and Cy3 (Cy2 and Cy3) and the lower hinge region between Cy1 (Cy1) and Cy2 (Cy2). Although the boundaries of the Fc region may vary, the Fc region of a human IgG heavy chain is generally defined to contain residues C226 or P230 to the C terminus, where the numbering corresponds to the EC index, as in the work of Kabat. In some embodiments, amino acid modifications are made to the Fc region, for example, to alter binding to one or more FcyR receptors or an FcRn receptor. In some embodiments, the antibodies are full-length. As used herein, a full-length antibody refers to the structure constituting the natural biological form of an antibody, including variable and constant regions, optionally containing one or more modifications described in the present invention.

Alternatively, antibodies can be a variety of structures, including antigen binding fragments, monoclonal antibodies, bispecific antibodies, mini antibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as antibody mimetics), chimeric antibodies, humanized antibodies, antibody hybrids (sometimes called antibody conjugates) and antigen-binding fragments of each of these structures, respectively, but are not limited to these. Structures based on the use of a set of CDRs are included in the definition of an antibody.

In one embodiment, the antibody is an antigen binding moiety. Specific antigen-binding antibody fragments include (i) a Fab fragment consisting of VL, VH, CL and CH1 domains, (ii) an Fd fragment consisting of VH and CH1 domains, (iii) an Fv fragment consisting of VL and VH domains of one antibody; (iv) a dAb fragment (Ward et al., 1989, Nature 341:544-546, incorporated herein by reference in its entirety) consisting of a single variable region, (v) dedicated CDR regions, (vi) F( ab') ₂ -fragments - a divalent fragment containing two linked Fab fragments, (vii) single-chain Fv (scFv) molecules in which the VH domain and VL domain are linked by a peptide linker, allowing the two domains to associate to form an antigen-binding site (articles by Bird et al, 1988, Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883, incorporated herein by reference in their entirety), (viii) bispecific single chain Fv fragment (WO 03/11161, incorporated herein by reference) and (ix) but not limited to, diabodies or tribodies, multivalent or multispecific fragments constructed by gene fusion (Tomlinson et. Al., 2000, Methods Enzymol. 326:461 -479; WO 94/13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448, all references cited are incorporated herein by reference in their entirety).

Chimeric and humanized antibodies

In some embodiments, the antibody may be a mixture of antibody fragments from different animal species, such as a chimeric antibody and/or a humanized antibody. Those. in the present invention, sets of CDRs can be used with framework and constant regions other than the regions specifically described in the sequences in the present invention.

In general, both chimeric antibodies and humanized antibodies refer to antibodies that combine regions of molecules from more than one animal species. For example, chimeric antibodies traditionally include mouse (or in some cases rat) variable region(s) and human constant region(s). Humanized antibodies generally refer to antibodies of non-human origin in which the variable domain framework regions are replaced with sequences present in human antibodies. Typically, in a humanized antibody, all of the antibody except the CDR is encoded by a polynucleotide of human origin or is identical to such an antibody except for its CDR. CDRs, some or all of which are encoded by nucleic acids derived from a non-human animal, are grafted onto a human antibody variable region beta sheet scaffold to create an antibody whose specificity is determined by the grafted CDRs. The creation of such antibodies is described, for example, in WO 92/11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al, 1988, Science 239:1534-1536; all references cited are incorporated herein by reference in their entirety. In one embodiment, the antibodies of the present invention may be multispecific antibodies, and in particular, bispecific antibodies, also sometimes referred to as diabodies. They are antibodies that bind to two (or more) different antigens or different epitopes on the same antigen. Diabodies can be produced by various methods known in the art (Holliger and Winter, 1993, Current Opinion Biotechnol. 4:446-449, incorporated by reference in their entirety), for example, chemically or from hybridomas.

In one embodiment of the invention, the antibody is a mini-antibody. Miniantibodies are minimized antibody-like proteins containing an scFv attached to a CH3 domain. Hu et al., 1996, Cancer Res. 56:3055-3061, incorporated herein by reference in its entirety. In some cases, the scFv may be attached to the Fc region and may include all or part of the hinge region. It should be noted that miniantibodies are included in the definition of antibody even though they do not contain the full set of CDRs.

Antibodies of the present invention are typically isolated or recombinant.

In some embodiments, the antibodies of the present invention are recombinant proteins, isolated proteins, or substantially pure proteins. The isolated protein is free of at least some of the material with which it is normally associated in its natural state, eg, it constitutes at least about 5 or at least about 50 weight percent of the total protein in the sample. It should be understood that the isolated protein may range from 5 to 99.9% by weight of the total protein content, depending on the circumstances. For example, the protein can be produced at significantly increased concentrations by using an inducible promoter or a highly expressed promoter allowing for increased protein concentrations. In the case of recombinant proteins, this definition includes the production of an antibody in a wide variety of organisms and/or host cells known in the art that do not produce it naturally. Typically, the isolated polypeptide is obtained using at least one purification step. An isolated antibody refers to an antibody that is substantially free of other antibodies having different specificities for an antigen. For example, an isolated antibody that specifically binds to CRTAM is substantially free of antibodies that specifically bind antigens other than CRTAM.

Isolated monoclonal antibodies, characterized by different specificities, can be combined into a well-defined composition. Thus, for example, an antibody of the present invention may optionally be separately included or excluded from the formulation, as further discussed below.

Specific binding to a particular antigen or epitope can be demonstrated, for example, by an antibody having a KD to the antigen or epitope of at least about 10 ^-4 , at least about 10 ^-5 , at least about 10 ^-6 , at least about 10 ^-7 , at least about 10 ^-8 , at least about 10 ^-9 , alternatively at least about 10 ^-10 , at least about 10 ^-11 , at least about 10 ^{- 12} M or more, where KD is the dissociation rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds an antigen has a KD that is 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5000-fold, 10,000-fold, or a greater number of times lower relative to the antigen or epitope than the control molecule .

In addition, specific binding to a particular antigen or epitope can be demonstrated, for example, by an antibody having a K _A or Ka relative to the antigen or epitope of at least 20, 50, 100, 500, 1000, 5000 -, 10,000-fold or more times greater relative to the epitope than to the control, where K _A or Ka is the rate of association of a particular antibody-antigen interaction.

Standard assays to assess the binding capacity of antibodies to CRTAM can be performed at the protein or cellular level; such assays are known in the art and include, for example, ELISA, Western blotting, RIA, Octet®, Biacore®, and flow cytometric assays. Suitable assays are described in detail in the Examples section. The binding kinetics (e.g., binding affinity) of antibodies can also be assessed using standard

- 10 043510 assays known in the art, for example Biacore® or Octet® system analysis.

CRTAM antibodies.

The present invention provides anti-CRTAM antibodies that bind to a CRTAM polypeptide or a fragment thereof. An example amino acid sequence of CRTAM is provided in SEQ ID NO:11. Subject anti-CRTAM antibodies may induce or enhance immune cell activation, such as T cell activation and/or NK cell activation, enhancing the immune response in the tumor. In the present invention, these antibodies are referred to as anti-CRTAM antibodies or, for ease of description, CRTAM antibodies.

In some embodiments, a subject anti-CRTAM antibody may induce and/or enhance cytokine release or proliferation upon contact with T cells, particularly CD8+ T cells expressing CRTAM on their surface. Cytokine release or T cell proliferation in this context can be measured in several ways. In one embodiment, an anti-CRTAM antibody of the present invention is contacted with activated T cells using standard assays, such as ELISA. In a further embodiment, an anti-CRTAM antibody can induce and/or enhance the activation and cytotoxic activity of NK cells.

In one embodiment, the antibody is an antibody comprising the following CDRs; In addition, as discussed below, these CDR sequences may also contain a limited number of amino acid variants, as described previously:

CDR SEQ Π)NO: 5A11VHCDR1 SEQIDNO:5 5A11VHCDR2 SEQ ID NO:6 5A11VHCDR3 SEQ ID NO:7 5A11VLCDR1 SEQ ID NO: 15 5A11VLCDR2 SEQ ID NO:9 5A11VLCDR3 SEQ ID NO: 16

In some embodiments, the antibody comprises an amino acid sequence of at least one or more of the CDR sequences set forth in SEQ ID NOs: 5, 6, 7, 15, 9, and 16. In some embodiments, the antibody comprises an amino acid sequence of at least about 75 , 80, 85, 90, 95, 96, 97, 98 or 99% identical in amino acid sequence to one or more of the CDR sequences presented in SEQ ID NO: 5, 6, 7, 15, 9 and 16.

The present invention also describes the heavy and light chain variable regions that comprise the CDR sets of the present invention, as well as full-length heavy and light chains (eg, also containing constant regions). Those skilled in the art will appreciate that sets of CDRs according to the present invention can be included in mouse, human, or humanized constant regions (including framework regions). Aspects of the present invention include heavy chain variable regions and light chain variable regions that are at least about 80, 85, 90, 95, 96, 97, 98, or 99% identical to the heavy chain variable region sequence (SEQ ID NO:13) and sequence light chain variable region (SEQ ID NO:14) described in the present invention.

Some embodiments of the present invention provide antibodies that bind to the same epitope on a human CRTAM as or that compete with the inventive CRTAM monoclonal antibody described herein (i.e., antibodies that are capable of cross-competing for binding with the CRTAM protein with the monoclonal antibody of the present invention described in the present invention). In some embodiments, the reference antibody for cross-competition studies may be the 5A11 antibody described in the present invention. Such cross-competing antibodies can be identified based on their ability to cross-compete with antibody 5A11. It should be understood that for an antibody to be considered cross-competitive, it does not necessarily completely block binding of the reference antibody. In some embodiments, reference antibody binding is reduced by at least about 10, 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 97, 98, or 99%.

In some embodiments, the present invention provides an antibody or antigen binding fragment thereof that competes for binding to CRTAM with the necl2 ligand.

Antibody modifications

The present invention further provides variant antibodies, sometimes referred to as antibody derivatives or antibody analogs. Those. there are a number of modifications that can be made to the antibodies of the present invention, including amino acid modifications in the CDR

- 11 043510 (affinity maturation), amino acid modifications in the Fc region, glycosylation variants and other types of covalent modifications (for example, for the addition of drug conjugates, etc.), but not limited to those.

By variant is meant a polypeptide sequence that differs from the sequence of the original polypeptide by at least one amino acid modification. In some embodiments, the parent polypeptide is either a full-length heavy or light chain variable region set forth in SEQ ID NO:1 or 2, 13 or 14, or is one or more CDR sequences described in any of SEQ ID NO:5-10 , 15 or 16. In some embodiments, the amino acid modification may include a substitution, insertion, and/or deletion, with substitution being preferred in many cases. In some embodiments, the replacement may be a conservative replacement.

In general, the variants may contain any number of modifications as long as the function of the antibody described in the present invention is maintained. For example, the antibody must still specifically bind to human CRTAM. Likewise, when generating amino acid variants, for example, in the Fc region, the variant antibodies must support the required receptor binding functions for the particular application or indication of the antibody.

Variants of subject antibodies may contain amino acid modifications described in the present invention in one or more of the listed CDR sequences, in one or more framework regions, or in one or more constant regions (eg, in the Fc region) of the antibody.

In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications are typically used over the original sequence, since the goal is often to change function with a minimum number of modifications. Some embodiments contain 1 to 5 (1, 2, 3, 4, or 5) modifications (e.g., individual amino acid substitutions, insertions, and/or deletions), with many embodiments also using 1 to 2, 1 to 3 and from 1 to 4 modifications. For example, in some embodiments, one or more CDR sequences of antibodies of the present invention may individually contain one or more, for example, 1, 2, 3, 4 or 5 amino acid modifications, preferably 1-4, 1-3, 1 or 2 modifications . Typically, no more than 4, 5, 6, 7, 8, 9, or 10 changes are made to a CDR set.

It should be noted that a number of amino acid modifications may occur within functional domains: for example, it may be desirable to have 1 to 5 modifications in the Fc region of a wild-type or engineered protein, and, for example, 1 to 5 modifications in the Fv region. The variant polypeptide sequence preferably has at least about 75, 80, 85, 90, 91, 92, 93, 93, 94, 95, 96, 97, 98, or 99% identity to the parent sequences (e.g., variable region sequences, constant region sequences and/or heavy and light chain sequences and/or CDRs, for example, antibody 5A11).

In the present invention, the term amino acid substitution or substitution refers to the replacement of an amino acid at a specific position in the original polypeptide sequence with another amino acid. In the present invention, the term amino acid insertion or insertion refers to the addition of an amino acid at a specific position in the original polypeptide sequence. In the present invention, the term amino acid deletion or deletion refers to the removal of an amino acid at a specific position in the original polypeptide sequence.

As used herein, the term parent polypeptide, parent protein, precursor polypeptide, or precursor protein refers to an unmodified polypeptide that is subsequently modified to form a variant polypeptide. Typically, in the present invention, the parent polypeptide may be 5A11 polypeptides, for example, VH or VL chains or 5A11 CDR sequences. Accordingly, in the present invention, a parent antibody means an antibody modified to produce a variant antibody.

In the present invention, the term wild type or wt. or native refers to an amino acid sequence or nucleotide sequence occurring in nature, including allelic variants thereof. Protein, polypeptide, antibody, immunoglobulin, IgG, etc. wild type contains an amino acid sequence or nucleotide sequence that has not been intentionally modified.

In the present invention, the term variant Fc region means an Fc sequence that differs from the wild-type Fc sequence due to at least one amino acid modification. An Fc variant may refer to the Fc polypeptide itself, compositions containing the variant Fc polypeptide, or an amino acid sequence.

In some embodiments, the anti-CRTAM antibody of the present invention contains a variant Fc domain. As is known in the art, the Fc region of an antibody interacts with a number of Fc receptors and ligands to impart a number of important functional properties to the antibody, referred to as effector functions. Suitable modifications and, in particular, specific amino acid substitutions can be made to one or more positions to reduce or inhibit binding to Fc receptors.

In addition to the modifications described above, other modifications can be made. For example, molecules can be stabilized by the inclusion of disulfide bridges linking the VH and VL domains (Reiter et al., 1996, Nature Biotech. 14:1239-1245, incorporated herein by reference in its entirety).

In addition, modifications at cysteine residues are particularly useful in the use of antibody-drug conjugates (ADCs), further described below. In some embodiments, the antibody constant region can be designed to contain one or more cysteine residues that are particularly reactive with thiols to provide more specific and controlled placement of the drug moiety. See, for example, US Pat. No. 7,521,541, which is incorporated herein by reference in its entirety. In addition, there are many covalent modifications of antibodies that can be made, as described below.

The essence of the present invention includes covalent modifications of antibodies, which typically (but not always) occur after translation. For example, covalent modifications of several types of antibodies are introduced into the molecule by reacting specific amino acid residues of the antibody with an organic modifying agent that is capable of reacting with selected side chains or N- or C-terminal residues.

In addition, those skilled in the art will understand that labels (including fluorescent, enzymatic, magnetic, radioactive, etc.) as well as other compositions of the present invention can be added to antibodies.

Bispecific molecules.

In yet another aspect, the present invention includes bispecific and multispecific molecules comprising an anti-CRTAM antibody or fragment thereof according to the present invention. An antibody of the present invention or an antigen binding fragment thereof can be modified or linked to another functional molecule, such as another peptide or protein (eg, another antibody or receptor ligand) to produce a bispecific molecule that binds to two different binding sites or target molecules. In some embodiments, an antibody of the present invention or an antigen-binding fragment thereof can be modified or linked to at least two functional molecules, such as other peptides or proteins (eg, other antibodies or receptor ligands) to provide a multispecific molecule that binds to at least three different binding sites or target molecules. To create a bispecific or multispecific molecule of the present invention, an antibody of the present invention can be operably linked (e.g., by chemical linkage, genetic association, non-covalent association, or otherwise) to one or more other binding molecules, e.g., another antibody, antibody fragment peptide, or mimetic binding, producing a bispecific or multispecific molecule.

Accordingly, the present invention includes bispecific molecules comprising at least one first specific binding fragment for a first target epitope (ie, CRTAM) and a second specific binding fragment for a second target epitope. The second target epitope may be present on the same target protein as the protein bound by the first specific binding fragment; or the second target epitope may be present in a target protein different from the protein bound by the first specific binding fragment. The second target epitope may be present on the same cell as the first target epitope (ie, CRTAM); or the second target epitope may be present on a target not exposed by a cell exhibiting the first target epitope. In the present invention, the term specific binding fragment refers to a fragment containing at least one antibody variable domain.

In another embodiment of the present invention, the second target epitope is present on the tumor cell. Therefore, aspects of the present invention include bispecific molecules capable of binding to both CRTAM-expressing effector cells (eg, CRTAM-expressing cytotoxic T cells) and tumor cells expressing a second target epitope.

In one embodiment, a bispecific antibody of the present invention may comprise a total of two or three antibody variable domains, wherein the first fragment of the bispecific antibody is capable of recruiting the activity of a human immune effector cell by specifically binding to an effector antigen located on the human immune effector cell in which the effector the antigen is a CRTAM, wherein said first fragment consists of one variable domain of an antibody, and the second bispecific antibody fragment is capable of specifically binding to a target antigen other than an effector antigen, wherein said target antigen is located on a target cell other than said immune effector cell human, and said second fragment contains one or two anti-13 043510 body variable domains.

In an embodiment of the present invention in which the binding protein is multispecific, the molecule may further comprise a third specific binding moiety in addition to the antitumor specific binding moiety and the anti-CRTAM specific binding moiety. In one embodiment, the third specific binding moiety is an enhancing factor (EF) antagonist moiety, for example, a molecule that binds to a surface protein involved in cytotoxic activity and thereby enhances the immune response against the target cell. The enhancing factor antagonist fragment may be an antibody, a functional antibody fragment, or a ligand that binds to a given molecule, such as an antigen or receptor, and thereby results in an enhanced effect of the binding determinants on a target cell antigen. The enhancing factor antagonist moiety can bind a target cell antigen. Alternatively, the enhancing factor antagonist fragment may bind to an entity different from the entity to which the first and second specific binding fragments bind. For example, the enhancing factor antagonist moiety may bind a cytotoxic T cell (eg, via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1, or other immune cell, resulting in an enhanced immune response against the target cell).

In one embodiment, the bispecific protein of the present invention comprises, as a specific binding moiety, at least one antibody or antigen-binding fragment thereof, including, for example, a Fab, Fab', F(ab') ₂ , Fv, Fd, dAb, or single-chain Fv. The antibody may also be a dimer of a light chain or a heavy chain, or any minimal fragment thereof, such as an Fv fragment or single chain construct as described in US Pat. No. 4,946,778, the contents of which are specifically incorporated herein by reference.

In some embodiments, the antibody that can be used in the bispecific molecule of the present invention is a murine, human, chimeric, or humanized monoclonal antibody.

The binding of bispecific molecules to their specific targets can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS assay, biological assay (eg, growth inhibition), or Western blotting. Each of these assays typically detects the presence of protein-antibody complexes of particular interest using a labeled reagent (eg, antibody) specific for the complex of interest.

Glycosylation

Another type of covalent modification is changes in glycosylation. For example, an aglycosylated antibody (ie, an antibody that lacks glycosylation) can be produced. Glycosylation can be modified, for example, to increase the affinity of an antibody for an antigen. Such modifications of carbohydrates can be accomplished, for example, by modifying one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions may be made to remove one or more glycosylation sites in the variable region framework, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. This approach is described in detail by So et al. in US patents No. 5714350 and 6350861; it can be realized by removing the asparagine at position 297.

Another type of covalent modification of an antibody involves linking the antibody to various non-protein polymers, including various polyols, such as, but not limited to, polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in a manner set forth, for example, in the 2005-2006 PEG Catalog from Nektar Therapeutics (available on the web - Nektar website), US patents 4640835; 4496689; 4301144; 4670417; 4791192 or 4179337, which are incorporated herein by reference in their entirety. In addition, as is known in the art, amino acid substitutions can be made at various positions within the antibody to facilitate the addition of polymers, such as PEG. See, for example, US Publication No. 2005/0114037A1, which is incorporated herein by reference in its entirety.

In additional embodiments, for example, when using the antibodies of the present invention for diagnostic or detection purposes, the antibodies may contain a label. In the present invention, by the term labeled is meant that a compound contains at least one fragment, element, isotope, or chemical compound attached to enable detection of the compound, as described in the 6th edition of the Molecular Probes Handbook, Richard P. Haugland, expressly incorporated the present invention by reference.

Methods for producing antibodies according to the present invention

The present invention also provides methods for producing the disclosed anti-CRTAM antibodies. These methods involve culturing a host cell containing isolated nucleic acid(s) encoding an antibody of the present invention. Those skilled in the art will appreciate that this can be done in a variety of ways depending on the nature of the antibody. In some embodiments, when the antibodies of the present invention are full-length conventional antibodies, for example, a host cell containing a nucleic acid encoding a heavy chain variable region and a light chain variable region can be cultured under conditions that permit production and isolation of the antibody.

The variable regions of the heavy and light chains of antibodies according to the present invention are described in the present invention (both protein and nucleotide sequences); it should be appreciated that they can be supplemented in the art to produce full-length heavy and light chains. Those. In the presence of DNA fragments encoding the VH and VL segments described in the present invention, these DNA fragments can be further manipulated using standard recombinant DNA techniques, for example, to convert variable region genes into full-length antibody chain genes, into Fab fragment genes, or into scFv. In these manipulations, a DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used herein, the term operably linked is intended to mean the joining of two DNA fragments while maintaining the reading frame of the amino acid sequences encoded by the two DNA fragments.

The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the DNA encoding the VH to another DNA molecule encoding the heavy chain constant regions ( _CH1 , CH2 and CH3). Mouse heavy chain constant region gene sequences are known in the art (see, e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments covering these regions can be obtained using standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. In one preferred embodiment, the heavy chain constant region is an IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene, DNA encoding _VH can be operably linked to another DNA molecule encoding only the heavy chain constant region _CH1 .

The isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region C _L . Mouse light chain constant region gene sequences are known in the art (see, e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments covering these regions can be obtained using standard NLR amplification. In one preferred embodiment, the light chain constant region is a kappa or lambda constant region.

To create a polynucleotide sequence encoding the scFv fragment of an antibody, DNA fragments encoding VH and VL are operably linked to another fragment encoding a flexible linker, for example, encoding the amino acid sequence (Gly4-Ser) ₃ , allowing expression of the _VH and VL sequences in as a continuous single-chain protein in which the VL and VH regions are connected by a flexible linker (see, for example, Bird et al. (1988) Science 242:423426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty et al., (1990) Nature 348:552554).

Aspects of the present invention include nucleic acids encoding antibodies of the present invention. Such polynucleotides encode both heavy and light chain variable and constant regions, although the present invention also contemplates other combinations consistent with the compositions described herein. Aspects of the present invention include oligonucleotide fragments derived from the described polynucleotides and nucleotide sequences complementary to these polynucleotides.

Polynucleotides in accordance with embodiments of the present invention may be or contain RNA, DNA, cDNA, genomic DNA, nucleic acid analogs, and synthetic DNA. In some embodiments, the DNA molecule may be double-stranded or single-stranded, and in the case of a single-stranded molecule, it may be a coding (sense) or non-coding (antisense) strand. The coding sequence that encodes the polypeptide may be identical to the coding sequence provided by the present invention, or may be a different coding sequence that, due to redundancy or degeneracy of the genetic code, encodes the same polypeptides as the DNA provided by the present invention.

In some embodiments, the nucleic acid(s) encoding the antibodies of the present invention are included in expression vectors, which may be extrachromosomal or designed to integrate into the genome of the host cell into which they are introduced. Expression vectors may contain any number of appropriate regulatory sequences (including, but not limited to, sequences for transcription and translation control, promoters, ribosome binding sites, enhancers, origins of replication, etc.) or other components (selectable genes etc.), all of which are operably linked, as is well known in the art. In some cases, two nucleic acids are used, each placed in a separate expression vector (e.g., the heavy chain in a first expression vector, the light chain in a second expression vector) or, alternatively, they can be included in the same expression vector. vector. Those skilled in the art will appreciate that the design of the expression vector(s), including the choice of regulatory sequences, may depend on factors such as the choice of host cell, the desired level of protein expression, etc.

Typically, nucleic acids and/or expression vectors can be introduced into a suitable host cell, producing a recombinant host cell using any method appropriate for the selected host cell (e.g., transformation, transfection, electroporation, infection), operably linking the molecule(s) a nucleic acid with one or more expression control elements (eg, in a vector, in a construct formed by processes in a cell, integrated into the genome of a host cell). The resulting recombinant host cell can be maintained under conditions suitable for expression (e.g., in the presence of an inducer, in a suitable non-human animal, in suitable culture media supplemented with appropriate salts, growth factors, antibiotics, nutritional supplements, etc. ), thereby producing the encoded polypeptide(s). In some embodiments, the heavy chain and the light chain are produced by the same host cell. In some embodiments, the heavy chain is produced by one host cell and the light chain is produced by another host cell.

Mammalian cell lines available as expression hosts are known in the art and include a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), Manassas, VA, USA, including Chinese hamster ovary (CHO) cells, HEK 293 cells, NSO cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (eg, Hep G2) and a number of other cell lines, but not limited to those. Non-mammalian cells, including but not limited to bacteria, yeast, insect and plant cells, can also be used to express recombinant antibodies. In some embodiments, antibodies can be produced in transgenic animals, such as cows or chickens.

General approaches in the field of molecular biology, expression, purification and screening of antibodies are well known, for example, see US patents No. 4816567, 4816397, 6331415 and 7923211, as well as Antibody Engineering, edited by Kontermann & Dubel, Springer, Heidelberg, 2001, 2010 Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2:339-76; and Morrison, S. (1985) Science 229:1202.

Pharmaceutical compositions.

Aspects of the present invention include a composition, for example a pharmaceutical composition, comprising one or more (or a combination) of anti-CRTAM antibodies or antigen binding fragment(s) thereof according to the present invention, formulated together with a pharmaceutically acceptable carrier. Such compositions may include one antibody or a combination (eg, two or more different) antibodies or bispecific molecules of the present invention. For example, the pharmaceutical composition of the present invention may contain a combination of antibodies that bind to different epitopes on the target antigen or have complementary activities.

The pharmaceutical compositions of the present invention can also be administered as part of a combination therapy, i.e. in combination with other agents. For example, combination therapy may include an antibody of the present invention in combination with at least one other antitumor agent or anti-inflammatory agent or immunosuppressant. Examples of therapeutic agents that can be used in combination therapy are described in detail below in the section on the use of antibodies according to the present invention.

In the present invention, the term pharmaceutically acceptable carrier includes various solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, and the like. physiologically compatible substances. The carrier is preferably suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion). Depending on the route of administration, the active compound, i.e. An antibody or antibody fragment may be coated with a material to protect the compound from acids and other natural conditions that may inactivate the compound.

The pharmaceutical compounds of the present invention may include one or more pharmaceutically acceptable salts. A pharmaceutically acceptable salt refers to a salt that retains the desired biological activity of the parent compound and does not have any adverse effects.

- 16 043510 adverse toxicological effects (see, for example, Berge, S.M., et al. (1977) J. Pharm. Sci. 66:119). The pharmaceutical composition of the present invention may also include a pharmaceutically acceptable antioxidant. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (for example, glycerin, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils, for example, olive oil and Injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

These compositions may also contain adjuvants, for example, preservatives, humectants, emulsifiers and dispersing agents. Prevention of the presence of microorganisms can be achieved both through sterilization procedures (see above) and through the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, etc. In addition, it may be desirable to include isotonic agents, such as carbohydrates, sodium chloride, and the like, in the compositions. In addition, prolonged absorption of the pharmaceutical injection form can be achieved by the inclusion of absorption delaying agents such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. In the pharmaceutical compositions of the present invention, the use of conventional vehicles or agents is provided, unless they are incompatible with the active compound. Additional active compounds may also be included in the composition.

Dosing regimens are adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated depending on the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease and uniformity of administration. In the present invention, unit dosage form refers to physically discrete units suitable for use as unit doses in subjects to be treated; each dosage form contains a predetermined amount of active compound calculated to obtain the desired therapeutic effect, in combination with the necessary pharmaceutical carrier. The desired characteristics of the unit dosage forms of the present invention are determined by and are directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect achieved, and (b) the limitations inherent in the formulation of such active compounds for the treatment of sensitivity in individuals.

When administering an antibody, the dosage may be from about 0.0001 to 100, from about 0.001 to 50, from about 0.001 to 10, from about 0.01 to 10 or more, typically from 0.01 to 5 mg/kg of host body weight. For example, dosages may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.75 mg/kg body weight, 1, 3, 4, 5, 7.5 body weight or 10 mg/kg body weight. kg body weight or within the range of 0.1-5 mg/kg or 1-10 mg/kg. An example treatment regimen includes once daily, every other day, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every 3-6 months. Preferred dosage regimens for the anti-CRTAM antibody of the present invention include 1 mg/kg body weight, 3, 5, or 10 mg/kg body weight intravenously, with the antibody administered using one of the following dosing schedules: (i) six doses every week, then every month; (ii) every week; (iii) a single dose of 3 mg/kg body weight, followed by 1 mg/kg body weight once a week.

In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered must fall within the specified ranges. In some embodiments, the antibody is administered multiple times. The intervals between single doses can be, for example, a week, a month, three months or a year. The intervals may also be irregular, according to the measurement of the level of antibodies to the target antigen in the patient's blood. In some embodiments, the dosage is adjusted to achieve a plasma antibody concentration of approximately 1-1000 μg/ml, and in some methods, approximately 25-300 μg/ml.

In some embodiments, the antibody may be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency depend on the half-life of the antibody in the patient. In general, human antibodies have the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and frequency of administration depend on whether the treatment is prophylactic or therapeutic. For prophylactic purposes, relatively low doses are administered at relatively long intervals over a long period of time.

- 17 043510

Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, it is sometimes necessary to administer relatively high doses at relatively short intervals until the progression of the disease is reduced or stopped and, preferably, until the patient's symptoms of the disease partially or completely improve. After this, the patient can be administered the composition according to the prophylactic regimen.

The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and route of administration and is not toxic to the patient. The dosage level selected depends on a variety of pharmacokinetic factors, including the potency of the particular compositions of the present invention or their esters, salts or amides used, the route of administration, the time of administration, the rate of elimination of the particular compound used from the body, the duration of treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, age, sex, body weight, medical condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.

A therapeutically effective dosage of an anti-CRTAM antibody according to the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of periods of asymptomatic disease, or the prevention of disability due to the disease. For example, a therapeutically effective dosage preferably inhibits cell or tumor growth by at least about 10%, at least about 20%, at least about 30%, more preferably at least about 40%, at least about 50%, even more preferably at least about 60, at least about 70, and even more preferably at least about 80, at least about 90, or at least about 95% compared to untreated subjects . The ability of a compound to inhibit tumor growth can be assessed in an animal model to predict efficacy in human tumors. Alternatively, this property of the composition can be assessed by assessing the ability of the compound to inhibit cell growth, which inhibition can be measured in vitro using assays known to one skilled in the art. A therapeutically effective amount of a therapeutic compound may reduce tumor size or otherwise reduce symptoms in a subject. One skilled in the art can determine such amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration chosen.

The composition of the present invention can be administered by one or more routes of administration, using one or more of a variety of methods known in the art. As will be appreciated by one skilled in the art, the route and/or mode of administration will depend on the desired results. Preferred routes of administration of the antibodies of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes, such as by injection or infusion. As used herein, the phrase parenteral administration means methods of administration other than enteral and topical administration, typically by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, intradermal , intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

Alternatively, the antibody of the present invention can be administered by a non-parenteral route, for example, by the topical, epidermal or mucosal route, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

It is possible to formulate the active compounds with carriers that protect the compound from immediate release, for example, in the form of a controlled release formulation, including implants, transdermal patches and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Many methods for preparing such formulations are patented or well known to those skilled in the art (see, for example, Sustained and Controlled Release Drug Delivery Systems (1978) J.R. Robinson, ed., Marcel Dekker, Inc., N.Y).

In certain embodiments, the monoclonal antibody of the present invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) does not allow many highly hydrophilic compounds to pass through. To ensure passage of the therapeutic compounds of the present invention across the BBB, they can (if desired) be formulated, for example, in liposomes. For methods of preparing liposomes, see, for example, US Pat. No. 4,522,811; 5374548; and 5399331. Liposomes may contain one or more fragments

- 18 043510 molecules selectively transported to specific cells or organs, which improves targeted drug delivery (see, for example, V.V. Ranade (1989) J. Clin. Pharmacol. 29:685). Typical targeting moieties include folate or biotin (see, for example, US Pat. No. 5,416,016); mannosides (Umezawa et al. (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346:123; J.J. Killion; I.J. Fidler (1994) Immunomethods 4:273.

Application and methods

The antibodies, antibody compositions and methods of the present invention find a variety of diagnostic and therapeutic uses in vitro and in vivo, including the diagnosis and treatment of immune-mediated disorders.

In some embodiments, these molecules can be administered to cells in culture, in vitro or ex vivo, or to human subjects, for example, in vivo, for the treatment, prevention and/or diagnosis of various disorders. In the present invention, the term subject is intended to include human and non-human animals. Non-human animals include all vertebrates, such as mammals, such as non-human primates and non-mammalian animals. Preferred subjects include human subjects. When administering anti-CRTAM antibodies along with another agent, the antibodies and agent can be administered in any order or simultaneously.

Given the specific binding of the antibodies of the present invention to CRTAM, the antibodies of the present invention can be used to specifically detect the expression of CRTAM on the surface of immune cells and, further, to purify CRTAM by immunoaffinity purification.

Additionally, given the expression of CRTAM on immune cells, the antibodies, antibody compositions, and methods of the present invention may be used to treat a subject with an oncogenic disorder, e.g., a disorder characterized by the presence of tumor cells, e.g., small cell lung cancer, non-small cell lung cancer (including squamous cell carcinomas) and adenocarcinoma), skin cancer, including melanoma, breast cancer (including TNBC), colorectal cancer, stomach cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer, including hepatocellular carcinoma, pancreatic cancer gland cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other types of uroepithelial cancer, stomach cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, bone cancer, gallbladder and bile duct cancer, uterine cancer, cancer adrenal glands, sarcomas, GISTs, neuroendocrine tumors and hematological malignancies.

In one embodiment, the antibodies of the present invention are used to treat cancer, for example, small cell lung cancer, non-small cell lung cancer (including squamous cell cancer and adenocarcinomas), skin cancer, including melanoma, breast cancer (including TNBC), colorectal cancer, stomach cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer, including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other types of uroepithelial cancer, stomach cancer , glioma, glioblastoma, testicular cancer, thyroid cancer, bone cancer, gallbladder and bile duct cancer, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumors and hematological malignancies.

In a further embodiment, the antibodies of the present invention are used in the preparation of a medicament for the treatment of cancer, for example, small cell lung cancer, non-small cell lung cancer (including squamous cell cancer and adenocarcinomas), skin cancer, including melanoma, breast cancer (including TNBC), colorectal cancer and rectal cancer, stomach cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer, including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other types of uroepithelial cancer, gastric cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, bone cancer, gallbladder and bile duct cancer, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumors and hematological malignancies.

In one embodiment, antibodies (e.g., monoclonal antibodies, antibody fragments, nanobodies, multispecific and bispecific molecules and compositions, etc.) of the present invention can be used to determine levels of CRTAM or levels of immune cells containing CRTAM on the surface of their membrane, levels which may be associated with certain symptoms of the disease that are important for diagnosis.

In yet another embodiment, antibodies (e.g., monoclonal antibodies, multispecific and bispecific molecules and compositions) according to the present invention can first

- 19 043510 be tested for binding activity associated with therapeutic or diagnostic use in vitro. For example, the compositions of the present invention can be tested using flow cytometry, as described in the examples below.

In some embodiments, the antibodies (eg, monoclonal antibodies, multispecific and bispecific molecules and compositions) of the present invention find additional use in the therapy and diagnosis of diseases. For example, monoclonal antibodies, multispecific or bispecific molecules can be used to detect in vivo or in vitro one or more of the following biological activities: to induce and/or enhance immune cell activation; mediating phagocytosis or ADCC of a cell in the presence of human effector cells expressing CRTAM, or blocking the binding of a CRTAM ligand to CRTAM.

In a specific embodiment, antibodies (eg, monoclonal antibodies, multispecific and bispecific molecules and compositions) are used in vivo for the treatment, prevention or diagnosis of various diseases. Examples of relevant diseases include, but are not limited to, human cancer tissues representing small cell lung cancer, non-small cell lung cancer (including squamous cell cancer and adenocarcinomas), skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, cancer stomach, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer, including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other types of uroepithelial cancer, stomach cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, bone cancer, gallbladder and bile duct cancer, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumors and hematological malignancies.

Suitable routes for administering antibody compositions (eg, monoclonal antibodies, multispecific and bispecific molecules and compositions) of the present invention in vivo and in vitro are well known in the art and can be selected by those skilled in the art. For example, antibody compositions can be administered by injection (eg, intravenous or subcutaneous). Suitable dosages of the molecules used depend on the age and body weight of the subject, as well as the concentration and/or composition of the antibodies.

As described previously, the anti-CRTAM antibodies of the present invention may be co-administered with one or more additional therapeutic agents, eg, an immunostimulant, a cytotoxic agent, a radiotoxic agent, or an immunosuppressant. The antibody can be associated with the agent (as part of an immunocomplex) or administered separately from the agent. In the latter case (separate administration), the antibody can be administered before, after, or simultaneously with the agent, or used in conjunction with other known therapeutic agents, such as an anticancer therapeutic agent, such as radiation therapy. Such therapeutic agents include, but are not limited to, antineoplastic agents such as doxorubicin (Adriamycin), cisplatin, bleomycin sulfate, carmustine, chlorambucil, cyclophosphamide, and hydroxyurea, which themselves are only effective at concentrations that are toxic or subtoxic to the patient. Other agents suitable for co-administration with the antibodies of the present invention include other agents used to treat cancer, for example, Avastin®, 5-FU and gemcitabine. Co-administration of anti-CRTAM antibodies or antigen-binding fragments thereof according to the present invention with chemotherapeutic agents provides the presence of two anti-cancer agents that act through different mechanisms and have a cytotoxic effect on human tumor cells. Such co-administration may overcome problems due to the development of drug resistance or changes in the antigenicity of tumor cells.

In addition, target-specific effector cells can be used as therapeutic agents, for example, effector cells bound to the compositions (eg, monoclonal antibodies, multispecific and bispecific molecules) of the present invention. The targeted effector cells may be human leukocytes, such as macrophages, neutrophils or monocytes. Other cells include eosinophils, natural killer cells, and other cells bearing the IgG or IgA receptor. If desired, effector cells can be obtained from the subject to be treated. Target-specific effector cells can be administered as a cell suspension in a physiologically acceptable solution. The number of introduced cells can be on the order of 108-10 ⁹ , but depends on the purpose of treatment.

Therapy using target-specific effector cells can be performed in combination with other techniques. For example, antitumor therapy using compositions (eg, monoclonal antibodies, multispecific and bispecific molecules) according to the present invention and/or effector cells in combination with these compositions can be used in combination with chemotherapy. In addition, combination immunotherapy can be used to promote tumor cell rejection using two different populations of cytotoxic effectors.

In addition, bispecific and multispecific molecules according to the present invention

- 20 043510 can be used to modulate FcyR or FcyR levels on effector cells, for example, by blocking and eliminating cell surface receptors. In addition, mixtures of antibodies against Fc receptors can be used for this purpose.

Aspects of the present invention include kits containing antibody compositions of the present invention (eg, monoclonal antibodies, bispecific or multispecific molecules) and instructions for their use, for example, in the treatment of cancer. The kit may further contain one or more additional reagents, for example, an immunosuppressant, a cytotoxic agent or a radiotoxic agent, or one or more additional antibodies according to the present invention (for example, an antibody having complementary activity and binding to an epitope of the CRTAM antigen that is different from the epitope for first antibody).

Accordingly, patients receiving treatment with antibody compositions of the present invention may be additionally administered (before, concurrently, or after administration of an antibody of the present invention) another therapeutic agent, such as a cytotoxic or radiotoxic agent, that enhances or enhances the therapeutic effect of the antibodies.

In other embodiments, the subject may further receive treatment with an agent that modulates, for example, enhances or inhibits the expression or activity of Fcy or Fcy receptors, such as treating the subject with a cytokine. Preferred cytokines for administration during treatment with a multispecific molecule include granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GMCSF), interferon-y (IFN-y) and tumor necrosis factor (TNF).

Compositions (eg, antibodies, multispecific and bispecific molecules) according to the present invention can also be used to target cells expressing FcyR or CRTAM, for example, to label such cells. For such use, the binding agent can be linked to a detectable molecule. Thus, the present invention provides methods for localizing ex vivo or in vitro cells expressing Fc receptors, for example, FcyR or CRTAM. The detectable label may be, for example, a radioactive isotope, a fluorescent compound, an enzyme, or an enzyme cofactor.

In a specific embodiment, the present invention provides methods for detecting the presence of CRTAM antigen in a sample or measuring the amount of CRTAM antigen, comprising contacting the sample and a control sample with a monoclonal antibody or an antigen-binding fragment thereof that specifically binds CRTAM, under conditions allowing the formation of a complex between the antibody or its fragment and CRTAM. Complex formation detection is then performed, with the difference in complex formation between the sample and the control sample indicating the presence of CRTAM antigen in the sample.

In other embodiments, the present invention provides methods for treating an immune-mediated disorder in a subject, for example, human cancers, small cell lung cancer, non-small cell lung cancer (including squamous cell cancer and adenocarcinomas), skin cancer, including melanoma, breast cancer (including TNBC), cancer colon and rectal cancer, stomach cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer, including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other types uroepithelial cancer, gastric cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, bone cancer, gallbladder and bile duct cancer, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumors and hematological malignancies.

All sources cited herein, including, but not limited to, all documents, publications, patents, patent applications, presentations, texts, reports, manuscripts, brochures, books, Internet publications, journal articles, periodicals, newsletters about products, etc., are incorporated herein by reference in their entirety. The discussion of sources in the present invention is intended solely to summarize the statements made by their authors; No source should be assumed to represent prior art, and applicants reserve the right to challenge the accuracy and appropriateness of cited sources.

Although the foregoing invention has been described in detail by way of illustrations and examples for purposes of clarity, those skilled in the art will, in light of the teachings of the present invention, understand that certain changes and modifications can be made thereto without departing from the spirit or scope of the dependent claims.

The present invention is further illustrated by the following examples, which should not be construed as further limitation. The following examples, sequences and figures are presented to facilitate understanding of the present invention, the true spirit of which is set forth in the appended claims. It should be understood that modifications can be made to the procedures set forth without departing from the spirit of the invention.

Examples.

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Example 1. Preparation and screening of antibodies.

Obtaining a hybridoma.

Rat monoclonal antibodies against CRTAM were generated by genetic immunization. CRTAM ECD (SEQ ID NO:12) was used to generate plasmid pB8-CRTAM-hum.ECD (Aldevron, Freiburg) for immunization. Plasmid pB8-CRTAM-hum.ECD was used to immunize three rats. Splenocytes from immunized rats were fused with myeloma cell lines to produce hybridomas using standard industrial methods. Rat immunization and titer test.

Animals were immunized with the vector (pB8-CRTAM-hum.ECD). Flow cytometry-based analysis (FACS test) was performed to detect the presence of target-specific antibodies (antibody titer) in the serum of immunized animals.

Hybridoma screening.

FACS analysis was used to identify overexpressing hybridoma cells. One hundred and eight hybridoma clones were identified as positive in the initial screening.

Secondary screening.

Recombinant hCRTAM/Fc chimeric protein (R&D Systems, cat. no. 1695-CR) was applied at 100 ng/well to high-binding assay plates (cat. no. 12-565-136, Thermo Scientific) after dilution with 1X saline with Dulbecco's phosphate buffer (DPBS) (cat. no. SH30028-03, Thermo Scientific). The plates were washed three times with wash buffer, 1X DPBS and 0.05% Tween 20 (cat. no. BP337-500, Fisher Scientific) using a Tecan Hydro speed 96-well plate washer.

Immobilized antigen plates were blocked with overblocking reagent solution (available from Thermo Fisher) for 1 h at room temperature (RT) after washing.

Add 100 μl of hybridoma supernatant to each well, diluted 1:5 with ELISA buffer (ELISA buffer, 1X DPBS, 0.05% Tween 20 and 1% bovine serum albumin, catalog no. SH30574.02, Thermo Scientific ). After primary antibody incubation, the plates were washed three times with wash buffer. HRP-conjugated secondary antibody (goat anti-rat IgG) diluted 1:5000 in ELISA buffer (heavy and light chain specific, Jackson Immunoresearch, catalog no. 112-035-167) was added to the plates for an hour at room temperature. The plate was washed three times in wash buffer. Ultra TMB-ELISA 1-Step ELISA reagent (cat. no. 34028, Thermo Scientific) was added to each well to develop a detectable signal. After complete signal development, 2 N was added to all wells. sulfuric acid (cat. no. 8140-16, Ricca Chemical Company) to stop the reaction.

The optical density (OD) of each sample was determined using a VERSA max microplate reader at a wavelength of 450 nm.

Example 2. Study of the structural characteristics of monoclonal antibodies to CRTAM.

cDNA sequences encoding the variable regions of the heavy and light chains of monoclonal antibodies were obtained using standard PCR techniques and sequenced using standard DNA sequencing techniques. The 5A11 heavy and light chain variable regions selected during the screening are shown in FIG. 1.

The nucleotide and amino acid sequences of the 5A11 heavy chain variable region are shown in SEQ ID NO:3 and 1, respectively.

The nucleotide and amino acid sequences of the 5A11 light chain variable region are shown in SEQ ID NO:4 and 2, respectively.

Further analysis of the VH 5A11 sequence using the Kabat CDR region determination system resulted in the delineation of the heavy chain CDR1, CDR2 and CDR3 regions shown in SEQ ID NO:5, 6 and 7, respectively. In fig. 1a shows the VH 5A11 sequences with CDR1, CDR2 and CDR3 enclosed in boxes. Further analysis of the VL 5A11 sequence using the Kabat CDR region determination system resulted in the delineation of the light chain CDR1, CDR2 and CDR3 regions shown in SEQ ID NO:8, 9 and 10, respectively. In fig. 1b shows the VL 5A11 sequences with CDR1, CDR2 and CDR3 enclosed in boxes.

Example 3. Specificity of monoclonal antibodies to CRTAM as determined using flow cytometric analysis. Preparation of in vitro activated T cells

Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat of human blood as described above. 2E6 PBMC cells/ml were cultured in T cell culture medium (AIM V medium with 5% FBS, cat. no. SH3008703, Fisher, Pittsburgh, PA, USA) and 300 μl IL-2 (cat. no. 200-02 , Peprotech, Rocky Hill, NJ, USA) in the presence of 100 ng/ml anti-CD3 antibody (OCT3) for 4 days. Cells were then cultured with activation beads containing anti-CO3 antibody/anti-CD28 antibody (cat. no. 11132D, Thermo Fisher, Waltham, MA, USA) for 24 h before FACS analysis (data not shown). Enzyme-linked immunosorbent assay (ELISA).

Fluorescence activated cell sorting (FACS) analysis was performed in 96 format

- 22 043510 well plate in accordance with standard protocols. Activated T cells were obtained as above. All subsequent steps of the staining protocol were performed on ice with reagents chilled on ice, except for the centrifugation steps, which were performed at room temperature. Three-fold serial dilutions of antibodies were prepared in FACS buffer to generate a 12-point titration curve with final concentrations ranging from 133 nM to 1 pM. The corresponding hybridoma supernatants, isotype controls, and positive control antibodies were diluted in ice-cold FACS buffer and then tested at a single final concentration of 30 nM. Each of the antibody dilutions was dispensed (100 μl/well) into one well for each cell line. One well was left unstained in FACS buffer. After 30 min of incubation with primary antibody or control, cells were washed twice by adding FACS buffer followed by centrifugation for 5 min at 1200 rpm. The supernatant was discarded and the cell pellet was saved. Goat anti-rat IgG secondary antibody (H+L)-RPE was diluted to a working concentration of 1 μg/ml and applied to all wells except one of the wells that was not stained with the primary antibody. The secondary antibody was incubated with cells for 30 min. Cells were washed twice by adding FACS buffer followed by centrifugation for 5 minutes at 1200 rpm. The final cell pellets were resuspended in 150 μl FACS buffer + 50 μl 4% paraformaldehyde and fixed until ready for recording on a flow cytometer. The plates were left at 4°C until data recording. The geometric mean fluorescence intensity (GMFI or geometric mean) of each sample was determined using a Guava Easycyte Plus high-throughput flow cytometer (96-well plates), and the raw data was analyzed using the Guava Cytosoft Pro software module, version 2.2.2 (Millipore, Billerica, AB). Massachusetts, USA). The geometric mean was plotted against antibody concentration using GraphPad™ Prism software to perform nonlinear regression, sigmoidal dose-response analysis, and calculate the EC50 for antibody binding to activated T cells. In fig. Figure 3 shows the specific binding of 5A11 to activated T cells in a dose-dependent manner compared to the commercially available anti-CRTAM antibody CR24.1 (BioLegend). As can be seen, antibody 5A11 showed excellent binding to activated T cells.

Example 4. Cross-reactivity of antibodies against CRTAM. Enzyme-linked immunosorbent assay (ELISA).

Enzyme-linked immunosorbent assay (ELISA) was performed in a 96-well plate format in accordance with standard protocols. A 96-well ELISA plate (cat. no. 12-565-136, Thermo Scientific) was coated with 100 μl of 1 μg/ml solutions of cynomolgus HIgG1-Fc CRTAM proteins in 1X phosphate-buffered saline overnight at 4°C. Dulbecco (DPBS) (cat. no. SH30028-03, Thermo Scientific). The plate was washed three times with wash buffer, 1X DPBS and 0.05% Tween 20 (catalog no. BP337-500, Fisher Scientific), blocked with 250 µl overblocking buffer (catalog no. 37515, Thermo Scientific) for 30 min at room temperature. , then washed again three times with wash buffer.

Anti-CRTAM antibodies were serially diluted 1:3 with ELISA buffer, 1X DPBS, 0.05% Tween 20, and 1% bovine serum albumin (cat. no. SH30574.02, Thermo Scientific), generating an 8-point titration curve with end point concentration from 0.003 to 2 μg/ml, and applied to the wells in duplicate. The last well was left as a blank (with ELISA buffer). After 60 min of incubation with the primary antibody, the plate was washed three times in wash buffer.

Peroxidase-conjugated goat anti-rat IgG (H+L) secondary antibody (AffiniPure) was diluted 1:5000 in ELISA buffer and applied to the anti-CRTAM and isotype control wells for 60 minutes. Peroxidase-conjugated goat anti-mouse IgG F(ab')2 fragment (H+L) (AffiniPure) was also diluted 1:5000 with ELISA buffer and applied to CR24.1 wells for 60 min.

The plate was washed three times in wash buffer. Ultra TMBELISA 1-Step ELISA Reagent (cat. no. 34028, Thermo Scientific) was added to each well to develop a detectable signal. After complete signal development, 2 N was added to all wells. sulfuric acid (cat. no. 8140-16, Ricca Chemical Company) to stop the reaction. The optical density (OD) of each sample was determined using a VERSA max microplate reader at a wavelength of 450 nm. The absorbance for each sample was averaged across two wells, plotted against antibody concentration, and the EC50 was calculated using nonlinear regression and sigmoidal dose-response analysis.

Anti-CRTAM antibody 5A11 was found to exhibit dose-dependent binding to cynomolgus CRTAM protein, whereas CR24.1 did not bind to cynomolgus CRTAM protein. In fig. Figure 4 shows the cross-reactivity of 5A11.

- 23 043510

Example 5: Ability of Anti-CRTAM Antibodies to Activate T Cells and Stimulate IFN-γ Production.

Generation of T cells activated by anti-CRTAM and anti-CD3 antibodies in vitro.

96-well non-tissue culture plates were coated with 2 μg/ml OKT3 and various concentrations of anti-CRTAM antibodies/isotypes (11-fold titration from 20 μg/ml with a 1:2 dilution) at 4°C and incubated overnight. The plates were washed twice with PBS followed by AIMV blocking with 5% FBS for 30 min. After blocking, 0.1 million OKT3-primed T cells (day 4), prepared as described in Example 3, were resuspended in fresh AIMV medium and added to the plates. After one day of incubation at 37°C, supernatants were collected and diluted for use in the IFN-γ ELISA.

Enzyme-linked immunosorbent assay (ELISA) for the determination of IFN-γ.

IFN-γ was measured using the appropriate Ready-SET-Go! for solid-phase ELISA manufactured by eBiosciences (cat. no. 50-173-24 Fischer Scientific). Following the manufacturer's instructions, a high-binding plate (Corning 3690) was coated with capture antibody (diluted 1:250 in immobilization buffer) overnight at 4°C, then washed three times with wash buffer (PBS + 0.05% Tween- 20). The plate was blocked with assay diluent for 1 h on a shaker. The cell culture supernatant was added to the plates at an appropriate dilution (1:400 for IFN-γ) and incubated for 2 hours on a shaker, followed by washing 3 times. Biotinylated detection antibodies (provided in the kit) diluted 1:250 in assay diluent were added and incubated for 1 hour on a plate shaker. The plate was washed with wash buffer and HRP-conjugated streptavidin (provided in the kit) diluted 1:1000 with assay diluent was added. After 30 minutes of incubation on a plate shaker, the plate was washed and the TMB substrate solution (provided in the kit) was added. The reaction was stopped after approximately 5-10 minutes by adding 2 N. H ₂ SO ₄ solution, and the absorbance was read on a plate reader at 450 nm (OD450).

Results.

Antibody 5A11 showed increased activity in pre-activated OKT3 T cells, which was reflected in increased production of IFN-γ compared to antibody CR24.1 (Fig. 5). This indicates that anti-CRTAM antibodies should have a therapeutic effect in patients with an inhibited immune system.

Example 6. Humanization of antibody 5A11.

Rat monoclonal antibody 5A11 was humanized using CDR grafting technology. To guide the humanization process and facilitate the decision to retain the original rat antibody residues or replace them with human germline analogues, a homologous molecular model of the Fv fragment of the rat monoclonal antibody 5A11 was constructed.

The definition of CDR is based on Kabat's nomenclature. The selection of human acceptor framework regions into which rat 5A11 antibody CDR regions should be grafted was accomplished by searching for rat and human V genes in the IMGT database using IgBLAST software developed at NCBI to facilitate sequence analysis of immunoglobulin V regions using sequences the variable region of the rat 5A11 antibody as input. The strategy used was to use human germline sequences, which are natural human sequences that do not contain the specific somatic mutations found in individual human antibody sequences.

Homologous molecular model of rat 5A11 antibody.

Methodology for constructing a homologous model.

The antibody model BUH-5A11-F2 ('5A11') was constructed according to established protocols (Ramos OHP. Computer-assisted modeling of antibody variable domains. Methods Mol Biol 2012; 907: 39-55). The heavy (VH) and light (VL) chain variable region sequences were separately numbered/annotated according to IMGT convention to separate framework and CDR sequences.

VL framework residues were used to search for sequences of resolved antibody structures using BLAST for proteins. The best match for VL was provided by structure 4AIZ (1.75 A resolution) (65 of 108 identical residues). This was chosen as a template, but the slightly lower ranking match provided for VL by the 3G6D structure (3.20 A resolution, 64 of 107 identical residues) was used to correct the conformation of the N-terminal serine missing in 4AIZ. The CDR2 sequence of VL was identical to that of 4AIZ; thus, there was no need to search for the CDR2 matrix. The VL CDR1 and CDR3 sequences, with two residues added at each end, were used to search for sequences of resolved antibody structures using BLAST for proteins. The best match for CDR3 VL was provided by the 1NFD structure (2.80 A resolution, 11 of 15 identical residues). No mouse or rat antibody structure matched the CDR1 sequence with two residues added at each end;

- 24 043510 search for the structures of mouse or rat antibodies with the VL framework also did not give matches with a good approximation. The best match for CDR1 in the human structure search was provided by the human lambda variable region at 4AIZ (5 of 10 identical residues), which was used as the VL template. PyMol software (PyMOL Molecular Graphics System, version 1.3r1, Schrodinger, LLC.) was used to fit the CDR3 matrix to the framework matrix using two sticky ends to attach a piece of the CDR matrix to the VL framework matrix. The assembled partial VL model was manually subjected to mutagenesis (in PyMol), selecting optimal rotamers corresponding to the VL 5A11 sequence.

VH framework residues were used to search sequences of resolved antibody structures using BLAST for proteins. The best match was provided by the VH structures of the rat 5AUM antibody (resolution 2.05 A, 85 of 116 identical residues); this antibody is from the same IGHV10-5 germline as 5A11 and was therefore chosen as a template. The CDR1, CDR2, and CDR3 VH sequences with the addition of two residues at each end were used to search for sequences of resolved antibody structures using BLAST for proteins. The best match for CDR1 VH was provided by the 4HPY structure (1.50 A resolution, 9 of 11 identical residues). The best match for CDR2 VH was provided by structure 5AUM (11 of 14 identical residues). The best match for VH CDR3, 1Q72, was not selected as a matrix due to the presence of 4 alignment gaps; the 4BZ1 structure was selected instead, providing a lower ranking match (1.50 A resolution, 7 of 14 identical residues); the reason for this was that the overlap of the CDR3 fragments 1Q72 and 4BZ1 showed that the N- and C-terminal regions matched the framework template and each other well, while 4BZ1 provided satisfactory closure of the insertion sequence found in 1Q72. The CDR matrices were fitted to the scaffold matrix (in PyMol) using two sticky ends to attach each CDR template fragment to the scaffold region matrix. Finally, the assembled partial VH model was manually subjected to mutagenesis (in PyMol), selecting optimal rotamers corresponding to the VH sequence 5A11. The best location of the VH and VL partial models in the tertiary structure was then selected to assemble the final model. Sequences of VH and VL matrices were sent to the PAPS server (packing angle prediction server - Abhinandan KR, Martin ACR Analysis and prediction of vh/vl packing in antibodies. Protein Eng Des Sel 2010; 23: 689-697) to find the optimal predicted tertiary structure. The PAPS server predicted that the resolved structure of the 1D5B antibody with a relative packing angle of 44.5 degrees would provide the best arrangement of VH and VL in the tertiary structure. Thus, the final model was assembled by fitting the coordinates of the conserved reference skeletal segments (residues 41–44 and 101–104) of the partial models of VH and VL to 1D5B (in PyMol). Finally, the coordinates of this final model were subjected to a run of energy minimization calculations using GROMACS (Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJC. Gromacs: fast, flexible, and free. J Comput Chem 2005; 26: 1701-1718) with the GROMOS96 force field (Scott WRP, Hunenberger PH, Tironi IG, Mark AE, Billeter SR, Fennen J, Torda AE, Huber T, Kruger P, van Gunsteren WF. The gromos biomolecular simulation program package. J Phys Chem A 1999;103:3596-3607).

Construction of the heavy chain.

Amino acid sequence differences from the most homologous rat germline IGHV10-5*01.

Amino acid sequence of VH isolated from rat hybridoma 5A11 (CDR regions according to the Kabat numbering scheme are in bold).

FRl CDRl FR2 CDR2

AVQLVESGGGLVQPKESLKISCAASGFTFSDAAMYWVRQAPGKGLEWVARIRTKTNN

ΥΛΛΙΙ

FR3 CDR3 FR4

YVESVKGRFTVSRDDSKSMVYLQMDNLKTDDTAMYYCTSVPQGTQDYWGQGVMVT

VSS

91% identity (91 identical residues out of a total of 100 amino acids) between the rat heavy chain variable region 5A11 and rat germline immunoglobulin VH 10-5*01 (IGHV10-5*01).

<------------FR1-----------XCDRX——FR2—->

5A11VH 1 AVQLVESGGGLVQPKESLKISCAASGFTFSDAAMYWVRQAPGKGLEWVA49

IGHV10-5*01 1........................N49 <-------CDR2---- --><--------------FR3>

5А11 VH 50 RIRTKTNNYAAHYVESVKGRFTVSRDDSKSMVYLQMDNLKTDDTAMYYCTS 100 IGHV10-5*01 50.....P....TY.AD......I...... EA 100

Selection of human VH acceptor framework regions.

The selection of human VH acceptor framework regions onto which the rat 5A11 antibody CDR regions were transplanted was carried out by searching for human VH genes in the IMGT database using Ig

- 25 043510

BLAST using the amino acid sequence of the rat VH region as input. Based on the sequence alignment of the parent antibody and human germlines, the most matching entries were identified. Identification of the optimal human germline for use as an acceptor was performed based on the following ordered criteria: sequence identity in reading frame according to Kabat numbering and identity and/or compatibility of interstrand interaction region residues and supporting loops with the canonical conformations of the original CDRs. In this analysis, human germline IGHV3-73*01 was found to be the best candidate for use as human acceptor frameworks, providing an overall percent identity of 75% (75 residues out of 100). Thus, this human germline was used to construct a humanized version. The rat germline J2 gene (IGHJ2*01) was identified as the segment gene most homologous to the corresponding rat J VH antibody 5A11 gene. The sequence of FR4 VH 5A11 is identical to the sequence of the rat germline gene IGHJ2*01. The rat J2 gene was compared with the human J genes at CDR3 and FR4, and selection for the human J segment IGHJ4 (IGHJ4*01) was performed.

Construction using human germline IGHV3-73*01 as acceptor scaffold regions.

Humanized version of A.

The rat CDR (in bold), defined according to Kabat nomenclature, was grafted onto IGHV3-73*01, yielding the sequence detailed below. The underlined residues represent rat framework residues (outside the CDR residues), i.e. preserved remnants of the original VH sequence of the rat 5A11 antibody; they were retained because they may be structurally important for maintaining full antibody activity.

Version A.

FR1 CDR1 FR2

EVQLVESGGGLVQPGGSLKLSCAASGFTFSDAAMYWVRQASGKGLEWVA

CDR2 FR3

RIRTKTNNYAAHYVESVKGRFTVSRDDSKNTVYLQMNSLKTEDTAMYYCTS

85% identity (85/100) between the humanized version of A and the human germline. IGHV3-73*01 <------------FR1------------XCDRX—-FR2—>

5A11-373-VHA 1 EVQLVESGGGLVQPGGSLKLSCAASGFTFSDAAMYWVRQASGKGLEWVA 49

IGHV3-73*01 1 ................................GS..H......... ...G49 <------CDR2-------><--------------FR3------------->

5A11-373-VHA 50 RIRTKTNNYAAHYVESVKGRFTVSRDDSKNTVYLQMNSLKTEDTAMYYCTS 100

IGHV3-73*01 50 ...S.A.S..TA.AA......I........A............V....R 100

In framework region 2 (FR2), the H49 Ala residue according to Kabat numbering is known as a Vernier residue, it is adjacent to VH-CDR2 and may influence CDR conformation and fine-tuning of antigen recognition. In framework region 3 (FR3), residues H69 Val and H78 Val are known as Vernier residues according to Kabat numbering. They are adjacent to the CDR and can influence the conformation of the CDR and the fine tuning of antigen recognition.

Light chain design.

Amino acid sequence differences from the most homologous rat germline IGLV4-S1*01.

Amino acid sequence of rat VL antibody 5A11 (CDR regions highlighted).

FR1 CDR1 FR2

SYELIQPPSASVTLGNTVSLTCVGDELSKRYVQWSQQKPDKTIVSVIY

CDR2 FR3 CDR3 FR4

KDSERPSGISDRFSGSSSGTTATLTIHGTLAEDEADYYCLSTYSDDNLPVFGGGTKLTVL

97.9% (94/96) identity (94 identical residues out of a total of 96 amino acids) between the rat VL antibody 5A11 and the rat germline immunoglobulin lambda variable region IGLV4-S1*01.

<---------FR1--------x-CDRl— x-—FR2-—>

5A11VL 1 SYELIQPPSASVTLGNTVSLTCVGDELSKRYVQWSQQKPDKTIVSVIY48

IGLV4-S1*O1 1 ......................A..Y......... ....48 <CDR2-x--------------FR3------------X-CDR3- 96

5А11 VL 49 KDSERPSGISDRFSGSSSGTTATLTIHGTLAEDEADYYCLSTYSDDNL 96

IGLV4-S1*O1 49................................................. ....96

- 26 043510

Selection of human VL acceptor framework regions.

The selection of human VL acceptor frameworks onto which rat 5A11 antibody CDRs were grafted was accomplished by searching the IMGT database for human VL genes using IgBLAST using the amino acid sequence of the rat VL region as input. Based on the sequence alignment of the parent antibody and human germlines, the most matching entries were identified. Identification of the optimal human germline for use as an acceptor was performed based on the following ordered criteria: sequence identity in reading frame according to Kabat numbering and identity and/or compatibility of interstrand interaction region residues and supporting loops with the canonical conformations of the original CDRs. In this analysis, the human germline IGLV3-27*01 was found to be the best candidate for use as human acceptor frameworks, providing an overall percent identity of 64.6% (62 amino acid residues out of 96). Thus, this human germline was used to construct humanized versions.

The rat germline J1 gene (IGKJ1*01) was identified as the segment gene most homologous to the corresponding rat VL antibody 5A11 J gene. The rat L-segment gene was compared with the human J-segment genes at CDR3 and FR4, and the selection of the human J-segment IGKJ2 (IGKJ2*01), which had the maximum homology, was performed. Construction using human germline IGLV3-27*01 as acceptor scaffold regions.

Humanized version.

The rat CDR, defined according to Kabat nomenclature, was grafted onto IGLV3-27*01, yielding the sequence detailed below. A number of rat framework residues, structurally important for maintaining full antibody activity, were preserved. This resulted in a humanized version with 87.5% identity (84 amino acid residues out of 96) compared to the human germline IGLV3-27*01.

<---------FR1--------x-CDRl— x——FR2— ->

5А11-327-VLC SYELTQPSSVSVSPGQTARITCSGDVLSKRYAQWSQQKPGQAIVSVIY < CDR2-X--------------FR3-------------X-CDR3 -

5А11-327-VLC KDSERPSGIPERFSGSSSGTTATLTISGAQVEDEADYYCLSTYADDNL <---------FRi--------x-CDRl— x—-FR2—->

5A11-327-VLC SYELTQPSSVSVSPGQTARITCSGDVLSKRYAQWSQQKPGQAIVSVIY IGLV3-27*01 ...........................A.K..R.F......P.L...

<CDR2-x--------------FR3------------X-CDR3 5A11-327-VLC KDSERPSGIPERFSGSSSGTTATLTISGAQVEDEADYYCLSTYADDNL

IGLV3-27*01 ...................V................Y.A-..N. Example 7 Perforin/Granzyme Assay.

The cytotoxic potential induced by anti-CRTAM antibodies was assessed by measuring the production of perforin and granzyme B by cytotoxic CD8+ lymphocytes after stimulation. Human CD8 T cells were negatively isolated from PBMCs from healthy donors using a commercially available kit (Miltenyi Biotec). Isolated CD8 cells were stimulated with plate-immobilized CD3 (OCT3, 0.1 μg/ml) and various doses of 5A11 for 4 days. After stimulation on the plate, CD8 cells were treated with PMA (25 ng/ml), ionomycin (10 μg/ml), and brefeldin (10 μg/ml) for 4 hours. Cells were then washed, fixed with 4% PFA for 10 min, and stained with anti-perforin and anti-granzyme B antibodies for flow cytometry diluted in PBS containing 0.5% saponin for 15 min. After staining, cells were washed twice with PBS containing 0.5% saponin and once with FACS buffer (PBS containing 2% FCS and 2 mM EDTA). Washed cells were resuspended in FACS buffer and analyzed using an Attune NxT flow cytometer (Fisher Scientific). As shown in detail in FIG. 7 and 8, escalating dose stimulation with 5A11 increased the cytotoxic potential of CD8 T cells in a dose-dependent manner.

Example 8 T Cell Cytotoxicity Assay.

T cell cytotoxicity to MCF-7 breast cancer cells using anti-CRTAM antibodies was assessed using a bispecific anti-Her2/CD3 antibody as an anchor for T cells and MCF-7 cells. Additional cytotoxicity was observed when anti-CRTAM antibodies were added to assay wells containing tumor cells, PBMCs, and anti-Her2/CD3 bispecific antibody. The percentage of cytotoxicity associated with anti-CRTAM antibody was calculated as the percentage of killing compared to cells not treated with anti-CRTAM antibody.

Freshly isolated PBMCs were collected by centrifugation for 5 min at 1000 x g and diluted with complete DMEM medium to a concentration of 1.5E ⁶ cells/ml. PBMCs were aliquoted and the test antibodies (antibody 5A11) were added to a concentration of 20 μg/ml. The cells were incubated on ice for 27 043510 10-20 minutes. MCF-7 cells were collected and diluted to a density of 1.5E ⁶ cells/ml.

µl of the resulting cell suspensions of both PBMC and MCF-7 were added to each well so that each well contained 75,000 PBMC and MCF-7 cells. Anti-Her2/CD3 bispecific antibody was serially diluted with complete DMEM, and 10 μl of this dilution was added to each well, resulting in a final concentration of 5 μg/ml in each assay well.

Untreated wells containing 50 μl of culture medium/well were negative controls. Assay plates were incubated for 96 hours at 37°C in an atmosphere of 5% CO ₂ . After incubation, assay plates were equilibrated to RT for 30 min. PBMCs were removed and discarded, leaving MCF-7 cells in the wells for analysis. The plates were washed three times with PBS at RT, and 100 μL of room temperature PBS was added to each well. Cell Titer-Glo® reagent (cat. no. G7572, Promega, Madison, WI, USA) was prepared according to the manufacturer's instructions and dispensed at 100 μl/well. The reagent was pipetted into and out of the assay wells several times to mix and lyse the cells. The assay plate was incubated for 15 min in the dark. Luminescence was recorded using a GloMax® luminometer (Promega) according to the manufacturer's recommendations.

The number of viable cells was proportional to the luminescence units, their average value was calculated for each concentration in the analyzed wells. Percent viability was calculated using wells without test antibody as a control and plotted against antibody concentration.

As can be seen in FIG. 9, the addition of the 5A11 antibody significantly increased the level of cytotoxicity compared to the control without the 5A11 antibody.

Example 9. ELISPOT with tumor-infiltrating lymphocytes.

Primary tumor-infiltrating lymphocytes (TILs) derived from NSCLC (Figure 10) or breast cancer (Figure 11) tumors were stimulated for 96 hours with CR24.1, 5A11 or pembrolizumab diluted to the indicated concentrations and OKT3 diluted up to 1 µg/ml with complete RPMI medium. After stimulation, TILs were collected, counted, and seeded onto an IFN-γ ELISPOT plate (Mabtech) at 100,000 cells/well. The plate was incubated at 37°C for 24 h and subsequently developed according to the manufacturer's instructions. Spot counts were read using a Series 5 ELISPOT ImmunoSpot® analyzer and data were analyzed using GraphPad Prism software.

In fig. 10 shows that antibody 5A111 activates more NSCLC-derived TILs, reflecting significantly increased production of IFN-γ compared to commercial antibody CR24.1 or pembrolizumab.

In fig. 11 shows that antibody 5A11 and pembrolizumab activate IFN-γ production in similar numbers of breast cancer-derived TILs.

Example 10. ELISPOT dose-response relationships with tumor-infiltrating lymphocytes.

Primary tumor-infiltrating lymphocytes (TILs) derived from NSCLC tumors were stimulated for 96 hours with 0.1, 1.0, or 10 μg/ml 5A11, pembrolizumab (10 μg/ml), or OKT3 diluted to 1 μg/ml. ml complete RPMI medium. After stimulation, TILs were collected, counted, and seeded onto an IFN-γ ELISPOT plate (Mabtech) at 100,000 cells/well. The plate was incubated at 37°C for 24 h and subsequently developed according to the manufacturer's instructions. Spot counts were read using a Series 5 ELISPOT ImmunoSpot® analyzer and data were analyzed using GraphPad Prism software. In fig. Figure 12 shows that all doses of 5A11 activated the production of increased amounts of IFN-γ in TILs compared to pembrolizumab.

Example 11 MIC-mediated cytotoxicity using the Delfia® kit.

K562 cells were stained as described in the Delfia® kit (Promega). Stained target K562 cells were mixed with PBMC from healthy donors (precultured for 72 hours with IL-2) at a target:effector ratio of 1:20 in the presence of 5A11, CR24.1, or pembrolizumab diluted to 10 μg/ml . IL-2 at a concentration of 50 IU/ml was used as a positive control for the assay. All conditions were repeated in triplicate. The co-culture was incubated at 37°C for 3 hours. After 3 hours, 20 µl of the co-culture supernatant was collected and analyzed according to the manufacturer's instructions (Delfia® Promega). Results were read using a SpectraMax M5 plate reader, recording time-resolved fluorescence (TRF) at an excitation wavelength of 320 nm, an emission wavelength of 615 nm, a delay of 100 microseconds, and an integration time of 100 microseconds. All controls recommended by the manufacturer to enable specific lysis calculations were included in the analysis, and calculations were performed as detailed in the kit instructions.

In fig. 13 shows that antibody 5A11 caused significantly increased cell lysis compared to commercial antibody CR24.1 or pembrolizumab.

Example 12. In vivo assessment of the antitumor efficacy of antibody 5A11 in a model of NSCLC.

- 28 043510 lovec HCC827 in the Mixeno NCG mouse.

Isotype control (10 mg/kg; BIWx3) and antibody 5A11 (10 mg/kg; BIWx3) were tested in an in vivo model; each group contained 10 mice. Each cohort was divided in half, resulting in 5 mice receiving intraperitoneal injections of PBMCs isolated and obtained from one of two healthy human donors. Three days later, the mice were injected subcutaneously with HCC827 tumor cells. A day later, treatment with test antibodies began in accordance with the dosage regimen described above. Body weight (BW) was monitored and tumors were measured three times a week. The mice were frequently monitored for health status and side effects. Mice were sacrificed if there were signs of graft-versus-host disease. Treatment outcome was determined by tumor growth inhibition (TGI), a measure of the difference in mean tumor volume in the experimental group compared to the control group measured on the designated study day. In fig. 14 demonstrated that after 15 days, tumors in mice treated with the 5A11 antibody were significantly smaller than in animals treated with an isotype control antibody.

Example 13 Binding affinity of humanized antibody 5A11.

The binding affinity of the humanized antibody 5A11 was determined using Octet® (ForteBio) using anti-hIgG antibody capture biosensors (cat. no. 18-5060).

ForteBio biosensors for capturing antibodies against human IgG-Fc (AH) were loaded with test antibodies at a concentration of 1 μg/ml for 300 s.

Loaded biosensors were incubated with 5 concentrations of recombinant human hCRTAM/Fc chimeric protein, starting at 200 nM, which were serially diluted in a ratio of 1:3; the control well contained only kinetic buffer (to correct for drift).

Data for all concentrations were fitted using a global 1:1 fit for each sample. Whenever possible, kinetic rates of association and dissociation and KD values were determined.

The analysis showed that the humanized antibody 5A11 had the characteristics shown in the table below. 2.

table 2

Sample Kd(nM) kopeck (1/Ms) koff(l/c) Imax (nm) Full X ² Full R ² 5A11 0.439 2.49 x 10 ⁵ 1.10 x Yu ⁴ 0.27 0.06 1.00

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List of sequences

SEQ ID Description Subsequence 1 5All_VH_aa AVQLVESGGGLVQPKESLKISCAASGFTFSDAAMYWVRQAPGKG LEWVARIRTKTNNYAAHYVESVKGRFTVSRDDSKSMVYLQMDNL KTDDTAMYYCTSVPQGTQDYWGQGVMVTVSS 2 5All_VL_aa SYELIQPPSASVTLGNTVSLTCVGDELSKRYVQWSQQKPDKTIVSV IYKDSERPSGISDRFSGSSSGTTATLTIHGTLAEDEADYYCLSTYSD DNLPVFGGGTKLTVL 3 5All_VH_nt atgttggtgctgcagtgggttttggtgactgctctttttcaaggtgttcattgtgcggtacagcttgttgagtctg gtggaggattggtgcagcctaaggagtcattgaaaatctcatgtgcagcctctggattcacgttcagtgatg ctgccatgtactgggtccgccaggctccaggaa agggtctggagtgggttgcgcgcatacgaactaaaa ctaataattatgcagcgcattatgttgagtcagtgaaaggcagattcaccgtctccagagatgattcaaaaa gcatggtctacctacaaatggataacttgaaaactgatgacacagccatgtattactgtacgtcagtccccc aaggaacgcaggattactggggccaa ggagtcatggtcacagtctcctca 4 5All_VL_nt atggcctgggtctctcttgttctacctctgctctctctgtatgcaggttatgtgaccagctatgagttgatccaa ccaccttcggcatcagtcactctgggaaatactgtctcactcacttgtgtcggagatgaattatcaaaaagat atgttcagtggtctcaacaaagccagacaagaccattgtgtccgtgat atacaaagatagcgagcggcc ctcaggcatctctgaccgattctctggttccagctccgggacaacagccactctgacaatccatggcaccct ggctgaggatgaggctgattattactgtttgtcaacatatagtgatgataatctccctgtgttcggtggtggaa ccaagctcactgtccta 5 5All_VH_CDRl_a a DAAMY 6 5All_VH_CDR2_a a RIRTKTNNYAAHYVESVKG 7 5All_VH_CDR3_a a VPQGTQDY 8 5All_VL_CDRl_a a VGDELSKRYVQ 9 5All_VL_CDR2_a a KDSERPS 10 5All_VL_CDR3_a a LSTYSDDNLPV eleven CRTAM (095727) MWWRVLSLLAWFPLQEASLTNHTETITVEEGQTLTLKCVTSLRKN SSLQWLTPSGFTIFLNEYPALKNSKYQLLHHSANQLSITVPNVTLQ DEGVYKCLHYSDSVSTKEVKVIVLATPFKPILEASVIRKQNGEEHV VLMCSTMRSKPPPQITWLLGNSMEVSGGTLHEFETDGKKCNTTST LIIHTYGKNS TVDCIIRHRGLQGRKLVAPFRFEDLVTDEETASDALE RNSLSSQDPQQPTSTVSVTEDSSTSEIDKEEKEQTTQDPDLTTEANP QYLGLARKKSGILLLTLVSFLIFILFIIVQLFIMKLRKAHVIWKKENE VSEHTLESYRSRSNNEETSSEEKNGQSSHPMRCMNYITKLYSEAKT KRKENVQHSKLEEKHIQVPESIV 12 CRTAM ECD (AK 18 - 287 SEQ ID NO:11) SLTNHTETITVEEGQTLTLKCVTSLRKNSSLQWLTPSGFTIFLNEYP ALKNSKYQLLHHSANQLSITVPNVTLQDEGVYKCLHYSDSVSTKE VKVIVLATPFKPILEASVIRKQNGEEHVVLMCSTMRSKPPPQITWLL GNSMEVSGGTLHEFETDGKKCNTTSTLIIHTYGKNSTVDCIIRHRGL QGRKLV APFRFEDLVTDEETASDALERNSLSSQDPQQPTSTVSVTE DSSTSEIDKEEKEQTTQDPDLTTEANPQYLGLARKKSG 13 5All_VHA_aa EVQLVESGGGLVQPGGSLKLSCAASGFTFSDAAMYWVRQASGKG LEWVARIRTKTNNYAAHYVESVKGRFTVSRDDSKNTVYLQMNSL KTEDTAMYYCTSVPQGTQDYWGQGTLVTVSS 14 5A11VLC327 aa

SYELTQPSSVSVSPGQTARITCSGDVLSKRYAQWSQQKPGQAIVSV IYKDSERPSGIPERFSGSSSGTTATLTISGAQVEDEADYYCLSTYAD DNLPVFGGGTKLTVL 15 5A11VLC327 CD R.I. SGDVLSKRYAQ 16 5A11VLC327 CD R3 LSTYADDNLPV 17 5All_VHA_Fc_aa EVQLVESGGGLVQPGGSLKLSCAASGFTFSDAAMYWVRQA SGKGLEWVARIRTKTNNYAAHYVESVKGRFTVSRDDSKNT VYLQMNSLKTEDTAMYYCTSVPQGTQDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK** 18 5All_VLC327_Fc_aa SYELTQPSSVSVSPGQTARITCSGDVLSKRYAQWSQQKPGQAIVSV IYKDSERPSGIPERFSGSSSGTTATLTISGAQVEDEADYYCLSTYAD DNLPVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLIS DFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTP EQWKSHRSYSCQ VTHEGSTVEKTVAPTECS*

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Claims (16)

CLAIM 1. An antibody or antigen-binding fragment thereof that binds to CRTAM, wherein said antibody or antigen-binding fragment comprises a heavy chain variable region comprising: CDR-H1 sequence containing SEQ ID NO:5; CDR-H2 sequence containing SEQ ID NO:6; and a CDR-H3 sequence containing SEQ ID NO:7, and a light chain variable region containing: a CDR-L1 sequence comprising a sequence selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:15; CDR-L2 sequence containing SEQ ID NO:9; and a CDR-L3 sequence comprising a sequence selected from the group consisting of SEQ ID NO:10 and SEQ ID NO:16. 2. An antibody or antigen-binding fragment according to claim 1, characterized in that the light chain variable region contains: CDR-L1 containing the sequence SEQ ID NO:8; CDR-L2 containing the sequence SEQ ID NO:9; and CDR-L3 containing the sequence SEQ ID NO:10. 3. The antibody or antigen-binding fragment according to claim 1, characterized in that the light chain variable region contains: CDR-L1 containing the sequence SEQ ID NO:15; CDR-L2 containing the sequence SEQ ID NO:9; and CDR-L3 containing the sequence SEQ ID NO:16. 4. An antibody or antigen-binding fragment thereof that binds to CRTAM, wherein said antibody or antigen-binding fragment comprises: i) 3 heavy chain CDRs from SEQ ID NO:1 and 3 light chain CDRs from SEQ ID NO:2 or ii) 3 heavy chain CDRs from SEQ ID NO:13 and 3 light chain CDRs from SEQ ID NO:14; where CDRs are given according to the Kabat or Chothia numbering system. 5. Antibody or antigen-binding fragment thereof according to any one of paragraphs. 1-4, containing: a heavy chain variable region containing a sequence at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the sequence of SEQ ID NO:13, and a light chain variable region containing a sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the sequence of SEQ ID NO:14. 6. The antibody or antigen binding fragment thereof according to claim 5, comprising a heavy chain variable region comprising SEQ ID NO:13 and a light chain variable region comprising SEQ ID NO:14. 7. The antibody or antigen-binding fragment thereof according to any of the preceding claims, characterized in that the antibody or antigen-binding fragment is a monoclonal antibody. 8. The antibody or antigen binding fragment thereof according to claim 7, wherein the antibody or antigen binding fragment is a chimeric, humanized antibody or antigen binding fragment, or a human antibody or antigen binding fragment. 9. The antibody or antigen binding fragment thereof as claimed in any one of the preceding claims, wherein the antibody or antigen binding fragment is a recombinant IgG1 antibody or antigen binding fragment produced under Fc silencing conditions and characterized by reduced or absent binding to one or more Fc receptors. 10. An antibody or an antigen-binding fragment thereof according to any of the previous claims, characterized in that the antibody or antigen-binding fragment is capable of inducing and/or enhancing the cytotoxicity of T cells. 11. An antibody or an antigen-binding fragment thereof according to any of the previous paragraphs, characterized in that the antigen-binding fragment is selected from the group consisting of: Fab, Fab', F(ab) ₂ , F(ab') ₂ , Fv, scFv and single-domain antibody . 12. A polynucleotide encoding the variable region of the heavy chain of an antibody or antigen-binding fragment according to any of the previous paragraphs. 13. A polynucleotide encoding the variable region of the light chain of an antibody or antigen-binding fragment according to any of the previous paragraphs. 14. An expression vector containing a polynucleotide according to claim 12 and a polynucleotide according to claim 13. 15. Host cell containing: i) an expression vector containing a polynucleotide according to claim 12 and a polynucleotide according to claim 13; or ii) a first expression vector containing a polynucleotide according to claim 12, and a second expression vector containing a polynucleotide according to claim 13. 16. A method for producing an antibody or an antigen-binding fragment thereof, comprising culturing a host cell according to claim 15 under conditions in which the antibody or antigen-binding fragment is expressed in the host cell. -