CN117980324A

CN117980324A - Antigen binding proteins that specifically bind CT45

Info

Publication number: CN117980324A
Application number: CN202280064076.3A
Authority: CN
Inventors: S·优素福; F·布伦克; A·莫里茨; S·邦克; C·瓦格纳; D·毛雷尔; F·翁弗多尔本
Original assignee: Immatics Biotechnologies GmbH
Current assignee: Immatics Biotechnologies GmbH
Priority date: 2021-07-27
Filing date: 2022-07-27
Publication date: 2024-05-03

Abstract

The present invention provides an antigen binding protein that specifically binds to a CT45 antigen peptide, the CT45 antigen peptide being in a complex with a Major Histocompatibility Complex (MHC) protein, wherein the CT45 antigen peptide comprises or consists of the amino acid sequence of SEQ ID No. 138 (KIFEMLEGV) and wherein the antigen binding protein comprises a first polypeptide comprising a variable domain VA comprising complementarity determining regions CDRa1, CDRa2 and CDRa3 and a second polypeptide comprising a variable domain V _B comprising CDRb1, CDRb2 and CDRb 3. Nucleic acids encoding the antigen binding proteins, vectors comprising the nucleic acids, recombinant cells expressing the antigen binding proteins, and pharmaceutical compositions comprising the antigen binding proteins are also provided. The invention further provides said antigen binding proteins for use in medicine and a method of producing said antigen binding proteins.

Description

Antigen binding proteins that specifically bind CT45

The present invention relates to antigen binding proteins directed against antigens derived from CT45 protein, in particular antigen binding proteins which specifically bind to CT45-IP antigen peptides expressed by tumors in the form of complexes with MHC. Antigen binding proteins are provided for use in the diagnosis, treatment and prevention of proliferative diseases that express CT 45. Further provided are nucleic acids encoding the antigen binding proteins, vectors comprising the nucleic acids, recombinant cells expressing the antigen binding proteins, and pharmaceutical compositions comprising the antigen binding proteins.

Background

T cell-based immunotherapy targets peptide epitopes derived from tumor-associated or tumor-specific proteins, which are presented by molecules of the Major Histocompatibility Complex (MHC). These tumor-associated antigens (TAAs) may be peptides derived from all classes of proteins (e.g., enzymes, receptors, transcription factors, etc.) that are specifically expressed by and/or up-regulated in cancer cells. Unlike CAR-T therapies and current antibody-based approaches that target only cell surface proteins, T cell-based immunotherapy enables targeting of otherwise unreachable intracellular proteins and thus significantly increases the number and diversity of targets.

"Cancer testis antigen 45 (CT 45)" is a polygene family of nine nearly identical genes (commonly designated A1, A2, A3, A5, A6, A7, A8, A9 and A10) that are directly tandem repeats. All nine CT45 genes encode a putative 189 amino acid protein. CT45A1 protein, which is usually expressed only in testicular germ cells, is shown to be also expressed in lung, breast and ovarian cancers (Chen, Y.T. et al, int.J Cancer 124 (2009): 2893-2898). CT45A1 has also been shown to correlate with poor prognosis and poor outcome for multiple myeloma (Andrade, V.C. et al, exp. Hematol.37 (2009): 446-449). CT45A1 is described as a gene that upregulates epithelial-mesenchymal transition (EMT) and a metastatic gene that promotes EMT and tumor spread. In addition, CT45A1 is described as involving initiation or maintenance of cancer stem cell-like cells, thereby promoting tumorigenesis and malignant progression (Yang, P. Et al, curr. Pharm. Des 21 (2015): 1292-1300). CT45A1 overexpression has been shown to lead to upregulation of various oncogenes and metastatic genes, constitutively activated ERK and CREB signaling pathways, and increased tumorigenesis, invasion and metastasis in breast cancer models. Silencing of CT45A1 was shown to reduce cancer cell migration and invasion. CT45A2 was shown to be a novel splicing MLL fusion partner in pediatric patients with neonatal dual phenotype acute leukemia, and thus may be involved in leukemia development (Cerveira, N.et al, BMC. Cancer 10 (2010): 518). CT45 was shown to be frequently expressed in cancer cell lines and lung cancer samples (Chen, L. Et al CANCER RES (2005): 5599-5606). CT45 is an attractive target for immunotherapeutic intervention because it is expressed in normal adult tissue with limited or no expression.

The development of new anticancer agents that specifically recognize intracellular targets in complex with MHC is one of the most important keys to address difficult to treat cancers, particularly solid tumors. Thus, there is a need to develop new anticancer agents that specifically target intracellular proteins that are highly specific for cancer cells. The present invention addresses this need by providing: novel antigen binding proteins that specifically bind to CT45 antigen peptide comprising the amino acid sequence of SEQ ID NO 138 (KIFEMLEGV), and methods of using such molecules in the treatment of proliferative diseases, particularly cancer. The antigen binding proteins of the invention are characterized by high stability, high affinity, high functional avidity, high efficacy and high specificity. In contrast to the previously described antigen binding proteins that bind to CT45 antigen peptides, the antigen binding proteins of the invention exhibit at least one of the following: increased stability, increased affinity, increased functional affinity, increased efficacy, and/or increased specificity. Thus, the antigen binding proteins of the invention are both more potent and safer than the antigen binding proteins of the prior art.

Disclosure of Invention

In a first aspect, the invention relates to an antigen binding protein that specifically binds to a CT45 antigen peptide, said CT45 antigen peptide being in a complex with a Major Histocompatibility Complex (MHC) protein, wherein said CT45 antigen peptide comprises or consists of the amino acid sequence of SEQ ID No. 138 (KIFEMLEGV), and wherein said antigen binding protein comprises a first polypeptide comprising a variable domain V _A comprising Complementarity Determining Regions (CDRs) CDRa1, CDRa2 and CDRa3, and a second polypeptide comprising a variable domain V _B comprising CDRb1, CDRb2 and CDRb3, wherein

CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 80, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 82, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 85, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 87,

CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 71, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 72, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 75, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 77,

CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 24, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 63, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 66, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 68,

CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 90, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 92, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 66, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 96,

CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO.2, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 4, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 8, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 10,

CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 53, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 55, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 58, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 60,

CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 24, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 133, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 75, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 136,

CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 99, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 101, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 75, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 104,

CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 14, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 16, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 19, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 21,

CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 107, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 109, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 112, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 114,

CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 125, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 127, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 112, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 130, or

CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 117, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 119, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 58, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 122,

CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 24, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 35, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 38, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 40,

CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 24, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 26, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 29, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 31, or

CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 43, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 45, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 48, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 50,

Wherein the antigen binding protein comprises the CDRa1, CDRa3, CDRb1 and CDRb3 sequences having no more than one, two or three amino acid mutations.

In a second aspect, the invention relates to a nucleic acid comprising a sequence encoding the antigen binding protein of the first aspect of the invention.

In a third aspect, the invention relates to a vector comprising a nucleic acid of the second aspect of the invention.

In a fourth aspect, the invention relates to a host cell comprising an antigen binding protein of the first aspect of the invention, a nucleic acid of the second aspect of the invention, or a vector of the third aspect of the invention.

In a fifth aspect, the invention relates to a pharmaceutical composition comprising an antigen binding protein of the first aspect of the invention, a nucleic acid of the second aspect of the invention, a vector of the third aspect of the invention or a host cell of the fourth aspect of the invention and optionally a pharmaceutically acceptable carrier.

In a sixth aspect, the present invention relates to a method of producing an antigen binding protein according to the first aspect of the invention, the method comprising the steps of: (a) providing a host cell, (b) providing a genetic construct comprising a coding sequence encoding an antigen binding protein of any of the first aspects of the invention, (c) introducing the genetic construct into the host cell, and (d) expressing the genetic construct from the host cell.

In a seventh aspect, the invention relates to an antigen binding protein of the first aspect of the invention, a nucleic acid of the second aspect of the invention, a vector of the third aspect of the invention, a host cell of the fourth aspect of the invention, or a pharmaceutical composition of the fourth aspect of the invention for use in medicine.

In an eighth aspect, the invention relates to an antigen binding protein of the first aspect of the invention, a nucleic acid of the second aspect of the invention, a vector of the third aspect of the invention, a host cell of the fourth aspect of the invention, or a pharmaceutical composition of the fourth aspect of the invention, for use in a method of treating and/or diagnosing a proliferative disorder.

In a ninth aspect, the invention relates to an in vitro method for detecting cancer in a biological sample, said in vitro method comprising the steps of: (a) Contacting the biological sample with an antigen binding protein of the first aspect of the invention, and (b) detecting binding of the antigen binding protein to the biological sample.

Definition of the definition

"CT45 antigenic peptide" comprises or consists of amino acid sequence KIFEMLEGV (SEQ ID NO: 138) corresponding to amino acids 143-151 of CT45A1 (as available under Uniprot accession number Q5HYN 5: 749), CT45A2, CT45A3, CT45A5, CT45A6, CT45A7, CT45A8, CT45A9 and CT45A 10. CT45 antigen peptides are also referred to herein as "CT45 peptides" or "CT45-IP". CT45 antigenic peptides are peptide epitopes derived from tumor-associated proteins or tumor-specific proteins and are presented on the cell surface by molecules of the Major Histocompatibility Complex (MHC), preferably MHC I. More particularly, CT45 antigen peptides are presented on the cell surface as complexes with HLA proteins (preferably HLA-A, more preferably HLA-A.times.02). In a most preferred embodiment, the CT45 antigen peptide consists of amino acid sequence KIFEMLEGV (SEQ ID NO: 138). In case the CT45 antigen peptide comprises other amino acids than amino acid sequence KIFEMLEGV (SEQ ID NO: 138), it is preferred that the total length of the CT45 antigen peptide is not more than 30 or 20 amino acids, more preferably not more than 15 amino acids, even more preferably not more than 12 amino acids. In the case where the CT45 antigen peptide comprises other amino acids than SEQ ID NO:138, when the antigen peptide is in a complex with an MHC protein, the amino acid of SEQ ID NO:138 is preferably located within the peptide binding groove of the MHC protein. Those skilled in the art will appreciate that antigen peptides presented on MHC I are typically no more than 12 amino acids. However, in the case where peptides are artificially loaded on MHC proteins, it seems reasonable that the antigen peptides artificially loaded on MHC I may be longer than 12 amino acids. The term "antigen" or "target antigen" as used herein refers to a molecule or part of a molecule or complex that is capable of being bound by an antigen binding site, wherein the antigen binding site is present in an antigen binding protein (preferably an antigen binding protein of the invention). In the context of the present invention, the antigen is a CT45 antigenic peptide, more particularly a CT45 antigenic peptide in a complex with an MHC protein (such as an HLA protein, for example HLA-a x 02).

As used herein, "cells presenting CT45-IP: MHC complex" refers to cells presenting CT45-IP as a complex with an MHC molecule on their surface. In a preferred embodiment, the cells presenting the CT45-IP: MHC complex are tumor cells, wherein the tumor is preferably a cancer as defined below in section "methods of treatment and uses". In the context of the present invention, the complex is over-presented on the cell surface of cells presenting the CT45-IP: MHC complex, compared to the level of the CT45-IP: MHC complex on the surface of cells in normal (healthy) tissue (also referred to as "healthy cells") or on the surface of control cells carrying different antigen presenting peptides or not carrying peptides. By "over-presentation" is meant that the CT45-IP: MHC complex is present at a level of at least 2-fold (preferably between 5-fold and 10-fold) of the level present in healthy tissue or control cells.

Examples of "cells presenting CT45-IP: MHC complex" are T2 cells carrying CT45-IP or NCIH.sup.1703 or A375 tumor cells used in the examples of the present application.

A "domain" may be any region of a protein, generally defined based on sequence homology, and often refers to a particular structural or functional entity.

In the context of the present invention, the term "immunoglobulin (Ig) domain" refers to a protein domain consisting of a 2-layer sandwich structure of 7-9 antiparallel β -strands arranged in a greek key topology into two β -sheets. Ig domains are probably the most commonly used "building blocks" in naturally occurring proteins. Proteins containing Ig domains are included in the immunoglobulin superfamily, including, for example, antibodies, T Cell Receptors (TCRs), and cell adhesion molecules. Examples of Ig domains are the variable and constant domains of antibodies and TCRs.

In the context of the present invention, "V _A" refers to a TCR variable domain comprising a TCR-derived CDR sequence and a TCR-derived framework sequence. The CDR and framework sequences may be derived from the variable domains of the TCR alpha chain (V _α), beta chain (V _β), gamma chain (V _γ) or delta chain (V _δ), preferably from V _α. In the context of the present invention, the CDR and framework sequences of the V _A domain do not necessarily have to be derived from the same TCR chain. In some embodiments, CDRs derived from one TCR variable domain (of the donor TCR) are grafted onto another TCR variable domain (of the recipient TCR). For example, the donor TCR may comprise V _α encoded by TRAV5 and TRAJ17, and the acceptor TCR may comprise V _A encoded by TRAV14 and TRAJ 33. If CDR1, CDR3 and optionally CDR2 of the donor TCR are grafted onto the acceptor TCR, the CDRs will be present in the context of the different framework regions, but the affinity and specificity of the antigen peptide delivered by the CDRs will not change, i.e., after grafting, the variable domain of the acceptor TCR will have substantially the same affinity and specificity of the antigen peptide as the variable domain of the donor TCR.

In the context of the present invention, "V _B" refers to a variable domain comprising a TCR-derived CDR sequence and a TCR-derived framework sequence. The CDR and framework sequences may be derived from the variable domains of the TCR alpha chain (V _α), beta chain (V _β), gamma chain (V _γ) or delta chain (V _δ), preferably from V _β. In the context of the present invention, the CDR and framework sequences of the V _B domain do not necessarily have to originate from the same TCR. In some embodiments, CDRs derived from one TCR variable domain (of the donor TCR) are grafted onto another TCR variable domain (of the recipient TCR). For example, the donor TCR can comprise V _v encoded by TRBV2 and TRBJ2-1, and the recipient TCR can comprise V _A encoded by TRBV27 and TRBJ 1-5. If CDR1, CDR3 and optionally CDR2 of the donor TCR are grafted onto the acceptor TCR, the CDRs will be present in the context of the different framework regions, but the affinity and specificity of the antigen peptide delivered by the CDRs will not change, i.e., after grafting, the variable domain of the acceptor TCR will have substantially the same affinity and specificity of the antigen peptide as the variable domain of the donor TCR.

CDRs may be exchanged/grafted not only between different α variable domains or between different β variable domains, but also from tcrα to tcrβ, γ or δ variable domains, or from tcrβ to tcrα, γ or δ variable domains.

In the context of the present invention, V _α refers to the variable domain of the TCR alpha chain.

In the context of the present invention, V _β refers to the variable domain of the TCR β chain.

In the context of the present invention V _γ refers to the variable domain of the TCR gamma chain.

In the context of the present invention, V _δ refers to the variable domain of the TCR delta chain.

In the context of the present invention, V _L refers to the variable domain of an antibody light chain.

In the context of the present invention, V _H refers to the variable domain of an antibody heavy chain.

In the context of the present invention, C _L refers to the constant domain of an antibody light chain.

In the context of the present invention, C _H1、C_H2 and C _H3 refer to constant domains of antibody heavy chains, in particular IgG heavy chains.

The term "epitope", also known as an antigenic determinant, is the part of an antigen that is recognized by the immune system. As used herein, the term epitope includes the terms "structural epitope" and "functional epitope". "structural epitopes" are those amino acids in an antigen (e.g., peptide-MHC complex) that are covered by an antigen binding protein when the antigen binding protein binds to an antigen. Typically, the antigen is at any atom of the amino acid of the antigen binding proteinAll amino acids within are considered to be covered. Structural epitopes of antigens can be determined by methods known in the art, including X-ray crystallography or NMR analysis. The structural epitope of an antibody typically comprises 20 to 30 amino acids. The structural epitope of the TCR typically comprises 20 to 30 amino acids. "functional epitopes" as defined herein are a subset of those amino acids forming structural epitopes and comprise amino acids in an antigen that are critical for the formation of an interface with the antigen binding protein of the invention or functional fragment thereof (either by direct formation of non-covalent interactions such as H-bonds, salt bridges, aromatic ring stacking or hydrophobic interactions, or by indirect stabilization of the binding conformation of the antigen) and are determined, for example, by mutation scanning. In the context of the present invention, a functional epitope is also referred to as a "binding motif". Typically, the functional epitope in an antigen bound by an antibody comprises between 4 and 6 amino acids. Typically, the functional epitope of the peptide-MHC complex comprises between 2 and 6 or 7 amino acids of the peptide and between 2 and 7 amino acids of the MHC molecule. Since peptides presented by MHC I typically have a length of between 8 amino acids and 10 amino acids, only a subset of the amino acids of each given peptide are part of the functional epitope of the peptide-MHC complex. The epitopes bound by the antigen binding proteins of the invention, in particular functional epitopes, comprise or consist of the amino acids in the antigen required to form a binding interface.

A "major histocompatibility complex" (MHC) is a group of cell surface proteins necessary for the recognition of foreign molecules by the adaptive immune system in vertebrates, which in turn determine tissue compatibility. The main function of MHC molecules is to bind to antigens derived from pathogens and display them on the cell surface for proper T cell recognition. Human MHC is also known as HLA (human leukocyte antigen) complex (or HLA alone). Thus, in a preferred embodiment, the MHC is an HLA. MHC gene families are divided into three subgroups: class I, class II and class III. Complexes of peptides with MHC class I molecules (MHC I) are typically recognized by CD8 positive T cells (cd8+ T cells) carrying the appropriate T Cell Receptor (TCR), while complexes of peptides with MHC class II molecules (MHC II) are typically recognized by CD4 positive helper T cells (cd4+ T cells) carrying the appropriate TCR. CD4 and CD8 generally function as accessory receptors for TCRs in binding to MHC I and MHC II, respectively. In some exceptional cases, complexes of peptides with MHC I are recognized by CD8 negative (in particular CD8 negative, CD4 positive) T cells (Soto et al, 2013,Cancer Immunol Immunother.2013, month 2; 62 (2): 359-369). Since the responses of CD 8-positive and CD 4-positive T cells promote anti-tumor effects in combination and synergistically, the identification and characterization of tumor-associated antigens and corresponding T cell receptors is of great importance in the development of cancer immunotherapy (e.g. vaccines) and cell therapies. The HLA-A gene is located on the short arm of chromosome 6 and encodes the larger component alpha chain of HLA-A. Variation of HLA-A alpha chains is critical for HLa function. This variation promotes genetic diversity in the population. Because each HLA has a different affinity for certain structural peptides, a larger class of HLA means that a larger class of antigen is "presented" on the cell surface. In the context of the present disclosure, MHC class I HLA proteins may be HLA-a, HLA-B or HLA-C proteins, suitably HLA-a proteins, e.g. HLA-a x 02. In an MHC class I-dependent immune response, peptides must not only be able to bind to certain MHC class I molecules expressed by tumor cells, they must then also be recognized by T cells carrying specific T Cell Receptors (TCRs).

By "antigenic peptide in complex with an MHC protein" is meant herein an antigenic peptide that is non-covalently bound to an MHC molecule. In particular, the antigenic peptides are localized to the "peptide binding groove" formed by the MHC molecules. Complexes of MHC molecules with antigenic peptides are also referred to herein as "peptide-MHC complexes" or "pMHC complexes". In the case of CT45 antigen peptide, the complex is also referred to as "CT45 antigen peptide-MHC complex" or "CT45-IP: MHC complex".

"HLA-A x 02" denotes a particular HLa allele, wherein the letter a denotes the allele and the prefix ". Times.02 prefix" denotes the A2 serotype.

The term "antigen binding protein" herein refers to a polypeptide or a complex of two or more polypeptides comprising an antigen binding site capable of specifically binding to an antigen, in particular an antigenic peptide in complex with MHC. As used in the context of this specification, the term antigen binding protein includes a variety of different forms of antigen binding proteins, including soluble antigen binding proteins, membrane-bound antigen binding proteins, monovalent, bivalent and multivalent antigen binding proteins, monospecific, bispecific and multispecific antigen binding proteins, single-chain antigen binding proteins and antigen binding proteins comprising two or more chains, fusion proteins and chimeric proteins, as described below. The term includes antigen binding proteins having the overall structure of a TCR, antibody or Chimeric Antigen Receptor (CAR). The antigen binding proteins of the invention comprise TCR-derived CDRs, in particular variable domain V _A comprising TCR-derived CDRa1, CDRa3 and optionally CDRa2, and variable domain V _B comprising TCR-derived CDRb1, CDRb3 and optionally CDRb 2. In particular embodiments, the complete V _A domain and/or the complete V _B domain is TCR-derived and thus is a TCR α, β, γ or δ variable domain (V _α、V_β、V_γ or V _δ). In a preferred embodiment, the antigen binding protein is a TCR. In some embodiments, the antigen binding proteins of the invention comprise V _A and V _B as defined herein, and further comprise an additional domain fused directly or indirectly to V _A or V _B. Such antigen binding proteins may be referred to as "fusion proteins". Examples of additional domains contained in the antigen binding proteins of the invention as "fusion proteins" are listed below. If the antigen binding protein is a bispecific or multispecific antigen binding protein, it comprises (in addition to V _A and V _B as defined herein) at least one further variable domain, preferably two variable domains and optionally a constant domain, wherein the variable domain and/or the constant domain may be derived from an antibody or TCR. The antigen binding protein thus comprises two different antigen binding sites (one formed by V _A and V _B and one formed by another at least one (preferably two) variable domain) and is capable of binding specifically to two different antigens simultaneously, as is known from e.g. bispecific antibodies. In some embodiments, the antigen binding protein comprises TCR-derived V _A and V _B and two additional antibody-derived variable domains (particularly V _L and V _H). Such constructs comprising elements of both antibody and TCR represent hybrid forms and may for example be referred to as "bispecific TCR-antibody fusion proteins". In such bispecific fusion proteins, the variable domains can be arranged in various orientations. Techniques for producing such bispecific fusion proteins are known to those skilled in the art, and thus the variable domains as defined herein can be readily used by the skilled artisan to generate and produce various forms of bispecific antigen binding proteins. Those skilled in the art are well able to select an appropriate linker to ensure folding in the desired conformation.

"At least one" herein refers to one or more specified objects, such as 1,2, 3, 4, 5, or 6 or more specified objects. For example, at least one binding site herein refers to 1,2, 3, 4, 5, or 6 or more binding sites.

In the context of the present invention, the term "bispecific" refers to an antigen binding protein having at least two valencies and binding specificities for two different antigens, thus comprising at least two antigen binding sites. The term "valency" refers to the number of binding sites in an antigen binding protein, e.g., a bivalent antigen binding protein refers to an antigen binding protein having two binding sites. The binding sites may bind to the same or different targets, i.e. the bivalent antigen binding protein may be monospecific, i.e. bind to one target, or bispecific, i.e. bind to two different targets. The antigen binding molecules of the invention comprise at least one antigen binding site comprising TCR-derived CDRs. In a preferred embodiment, the antigen binding molecules of the invention comprise at least one TCR-derived antigen binding site.

Preferably, the antigen binding protein is a TCR. The term "TCR" as used herein includes native TCRs and engineered TCRs.

"Native TCR" refers to a wild-type TCR that can be isolated from nature. Native TCRs are heterodimeric cell surface proteins of the immunoglobulin superfamily that are associated with invariant proteins of the CD3 complex involved in mediating signal transduction. Native heterodimeric TCRs exist in αβ and γδ forms that are structurally similar but have different positions and possible functions. The native, full length αβ heterodimeric TCR consists of an α chain and a β chain. The alpha chain comprises a variable region (V region) encoded by the TRAV gene, a junction region (J region) encoded by the TRAJ gene and a constant region (C region) encoded by the TRAC gene. The beta strand comprises a variable region (V region) encoded by the TRBV gene, a junction region (J region) encoded by the TRBJ gene, and a constant region (C region) encoded by the TRBC gene, and a short variable region (D region) encoded by the TRBD gene, typically between the V region and the J region, but this D region is often considered part of the J region (Lefranc, (2001), curr Protoc Immunol Appendix 1:appdix 10). Genes encoding the various alpha and beta chain variable, linking and constant regions are referred to by unique numbers in the IMGT nomenclature (Folch and Lefranc, (2000), exp Clin Immunogenet (1): 42-54; scaniner and Lefranc, (2000), exp Clin Immunogenet (2): 83-96; lebranc and LeFranc, (2001), "T cell Receptor Factsbook", ACADEMIC PRESS). Additional information about the TCR gene can be found in international ImMunoGeneTics informationLefranc M-P et al (Nucleic Acids Res.2015, month 1; 43 (Database issue): D413-22; and http:// www.imgt.org /).

At the protein level, TCR alpha, beta, gamma and delta chains comprise two immunoglobulin domains, i.e. a variable domain and a constant domain. The variable domain corresponds to the V (D) J region. The constant domain corresponds to the C region. The constant domain is a membrane proximal domain and in the context of the present invention also includes a Transmembrane (TM) domain and a short cytoplasmic tail. The constant domain and the variable domain each comprise an intrachain disulfide bond. The variable domains (V _α and V _β in αβ TCRs and V _γ and V _δ in γδ TCRs) contain highly polymorphic loops comprising Complementarity Determining Regions (CDRs).

Each TCR variable domain comprises three "TCR Complementarity Determining Regions (CDRs)" embedded in a framework sequence, one hypervariable region designated CDR 3. In the context of the present invention, CDRa1, CDRa2 and CDRa3 represent the α chain CDRs and CDRb1, CDRb2 and CDRb3 represent the β chain CDRs. The sequences encoding CDRa1 and CDRa2 are contained in TRAV, the sequences encoding CDRa3 are contained in TRAV and TRAJ, the sequences encoding CDRb1 and CDRb2 are contained in TRBV, and the sequences encoding CDRb3 are contained in TRBV, TRBD, and TRBJ. In TCRs, CDR1 and CDR3 amino acid residues are contacted with the antigenic peptide, while CDR2 amino acid residues are predominantly contacted with the HLA molecule (STADINSKI et al, JImmunol.2014, month 6, 15; 192 (12): 6071-6082; cole et al, J Biol chem.2014, month 1, 10; 289 (2): 628-38). The antigen specificity of a TCR is thus defined by CDR3 and CDR1 sequences. CDR2 sequences are not required for determination of antigen specificity, but may play a role in the overall affinity of the TCR for peptide-MHC complexes.

"TCR framework regions" (FR) refer to amino acid sequences inserted between CDRs, i.e., those portions of the variable domain that are conserved to some extent between different TCRs. The α, β, γ and δ chain variable domains each have four FRs, designated herein as FR1-a, FR2-a, FR3-a, FR4-a (for the α or γ chain), and FR1-b, FR2-b, FR3-b, FR4-b (for the β or δ chain), respectively. Thus, an α -chain or γ -chain variable domain can be described as (FR 1-a) - (CDRa 1) - (FR 2-a) - (CDRa 2) - (FR 3-a) - (CDRa 3) - (FR 4-a), and a β -chain or δ -chain variable domain can be described as (FR 1-b) - (CDRb 1) - (FR 2-b) - (CDRb 2) - (FR 3-b) - (CDRb 3) - (FR 4-b). In the context of the present invention, the CDR/FR sequences in the variable domains of the alpha, beta, gamma or delta chain are determined based on the IMGT definition (Lefranc et al, dev. Comp. Immunol.,2003,27 (1): 55-77; www.imgt. Org). Thus, when related to TCR or TCR-derived domains, CDR/FR amino acid positions are expressed according to the IMGT definition. Preferably, the IMGT positions of the CDR/FR amino acid positions of variable domain V _α are given by analogy with the IMGT numbers of TRAV24 x 01 and/or the IMGT positions of the CDR/FR amino acid positions of variable domain V _β are given by analogy with the IMGT numbers of TRBV12-3 x 01.

An "engineered TCR" may be a protein very similar to a native TCR, but comprising minor modifications in the variable and/or constant domains, e.g. a humanized TCR or a TCR with improved heterodimerization or expression levels, or may be a single chain TCR, a soluble TCR, a monovalent, divalent or multivalent TCR, a monospecific, bispecific or multispecific TCR, a functional fragment of a TCR, or a fusion or chimeric protein comprising a functional fragment of a TCR.

"Functional fragment of a TCR" refers to a TCR fragment that retains or substantially retains the affinity, functional avidity, and/or specificity of the parent TCR from which it is derived for a target antigen. In this context, the "parent TCR" refers to the full length TCR from which the functional fragment is derived. Because binding to the target antigen peptide is defined by CDR1 and CDR3 sequences and binding to the target antigen peptide MHC complex is defined by CDR1, CDR2 and CDR3, an antigen binding protein comprising CDR1 and CDR3 and optionally CDR2 sequences of the parent TCR retains the affinity, functional avidity and/or specificity of the parent TCR for the target antigen. Those skilled in the art know that CDRs must be interspersed with Framework Regions (FR), however the specific amino acid sequences of the framework regions are not directly involved in target antigen specificity. The ions of the functional TCR fragment include a single variable domain, such as a TCR α, β, γ, or δ variable domain, or a fragment of such an α, β, δ, or γ chain, such as an α, β, δ, or γ chain without a transmembrane domain and a short cytoplasmic tail. The term "fragment" as used herein refers to naturally occurring fragments (e.g., splice variants or peptide fragments) as well as artificially constructed fragments, particularly those obtained by genetic means.

For example, a functional fragment of a TCR is considered to retain or substantially retain affinity for a target antigen if K _D binding to the target antigen is the same as or increases or decreases (preferably decreases) by no more than 10x, 5x, 3x or 2x as measured as outlined below with K _D of the parent TCR.

For example, a functional fragment of a TCR is considered to retain or substantially retain functional avidity for a target antigen if the functional avidity for the target antigen is the same as, or increases or decreases (preferably decreases) by no more than 50%, 40%, 30%, 20%, 15%, 10%, 8%, 5%, 3%, 2% or 1% of the parent TCR. Specifically, for example, a functional fragment of a TCR is considered to retain or substantially retain functional affinity for a target antigen if the cytotoxic activity of the target in response to the parent protein is the same as or increases or decreases (preferably decreases) by no more than 50%, 40%, 30%, 20%, 15%, 10%, 8%, 5%, 3%, 2% or 1% (preferably 10%, 8%, 5%, 3%, 2% or 1%) as measured in a cytotoxicity assay (preferably a luciferase release assay) as described below.

A functional fragment of a TCR is considered to retain or substantially retain specificity for (i.e., the ability to specifically bind to) a target antigen if the functional fragment does not bind significantly to a peptide other than the target antigen peptide of the parent TCR.

The term "α/β TCR" or "γ/δ TCR" refers to a TCR comprising an α chain and a β chain, or a γ chain and a δ chain, respectively, as described above. Such TCRs may also be described as "full length TCRs" or "conventional TCRs. The α/β TCR or γ/δ TCR may be a native TCR or may be an engineered TCR that retains the structure of a native TCR, i.e., an engineered TCR comprising minor modifications in the variable and/or constant domains as described above, such as a humanized TCR.

As used herein, "single chain TCR (scTCR)" refers to a TCR in which the variable domain of the TCR is located on a single polypeptide. Typically, the variable domains in sctcrs are separated by a linker, wherein the linker typically comprises 10 to 30 amino acids, such as 25 amino acids.

"Chimeric protein" herein refers to a protein comprising sequences from multiple species. "chimeric TCR" herein refers to a TCR comprising sequences from multiple species. Preferably, a chimeric TCR in the context of the present invention may comprise an alpha chain comprising at least one domain from a human and one domain from a mouse. More preferably, a chimeric TCR in the context of the present invention may comprise an alpha chain comprising a variable domain of a human alpha chain and a constant domain of, for example, a murine TCR alpha chain.

The term "antibody" as used herein is intended to include both natural antibodies and engineered antibodies. The term "engineered antibody" includes functional antibody fragments, single chain antibodies, single domain antibodies, bispecific or multispecific antibodies.

A natural "antibody" comprises two heavy chains and two light chains, wherein the heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by disulfide bonds. There are two types of light chains, namely Lanbuda (lambda) and Kapa (kappa). There are five major heavy chain classes (or isotypes) that determine the functional activity of an antibody molecule: igM, igD, igG, igA and IgE. Each chain contains a different domain (also referred to as a region). The light chain comprises two domains, the variable domain (V _L) and the constant domain (C _L). The heavy chain comprises four or five domains, depending on the antibody isotype; i.e., a variable domain (V _H) and three or four constant domains (C _H1、C_H2 and C _H3, and optionally C _H4, collectively C _H). The variable domains of both the light chain (V _L) and the heavy chain (V _H) determine the binding recognition and specificity to an antigen. The constant domains of the light chain (C _L) and heavy chain (C _H) confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding and binding to the F _c receptor (F _c R).

The specificity of an antibody exists in the structural complementarity between the binding site of the antibody and the epitope. The antibody binding site is composed of residues primarily from the "complementarity determining regions" (CDRs) or hypervariable regions of the antibody. Occasionally, residues from non-hypervariable regions or Framework Regions (FR) affect the overall domain structure and thus the binding site. CDRs refer to amino acid sequences that together define the binding affinity and specificity of the native Fv region of the native antibody binding site. The light and heavy chains of antibodies each have three CDRs, designated CDR1-L, CDR2-L, CDR-L and CDR1-H, CDR-H, CD R3-H, respectively. Thus, an antibody antigen binding site comprises six CDRs, including sets of CDRs from each of the heavy and light chain V regions. "antibody framework regions" (FRs) refer to amino acid sequences inserted between CDRs, i.e., those portions of the antibody light and heavy chain variable regions that are relatively conserved between different antibodies of a single species. The light and heavy chains of the antibody each have four FRs, designated FR1-L, FR2-L, FR3-L, FR-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Thus, the light chain variable domain may be described as (FR 1-L) - (CDR 1-L) - (FR 2-L) - (CDR 2-L) - (FR 3-L) - (CDR 3-L) - (FR 4-L), and the heavy chain variable domain may be described as (FR 1-H) - (CDR 1-H) - (FR 2-H) - (CD R2-H) - (FR 3-H) - (CDR 3-H) - (FR 4-H). As used herein, a "human framework region" is a framework region that is substantially identical (about 85% or more, particularly 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody. In the context of the present invention, the CDR/FR definitions in the antibody light or heavy chain variable domains are determined based on the IMGT definition (Lefranc et al, dev. Comp. Immunol.,2003,27 (1): 55-77; www. IMGT. Org). Thus, the amino acid sequences of CDR1, CDR2 and CDR3 and the amino acid sequences of FR1, FR2, FR3 and FR4 of a given variable chain are expressed according to the IMGT definition.

Knowing the amino acid sequence of the CDRs of an antibody, TCR or antigen-binding protein of the invention, one skilled in the art can readily determine framework regions, such as TCR framework regions or antibody framework regions. In the case where no CDR is indicated, one skilled in the art can first determine the CDR amino acid sequence based on the IMGT definition of the TCR or the IMGT definition of the antibody, and then determine the amino acid sequence of the framework region.

Engineered antibody forms include functional antibody fragments, single chain antibodies, single domain antibodies, and chimeric, humanized, bispecific or multispecific antibodies. Engineered antibody forms further include constructs in which the light chain variable domain of the antibody may be replaced by the alpha chain variable domain of the TCR and the heavy chain variable domain may be replaced by the beta chain variable domain of the TCR, or vice versa. "functional antibody fragment" refers to a portion of a full-length antibody that retains the ability to bind to its target antigen (particularly affinity and/or specificity for its target antigen). Preferably, the functional antibody fragment comprises the antigen binding or variable region of a full length antibody. Examples of functional antibody fragments include Fv, fab, F (ab ') 2, fab', dsFv, (dsFv) 2, scFv, sc (Fv) 2 and diabodies. The functional antibody fragment may also be a single domain antibody, such as a heavy chain antibody. The term "Fab" refers to an antibody fragment having a molecular weight of about 50,000 daltons and antigen binding activity, wherein about half of the N-terminal side of the H chain and the entire L chain are bound together via disulfide bonds in a fragment obtained by treating IgG with a protease (e.g., papain). Fv fragments are the N-terminal part of antibody Fab fragments and consist of a variable part of one light chain and one heavy chain.

As used herein, the "form" of an antigen binding protein specifies a defined spatial arrangement of domains, in particular variable domains and optionally constant domains. Important features of such antigen binding protein forms are the number of polypeptide chains (single chain, double chain or multiple chain), the type and length of the linker connecting the different domains, the number of variable domains (and thus the number of valences), the number of different variable domains (and thus the number of specificities for different antigens, e.g. bispecific, multispecific), and the order and orientation of the variable domains (e.g. crossed, parallel).

The term "humanized antibody" refers to antibodies that are of wholly or partially non-human origin and that have been modified by substitution of certain amino acids, particularly in the framework regions of the heavy and light chains, to avoid or minimize the immune response in humans. The constant domains of humanized antibodies are mainly the human C _H and C _L domains. Various methods for humanization of antibody sequences are known in the art; see, for example, almagro and Franson (2008) Front biosci.13:1619-1633 for reviews.

In the context of the present application, a sequence "at least 85% identical" to a reference sequence is a sequence which has 85% or more, in particular 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity over its entire length to the entire length of the reference sequence. Proteins consisting of amino acid sequences that are "at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical" to a reference sequence may comprise mutations, such as deletions, insertions and/or substitutions, as compared to the reference sequence. In the case of substitution, a protein consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence may correspond to a homologous sequence derived from another species different from the reference sequence.

In the context of the present application, the "percent identity" may be calculated using global alignment (i.e., comparing two sequences over their entire length). Methods for comparing identity of two or more sequences are well known in the art. For example, a "needle" program can be used that uses the Needman-tumbler global alignment algorithm (Needleman-Wunsch global alignment algorithm) (Needleman and Wunsch,1970J. Mol. Biol. 48:443-453) to find the optimal alignment (including gaps) of the sequences when considering the entire length of the two sequences. The needle program is available, for example, on the ebi.ac.uk web site and is further described in the following publications (EMBOSS:The European Molecular Biology Open Software Suite(2000)Rice,P.Longden,I.and Bleasby,A.Trends in Genetics 16,(6) at pages 276-277). According to the application, the percentage identity between two polypeptides is calculated using EMBOSS: a program in which the "vacancy open" parameter is equal to 10.0, the "vacancy extend" parameter is equal to 0.5, and the Blosum62 matrix is used.

An "amino acid mutation" may be a deletion, insertion, or substitution.

"Amino acid substitutions" may be conservative or non-conservative. In embodiments, a substitution is a conservative substitution, wherein one amino acid is substituted for another amino acid having similar structural and/or chemical properties.

In one embodiment, conservative amino acid substitutions may include substitution of another amino acid with an amino acid of the same class, e.g., (1) non-polar: ala, val, leu, ile, pro, met, phe, trp; (2) uncharged polarity: gly, ser, thr, cys, tyr, asn, gln; (3) acidity: asp, glu; and (4) alkaline: lys, arg, his. Other conservative amino acid substitutions may also be made as follows: (1) aromatic: phe, tyr, his; (2) proton donor: asn, gln, lys, arg, his, trp; and (3) proton acceptor: glu, asp, thr, ser, tyr, asn, gln (see, e.g., U.S. patent No. 10,106,805, the contents of which are incorporated by reference in their entirety).

In another embodiment, conservative substitutions may be made according to table 1. Methods for predicting tolerance to protein modifications can be found, for example, in Guo et al, proc.Natl.Acad.Sci., USA,101 (25): 9205-9210 (2004), the contents of which are incorporated by reference in their entirety.

Table 1: conservative amino acid substitutions

The antigen binding proteins of the invention may comprise synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and may include, for example, aminocyclohexane carboxylic acid, norleucine, α -amino-N-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β -phenylserine β -hydroxyphenylalanine, phenylglycine, α -naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid monoamide, N ' -benzyl-N ' -methyl-lysine, N ' -dibenzyl-lysine, 6-hydroxylysine, ornithine, α -aminocyclopentane carboxylic acid, α -aminocyclohexane carboxylic acid, α - (2-amino-2-norbornane) -carboxylic acid, α, γ -diaminobutyric acid, α, β -diaminopropionic acid, homophenylalanine and α -tert-butylglycine.

In one embodiment, the antigen binding proteins of the invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, for example, a disulfide bridge, or converted to an acid addition salt and/or optionally dimerized or multimerized, or conjugated.

"Covalent linkage" as used herein refers to, for example, disulfide bonds or peptide linkages or covalent linkages via a linker or linker sequence (e.g., a polypeptide linker).

As used herein, the term "linker" refers to one or more amino acid residues inserted between domains or between a domain and an agent that provides sufficient flexibility for a domain or element (e.g., a variable domain of bispecific antigen binding) to fold correctly to form an antigen binding site.

In some embodiments, a linker consisting of 0 amino acids means that the linker is absent. At the amino acid sequence level, linkers are inserted at the transition between the variable domains or between the variable domain and the constant domain (or dimerization domain), respectively. Transitions between domains can be identified because the general size of the antibody domain as well as the TCR domain is well understood. The exact location of the domain transition can be determined by locating peptide stretches that do not form secondary structural elements (e.g., β -sheet or α -helix), as confirmed by experimental data or as can be assumed by modeling or techniques of secondary structure prediction.

The length of the linker may be at least 1 to 30 amino acids, provided that no additional provision is made in the corresponding context. In some embodiments, the linker may be 2-25, 2-20, or 3-18 amino acids long. In some embodiments, the linker may be a peptide of no more than 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids in length. In other embodiments, the linker may be 5-25, 5-15, 4-11, 10-20, or 20-30 amino acids long. In other embodiments, the linker may be about 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids long. In particular embodiments, the linker length may be less than 24, less than 20, less than 16, less than 12, less than 10 (e.g., from 5 to 24, 10 to 24, or 5-10) amino acid residues. In some embodiments, the linker is equal to 1 or more amino acid residues in length, such as more than 1, more than 2, more than 5, more than 10, more than 20 amino acid residues in length, and more than 22 amino acid residues in length. In a preferred embodiment, the linker is a glycine/serine linker, i.e. a linker consisting of or consisting essentially of glycine and serine residues.

The antigen binding proteins of the present disclosure may be synthetic, recombinant, isolated, engineered, and/or purified.

An "engineered" antigen binding protein (particularly an engineered TCR in the context of the present invention) refers to a protein that has been modified by biotechnology methods (particularly by introducing amino acid mutations into the native protein sequence). Such biotechnological methods are well known to those skilled in the art.

By "purified" is meant that the indicated molecule is present, but essentially no other biological macromolecules of the same type are present, in relation to a polypeptide (e.g., an antigen binding protein of the invention) or nucleotide sequence (e.g., encoding an antigen binding protein or functional fragment thereof described herein). The term "purified" as used herein particularly means that at least 75%, 85%, 95% or 98% by weight of the same type of biomacromolecule is present. The term "purified" as used herein may further indicate that the antigen binding protein does not contain DNA, RNA, proteins, polypeptides or cells that would interfere with its therapeutic, diagnostic, prophylactic, research or other uses.

Purified nucleic acid molecule encoding a particular polypeptide means that the nucleic acid molecule is substantially free of other nucleic acid molecules not encoding the subject polypeptide; however, the molecule may include some additional bases or moieties that do not adversely affect the essential characteristics of the composition.

The term "isolated" means altered or removed from a natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide, partially or completely isolated from coexisting materials in its natural state, is "isolated. The isolated nucleic acid or protein may be present in a substantially purified form, or may be present in a non-natural environment (such as, for example, a host cell). The isolated antigen binding protein is substantially free of other antigen binding proteins having different antigen specificities (e.g., an antigen binding protein that specifically binds CT45-IP is substantially free of antigen binding proteins that specifically bind antigens other than CT 45-IP). In addition, the isolated antigen binding protein may be substantially free of other cellular material and/or chemicals.

A "recombinant" molecule is a molecule that has been prepared, expressed, produced, or isolated by recombinant means. Recombinant molecules do not exist in nature.

The term "gene" means a DNA sequence encoding or corresponding to a particular amino acid sequence comprising all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, that determine, for example, the conditions under which the gene is expressed. Some genes (not structural genes) may be transcribed from DNA to RNA but are not translated into amino acid sequences. Other genes may function as regulators of structural genes or as regulators of DNA transcription. In particular, the term gene may be intended for genomic sequences encoding proteins, i.e. sequences comprising regulatory, promoter, intron and exon sequences.

In the context of the present invention, "affinity" is defined by the equilibrium binding between an antigen binding protein and its antigen (i.e. CT45-IP peptide in a complex with an MHC protein). Affinity is typically expressed as equilibrium dissociation constant (K _D).

"K _D" is the equilibrium dissociation constant between an antigen binding protein and its antigen, i.e., the ratio of K _off/k_on. K _D is inversely related to affinity. The K _D value correlates with the concentration of antigen binding protein, and the lower the K _D value, the higher the affinity of the antigen binding protein. The K _D value can be assessed experimentally by a variety of known methods, such as measuring association and dissociation rates using Surface Plasmon Resonance (SPR) or Biological Layer Interferometry (BLI). As known to those skilled in the art, the experimental conditions, such as the buffers used, the protein concentration, used in those experiments can significantly affect the results.

In the context of the present invention, "functional affinity" is defined as a parameter describing the ability of an antigen binding protein (preferably a TCR) to activate effector cells (preferably T cells) upon binding to its target antigen peptide in complex with MHC. Activation of effector cells, preferably T cells, can be measured in a functional assay (in particular a cytokine production assay or a cytotoxicity assay) as described below. In some embodiments, an antigen binding protein is considered to have high functional affinity if EC ₅₀ as determined in a functional assay is low, such as less than about 60nM, less than about 10nM, or less than about 1nM in a cytotoxicity assay as described below, and/or activity as determined in a functional assay is high, such as at least 50%, at least 60%, at least 70%, at least 75%, preferably at least 80%, at least 85%, at least 90%, or at least 95% of the maximum activity as defined in the corresponding functional assay. Depending on the functional assay, the maximum activity may be the activity of a reference protein with a known high functional affinity or the activity of a "maximum cleavage control" as described below.

In the context of the present invention, "efficacy" is defined as a parameter describing the ability of an antigen binding protein (preferably a TCR) to activate effector cells (preferably T cells) to kill cancer cells that present on their surface the target antigenic peptide in complex with MHC. Efficacy can be determined in functional assays, particularly in live cell monitoring cytotoxicity assays as described below.

In a "functional assay", the antigen binding protein is expressed, for example, in "effector cells (E)", and the effector cells are co-cultured with "target cells (T)" (i.e., with antigen presenting cells presenting peptide-MHC complexes). Thus, functional assays can also be described as "co-culture assays". For all cell culture assays described herein, the cell culture temperature is preferably about 37 ℃. Preferably, the antigen binding protein is a TCR and the effector cell is a T cell. The target cell may be a cell artificially loaded with the antigen peptide (e.g., T2 cell), or may be a cell that endogenously presents the target antigen peptide on its surface (e.g., a cancer cell expressing CT 45). Binding of the antigen binding protein to the peptide-MHC complex results in activation of effector cells. Depending on the type of functional assay, there are different readings of the degree of activation measured. In cytokine production assays, the production of cytokines (e.g., TNF- α, IFN- γ, CD107a+, IL-2, and/or granzyme B) by effector cells is determined. In a cytotoxicity assay, killing of target cells by effector cells is determined, for example, by measuring a decrease in proliferation of target cells (particularly cancer cells), for example in a live cell monitoring cytotoxicity assay, or by measuring the release of intracellular proteins of target cells. Suitable intracellular proteins to be measured in a cytotoxicity assay may be endogenous proteins (e.g. LDH) or transgenic proteins expressed by antigen presenting cells (e.g. luciferase).

In the context of the present invention, the term "T2 cell" refers to a cell expressing an MHC I molecule (HLA-A 2) lacking TAP function. T2 cells can be easily artificially loaded with different concentrations of exogenous antigenic peptides. T2 cells are described, for example, in (Hosken and Bevan, science 1990, month 4, day 20; 248 (4953): 367-70). T2 cells are commercially available, for example, from ATCC (American type culture Collection (AMERICAN TYPE Culture Collection)). Loading of T2 cells can be accomplished by incubating T2 cells with the desired concentration of antigenic peptide for about 2 hours under standard cell culture conditions known to those skilled in the art. In the context of the present invention, T2 cells incubated with a concentration of antigenic peptide (e.g. 1 μm, 100nM, 10nM, 1nM, 100pM, 10pM, 1 pM) are referred to as T2 cells loaded with said concentration of antigenic peptide, e.g. T2 cells incubated with a10 μm antigenic peptide instrument are referred to as T2 cells loaded with 10 μm antigenic peptide.

The term "E:T ratio" refers to the ratio of effector cells (i.e., immune cells, particularly T cells, expressing an antigen binding protein, particularly TCR) to target cells. In some embodiments, the E:T ratio corresponds to the inoculation ratio, i.e., the ratio of the total number of immune cells (particularly T cells) to the target cells. In some embodiments, the E:T ratio is lower than the inoculation ratio. This applies to the case where not all immune cells express antigen binding proteins, i.e. not all immune cells are effector cells, for example due to low electroporation efficiency. In some embodiments, the inoculation ratio is used as an approximation of the E:T ratio. In some embodiments, the E:T ratio is determined by adjusting the inoculation ratio to account for electroporation efficiency.

Exemplary luciferase release assays are described in the methods section and performed in examples 1 and 2. In a particular embodiment of the luciferase release assay, the effector cell is a T cell transiently or stably expressing a TCR (preferably a pre-stimulated T cell), such as a T cell electroporated with mRNA encoding the TCR or stably transduced with nucleic acid encoding the TCR, e.g. a T cell transduced with a lentiviral vector comprising nucleic acid encoding the TCR. These effector cells are co-cultured with target cells expressing luciferase. Preferably, the target cell is a T2 cell loaded with the antigenic peptide. Preferably, effector cells and target cells are seeded at a ratio between 2:1 and 1:2 (preferably 1:1). After a defined co-incubation time (e.g. 12-38 hours, preferably 18-30 hours, more preferably about 24 hours), the amount of luciferase in the supernatant is measured, wherein a high luciferase concentration indicates a high killing activity of the antigen binding protein (preferably TCR) and thus a high functional affinity for the presented peptide. An antigen binding protein is considered to have a high functional affinity if in a cytotoxicity assay (preferably a luciferase release assay as defined above) the cytotoxic activity of the effector cell against the target cell is at least 50%, at least 60%, at least 70%, at least 75%, preferably at least 80%, at least 85%, at least 90% or at least 95% of the cytotoxic activity of the control toxic agent. Those skilled in the art know that cytotoxic activity may be greater than 100%. This is due to the fact that: 100% cytotoxic activity is defined by a "maximum lysis control" which refers to incubating a target cell with a toxic agent. In some embodiments, the toxic agent is a detergent, such as Triton-X100, tween-20, tween-80 or NP-40, that effects lysis of the target cells. In some specific examples, the maximum lysis control comprises adding a 0.2% Triton-X100 solution to the target cell culture. The cytotoxic activity of a toxic agent (i.e., the number of target cells killed by the toxic agent) is defined as 100%. Because the target cells can still proliferate during co-culture, effector cells may eventually kill even greater numbers of target cells during the cytotoxicity assay than the number of target cells that toxic agents kill during the maximum lysis control. In such cases, the calculated cytotoxic activity will be higher than 100%.

An exemplary cytokine production assay is described in the methods section and is performed in example 3. In a particular embodiment of the cytokine production assay, the effector cell is a T cell expressing a TCR (preferably a pre-stimulated T cell), such as a T cell transiently transfected or stably transduced with a nucleic acid encoding a TCR, preferably a T cell electroporated with mRNA encoding a TCR. These effector cells are co-cultured with target cells, preferably T2 cells loaded with antigenic peptides. Preferably, effector cells and target cells are seeded at a ratio between 2:1 and 1:2 (preferably 1:1). The co-cultivation is preferably carried out in the presence of a secretion blocking agent. After a determined co-culture time (e.g., 3-7 hours, preferably about 5 hours), the effector cells are stained for at least one intracellular cytokine selected from, for example, cd107a+, IFN- γ, tnfα, IL-2, and granzyme B to determine the amount of effector cells that produce the cytokine. An antigen is considered to have a high functional affinity if it is capable of activating effector cells in a cytokine production assay as defined above, in particular if the number of effector cells that produce cytokines after co-culture with target cells is at least 2%, at least 2.5%, preferably at least 3% of each immune cell population (e.g. live CD4 ⁺、CD8⁺ and/or CD3 ⁺ cells). Preferably, the number of effector cells that produce cytokines after co-culture with the target cells is at least 10%, at least 25%, at least 50% of the total number of effector cells. As explained above, not all immune cells express antigen binding proteins, i.e. not all immune cells vaccinated in a co-culture assay are effector cells, e.g. due to low electroporation efficiency.

An exemplary live cell monitoring cytotoxicity assay is described in the methods section and performed in example 5. In a particular embodiment of the live cell monitoring cytotoxicity assay, the effector cell is a T cell (preferably a pre-stimulated T cell) that transiently or stably expresses the TCR, such as a T cell electroporated with mRNA encoding the TCR. These effector cells are co-cultured with tumor cells that endogenously express and present the CT45-IP antigen peptide and optionally additionally loaded with the CT45-IP antigen peptide. In some embodiments, the tumor cell is an A375 cell or NCIH-1703 cell. The tumor cells are preferably fluorescently labeled. In some embodiments of the live cell monitoring cytotoxicity assay, the seeding ratio of total T cells (including TCR-expressing T cells (=effector cells) and T cells that do not express TCR) is between 9:1 and 0.5:1, such as 9:1, 6:1, 3:1, 2:1, or 1:1. In some embodiments, the E:T ratio is between 6:1 and 0.2:1. An antigen binding protein is considered to be highly potent if killing of tumor cells (as determined by a reduction in tumor cell proliferation) is observed at an E: T ratio of 6:1 or less, 5:1 or less, 4:1 or less, 3:1 or less, preferably 2:1 or less, more preferably 1:1 or less, even more preferably 0.5:1 or less in a live cell monitoring cytotoxicity assay as defined above.

"Half maximum effective concentration," also referred to as "EC ₅₀," generally refers to the concentration of a molecule that induces a half of the response between baseline and maximum after a specified exposure time. The lower the EC ₅₀ value, the higher the functional affinity of the molecule. EC ₅₀ values can be assessed experimentally by a variety of known methods, for example using the functional assays described above or other ELISA or flow cytometry based killing assays.

In order to determine EC ₅₀ in the functional assays described above, it is necessary to use different concentrations of antigen peptide loaded on antigen presenting cells (e.g., T2 cells) in a "peptide titration experiment". An exemplary luciferase release assay with peptide titration is described in the methods section and performed in example 1. In a particular embodiment, "EC ₅₀" refers to the concentration of the antigen peptide loaded on a target cell (in particular a T2 cell loaded with CT45 antigen peptide) that induces a reaction halfway between baseline and maximum when the target cell is co-cultured with effector cells in luciferase release as defined above. An antigen binding protein is considered to have high functional affinity if EC ₅₀, as determined in a cytotoxicity assay (preferably a luciferase release assay as defined above), is less than about 60nM, less than about 50nM, less than about 30nM, less than about 25nM, preferably less than about 20nM, less than about 15nM, less than about 10nM, more preferably less than about 5nM, less than about 2.5nM, less than about 1.5nM, or less than about 1 nM.

"Dextramer staining" involves contacting cells expressing an antigen binding protein with a fluorescently labeled multimer comprising ten CT45-IP: MHC complexes.

In the context of the present invention, the term "specific" refers to the ability of an antigen binding protein to distinguish its target peptide from peptides having different amino acid sequences (e.g., similar peptides as defined below). An antigen binding protein is considered specific for a target peptide if binding to the target peptide occurs with significantly higher affinity and/or higher functional avidity than binding to a similar peptide. The specificity of the antigen binding protein is determined by the amino acid sequences of CDRa1, CDRa3, CDRb1 and CDRb 3. The amino acid sequences of CDRa2 and CDRb2 contact MHC molecules and are not necessary for antigen specificity.

In the context of the present invention, "analogous peptide" herein refers to a potentially off-target peptide, i.e., a peptide that may potentially be bound by an antigen binding protein of the present invention based on its biochemical/biophysical characteristics (including but not limited to homologous sequences or analogous motifs). The analogous peptides typically comprise 8 to 12 amino acids in length. In the context of the present invention, a similar peptide is usually presented by MHC (in particular MHC I). Furthermore, in the context of the present invention, similar peptides include peptides comprising or consisting of an amino acid sequence similar to the amino acid sequence of the CT45 antigen peptide, more particularly peptides comprising epitopes in which some or all of the amino acids have the same and/or similar biochemical/biophysical characteristics as the amino acids constituting the epitope of the CT45 antigen peptide, compared to the epitope of the CT45 antigen peptide. In some examples, the analogous peptides studied in the context of the present invention are databases of HLA-A x 02 binding peptides presented from tumor and normal tissues using a similarity score in the binding-associated position of CT45-IP and a requirement for at least one detection on normal tissuesDatabase) is selected. Binding of antigen binding proteins to similar peptides presented by MHC proteins may lead to adverse reactions. Such adverse effects may be "extra-neoplastic" side effects, such as cross-reactivity of specific TCRs with similar peptides in healthy tissues, as reported in the following documents: lowdell et al, cytotherapy,2018, 12, 4.

Specifically, in the context of the present invention, the following peptides are analogous peptide ：SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO 155 (SP-05-0010).

One skilled in the art will recognize that among similar peptides, there are some peptides that do not bind to the antigen binding proteins of the invention to a detectable extent, e.g., peptides that do not detect binding signals or reactions above background levels in a functional assay. In this case, "background level" refers to the binding signal or response observed in a functional assay for a non-homologous, "non-similar" peptide (e.g., control peptide NYESO-001) or in the absence of peptide.

For other similar peptides, low but insignificant binding could be detected. These latter-mentioned analogous peptides can also be described as "potentially related" analogous peptides. An antigen binding protein is considered not to bind significantly to a similar peptide and to have specificity for its target antigen peptide if at least one of the following applies when compared to the binding of the similar peptide to the target antigen peptide under similar (preferably identical) experimental conditions:

the functional avidity in response to the analogue determined in the functional assay as described above is 25% or less, 20% or less, 15% or less, 10% or less of the functional avidity in response to the target antigen peptide CT 45-IP.

The cytotoxic activity in response to the analogue determined in the cytotoxicity assay as described above is 25% or less, 20% or less, 15% or less, 10% or less of the cytotoxic activity in response to the target antigen peptide CT 45-IP.

The EC ₅₀ of the analogous peptide determined in the functional assay (preferably the cytotoxicity assay) described above is increased by at least 50, at least 100, at least 200 or at least 500 fold compared to the EC ₅₀ of the target antigen peptide CT 45-IP.

-At least 25, at least 30, at least 40, at least 50, at least 75 or at least 100-fold increase in K _D of the similar peptide compared to K _D of the target antigen peptide CT 45-IP.

In the context of the present specification, the term "about" is intended to indicate that, in relation to a particular value, the value may deviate from ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1%.

Antigen binding proteins

1) CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 14, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 489 (preferably SEQ ID NO. 519), CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 19, and CDRB3 comprises the amino acid sequence of SEQ ID NO. 504,

2) CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 24, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 490 (preferably SEQ ID NO. 520), CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 75, and CDRB3 comprises the amino acid sequence of SEQ ID NO. 505 (preferably SEQ ID NO. 526),

3) CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 24, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 491, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 66, and CDRB3 comprises the amino acid sequence of SEQ ID NO. 506 (preferably SEQ ID NO. 527),

4) CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 90, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 492, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 66, and CDRB3 comprises the amino acid sequence of SEQ ID NO. 507 (preferably SEQ ID NO. 528),

5) CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 2, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 493, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 8, and CDRB3 comprises the amino acid sequence of SEQ ID NO. 508 (preferably SEQ ID NO. 529),

6) CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 53, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 494 (preferably SEQ ID NO. 521), CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 58, and CDRB3 comprises the amino acid sequence of SEQ ID NO. 509 (preferably SEQ ID NO. 530),

7) CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 71, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 495, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 75, and CDRB3 comprises the amino acid sequence of SEQ ID NO. 510 (preferably SEQ ID NO. 531),

8) CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 99, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 496 (preferably SEQ ID NO. 522), CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 75, and CDRB3 comprises the amino acid sequence of SEQ ID NO. 511,

9) CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 80, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 497, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 85, and CDRB3 comprises the amino acid sequence of SEQ ID NO. 512 (preferably SEQ ID NO. 532),

10 CDRa1 comprises or consists of the amino acid sequence of SEQ ID No. 107, CDRa3 comprises or consists of the amino acid sequence of SEQ ID No. 498 (preferably SEQ ID No. 523), CDRb1 comprises or consists of the amino acid sequence of SEQ ID No. 112, and CDRb3 comprises the amino acid sequence of SEQ ID No. 513 (preferably SEQ ID No. 533),

11 CDRa1 comprises or consists of the amino acid sequence of SEQ ID No. 125, CDRa3 comprises or consists of the amino acid sequence of SEQ ID No. 499, CDRb1 comprises or consists of the amino acid sequence of SEQ ID No. 112, and CDRb3 comprises the amino acid sequence of SEQ ID No. 514 (preferably SEQ ID No. 534),

12 CDRa1 comprises or consists of the amino acid sequence of SEQ ID No. 117, CDRa3 comprises or consists of the amino acid sequence of SEQ ID No. 500, CDRb1 comprises or consists of the amino acid sequence of SEQ ID No. 58, and CDRb3 comprises the amino acid sequence of SEQ ID No. 515 (preferably SEQ ID No. 535),

13 CDRa1 comprises or consists of the amino acid sequence of SEQ ID No. 24, CDRa3 comprises or consists of the amino acid sequence of SEQ ID No. 501 (preferably SEQ ID No. 524), CDRb1 comprises or consists of the amino acid sequence of SEQ ID No. 38, and CDRb3 comprises the amino acid sequence of SEQ ID No. 516 (preferably SEQ ID No. 536),

14 CDRa1 comprises or consists of the amino acid sequence of SEQ ID No. 24, CDRa3 comprises or consists of the amino acid sequence of SEQ ID No. 502 (preferably SEQ ID No. 525), CDRb1 comprises or consists of the amino acid sequence of SEQ ID No. 29, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID No. 517 (preferably SEQ ID No. 537), or

15 CDRa1 comprises or consists of the amino acid sequence of SEQ ID No. 43, CDRa3 comprises or consists of the amino acid sequence of SEQ ID No. 503, CDRb1 comprises or consists of the amino acid sequence of SEQ ID No. 48, and CDRb3 comprises the amino acid sequence of SEQ ID No. 518 (preferably SEQ ID No. 538),

The above mentioned amino acid sequences comprised in CDRa3 and CDRb3 are the central amino acids of CDRa3 and CDRb3 (also referred to herein as "CDR3 core"). The inventors have found that within the CDR3 sequence, a core sequence can be defined that comprises the amino acids most relevant for specific binding to an antigen peptide, while amino acids outside the CDR3 core are less relevant for specific binding to an antigen peptide (data not shown). The CDR3 core comprises the central 8 amino acids of CDR 3. In some embodiments, the CDR3 core consists of the central 8 amino acids of CDR3, particularly where the CDR3 sequence is no longer than 12 amino acids. In the case of CDR3 sequences longer than 12 amino acids, the CDR3 core may comprise further amino acids, in particular the central 9-13 amino acids.

All embodiments described herein as embodiment 1 (of 15) are preferably combined with other embodiment 1 (of 15). In the same way, all embodiments described herein as embodiments 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 (of 15) respectively are preferably combined with other embodiments 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 (of 15) respectively. For example, the CDR1 and CDR3 sequences of embodiment 1 (of 15) are preferably combined with the CDR2 sequences of embodiment 1 (of 15).

In a preferred embodiment of the present invention, in a preferred embodiment,

1) CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 16 and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 21,

2) CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 133 and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 136,

3) CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 63 and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 68,

4) CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 92 and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 96,

5) CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 4 and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 10,

6) CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 55 and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 60,

7) CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 72 and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 77,

8) CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 101 and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 104,

9) CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 82 and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 87,

10 CDRa3 comprises or consists of the amino acid sequence of SEQ ID No. 109 and CDRb3 comprises or consists of the amino acid sequence of SEQ ID No. 114,

11 CDRa3 comprises or consists of the amino acid sequence of SEQ ID No. 127 and CDRb3 comprises or consists of the amino acid sequence of SEQ ID No. 130, or

12 CDRa3 comprises or consists of the amino acid sequence of SEQ ID No. 119 and CDRb3 comprises or consists of the amino acid sequence of SEQ ID No. 122,

13 CDRa3 comprises or consists of the amino acid sequence of SEQ ID No. 35 and CDRb3 comprises or consists of the amino acid sequence of SEQ ID No. 40,

14 CDRa3 comprises or consists of the amino acid sequence of SEQ ID No. 26 and CDRb3 comprises or consists of the amino acid sequence of SEQ ID No. 31, or

15 CDRa3 comprises or consists of the amino acid sequence of SEQ ID No.45 and CDRb3 comprises or consists of the amino acid sequence of SEQ ID No. 50,

An antigen binding protein comprising CDRa1, CDRa3, CDRb1 and CDRb3 sequences having no more than one, two or three amino acid mutations refers herein to an antigen binding protein that may comprise one, two or three amino acid mutations in each of CDRa1, CDRa3, CDRb1 and/or CDRb 3.

In a preferred embodiment of the antigen binding protein,

1) CDRA2 comprises or consists of the amino acid sequence of SEQ ID NO. 15 and CDRB2 comprises or consists of the amino acid sequence of SEQ ID NO. 20,

2) CDRA2 comprises or consists of the amino acid sequence of SEQ ID NO. 25 and CDRB2 comprises or consists of the amino acid sequence of SEQ ID NO. 76,

3) CDRA2 comprises or consists of the amino acid sequence of SEQ ID NO. 25 and CDRB2 comprises or consists of the amino acid sequence of SEQ ID NO. 67,

4) CDRA2 comprises or consists of the amino acid sequence of SEQ ID NO. 91 and CDRB2 comprises or consists of the amino acid sequence of SEQ ID NO. 95,

5) CDRA2 comprises or consists of the amino acid sequence of SEQ ID NO. 3 and CDRB2 comprises or consists of the amino acid sequence of SEQ ID NO. 9

6) CDRA2 comprises or consists of the amino acid sequence of SEQ ID NO. 54 and CDRB2 comprises or consists of the amino acid sequence of SEQ ID NO. 59,

7) CDRA2 comprises or consists of the amino acid sequence of SEQ ID NO. 15 and CDRB2 comprises or consists of the amino acid sequence of SEQ ID NO. 76,

8) CDRA2 comprises or consists of the amino acid sequence of SEQ ID NO. 100 and CDRB2 comprises or consists of the amino acid sequence of SEQ ID NO. 76,

9) CDRA2 comprises or consists of the amino acid sequence of SEQ ID NO. 81 and CDRB2 comprises or consists of the amino acid sequence of SEQ ID NO. 86,

10 CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 108 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 113,

11 CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 126 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 113,

12 CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 118 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 59

13 CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 25 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 39,

14 CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 25 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No.30, or

15 CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 44 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 49,

Wherein the antigen binding protein comprises the CDRa2 and CDRb2 sequences having no more than one, two, three or four amino acid mutations.

An antigen binding protein comprising a CDRa2 and CDRb2 sequence having no more than one, two, three or four amino acid mutations refers herein to an antigen binding protein that may comprise one, two, three or four amino acid mutations in each of CDRa2 and/or CDRb 2.

In all embodiments of the antigen binding proteins of the invention, the amino acid mutations (if present) within the sequences of CDRa1, CDRa2, CDRa3, CDRb1, CDRb2 and CDRb3 are preferably amino acid substitutions, more preferably conservative amino acid substitutions (see table 1). Preferably, the CDR sequences comprise no more than two (preferably no more than one) amino acid mutations. It is further preferred that the amino acid mutation (if present) is at the first or last position of the corresponding CDR sequence. In a most preferred embodiment, the CDR sequences do not comprise any amino acid mutations.

The introduction of mutations into known amino acid sequences is a routine of standard procedures and the routine work of the skilled person, which are well known in the art. Corresponding methods are known in the art (e.g., quikChange site-directed mutagenesis kit from Stratagene in 2007). Thus, it is well within the ability of those skilled in the art to introduce specific mutations (e.g., substitutions) generally into amino acid sequences, and particularly into CDR sequences.

Screening of variants of CDRs that bind to their targets is also a standard procedure applied by the skilled person. The present application refers to functional assays (including cytokine production and luciferase release assays) to determine binding of the antigen binding proteins of the application to CT45-IP peptides. Binding of the antigen binding proteins of the application to CT45-IP peptides can also be determined by dextramer staining.

While the outcome of amino acid mutations in CDRs may not be easily predicted, the skilled artisan will be able to well generate and screen multiple mutants without undue burden. Thus, the skilled artisan will be able to produce antigen binding proteins carrying one, two or three amino acid mutations within their CDRs and subsequently identify antigen binding proteins having the same binding characteristics as antigen binding proteins comprising the CDR sequences of table 3.

In a preferred embodiment, no more than 1 or 2 amino acid mutations, preferably no more than 1 amino acid mutation, more preferably no more than 1 amino acid substitution, most preferably no more than 1 conservative amino acid substitution are included within the central 8 amino acids of CDRa3 and/or CDRb3 (i.e., within the CDR3 core). In the case where CDR3 comprises more than 12 amino acids, even more preferably no more than 1 or 2 amino acid mutations, preferably no more than 1 amino acid mutation, more preferably no more than 1 amino acid substitution, most preferably no more than 1 conservative amino acid substitution, are included within the central 9-13 amino acids of CDRa3 and/or CDRb3 (i.e., within the CDR3 core max (table 3)).

In some embodiments, the antigen binding protein induces an immune response, for example in a cell (preferably a lymphocyte, more preferably a T cell or NK cell, more preferably a T cell) expressing the antigen binding protein (if the antigen binding protein is membrane-bound). In some embodiments, an immune response may also be induced in cells recruited by the antigen binding proteins of the invention (if the antigen binding proteins are soluble, bispecific antigen binding proteins capable of binding to and thereby recruiting, for example, T cells or NK cells). Preferably, the immune response is characterized by increased production of Interferon (IFN) gamma and/or Tumor Necrosis Factor (TNF) alpha. The immune response is preferably directed against tumor cells presenting complexes of CT45 antigen peptides with MHC proteins on their surface.

In some embodiments, the antigen binding protein specifically binds to a complex of a CT45 antigen peptide and an MHC protein. In some embodiments, the CT45 antigen peptide consists of SEQ ID NO. 138. In some embodiments, the antigen binding protein specifically binds to the amino acid sequence of SEQ ID NO:138 as a complex with an MHC protein.

In all aspects of the invention, the CT45 antigenic peptide is preferably in a complex with an MHC class I HLA protein (e.g. HLA-A, HLA-B or HLA-C, preferably HLA-A, more preferably HLA-A.times.02).

The antigen binding proteins of the invention are characterized by high stability, high affinity for CT45-IP antigen peptide, high functional affinity for CT45-IP antigen peptide, high efficacy in killing tumor cells presenting CT45-IP antigen peptide, and/or high specificity for CT45-IP antigen peptide.

The antigen binding proteins of the invention have increased stability, increased binding affinity, increased functional avidity, increased efficacy and/or increased specificity, preferably increased binding affinity, increased functional avidity, increased efficacy and/or increased specificity, when measured under similar (preferably identical) experimental conditions as compared to a reference protein.

Reference protein herein refers to a protein that is compared to the antigen binding proteins of the invention. The comparison of the antigen binding proteins of the invention with a reference protein is performed under similar (preferably identical) experimental conditions, preferably in parallel. Depending on the parameters compared, such a reference protein may be, for example, a TCR binding to a CT45 antigenic peptide, a TCR binding to a different antigenic peptide derived from CT45 or a TCR binding to an unrelated antigenic peptide, such as antigenic peptide NYESO-001 (SEQ ID NO: 188), which does not comprise a CDR sequence as defined in the context of the present invention. The reference protein is preferably in the same form as the antigen binding protein to be compared. Where the antigen binding protein is a TCR, a suitable reference protein is also a TCR.

"Increased stability" herein refers to, for example, an increase in the expression level of an antigen binding protein as compared to a reference protein under the same experimental conditions. The antigen binding proteins of the invention have high expression levels, in particular increased expression levels compared to a reference protein when measured under similar (preferably identical) experimental conditions. The inventors show in example 4 that antigen binding proteins exhibit high expression levels in T cells. The expression level of the antigen binding protein may be measured, for example, by dextramer staining.

The antigen binding proteins of the invention have a high affinity for CT45-IP antigenic peptides, in particular an increased affinity compared to a reference protein when measured under similar (preferably identical) experimental conditions.

The antigen binding proteins of the invention have high functional affinity for CT45-IP antigenic peptides, in particular increased functional affinity compared to a reference protein when measured under similar (preferably identical) experimental conditions. The inventors show in example 1 that antigen binding proteins exhibit high functional affinity for CT45-IP antigen peptides. Functional affinity can be determined in functional assays, particularly cytotoxicity assays as described above. Measuring functional activity may include determining EC ₅₀ in a peptide titration experiment.

The antigen binding proteins of the invention have high efficacy in killing tumor cells presenting CT45-IP antigenic peptides, particularly when measured under similar (preferably identical) experimental conditions, an increased efficacy compared to the reference protein. The inventors show in example 5 that antigen binding proteins exhibit high efficacy in killing tumor cells presenting CT45-IP antigenic peptides. Efficacy can be determined in functional assays, particularly in live cell monitoring cytotoxicity assays as described above.

In a preferred embodiment, the antigen binding protein specifically binds to a structural epitope of CT 45-IP. In a more preferred embodiment, the antigen binding protein specifically binds to a functional epitope of CT 45-IP. The inventors performed experiments to identify residues in CT45-IP that are associated with binding of the antigen binding proteins of the invention (example 2, table 4). The inventors identified that amino acid positions 3,4, 5, 6 and 7 of SEQ ID NO:138 are associated with binding. Thus, in some embodiments, the antigen binding protein specifically binds to a functional epitope comprising or consisting of 2, 3 or 4 amino acid positions selected from positions 3,4, 5, 6 and 7 of SEQ ID No. 138. Residues associated with binding of antigen binding proteins may also be referred to as the "binding motif" of CT 45-IP. The person skilled in the art knows that the exact epitope or functional epitope determination may vary slightly depending on the method used and the cut-off value chosen. In the context of the present invention, epitopes have been determined in cytotoxicity assays (luciferase release) as described above. Experimental conditions are further defined in example 2.

The amino acid sequence according to SEQ ID NO. 138, in which at least one position is substituted, is referred to as "CT45-IP variant sequence" in the context of the present description. In particular, one position is substituted with alanine (SEQ ID NOS: 139-145). Peptides having CT45-IP variant sequences are also referred to herein as CT45-IP variant peptides. In one embodiment, the antigen binding proteins of the invention exhibit reduced functional affinity for CT45-IP variant peptides (wherein at least one of positions 3, 4,5, 6 and 7 of SEQ ID NO:138 is substituted with alanine), in particular reduced cytotoxic activity in a cytotoxicity assay, more in particular in a luciferase release assay as described above, in particular reduced functional affinity by more than 70%, more than 80%, more than 90% or more than 95% compared to functional affinity for CT 45-IP.

The antigen binding proteins of the invention have a high specificity for CT45-IP antigenic peptides, in particular an increased specificity compared to a reference protein when measured under similar (preferably identical) experimental conditions. The inventors demonstrate in example 2 that the antigen binding proteins of the invention bind to the target antigen with high specificity, i.e. the CT45 antigen peptide in complex with MHC proteins.

The inventors identified potential off-target peptides that are, for example, similar to the sequence and/or motif of CT45-IP and thus have an increased risk of binding by antigen binding proteins that bind to CT 45-IP.

In some embodiments, the antigen binding protein does not bind significantly to: at least 1, at least 2, at least 3, at least 4, at least 5 or all analogous peptides selected from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO 155 (SP-05-0010). In some embodiments, the antigen binding protein does not bind significantly to: at least 1, at least 2, at least 3, at least 4, at least 5 or all analogous peptides selected from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO 155 (SP-05-0010). In some embodiments, the antigen binding protein does not bind significantly to: at least 1, at least 2, at least 3, at least 4, at least 5 or all of the like peptides selected from the group consisting of SEQ ID NO:147 (SP-05-0002), SEQ ID NO:151 (SP-05-0006), SEQ ID NO:152 (SP-05-0007) and SEQ ID NO:155 (SP-05-0010).

In a preferred embodiment, V _A and V _B are TCR variable domains, in particular TCR α, β, γ or δ variable domains. In some embodiments, V _A is a TCR α, γ, or δ variable domain and V _B is a TCR β, γ, or δ variable domain. Preferably, V _A is a TCR α variable domain and V _B is a TCR β variable domain, or V _A is a TCR γ variable domain and V _B is a TCR δ variable domain, or V _A is a TCR α variable domain and V _B is a TCR γ variable domain, or V _A is a TCR δ variable domain and V _B is a TCR β variable domain. In a preferred embodiment, V _A is a tcra variable domain and V _B is a tcrp variable domain. In some embodiments, V _A is a TCR γ variable domain comprising CDR1 and CDR3, and optionally CDR2 derived from a TCR α variable domain, and/or V _B is a TCR δ variable domain comprising CDR1 and CDR3, and optionally CDR2 derived from a TCR β variable domain.

Preferably within V _A

1) FR1-a comprises or consists of SEQ ID NO 539, 554 or 569;

FR2-a comprises or consists of SEQ ID NO 584;

FR3-a comprises or consists of SEQ ID NO 599 or 614; and/or

FR4-a comprises or consists of SEQ ID NO 629; or alternatively

2) FR1-a comprises or consists of SEQ ID NO 540, 555 or 570;

FR2-a comprises or consists of SEQ ID NO: 585;

FR3-a comprises or consists of SEQ ID NO 600 or 615; and/or

FR4-a comprises or consists of SEQ ID NO 630; or alternatively

3) FR1-a comprises or consists of SEQ ID NO 541, 556 or 571;

FR2-a comprises or consists of SEQ ID NO 586;

FR3-a comprises or consists of SEQ ID NO 601 or 616; and/or

FR4-a comprises or consists of SEQ ID NO 631; or alternatively

4) FR1-a comprises or consists of SEQ ID NO 542, 557 or 572;

FR2-a comprises or consists of SEQ ID NO: 587;

FR3-a comprises or consists of SEQ ID NO 602 or 617; and/or

FR4-a comprises or consists of SEQ ID NO 632; or alternatively

5) FR1-a comprises or consists of SEQ ID NO 543, 558 or 573;

FR2-a comprises or consists of SEQ ID NO: 588;

FR3-a comprises or consists of SEQ ID NO 603 or 618; and/or

FR4-a comprises or consists of SEQ ID NO 633; or alternatively

6) FR1-a comprises or consists of SEQ ID NO 544, 559 or 574;

FR2-a comprises or consists of SEQ ID NO: 589;

FR3-a comprises or consists of SEQ ID NO 604 or 619; and/or

FR4-a comprises or consists of SEQ ID NO 634; or alternatively

7) FR1-a comprises or consists of SEQ ID NO 545, 560 or 575;

FR2-a comprises or consists of SEQ ID NO 590;

FR3-a comprises or consists of SEQ ID NO 605 or 620; and/or

FR4-a comprises or consists of SEQ ID NO 635; or alternatively

8) FR1-a comprises or consists of SEQ ID NO 546, 561 or 576;

FR2-a comprises or consists of SEQ ID NO 591;

FR3-a comprises or consists of SEQ ID NO 606 or 621; and/or

FR4-a comprises or consists of SEQ ID NO 636; or alternatively

9) FR1-a comprises or consists of SEQ ID NO 547, 562 or 577;

FR2-a comprises or consists of SEQ ID NO 592;

FR3-a comprises or consists of SEQ ID NO 607 or 622; and/or

FR4-a comprises or consists of SEQ ID NO 637; or alternatively

10 FR1-a comprises or consists of SEQ ID NO 548, 563 or 578;

FR2-a comprises or consists of SEQ ID NO 593;

FR3-a comprises or consists of SEQ ID NO 608 or 623; and/or

FR4-a comprises or consists of SEQ ID NO 638;

11 FR1-a comprises or consists of SEQ ID NO 549, 564 or 579;

FR2-a comprises or consists of SEQ ID NO. 594;

FR3-a comprises or consists of SEQ ID NO 609 or 624; and/or

FR4-a comprises or consists of SEQ ID NO 639; or alternatively

12 FR1-a comprises or consists of SEQ ID NO 550, 565 or 580;

FR2-a comprises or consists of SEQ ID NO. 595;

FR3-a comprises or consists of SEQ ID NO 610 or 625; and/or

FR4-a comprises or consists of SEQ ID NO. 640; or alternatively

13 FR1-a comprises or consists of SEQ ID NO 551, 566 or 581;

FR2-a comprises or consists of SEQ ID NO 596;

FR3-a comprises or consists of SEQ ID NO 611 or 626; and/or

FR4-a comprises or consists of SEQ ID NO 641; or alternatively

14 FR1-a comprises or consists of SEQ ID NO 552, 567 or 582;

FR2-a comprises or consists of SEQ ID NO 597;

FR3-a comprises or consists of SEQ ID NO 612 or 627; and/or

FR4-a comprises or consists of SEQ ID NO 642; or alternatively

15 FR1-a comprises or consists of SEQ ID No. 553, 568 or 583;

FR2-a comprises or consists of SEQ ID NO 598;

FR3-a comprises or consists of SEQ ID NO 613 or 628; and/or

FR4-a comprises or consists of SEQ ID NO. 643; and

In V _B

1) FR1-b comprises or consists of SEQ ID NO 644 or 659;

FR2-b comprises or consists of SEQ ID NO. 674 or 689;

FR3-b comprises or consists of SEQ ID NO 704 or 719; and/or

FR4-b comprises or consists of SEQ ID NO 734; or alternatively

2) FR1-b comprises or consists of SEQ ID NO 645 or 660;

FR2-b comprises or consists of SEQ ID NO 675 or 690;

FR3-b comprises or consists of SEQ ID NO 705 or 720; and/or

FR4-b comprises or consists of SEQ ID NO 735; or alternatively

3) FR1-b comprises or consists of SEQ ID NO 646 or 661;

FR2-b comprises or consists of SEQ ID NO 676 or 691;

FR3-b comprises or consists of SEQ ID NO 706 or 721; and/or

FR4-b comprises or consists of SEQ ID NO 736; or alternatively

4) FR1-b comprises or consists of SEQ ID NO 647 or 662;

FR2-b comprises or consists of SEQ ID NO:677 or 692;

FR3-b comprises or consists of SEQ ID NO 707 or 722; and/or

FR4-b comprises or consists of SEQ ID NO. 737; or alternatively

5) FR1-b comprises or consists of SEQ ID NO 648 or 663;

FR2-b comprises or consists of SEQ ID NO 678 or 693;

FR3-b comprises or consists of SEQ ID NO 708 or 723; and/or

FR4-b comprises or consists of SEQ ID NO: 738; or alternatively

6) FR1-b comprises or consists of SEQ ID NO 649 or 664;

FR2-b comprises or consists of SEQ ID NO:679 or 694;

FR3-b comprises or consists of SEQ ID NO 709 or 724; and/or

FR4-b comprises or consists of SEQ ID NO. 739; or alternatively

7) FR1-b comprises or consists of SEQ ID NO 650 or 665;

FR2-b comprises or consists of SEQ ID NO 680 or 695;

FR3-b comprises or consists of SEQ ID NO 710 or 725; and/or

FR4-b comprises or consists of SEQ ID NO 740; or alternatively

8) FR1-b comprises or consists of SEQ ID NO 651 or 666;

FR2-b comprises or consists of SEQ ID NO 681 or 696;

FR3-b comprises or consists of SEQ ID NO 711 or 726; and/or

FR4-b comprises or consists of SEQ ID NO 741; or alternatively

9) FR1-b comprises or consists of SEQ ID NO 652 or 667;

FR2-b comprises or consists of SEQ ID NO 682 or 697;

FR3-b comprises or consists of SEQ ID NO 712 or 727; and/or

FR4-b comprises or consists of SEQ ID NO 742; or alternatively

10 FR1-b comprises or consists of SEQ ID NO 653 or 668;

FR2-b comprises or consists of SEQ ID NO:683 or 698;

FR3-b comprises or consists of SEQ ID NO 713 or 728; and/or

FR4-b comprises or consists of SEQ ID NO. 743; or alternatively

11 FR1-b comprises or consists of SEQ ID NO 654 or 669;

FR2-b comprises or consists of SEQ ID NO:684 or 699;

FR3-b comprises or consists of SEQ ID NO 714 or 729; and/or

FR4-b comprises or consists of SEQ ID NO 744; or alternatively

12 FR1-b comprises or consists of SEQ ID NO 655 or 670;

FR2-b comprises or consists of SEQ ID NO:685 or 700;

FR3-b comprises or consists of SEQ ID NO 715 or 730; and/or

FR4-b comprises or consists of SEQ ID NO 745; or alternatively

13 FR1-b comprises or consists of SEQ ID No. 656 or 671;

FR2-b comprises or consists of SEQ ID NO:686 or 701;

FR3-b comprises or consists of SEQ ID NO 716 or 731; and/or

FR4-b comprises or consists of SEQ ID NO 746; or alternatively

14 FR1-b comprises or consists of SEQ ID No. 657 or 672;

FR2-b comprises or consists of SEQ ID NO:687 or 702;

FR3-b comprises or consists of SEQ ID NO 717 or 732; and/or

FR4-b comprises or consists of SEQ ID NO. 747; or alternatively

15 FR1-b comprises or consists of SEQ ID No. 658 or 673;

FR2-b comprises or consists of SEQ ID NO 688 or 703;

FR3-b comprises or consists of SEQ ID NO 718 or 733; and/or

FR4-b comprises or consists of SEQ ID NO. 748,

Wherein each of FR1-a, FR2-a, FR3-a, FR4-a, FR1-b, FR2-b, FR3-b and FR4-b can optionally comprise 8, 7, 6, 5, 4, 3, 2 or 1 amino acid mutation.

In some embodiments, the first and second substrates are, preferably,

1) FR1-a comprises or consists of SEQ ID NO. 539;

2) FR1-a comprises or consists of SEQ ID NO. 540;

3) FR1-a comprises or consists of SEQ ID NO. 541;

4) FR1-a comprises or consists of SEQ ID NO 542;

5) FR1-a comprises or consists of SEQ ID NO 543;

6) FR1-a comprises or consists of SEQ ID NO 544;

7) FR1-a comprises or consists of SEQ ID NO 545;

8) FR1-a comprises or consists of SEQ ID NO 546;

9) FR1-a comprises or consists of SEQ ID NO 547;

10 FR1-a comprises or consists of SEQ ID NO 548;

11 FR1-a comprises or consists of SEQ ID NO 549;

12 FR1-a comprises or consists of SEQ ID NO 550;

13 FR1-a comprises or consists of SEQ ID NO 551;

14 FR1-a comprises or consists of SEQ ID No. 552; or alternatively

15 553 Or consists of SEQ ID NO, wherein each of FR1-a, FR2-a, FR3-a and FR4-a may optionally comprise 8, 7, 6, 5, 4, 3, 2 or 1 amino acid mutations.

In some embodiments, the first and second substrates are, preferably,

1) FR3-a comprises or consists of SEQ ID NO. 599, optionally comprising 8, 7, 6, 5, 4, 3, 2 or 1 amino acid mutations outside position 10 of SEQ ID NO. 599;

2) FR3-a comprises or consists of SEQ ID NO. 600, optionally comprises 8, 7, 6, 5, 4, 3, 2 or 1 amino acid mutations outside position 11 of SEQ ID NO. 600;

3) FR3-a comprises or consists of SEQ ID NO. 601, optionally comprises 8, 7, 6, 5, 4, 3, 2 or 1 amino acid mutations outside position 5 of SEQ ID NO. 601;

4) FR3-a comprises or consists of SEQ ID NO. 602, optionally comprises 8, 7, 6, 5, 4, 3, 2 or 1 amino acid mutations outside position 12 of SEQ ID NO. 602;

5) FR3-a comprises or consists of SEQ ID NO:603, optionally comprising an 8, 7, 6, 5, 4, 3, 2 or 1 amino acid mutation outside position 22 of SEQ ID NO: 603;

6) FR3-a comprises or consists of SEQ ID NO 604, optionally comprises 8, 7, 6, 5, 4, 3, 2 or 1 amino acid mutations outside position 25 of SEQ ID NO 604;

7) FR3-a comprises or consists of SEQ ID NO. 605, optionally comprises 8, 7, 6, 5, 4, 3, 2 or 1 amino acid mutations outside position 21 of SEQ ID NO. 605;

8) FR3-a comprises or consists of SEQ ID NO. 606, optionally comprises a mutation of 8, 7, 6, 5, 4, 3, 2 or 1 amino acids outside position 18 of SEQ ID NO. 606;

9) FR3-a comprises or consists of SEQ ID NO:607, optionally comprises a mutation of 8, 7, 6, 5, 4, 3, 2 or 1 amino acids outside position 24 of SEQ ID NO: 607;

10 FR3-a comprises or consists of SEQ ID No. 608, optionally comprises 8, 7, 6, 5, 4, 3, 2 or 1 amino acid mutations outside position 21 of SEQ ID No. 608;

11 FR3-a comprises or consists of SEQ ID No. 609, optionally comprises 8, 7, 6, 5, 4, 3, 2 or 1 amino acid mutations outside position 7 of SEQ ID No. 609;

12 FR3-a comprises or consists of SEQ ID No. 610, optionally comprises 8, 7, 6, 5, 4, 3, 2 or 1 amino acid mutations outside position 30 of SEQ ID No. 610;

13 FR3-a comprises or consists of SEQ ID NO 611, optionally comprises 8, 7, 6, 5, 4, 3, 2 or 1 amino acid mutations outside position 18 of SEQ ID NO 611;

14 FR3-a comprises or consists of SEQ ID NO:612, optionally comprises 8, 7, 6, 5, 4, 3, 2 or 1 amino acid mutations outside position 19 of SEQ ID NO: 612; or alternatively

15 FR3-a comprises or consists of SEQ ID No. 613, optionally comprising 8, 7, 6, 5, 4, 3, 2 or 1 amino acid mutations outside position 23 of SEQ ID No. 613.

In some embodiments, the first and second substrates are, preferably,

1) FR2-b comprises or consists of SEQ ID NO. 674;

2) FR2-b comprises or consists of SEQ ID NO 675;

3) FR2-b comprises or consists of SEQ ID NO. 676;

4) FR2-b comprises or consists of SEQ ID NO: 677;

5) FR2-b comprises or consists of SEQ ID NO. 678;

6) FR2-b comprises or consists of SEQ ID NO: 679;

7) FR2-b comprises or consists of SEQ ID NO 680;

8) FR2-b comprises or consists of SEQ ID NO: 681;

9) FR2-b comprises or consists of SEQ ID NO 682;

10 FR2-b comprises or consists of SEQ ID No. 683;

11 FR2-b comprises or consists of SEQ ID No. 684;

12 FR2-b comprises or consists of SEQ ID No. 685;

13 FR2-b comprises or consists of SEQ ID No. 686;

14 FR2-b comprises or consists of SEQ ID No. 687; or alternatively

15 FR2-b comprises or consists of SEQ ID No. 688,

Wherein each of FR1-b, FR2-b, FR3-b and FR4-b can optionally comprise a mutation of 6, 5, 4, 3, 2 or 1 amino acids.

In some embodiments, the first and second substrates are, preferably,

1) FR2-b comprises or consists of SEQ ID NO:689, optionally comprising a mutation of 6, 5, 4, 3, 2 or 1 amino acids outside position 16 of SEQ ID NO: 689; or alternatively

2) FR2-b comprises or consists of SEQ ID NO 690, optionally comprises a mutation of 6, 5, 4, 3,2 or 1 amino acids outside position 17 of SEQ ID NO 690;

3) FR2-b comprises or consists of SEQ ID NO. 691, optionally comprises a mutation of 6, 5, 4,3, 2 or 1 amino acids outside position 5 of SEQ ID NO. 691;

4) FR2-b comprises or consists of SEQ ID NO 692, optionally comprising a mutation of 6, 5, 4, 3,2 or 1 amino acids outside position 15 of SEQ ID NO 692;

5) FR2-b comprises or consists of SEQ ID NO 693, optionally comprises a mutation of 6, 5, 4,3, 2 or 1 amino acids outside position 7 of SEQ ID NO 693;

6) FR2-b comprises or consists of SEQ ID NO 694, optionally comprises a mutation of 6, 5, 4, 3, 2 or 1 amino acids outside position 13 of SEQ ID NO 694;

7) FR2-b comprises or consists of SEQ ID NO. 695, optionally comprises a mutation of 6, 5, 4, 3, 2 or 1 amino acids outside position 11 of SEQ ID NO. 695;

8) FR2-b comprises or consists of SEQ ID NO 696, optionally comprises a mutation of 6, 5, 4, 3, 2 or 1 amino acids outside position 11 of SEQ ID NO 696;

9) FR2-b comprises or consists of SEQ ID NO 697, optionally comprises a mutation of 6, 5, 4, 3, 2 or 1 amino acids outside position 1 of SEQ ID NO 697;

10 FR2-b comprises or consists of SEQ ID No. 698, optionally comprises a6, 5, 4, 3,2 or 1 amino acid mutation outside position 1 of SEQ ID No. 698;

11 FR2-b comprises or consists of SEQ ID No. 699, optionally comprises a 6, 5, 4, 3, 2 or 1 amino acid mutation outside position 16 of SEQ ID No. 699;

12 FR2-b comprises or consists of SEQ ID No. 700, optionally comprises a 6, 5, 4, 3, 2 or 1 amino acid mutation outside position 7 of SEQ ID No. 700;

13 FR2-b comprises or consists of SEQ ID No. 701, optionally comprises a 6, 5, 4, 3, 2 or 1 amino acid mutation outside position 11 of SEQ ID No. 701;

14 FR2-b comprises or consists of SEQ ID No. 702, optionally comprises a 6, 5, 4, 3, 2 or 1 amino acid mutation outside position 1 of SEQ ID No. 702; or alternatively

15 FR2-b comprises or consists of SEQ ID No. 703, optionally comprises a 6, 5, 4, 3, 2 or 1 amino acid mutation outside position 7 of SEQ ID No. 703.

In a preferred embodiment of the present invention, in a preferred embodiment,

1) V _A comprises SEQ ID No. 189 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 189 and comprises CDRa1 comprising SEQ ID No. 14, CDRa3 comprising SEQ ID No. 489 and optionally CDRa2 comprising SEQ ID No. 15; and V _B comprises SEQ ID NO 339 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO 339 and comprises CDRB1 comprising SEQ ID NO 19, CDRB3 comprising SEQ ID NO 504 and optionally CDRB2 comprising SEQ ID NO 20, or

2) V _A comprises SEQ ID No. 190 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 190 and comprises CDRa1 comprising SEQ ID No. 24, CDRa3 comprising SEQ ID No. 490 and optionally CDRa2 comprising SEQ ID No. 25; and V _B comprises SEQ ID NO:340 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO:340 and comprises CDRB1 comprising SEQ ID NO:75, CDRB3 comprising SEQ ID NO:505 and optionally CDRB2 comprising SEQ ID NO:76, or

3) V _A comprises SEQ ID No. 191 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 191 and comprises CDRa1 comprising SEQ ID No. 24, CDRa3 comprising SEQ ID No. 491 and optionally CDRa2 comprising SEQ ID No. 25; and V _B comprises SEQ ID NO 341 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO 341 and comprises CDRB1 comprising SEQ ID NO 66, CDRB3 comprising SEQ ID NO 506 and optionally CDRB2 comprising SEQ ID NO 67, or

4) V _A comprises SEQ ID NO 192 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO 192 and comprises CDRA1 comprising SEQ ID NO 90, CDRA3 comprising SEQ ID NO 492 and optionally CDRA2 comprising SEQ ID NO 91; and V _B comprises SEQ ID NO:342 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO:342 and comprises CDRB1 comprising SEQ ID NO:66, CDRB3 comprising SEQ ID NO:507 and optionally CDRB2 comprising SEQ ID NO:95, or

5) V _A comprises SEQ ID No. 193 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 193 and comprises CDRa1 comprising SEQ ID No. 2, CDRa3 comprising SEQ ID No. 493 and optionally CDRa2 comprising SEQ ID No. 3; and V _B comprises SEQ ID NO. 343 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 343 and comprises CDRB1 comprising SEQ ID NO. 8, CDRB3 comprising SEQ ID NO. 508 and optionally CDRB2 comprising SEQ ID NO. 9, or

6) V _A comprises SEQ ID No. 194 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 194 and comprises CDRa1 comprising SEQ ID No. 53, CDRa3 comprising SEQ ID No. 494 and optionally CDRa2 comprising SEQ ID No. 54; and V _B comprises SEQ ID NO:344 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO:344 and comprises CDRB1 comprising SEQ ID NO:58, CDRB3 comprising SEQ ID NO:509 and optionally CDRB2 comprising SEQ ID NO:59, or

7) V _A comprises SEQ ID No. 195 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 195 and comprises CDRa1 comprising SEQ ID No. 71, CDRa3 comprising SEQ ID No. 495 and optionally CDRa2 comprising SEQ ID No. 15; and V _B comprises SEQ ID No. 345 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 345 and comprises CDRb1 comprising SEQ ID No. 75, CDRb3 comprising SEQ ID No. 510 and optionally CDRb2 comprising SEQ ID No. 76, or

8) V _A comprises SEQ ID No. 196 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 196 and comprises CDRa1 comprising SEQ ID No. 99, CDRa3 comprising SEQ ID No. 496 and optionally CDRa2 comprising SEQ ID No. 100; and V _B comprises SEQ ID NO. 346 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 346 and comprises CDRB1 comprising SEQ ID NO. 75, CDRB3 comprising SEQ ID NO. 511 and optionally CDRB2 comprising SEQ ID NO. 76, or

9) V _A comprises SEQ ID NO 197 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO 197 and comprises CDRa1 comprising SEQ ID NO 80, CDRa3 comprising SEQ ID NO 497 and optionally CDRa2 comprising SEQ ID NO 81; and V _B comprises SEQ ID NO. 347 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 347 and comprises CDRB1 comprising SEQ ID NO. 85, CDRB3 comprising SEQ ID NO. 512 and optionally CDRB2 comprising SEQ ID NO. 86, or

10 V _A comprises SEQ ID No. 198 or an amino acid sequence having at least 70%, 75%, 80%, 85%,90%, 95%, 98% or 99% identity to SEQ ID No. 198 and comprises CDRa1 comprising SEQ ID No. 107, CDRa3 comprising SEQ ID No. 498 and optionally CDRa2 comprising SEQ ID No. 108; and V _B comprises SEQ ID NO:348 or an amino acid sequence having at least 70%, 75%, 80%, 85%,90%, 95%, 98% or 99% identity to SEQ ID NO:348 and comprises CDRB1 comprising SEQ ID NO:112, CDRB3 comprising SEQ ID NO:513 and optionally CDRB2 comprising SEQ ID NO:113, or

11 V _A comprises SEQ ID NO 199 or an amino acid sequence having at least 70%, 75%, 80%, 85%,90%, 95%, 98% or 99% identity to SEQ ID NO 199 and comprises CDRa1 comprising SEQ ID NO 125, CDRa3 comprising SEQ ID NO 499 and optionally CDRa2 comprising SEQ ID NO 126; and V _B comprises SEQ ID NO. 349 or an amino acid sequence having at least 70%, 75%, 80%, 85%,90%, 95%, 98% or 99% identity to SEQ ID NO. 349 and comprises CDRB1 comprising SEQ ID NO. 112, CDRB3 comprising SEQ ID NO. 514 and optionally CDRB2 comprising SEQ ID NO. 113, or

12 V _A comprises SEQ ID No. 200 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 200 and comprises CDRa1 comprising SEQ ID No. 117, CDRa3 comprising SEQ ID No. 500 and optionally CDRa2 comprising SEQ ID No. 118; and V _B comprises SEQ ID NO:350 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO:350 and comprises CDRB1 comprising SEQ ID NO:58, CDRB3 comprising SEQ ID NO:515 and optionally CDRB2 comprising SEQ ID NO:59, or

13 V _A comprises SEQ ID No. 201 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 201 and comprises CDRa1 comprising SEQ ID No. 24, CDRa3 comprising SEQ ID No. 501 and optionally CDRa2 comprising SEQ ID No. 25; and V _B comprises SEQ ID NO:351 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO:351 and comprises CDRB1 comprising SEQ ID NO:38, CDRB3 comprising SEQ ID NO:516 and optionally CDRB2 comprising SEQ ID NO:39, or

14 V _A comprises SEQ ID No. 202 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 202 and comprises CDRa1 comprising SEQ ID No. 24, CDRa3 comprising SEQ ID No. 502 and optionally CDRa2 comprising SEQ ID No. 25; and V _B comprises SEQ ID NO. 352 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 352 and comprises CDRB1 comprising SEQ ID NO. 29, CDRB3 comprising SEQ ID NO. 517 and optionally CDRB2 comprising SEQ ID NO. 30, or

15 V _A comprises SEQ ID No. 203 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 203 and comprises CDRa1 comprising SEQ ID No. 43, CDRa3 comprising SEQ ID No. 503 and optionally CDRa2 comprising SEQ ID No. 44; and V _B comprises SEQ ID NO. 353 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 353 and comprises CDRB1 comprising SEQ ID NO. 48, CDRB3 comprising SEQ ID NO. 518 and optionally CDRB2 comprising SEQ ID NO. 49.

In a preferred embodiment of the present invention, in a preferred embodiment,

1) V _A comprises SEQ ID NO 189, 249, 264 or 279; and V _B comprises SEQ ID NO 339, 399, 414 or 429, or

2) V _A comprises SEQ ID NO 190, 250, 265 or 280; and V _B comprises SEQ ID NO 340, 400, 415 or 430, or

3) V _A comprises SEQ ID NO 191, 251, 266 or 281; and V _B comprises SEQ ID NO 341, 401, 416 or 431, or

4) V _A comprises SEQ ID NO 192, 252, 267 or 282; and V _B comprises SEQ ID NO 342, 402, 417 or 432, or

5) V _A comprises SEQ ID NO 193, 253, 268 or 283; and V _B comprises SEQ ID NO. 343, 403, 418 or 433, or

6) V _A comprises SEQ ID NO 194, 254, 269 or 284; and V _B comprises SEQ ID NO 344, 404, 419 or 434, or

7) V _A comprises SEQ ID NO 195, 255, 270 or 285; and V _B comprises SEQ ID NO 345, 405, 420 or 435, or

8) V _A comprises SEQ ID NO 196, 256, 271 or 286; and V _B comprises SEQ ID NO 346, 406, 421 or 436, or

9) V _A comprises SEQ ID NO 197, 257, 272 or 287; and V _B comprises SEQ ID NO 347, 407, 422 or 437, or

10 V _A comprises SEQ ID No. 198, 258, 273 or 288; and V _B comprises SEQ ID NO 348, 408, 423 or 438, or

11 V _A comprises SEQ ID NO 199, 259, 274 or 289; and V _B comprises SEQ ID NO 349, 409, 424 or 439, or

12 V _A comprises SEQ ID No. 200, 260, 275 or 290; and V _B comprises SEQ ID NO 350, 410, 425 or 440, or

13 V _A comprises SEQ ID No. 201, 261, 276 or 291; and V _B comprises SEQ ID NO 351, 411, 426 or 441, or

14 V _A comprises SEQ ID NO 202, 262, 277 or 292; and V _B comprises SEQ ID NO 352, 412, 427 or 442, or

15 V _A comprises SEQ ID No. 203, 263, 278 or 293; and V _B comprises SEQ ID NO 353, 413, 428 or 443.

In some embodiments of the present invention, in some embodiments,

1) V _A comprises SEQ ID NO 189; and V _B comprises SEQ ID NO:414, or

2) V _A comprises SEQ ID NO. 190; and V _B comprises SEQ ID NO:415, or

3) V _A comprises SEQ ID NO 191; and V _B comprises SEQ ID NO. 416, or

4) V _A comprises SEQ ID NO 192; and V _B comprises SEQ ID NO 417, or

5) V _A comprises SEQ ID NO 193; and V _B comprises SEQ ID NO:418, or

6) V _A comprises SEQ ID NO 194; and V _B comprises SEQ ID NO:419, or

7) V _A comprises SEQ ID NO 195; and V _B comprises SEQ ID NO:420, or

8) V _A comprises SEQ ID NO 196; and V _B comprises SEQ ID NO 421, or

9) V _A comprises SEQ ID NO 197; and V _B comprises SEQ ID NO 422, or

10 V _A comprises SEQ ID NO. 198; and V _B comprises SEQ ID NO:423, or

11 V _A comprises SEQ ID NO 199; and V _B comprises SEQ ID NO:424, or

12 V _A comprises SEQ ID NO. 200; and V _B comprises SEQ ID NO:425, or

13 V _A comprises SEQ ID NO. 201; and V _B comprises SEQ ID NO:426, or

14 V _A comprises SEQ ID NO 202; and V _B comprises SEQ ID NO:427, or

15 V _A comprises SEQ ID No. 203; and V _B comprises SEQ ID NO 428.

V _A preferably comprises or consists of: an amino acid sequence selected from SEQ ID NOs 13, 132, 62, 89, 1, 52, 70, 98, 79, 106, 124, 116, 34, 23 and 42, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NOs 13, 132, 62, 89, 1, 52, 70, 98, 79, 106, 124, 116, 34, 23 and 42, and comprising an amino acid sequence of CDRa1, CDRa2 and CDRa3 as defined in the context of an antigen binding protein of the invention, wherein said CDRa1, CDRa2 and CDRa3 sequences may comprise one, two or three amino acid mutations. V _B preferably comprises or consists of: an amino acid sequence selected from SEQ ID NOs 18, 135, 65, 94, 7, 57, 74, 103, 84, 111, 129, 121, 37, 28 and 47, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NOs 18, 135, 65, 94, 7, 57, 74, 103, 84, 111, 129, 121, 37, 28 and 47, and comprising an amino acid sequence of CDRa1, CDRa2 and CDRa3 as defined in the context of an antigen binding protein of the invention, wherein the CDRb1, CDRb2 and CDRb3 sequences may comprise one, two or three amino acid mutations.

Modifications and variations can be made in the amino acid sequence of the antigen binding proteins of the invention and in the corresponding DNA sequence, respectively, and still result in a functional antigen binding protein or polypeptide having the desired characteristics.

In a preferred embodiment of the present invention, in a preferred embodiment,

1) V _A comprises or consists of: the amino acid sequence of SEQ ID No. 13 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 13 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 18 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 18;

2) V _A comprises or consists of: the amino acid sequence of SEQ ID No. 132 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 132 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 135 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 135;

3) V _A comprises or consists of: the amino acid sequence of SEQ ID No. 62 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 62 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 65 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 65;

4) V _A comprises or consists of: the amino acid sequence of SEQ ID No. 89 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 89 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 94 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 94;

5) V _A comprises or consists of: the amino acid sequence of SEQ ID No. 1 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 1 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 7 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 7;

6) V _A comprises or consists of: the amino acid sequence of SEQ ID NO. 52 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 52 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 57 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 57;

7) V _A comprises or consists of: the amino acid sequence of SEQ ID No. 70 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 70 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 74 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 74;

8) V _A comprises or consists of: the amino acid sequence of SEQ ID No. 98 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 98 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 103 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 103;

9) V _A comprises or consists of: the amino acid sequence of SEQ ID No. 79 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 79 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 84 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 84;

10 V _A comprises or consists of: the amino acid sequence of SEQ ID NO. 106 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 106 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 111 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 111;

11 V _A comprises or consists of: the amino acid sequence of SEQ ID NO. 124 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 124 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 129 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 129;

12 V _A comprises or consists of: the amino acid sequence of SEQ ID NO. 116 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 116 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 121 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 121;

13 V _A comprises or consists of: the amino acid sequence of SEQ ID No. 34 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 34 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 37 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 37;

14 V _A comprises or consists of: the amino acid sequence of SEQ ID NO. 23 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 23 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 28 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 28; or alternatively

15 V _A comprises or consists of: the amino acid sequence of SEQ ID No. 42 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 42 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 47 or at least 70% with SEQ ID NO. 47,

75%, 80%, 85%, 90%, 95%, 98% Or 99% identical, wherein V _A and V _B comprise CDR sequences as defined in the context of an antigen binding protein of the invention, wherein the CDR sequences may comprise one, two or three amino acid mutations, preferably no amino acid mutations.

In a preferred embodiment of the present invention, in a preferred embodiment,

1) V _A comprises or consists of SEQ ID NO 13, 204, 219 or 234; and V _B comprises or consists of SEQ ID NO 18, 354, 369 or 384, or

2) V _A comprises or consists of SEQ ID NO 132, 205, 220 or 235; and V _B comprises or consists of SEQ ID NO 135, 355, 370 or 385, or

3) V _A comprises or consists of SEQ ID NO 62, 206, 221 or 236; and V _B comprises or consists of SEQ ID NO 65, 356, 371 or 386, or

4) V _A comprises or consists of SEQ ID NO 89, 207, 222 or 237; and V _B comprises or consists of SEQ ID NO 94, 357, 372 or 387, or

5) V _A comprises or consists of SEQ ID NO 1, 208, 223 or 238; and V _B comprises or consists of SEQ ID NO 7, 358, 373 or 388, or

6) V _A comprises or consists of SEQ ID NO 52, 209, 224 or 239; and V _B comprises or consists of SEQ ID NO 57, 359, 374 or 389, or

7) V _A comprises or consists of SEQ ID NO 70, 210, 225 or 240; and V _B comprises or consists of SEQ ID NO 74, 360, 375 or 390, or

8) V _A comprises or consists of SEQ ID NO 198, 211, 226 or 241; and V _B comprises or consists of SEQ ID NO 103, 361, 376 or 391, or

9) V _A comprises or consists of SEQ ID NO 79, 212, 227 or 242; and V _B comprises or consists of SEQ ID NO 84, 362, 377 or 392, or

10 V _A comprises or consists of SEQ ID No. 106, 213, 228 or 243; and V _B comprises or consists of SEQ ID NO 111, 363, 378 or 393, or

11 V _A comprises or consists of SEQ ID No. 124, 214, 229 or 244; and V _B comprises or consists of SEQ ID NO 129, 364, 379 or 394, or

12 V _A comprises or consists of SEQ ID No. 116, 215, 230 or 245; and V _B comprises or consists of SEQ ID NO 121, 365, 380 or 395, or

13 V _A comprises or consists of SEQ ID NO 34, 216, 231 or 246; and V _B comprises or consists of SEQ ID NO 37, 366, 381 or 396, or

14 V _A comprises or consists of SEQ ID No. 23, 217, 232 or 247; and V _B comprises or consists of SEQ ID NO 28, 367, 382 or 397, or

15 V _A comprises or consists of SEQ ID No. 42, 218, 233 or 248; and V _B comprises or consists of SEQ ID NO 47, 368, 383 or 398.

More preferably, the process is carried out,

1) V _A comprises or consists of the amino acid sequence of SEQ ID NO. 13 and V _B comprises or consists of the amino acid sequence of SEQ ID NO. 18;

2) V _A comprises or consists of the amino acid sequence of SEQ ID NO. 132, and V _B comprises or consists of the amino acid sequence of SEQ ID NO. 135;

3) V _A comprises or consists of the amino acid sequence of SEQ ID NO. 62, and V _B comprises or consists of the amino acid sequence of SEQ ID NO. 65;

4) V _A comprises or consists of the amino acid sequence of SEQ ID NO. 89, and V _B comprises or consists of the amino acid sequence of SEQ ID NO. 94;

5) V _A comprises or consists of the amino acid sequence of SEQ ID NO. 1 and V _B comprises or consists of the amino acid sequence of SEQ ID NO. 7;

6) V _A comprises or consists of the amino acid sequence of SEQ ID NO. 52 and V _B comprises or consists of the amino acid sequence of SEQ ID NO. 57;

7) V _A comprises or consists of the amino acid sequence of SEQ ID NO. 70, and V _B comprises or consists of the amino acid sequence of SEQ ID NO. 74;

8) V _A comprises or consists of the amino acid sequence of SEQ ID NO. 98 and V _B comprises or consists of the amino acid sequence of SEQ ID NO. 103;

9) V _A comprises or consists of the amino acid sequence of SEQ ID NO. 79 and V _B comprises or consists of the amino acid sequence of SEQ ID NO. 84;

10 V _A comprises or consists of the amino acid sequence of SEQ ID No. 106 and V _B comprises or consists of the amino acid sequence of SEQ ID No. 111;

11 V _A comprises or consists of the amino acid sequence of SEQ ID No. 124 and V _B comprises or consists of the amino acid sequence of SEQ ID No. 129;

12 V _A comprises or consists of the amino acid sequence of SEQ ID No. 116 and V _B comprises or consists of the amino acid sequence of SEQ ID No. 121;

13 V _A comprises or consists of the amino acid sequence of SEQ ID No. 34 and V _B comprises or consists of the amino acid sequence of SEQ ID No. 37;

14 V _A comprises or consists of the amino acid sequence of SEQ ID No. 23 and V _B comprises or consists of the amino acid sequence of SEQ ID No. 28; or alternatively

15 V _A comprises or consists of the amino acid sequence of SEQ ID No. 42 and V _B comprises or consists of the amino acid sequence of SEQ ID No. 47.

In a further embodiment of the present invention,

1) V _A comprises or consists of SEQ ID NO 13 and V _B comprises or consists of SEQ ID NO 369, or

2) V _A comprises or consists of SEQ ID NO 132 and V _B comprises or consists of SEQ ID NO 370, or

3) V _A comprises or consists of SEQ ID NO. 62 and V _B comprises or consists of SEQ ID NO. 371, or

4) V _A comprises or consists of SEQ ID NO 89 and V _B comprises or consists of SEQ ID NO 372, or

5) V _A comprises or consists of SEQ ID NO 1 and V _B comprises or consists of SEQ ID NO 373, or

6) V _A comprises or consists of SEQ ID NO. 52 and V _B comprises or consists of SEQ ID NO. 374, or

7) V _A comprises or consists of SEQ ID NO 70 and V _B comprises or consists of SEQ ID NO 375, or

8) V _A comprises or consists of SEQ ID NO:198 and V _B comprises or consists of SEQ ID NO:376, or

9) V _A comprises or consists of SEQ ID NO 79 and V _B comprises or consists of SEQ ID NO 377, or

10 V _A comprises or consists of SEQ ID NO:106 and V _B comprises or consists of SEQ ID NO:378, or

11 V _A comprising or consisting of SEQ ID No. 124 and V _B comprising or consisting of SEQ ID No. 379, or

12 V _A comprises or consists of SEQ ID NO 116 and V _B comprises or consists of SEQ ID NO 380, or

13 V _A comprises or consists of SEQ ID NO 34 and V _B comprises or consists of SEQ ID NO 381, or

14 V _A comprising or consisting of SEQ ID No. 23 and V _B comprising or consisting of SEQ ID No. 382, or

15 V _A comprises or consists of SEQ ID No. 42 and V _B comprises or consists of SEQ ID No. 383.

The antigen binding protein may be monovalent or multivalent, e.g., tetravalent, trivalent, or divalent.

Antigen binding proteins are monospecific or multispecific, e.g., tetraspecific, trispecific, or bispecific.

In some embodiments, the antigen binding protein is a soluble protein.

In the antigen binding proteins of the invention, the first polypeptide and the second polypeptide may be comprised in a single polypeptide chain. Such single chain constructs may be single chain TCRs (sctcrs) or single chain bispecific antigen binding proteins, in particular single chain bispecific TCRs or single chain bispecific TCR-antibody molecules.

Preferably, the first polypeptide and the second polypeptide are comprised in two polypeptide chains, i.e. V _A is comprised in the first polypeptide chain and V _B is comprised in the second polypeptide chain.

Preferably, the antigen binding protein is a TCR. The TCR may be selected from an alpha/beta TCR, gamma/delta TCR, single chain TCR, membrane bound TCR, soluble TCR, monovalent, divalent or multivalent TCR, monospecific, bispecific or multispecific TCR, a functional fragment of a TCR, a fusion protein comprising a functional fragment of a TCR, or a chimeric protein comprising a functional fragment of a TCR. In a preferred embodiment, the TCR is an α/β TCR or a γ/δ TCR, preferably an α/β TCR. In the context of the present invention, whenever it is stated that the antigen binding protein is preferably a TCR, this further means that most preferably the antigen binding protein is an α/β TCR or a γ/δ TCR, preferably an α/β TCR. In one embodiment, the TCR constant domain sequence may be derived from any suitable species, such as any mammal, e.g., human, rat, monkey, rabbit, donkey, or mouse, preferably human or mouse, more preferably human. In one embodiment, the TCR is an αβ TCR and comprises an α chain constant domain (TRAC) sequence according to SEQ ID NO:5, 750, 751 or 156 (preferably SEQ ID NO:5, 750 or 751), and a β chain constant domain (TRBC 1 or TRBC 2) sequence according to SEQ ID NO:11, 32 or 157 (preferably SEQ ID NO:11 or 32).

Preferably, the first polypeptide comprises or consists of: an amino acid sequence selected from the group consisting of SEQ ID NOs 17, 134, 64, 93, 6, 56, 73, 102, 83, 110, 128, 120, 36, 27, 46 and 158-172, preferably selected from the group consisting of SEQ ID NOs 17, 134, 64, 93, 6, 56, 73, 102, 83, 110, 128, 120, 36, 27 and 46, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NOs 17, 134, 64, 93, 110, 128, 120, 36, 27, 46 or 158-172, and comprising an amino acid sequence of CDRa1, CDRa2 and CDRa3 as defined in the context of an antigen binding protein of the invention, wherein the CDRa1, CDRa2 and a3 sequences may comprise one, two or three amino acid mutations, and the second polypeptide comprises or consists of: an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 137, 69, 97, 12, 61, 78, 105, 88, 115, 131, 123, 41, 33, 51 and 173-187, preferably selected from the group consisting of SEQ ID NOs 22, 137, 69, 97, 12, 61, 78, 105, 88, 115, 131, 123, 41, 33 and 51, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NOs 22, 137, 69, 97, 12, 61, 78, 105, 88, 115, 131, 123, 41, 33, 51 or 173-187, and comprising CDRa1, CDRa2 and CDRa3 as defined in the context of an antigen binding protein of the invention, wherein the CDRb1, CDRb2 and CDRa3 sequences may comprise one, two or three amino acid mutations.

More preferably, the first polypeptide comprises or consists of: an amino acid sequence selected from the group consisting of SEQ ID NOs 17, 134, 64, 93, 6, 56, 73, 102, 83, 110, 128, 120, 36, 27, 46 and 158-172, preferably selected from the group consisting of SEQ ID NOs 17, 134, 64, 93, 6, 56, 73, 102, 83, 110, 128, 120, 36, 27 and 46, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NOs 17, 134, 64, 93, 6, 56, 73, 102, 83, 110, 128, 120, 36, 27, 46 or 158-172 and comprising an amino acid sequence of CDRa1, CDRa2 and CDRa3 as defined in the context of an antigen binding protein of the invention without amino acid mutations, and the second polypeptide comprises or consists of: an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 137, 69, 97, 12, 61, 78, 105, 88, 115, 131, 123, 41, 33, 51 and 173-187, preferably selected from the group consisting of SEQ ID NOs 22, 137, 69, 97, 12, 61, 78, 105, 88, 115, 131, 123, 41, 33 and 51, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NOs 22, 137, 69, 97, 12, 61, 78, 105, 88, 115, 131, 123, 41, 33, 51 or 173-187 and comprising CDRa1, CDRa2 and CDRa3 as defined in the context of the antigen binding protein of the invention without amino acid mutations.

In a preferred embodiment of the present invention, in a preferred embodiment,

1) The first polypeptide comprises or consists of: the amino acid sequence of SEQ ID No. 17 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 17 and the second polypeptide comprises or consists of: the amino acid sequence of SEQ ID NO. 22 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 22;

2) The first polypeptide comprises or consists of: the amino acid sequence of SEQ ID No. 134 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 134 and the second polypeptide comprises or consists of: the amino acid sequence of SEQ ID NO. 137 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 137;

3) The first polypeptide comprises or consists of: the amino acid sequence of SEQ ID No. 64 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 64 and the second polypeptide comprises or consists of: the amino acid sequence of SEQ ID NO. 69 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 69;

4) The first polypeptide comprises or consists of: the amino acid sequence of SEQ ID No. 93 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 93 and the second polypeptide comprises or consists of: the amino acid sequence of SEQ ID NO. 97 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 97;

5) The first polypeptide comprises or consists of: the amino acid sequence of SEQ ID No. 6 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 6 and the second polypeptide comprises or consists of: the amino acid sequence of SEQ ID NO. 12 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 12;

6) The first polypeptide comprises or consists of: the amino acid sequence of SEQ ID No. 56 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 56 and the second polypeptide comprises or consists of: the amino acid sequence of SEQ ID NO. 61 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 61;

7) The first polypeptide comprises or consists of: the amino acid sequence of SEQ ID No. 73 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 73 and the second polypeptide comprises or consists of: the amino acid sequence of SEQ ID NO. 78 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 78;

8) The first polypeptide comprises or consists of: the amino acid sequence of SEQ ID No. 102 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 102 and the second polypeptide comprises or consists of: the amino acid sequence of SEQ ID NO. 105 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 105;

9) The first polypeptide comprises or consists of: the amino acid sequence of SEQ ID No. 83 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 83 and the second polypeptide comprises or consists of: the amino acid sequence of SEQ ID NO. 88 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 88;

10 A) the first polypeptide comprises or consists of: the amino acid sequence of SEQ ID No. 110 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 110 and the second polypeptide comprises or consists of: the amino acid sequence of SEQ ID NO. 115 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 115;

11 A) the first polypeptide comprises or consists of: the amino acid sequence of SEQ ID No. 128 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 128 and the second polypeptide comprises or consists of: the amino acid sequence of SEQ ID NO. 131 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 131;

12 A) the first polypeptide comprises or consists of: the amino acid sequence of SEQ ID No. 120 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 120 and the second polypeptide comprises or consists of: the amino acid sequence of SEQ ID NO. 123 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 123;

13 A) the first polypeptide comprises or consists of: the amino acid sequence of SEQ ID No. 36 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 36 and the second polypeptide comprises or consists of: the amino acid sequence of SEQ ID NO. 41 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 41;

14 A) the first polypeptide comprises or consists of: the amino acid sequence of SEQ ID No. 27 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 27 and the second polypeptide comprises or consists of: the amino acid sequence of SEQ ID NO. 33 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 33; or alternatively

15 A) the first polypeptide comprises or consists of: the amino acid sequence of SEQ ID No. 46 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 46 and the second polypeptide comprises or consists of: the amino acid sequence of SEQ ID NO. 51 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 51;

Wherein the first polypeptide and the second polypeptide comprise CDR sequences as defined in the context of the antigen binding proteins of the invention, wherein said CDR sequences may comprise one, two or three amino acid mutations, preferably no amino acid mutations.

In a preferred embodiment of the present invention, in a preferred embodiment,

1) The first polypeptide comprises or consists of SEQ ID NO. 17, 294, 309 or 324 and the second polypeptide comprises or consists of SEQ ID NO. 22, 444, 459 or 474;

2) The first polypeptide comprises or consists of SEQ ID NO 134, 295, 310 or 325 and the second polypeptide comprises or consists of SEQ ID NO 137, 445, 460 or 475;

3) The first polypeptide comprises or consists of SEQ ID NO. 64, 296, 311 or 326 and the second polypeptide comprises or consists of SEQ ID NO. 69, 446, 461 or 476;

4) The first polypeptide comprises or consists of SEQ ID No. 93, 297, 312 or 327 and the second polypeptide comprises or consists of SEQ ID No. 97, 447, 462 or 477;

5) The first polypeptide comprises or consists of SEQ ID NO. 6, 298, 313 or 328 and the second polypeptide comprises or consists of SEQ ID NO. 12, 448, 463 or 478;

6) The first polypeptide comprises or consists of SEQ ID NO. 56, 299, 314 or 329 and the second polypeptide comprises or consists of SEQ ID NO. 61, 449, 464 or 479;

7) The first polypeptide comprises or consists of SEQ ID NO 73, 300, 315 or 330 and the second polypeptide comprises or consists of SEQ ID NO 78, 450, 465 or 480;

8) The first polypeptide comprises or consists of SEQ ID NO. 102, 301, 316 or 331 and the second polypeptide comprises or consists of SEQ ID NO. 105, 451, 466 or 481;

9) The first polypeptide comprises or consists of SEQ ID NO 83, 302, 317 or 332 and the second polypeptide comprises or consists of SEQ ID NO 88, 452, 467 or 482;

10 110, 303, 318 or 333 or consists of SEQ ID No. s.115, 453, 468 or 483;

11 128, 304, 319 or 334 or consists of SEQ ID No. 131, 454, 469 or 484;

12 120, 305, 320 or 335 or consists of SEQ ID No. and the second polypeptide comprises or consists of SEQ ID No. 123, 455, 470 or 485;

13 36, 306, 321 or 336 or consists of SEQ ID No. s, and the second polypeptide comprises or consists of SEQ ID No. s 41, 456, 471 or 486;

14 27, 307, 322 or 337, and the second polypeptide comprises or consists of SEQ ID No. 33, 457, 472 or 487; or alternatively

15 A) the first polypeptide comprises or consists of SEQ ID No. 46, 308, 323 or 338 and the second polypeptide comprises or consists of SEQ ID No. 51, 458, 473 or 488.

More preferably, the process is carried out,

1) The first polypeptide comprises or consists of SEQ ID NO. 17 and the second polypeptide comprises or consists of SEQ ID NO. 22;

2) The first polypeptide comprises or consists of SEQ ID NO. 134 and the second polypeptide comprises or consists of SEQ ID NO. 137;

3) The first polypeptide comprises or consists of SEQ ID NO. 64 and the second polypeptide comprises or consists of SEQ ID NO. 69;

4) The first polypeptide comprises or consists of SEQ ID NO. 93 and the second polypeptide comprises or consists of SEQ ID NO. 97;

5) The first polypeptide comprises or consists of SEQ ID NO. 6 and the second polypeptide comprises or consists of SEQ ID NO. 12;

6) The first polypeptide comprises or consists of SEQ ID NO. 56 and the second polypeptide comprises or consists of SEQ ID NO. 61;

7) The first polypeptide comprises or consists of SEQ ID NO. 73 and the second polypeptide comprises or consists of SEQ ID NO. 78;

8) The first polypeptide comprises or consists of SEQ ID NO. 102 and the second polypeptide comprises or consists of SEQ ID NO. 105;

9) The first polypeptide comprises or consists of SEQ ID NO. 83 and the second polypeptide comprises or consists of SEQ ID NO. 88;

10 A first polypeptide comprising or consisting of SEQ ID NO. 110 and a second polypeptide comprising or consisting of SEQ ID NO. 115;

11 A first polypeptide comprising or consisting of SEQ ID NO. 128 and a second polypeptide comprising or consisting of SEQ ID NO. 131;

12 A first polypeptide comprising or consisting of SEQ ID NO. 120 and a second polypeptide comprising or consisting of SEQ ID NO. 123;

13 A first polypeptide comprising or consisting of SEQ ID NO. 36 and a second polypeptide comprising or consisting of SEQ ID NO. 41;

14 A first polypeptide comprising or consisting of SEQ ID NO. 27 and a second polypeptide comprising or consisting of SEQ ID NO. 33; or alternatively

15 A first polypeptide comprising or consisting of SEQ ID NO. 46 and a second polypeptide comprising or consisting of SEQ ID NO. 51.

In the case of a further embodiment of the present invention,

1) The first polypeptide comprises or consists of SEQ ID NO. 17 and the second polypeptide comprises or consists of SEQ ID NO. 459;

2) The first polypeptide comprises or consists of SEQ ID NO. 134 and the second polypeptide comprises or consists of SEQ ID NO. 460;

3) The first polypeptide comprises or consists of SEQ ID NO. 64 and the second polypeptide comprises or consists of SEQ ID NO. 461;

4) The first polypeptide comprises or consists of SEQ ID NO. 93 and the second polypeptide comprises or consists of SEQ ID NO. 462;

5) The first polypeptide comprises or consists of SEQ ID NO. 6 and the second polypeptide comprises or consists of SEQ ID NO. 463;

6) The first polypeptide comprises or consists of SEQ ID NO. 56 and the second polypeptide comprises or consists of SEQ ID NO. 464;

7) The first polypeptide comprises or consists of SEQ ID NO. 73 and the second polypeptide comprises or consists of SEQ ID NO. 465;

8) The first polypeptide comprises or consists of SEQ ID NO. 102 and the second polypeptide comprises or consists of SEQ ID NO. 466;

9) The first polypeptide comprises or consists of SEQ ID NO. 83 and the second polypeptide comprises or consists of SEQ ID NO. 467;

10 A first polypeptide comprising or consisting of SEQ ID NO. 110 and a second polypeptide comprising or consisting of SEQ ID NO. 468;

11 A first polypeptide comprising or consisting of SEQ ID NO. 128 and a second polypeptide comprising or consisting of SEQ ID NO. 469;

12 A first polypeptide comprising or consisting of SEQ ID NO. 120 and a second polypeptide comprising or consisting of SEQ ID NO. 470;

13 A first polypeptide comprising or consisting of SEQ ID NO. 36 and a second polypeptide comprising or consisting of SEQ ID NO. 471;

14A first polypeptide comprising or consisting of SEQ ID NO. 27 and a second polypeptide comprising or consisting of SEQ ID NO. 472; or alternatively

15 A) the first polypeptide comprises or consists of SEQ ID NO. 46 and the second polypeptide comprises or consists of SEQ ID NO. 473.

In some preferred embodiments, the antigen binding proteins of the invention may be engineered, for example, by introducing heterologous sequences, preferably mouse sequences, that may increase expression and stability. Moreover, other stabilizing mutations as known in the state of the art (e.g. WO 2018/104407, PCT/EP 2018/069151, WO 2011/044186, WO 2014/018863) may be introduced, such as substitution of unfavorable amino acids in the variable domains and/or the introduction of disulfide bonds (e.g. between constant domains of TCRs) and removal of unpaired cysteines.

In particular, the TCR constant domain sequence may be modified by truncation or substitution to delete the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC 2. One or more alpha and/or beta chain constant domain sequences may also be modified by substituting Thr 48 of TRAC and Ser57 of TRBC1 or TRBC2 with a cysteine residue that forms a disulfide bond between the alpha and beta constant domains of the TCR. TRBC1 or TRBC2 may additionally comprise a cysteine to alanine mutation at position 75 of the constant domain, and an asparagine to aspartic acid mutation at position 89 of the constant domain. The constant domain may additionally or alternatively contain other mutations, substitutions or deletions relative to the native TRAC and/or TRBC1/2 sequence. The terms TRAC and TRBC1/2 encompass natural polymorphic variants such as N to K at position 4 of TRAC (Bragado et al Int immunol.1994, month 2; 6 (2): 223-30).

In some embodiments, the antigen binding protein is monovalent or multivalent, e.g., tetravalent, trivalent, or divalent.

In some embodiments, the antigen binding protein is bispecific, particularly a bispecific TCR, bispecific antibody, or bispecific TCR-antibody molecule. One skilled in the art knows that where the antigen binding protein is a bispecific "antibody", one of the antigen binding sites comprises CDR1, CDR3 and optionally CDR2 sequences of TCR origin as defined in the context of the antigen binding protein of the invention, while the other antigen binding site may be entirely of antibody origin.

In some embodiments, the antigen binding protein is a soluble protein. In some embodiments, the antigen binding protein is a soluble TCR. As used herein, the term "soluble TCR" refers to a heterodimeric truncated variant of a native TCR, which comprises the extracellular portion of the TCR alpha and beta chains (e.g., linked by disulfide bonds), but which lacks the transmembrane and cytoplasmic domains of the native protein.

In one embodiment, the antigen binding protein is of human origin, which is understood to be produced from a human locus and thus comprises human sequences.

In some embodiments, the antigen binding protein is humanized, chimeric and/or murine.

In one embodiment, the antigen binding proteins of the invention further comprise one or more of the following:

(i) One or more additional antigen binding sites;

(ii) A transmembrane region, optionally comprising a cytoplasmic signaling region;

(iii) A diagnostic agent;

(iv) A therapeutic agent.

Where elements (i) to (v) listed above are polypeptides fused to an antigen binding protein of the invention, the antigen binding protein may also be referred to as a "TCR fusion protein".

The other antigen binding site, if present, is preferably of antibody origin.

The invention encompasses antigen binding proteins (particularly TCRs) having an alternative domain, such as a membrane anchoring domain, in place of the endogenous transmembrane region. Antigen binding proteins (particularly TCRs) having point mutations in the TCR variable domain or constant domain to improve TCR expression or stability and/or chain pairing are also contemplated.

In the context of the present invention, a "transmembrane region" may be, for example, a TCR α or β transmembrane domain.

The "cytoplasmic signaling region" may be, for example, a TCR α or β intracellular domain.

"Diagnostic agent" herein refers to a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule, or any other label known in the art that provides a signal (directly or indirectly).

"Fluorescent molecules" are known in the art and include Fluorescein Isothiocyanate (FITC), phycoerythrin (PE), fluorophores for blue lasers (e.g., perCP, PE-Cy7, PE-Cy5, FL3, and APC or Cy5, FL 4), fluorophores for red, violet, or UV lasers (e.g., pacific blue, pacific orange).

"Radioactive molecules" include, but are not limited to, radioactive atoms such as I ¹²³、I¹²⁴、In¹¹¹、Re¹⁸⁶、Re¹⁸⁸、Tc⁹⁹ for scintigraphy studies. The antigen binding proteins of the invention may also comprise spin labels for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Such diagnostic agents may be directly coupled (i.e., physically linked) to the antigen binding protein, or may be indirectly linked.

"Therapeutic agent" herein refers to an agent that has a therapeutic effect. The terms therapeutic agent and drug are used interchangeably herein. In one embodiment, the therapeutic agent may be a growth inhibitor, such as a cytotoxic agent or radioisotope.

"Growth inhibitory agent" or "antiproliferative agent" which may be used indifferently refers to a compound or composition which inhibits the growth of cells, in particular tumor cells, in vitro or in vivo.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cellular function and/or causes cellular destruction. The term "cytotoxic agent" is intended to include chemotherapeutic agents, enzymes, antibiotics and toxins (e.g., small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof) as well as various antineoplastic or anticancer agents disclosed below. In some embodiments, the cytotoxic agent is a taxane, a vinca, a taxane, a maytansinoid or a maytansinoid analog (e.g., DM1 or DM 4), a small drug, a tomaymycin or a pyrrolobenzodiazepineDerivatives, nostoc derivatives, leptomycin derivatives, australistatin or a dolastatin analog, prodrugs, topoisomerase II inhibitors, DNA alkylating agents, anti-tubulin agents, CC-1065 or CC-1065 analogs.

The term "radioisotope" is intended to include radioisotopes suitable for the treatment of cancer, such as At²¹¹、Bi²¹²、Er¹⁶⁹、I¹³¹、I¹²⁵、Y⁹⁰、In¹¹¹、P³²、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Sr⁸⁹ and radioisotopes of Lu. Such radioisotopes typically emit primarily beta radiation. In one embodiment, the radioisotope is an alpha emitter isotope, more precisely thorium 227, which emits alpha radiation.

In some embodiments, the antigen binding proteins of the invention are covalently attached to at least one growth inhibitor directly or via a cleavable or non-cleavable linker. An antigen binding protein attached to such at least one growth inhibitor may also be referred to as a conjugate. The cleavable linker facilitates release of the cytotoxic agent or growth inhibitory agent from the antigen binding protein in the cell. For example, an acid labile linker, a peptidase sensitive linker, an esterase labile linker, a photolabile linker, or a disulfide containing linker may be used (see, e.g., U.S. Pat. No. 5,208,020). The linker may also be a "non-cleavable linker" (e.g., an SMCC linker), which may in some cases lead to better tolerability.

The preparation of such conjugates (e.g., immunoconjugates) is described in applications WO2004/091668 or Hudecz, f., methods mol. Biol.298:209-223 (2005) and Kirin et al, inorg chem.44 (15): 5405-5415 (2005), the contents of which are incorporated herein by reference in their entirety, and can be transferred by one of skill in the art into the preparation of antigen binding proteins of the invention attached to such at least one growth inhibitor.

Alternatively, fusion proteins comprising an antigen binding protein of the invention and a cytotoxic or growth inhibitory polypeptide may be prepared by recombinant techniques or peptide synthesis. The length of DNA may comprise corresponding regions encoding the two portions of the conjugate, adjacent to each other or separated by a region encoding a linker peptide that does not disrupt the desired properties of the conjugate.

The antigen binding proteins of the invention may also be used in dependent enzyme mediated prodrug therapies by conjugating the polypeptide to a prodrug activating enzyme that converts a precursor (e.g., a peptide-based chemotherapeutic agent, see WO 81/01145) to an active anticancer agent (see, e.g., WO 88/07378 and U.S. patent No. 4,975,278).

In some embodiments, the antigen binding protein specifically binds to or consists of functional epitopes comprising amino acid positions 1, 3 and 4 of SEQ ID NO 138. In some preferred embodiments, these antigen binding proteins specifically bind to a functional epitope comprising or consisting of: amino acid positions 1, 3, 4 and 5, or 1, 3, 4 and 6, or 1, 3, 4, 5, 6 and 7 of SEQ ID NO. 138.

In some embodiments, the antigen binding protein specifically binds to or consists of functional epitopes comprising amino acid positions 4, 6 and 7 of SEQ ID NO 138. In some preferred embodiments, these antigen binding proteins specifically bind to a functional epitope comprising or consisting of: amino acid positions 1,4, 6 and 7, or 3,4, 6 and 7, or 1,3, 4, 6 and 7 of SEQ ID NO. 138.

In some embodiments, the antigen binding protein specifically binds to or consists of functional epitopes comprising amino acid positions 5 and 7 of SEQ ID NO 138. In some preferred embodiments, these antigen binding proteins specifically bind to a functional epitope comprising or consisting of: amino acid positions 5, 6 and 7, or 3, 4, 5, 6 and 7 of SEQ ID NO. 138.

In some embodiments, the antigen binding protein does not bind significantly to: a similar peptide of the group consisting of SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO 155 (SP-05-0010).

In some embodiments, the antigen binding protein does not bind significantly to: a similar peptide of the group consisting of SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO 155 (SP-05-0010).

Genes encoding the V and J regions contained in V _A and V _B of the antigen binding proteins of the invention are listed in table 2. The annotation is made by GeneData 11.0.1 using IMGT/GENE-DB (version: 28/11/2019) as a reference database.

In some embodiments, V _A comprises the V region encoded by TRAV14 (especially TRAV14/DV 4), as well as CDRA1 according to SEQ ID NO. 24 and CDRA2 according to SEQ ID NO. 25.

In some embodiments, V _B comprises the V region encoded by TRBV13, as well as CDRB1 according to SEQ ID NO. 75 and CDRB2 according to SEQ ID NO. 76.

In some embodiments, V _B comprises the V region encoded by TRBV4-1, as well as CDRB1 according to SEQ ID NO. 58 and CDRB2 according to SEQ ID NO. 59.

In some embodiments, V _B comprises the V region encoded by TRBV6-1, as well as CDRB1 according to SEQ ID NO. 112 and CDRB2 according to SEQ ID NO. 113.

The inventors have shown that the antigen binding proteins of the invention, if expressed in cd8+ T cells, are capable of activating said cd8+ T cells upon binding to CT45-IP presented on MHC by antigen presenting cells.

In addition to cd8+ T cells, cd4+ T cells (known as T helper cells) are also critical for the coordinated immune response that joins a variety of different immune cells. For complete activation of T cells after encountering their cognate peptide-MHC complex, additional binding of the corresponding accessory receptor is often required. In the case of cd8+ T cells, this assistance is provided by the CD8 co-receptor, and in the case of cd4+ T cells, this assistance is provided by the CD4 co-receptor. In only a few cases, TCRs derived from cd8+ T cells are able to elicit intracellular signaling strong enough to result in activation of cd4+ T cells when transferred to cd4+ T cells.

The inventors have shown that some antigen binding proteins of the invention, if expressed in cd4+ T cells (in particular cd4+cd8-T cells), are capable of activating said cd4+cd8-T cells after binding to CT45-IP presented on MHC by antigen presenting cells (example 3, data not shown).

In the context of the present invention, an antigen binding protein is considered "capable of activating T cells" if, in a cytokine production assay as defined above, T cells expressing the antigen binding protein (i.e. effector cells) produce at least one intracellular cytokine upon co-culture with target cells presenting the CT45-IP antigenic peptide, in particular if the number of T cells expressing the antigen binding protein and producing the at least one intracellular cytokine is at least 2%, at least 2.5%, preferably at least 3% per analyzed T cell population (e.g. CD4 ⁺ or CD8 ⁺ T cells).

Thus, in some embodiments, the antigen binding protein is capable of activating cd8+ T cells (particularly cd8+cd4-T cells) and/or cd4+ T cells (particularly cd4+cd8-T cells). In a preferred embodiment, the antigen binding protein is capable of activating CD4+ T cells, in particular CD4+ CD8-T cells. In other words, the antigen binding protein (preferably TCR) is capable of activating cd4+ T cells, independent of CD 8. In other words, the antigen binding protein (preferably TCR) is capable of binding to a complex of CT45 antigen peptide and MHC molecule in the absence of CD 8. In a most preferred embodiment, the antigen binding protein is capable of activating CD4+ T cells (particularly CD4+CD8-T cells) and CD8+ T cells (particularly CD8+CD4-T cells). In a preferred embodiment, the antigen binding protein is a TCR.

In the context of the present invention, an antigen binding protein is considered to be capable of activating a T cell population if cytokine production is detected in at least 2%, at least 2.5%, preferably at least 3% of said cd4+ or CD4-cd8+ T cell population in a functional cytokine production assay as described above. The secreted cytokine may be, for example, IFN-gamma and/or TNF-alpha.

Activation of cd4+ T cells via a TCR derived from cd8+ T cells can be enhanced by transferring the CD8 co-receptor into the cd4+ cells along with the TCR. The inventors have shown that co-transfection of cd4+ T cells with CT45-IP specific TCRs and CD8 as described herein significantly enhances killing of CT45-IP presenting tumor cells (example 8, data not shown). Joining cd4+ T cells together with cd8+ T cells offers many advantages for cellular immunotherapy. Cd4+ T cells not only can cause direct cytotoxicity against tumor cells, but also can bind other immune cells, promoting long-lasting anti-tumor effects. This helper function is exerted by the provision of cytokines, chemokines and costimulatory molecules, and includes the support of cytotoxic cd8+ T cells, the formation of effector and memory T cells, the activation and maturation of macrophages/dendritic cells, the licensing of dendritic cells (which in turn effectively stimulate cd8+ T cells and drive cd8+ T effector and memory cell formation), the activation of innate immune cells (such as NK cells), the formation of memory B cells and many other effects.

In some embodiments, the antigen binding protein has an average expression of at least 5%, at least 10%, at least 20%, at least 30%, or at least 40%, particularly after transient expression. Average expression of TCRs can be determined by TCR surface staining as described in the examples. Dyeing may be performed by techniques known in the art. For example, staining can be performed using a specific V _B antibody (in the case of a human TCR) or an anti-mTCRB antibody (in the case of a chimeric TCR) or a labeled CT45-IP: MHC multimer (e.g., tetramer or dextramer). Such staining may further be combined with staining for identifying specific cell populations.

Antigen binding protein 1

In some embodiments, the invention relates to an antigen binding protein that specifically binds to a CT45 antigen peptide, the CT45 antigen peptide being in a complex with a Major Histocompatibility Complex (MHC) protein, wherein the CT45 antigen peptide comprises or consists of the amino acid sequence of SEQ ID NO:138 (KIFEMLEGV) and wherein the antigen binding protein comprises or consists of a first polypeptide comprising or consisting of the variable domain V _A comprising Complementarity Determining Regions (CDRs) CDRa1, CDRa2 and CDRa3, and the second polypeptide comprising or consisting of the variable domain V _B comprising CDRa1, CDRa2 and CDRa3, wherein CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO:14, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:16, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:19, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:21, wherein the CDRa1, CDRa3 and/or two or three amino acids may comprise mutations. The antigen binding protein comprising the CDR sequences is also referred to hereinafter as "antigen binding protein 1". In a preferred embodiment of the antigen binding protein 1, CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 15 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 20, wherein the CDRa2 and/or CDRb2 sequences may comprise one, two, three or four amino acid mutations. Antigen binding protein 1 specifically binds to a functional epitope comprising or consisting of: position 1,3, 4, 5, 6 and 7 of SEQ ID NO. 138. The EC ₅₀ of cells that express the antigen binding protein 1 (e.g., in a luciferase release assay) that are induced to kill cells presenting the CT45-IP: MHC complex (e.g., CT45-IP loaded T2 cells) is measured to be less than about 30nM, less than about 25nM, less than about 20nM, less than about 15nM, or less than about 10nM. Antigen binding protein 1 did not bind significantly to: at least 1, at least 2, at least 3, at least 4, at least 5 or all analogous peptides selected from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO 155 (SP-05-0010). In a preferred embodiment of antigen binding protein 1, V _A comprises or consists of: the amino acid sequence of SEQ ID No. 13 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 13 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 18 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 18; wherein V _A and V _B comprise a CDR sequence as described above for antigen binding protein 1, wherein the CDR sequence may comprise one, two or three amino acid mutations, preferably no amino acid mutations. Preferably, the alpha chain variable region of antigen binding protein 1 is encoded by TRAV38-1 and/or the beta chain variable region of antigen binding protein 1 is encoded by TRBV 7-9. In preferred embodiments, antigen binding protein 1 is capable of activating CD8+ T cells (particularly CD8+ CD4-T cells), and/or CD4+ T cells (particularly CD4+ CD8-T cells).

Antigen binding protein 2

In some embodiments, the invention relates to an antigen binding protein that specifically binds to a CT45 antigen peptide, the CT45 antigen peptide being in a complex with a Major Histocompatibility Complex (MHC) protein, wherein the CT45 antigen peptide comprises or consists of the amino acid sequence of SEQ ID NO:138 (KIFEMLEGV) and wherein the antigen binding protein comprises a first polypeptide comprising or consisting of the variable domain V _A comprising Complementarity Determining Regions (CDRs) CDRa1, CDRa2 and CDRa3, and a second polypeptide comprising or consisting of the variable domain V _B comprising CDRa1, CDRa2 and CDRa3, wherein CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO:24, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:133, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:75, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:136, wherein the CDRa1, CDRa3 and/or two or three amino acids may comprise mutations. The antigen binding protein comprising the CDR sequences is also referred to hereinafter as "antigen binding protein 2". In a preferred embodiment of antigen binding protein 2, CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 25 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 76, wherein the CDRa2 and/or CDRb2 sequences may comprise one, two, three or four amino acid mutations. Antigen binding protein 2 specifically binds to a functional epitope comprising or consisting of: position 3, 4, 5, 6 and 7 of SEQ ID NO. 138. The EC ₅₀ of cells expressing the antigen binding protein 2 (e.g., T2 cells loaded with CT 45-IP) is measured to be less than about 30nM, less than about 25nM, less than about 20nM, less than about 15nM, less than about 10nM or less than about 5nM for the induction of antigen binding protein 2-expressing T cell killing of MHC complex (e.g., CT45-IP loaded T2 cells). Antigen binding protein 2 did not bind significantly to: at least 1, at least 2, at least 3, at least 4, at least 5 or all analogous peptides selected from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO 155 (SP-05-0010). In a preferred embodiment of antigen binding protein 2, V _A comprises or consists of: the amino acid sequence of SEQ ID No. 132 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 132 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 135 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 135; wherein V _A and V _B comprise a CDR sequence as described above for antigen binding protein 2, wherein the CDR sequence may comprise one, two or three amino acid mutations, preferably no amino acid mutations. Preferably, the alpha chain variable region of antigen binding protein 2 is encoded by TRAV14/DV4 and/or the beta chain variable region of antigen binding protein 2 is encoded by TRBV 13. In preferred embodiments, antigen binding protein 2 is capable of activating CD8+ T cells (particularly CD8+ CD4-T cells), and/or CD4+ T cells (particularly CD4+ CD8-T cells).

Antigen binding protein 3

In some embodiments, the invention relates to an antigen binding protein that specifically binds to a CT45 antigen peptide, the CT45 antigen peptide being in a complex with a Major Histocompatibility Complex (MHC) protein, wherein the CT45 antigen peptide comprises or consists of the amino acid sequence of SEQ ID NO:138 (KIFEMLEGV) and wherein the antigen binding protein comprises or consists of a first polypeptide comprising or consisting of the variable domain V _A comprising Complementarity Determining Regions (CDRs) CDRa1, CDRa2 and CDRa3, and the second polypeptide comprising or consisting of the variable domain V _B comprising CDRa1, CDRa2 and CDRa3, wherein CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO:24, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:66, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:68, wherein the CDRa1, CDRa3 and/or two or three amino acids may comprise mutations. The antigen binding protein comprising the CDR sequences is also referred to hereinafter as "antigen binding protein 3". In a preferred embodiment of antigen binding protein 3, CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 25 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 67, wherein CDRa2 and/or CDRb2 sequences may comprise one, two, three or four amino acid mutations. Antigen binding protein 3 specifically binds to a functional epitope comprising or consisting of: position 3, 4,5, 6 and 7 of SEQ ID NO. 138. The EC ₅₀ of cells that express the antigen binding protein 3 (e.g., in a luciferase release assay) that are induced to kill cells presenting the CT45-IP: MHC complex (e.g., CT45-IP loaded T2 cells) is measured to be less than about 15nM, less than about 10nM, less than about 5nM, less than about 2.5nM, or less than about 1.5nM. Antigen binding protein 3 did not bind significantly to: at least 1, at least 2, at least 3, at least 4, at least 5 or all of the like peptides selected from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO:155 (SP-05-0010), preferably from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO:155 (SP-05-0010). In a preferred embodiment of antigen binding protein 3, V _A comprises or consists of: the amino acid sequence of SEQ ID No. 62 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 62 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 65 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 65; wherein V _A and V _B comprise a CDR sequence as described above for antigen binding protein 3, wherein the CDR sequence may comprise one, two or three amino acid mutations, preferably no amino acid mutations. Preferably, the alpha chain variable region of antigen binding protein 3 is encoded by TRAV14/DV4 and/or the beta chain variable region of antigen binding protein 3 is encoded by TRBV 27. In preferred embodiments, antigen binding protein 3 is capable of activating CD8+ T cells (particularly CD8+ CD4-T cells), and/or CD4+ T cells (particularly CD4+ CD8-T cells).

Antigen binding protein 4

In some embodiments, the invention relates to an antigen binding protein that specifically binds to a CT45 antigen peptide, the CT45 antigen peptide being in a complex with a Major Histocompatibility Complex (MHC) protein, wherein the CT45 antigen peptide comprises or consists of the amino acid sequence of SEQ ID NO:138 (KIFEMLEGV) and wherein the antigen binding protein comprises or consists of a first polypeptide comprising or consisting of the variable domain V _A comprising Complementarity Determining Regions (CDRs) CDRa1, CDRa2 and CDRa3, and the second polypeptide comprising or consisting of the variable domain V _B comprising CDRa1, CDRa2 and CDRa3, wherein CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO:90, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:66, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:96, wherein the CDRa1, CDRa3 and/or two or three amino acids may comprise mutations. The antigen binding protein comprising the CDR sequences is also referred to hereinafter as "antigen binding protein 4". In a preferred embodiment of antigen binding protein 4, CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 91 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 95, wherein CDRa2 and/or CDRb2 sequences may comprise one, two, three or four amino acid mutations. Antigen binding protein 4 specifically binds to a functional epitope comprising or consisting of: position 3, 4, 6, 7 and 8 of SEQ ID NO. 138. The EC ₅₀ of cells expressing the antigen binding protein 4 (e.g., T2 cells loaded with CT 45-IP) that are tested (e.g., in a luciferase release assay) to kill cells presenting the CT45-IP MHC complex is less than about 25nM, less than about 20nM, less than about 15nM, less than about 10nM, less than about 5nM, less than about 2.5nM, or less than about 1.5nM. Antigen binding protein 1 did not bind significantly to: at least 1, at least 2, at least 3, at least 4, at least 5 or all of the like peptides selected from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO:155 (SP-05-0010), preferably from SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO:155 (SP-05-0010). In a preferred embodiment of antigen binding protein 4, V _A comprises or consists of: the amino acid sequence of SEQ ID No. 89 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 89 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 94 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 94; wherein V _A and V _B comprise a CDR sequence as described above for antigen binding protein 4, wherein the CDR sequence may comprise one, two or three amino acid mutations, preferably no amino acid mutations. Preferably, the alpha chain variable region of antigen binding protein 4 is encoded by TRAV3 and/or the beta chain variable region of antigen binding protein 4 is encoded by TRBV 6-2. In preferred embodiments, the antigen binding protein 4 is capable of activating CD8+ T cells (particularly CD8+ CD4-T cells), and/or CD4+ T cells (particularly CD4+ CD8-T cells).

Antigen binding protein 5

In some embodiments, the invention relates to an antigen binding protein that specifically binds to a CT45 antigen peptide, the CT45 antigen peptide being in a complex with a Major Histocompatibility Complex (MHC) protein, wherein the CT45 antigen peptide comprises or consists of the amino acid sequence of SEQ ID NO:138 (KIFEMLEGV) and wherein the antigen binding protein comprises a first polypeptide comprising or consisting of the variable domain V _A comprising Complementarity Determining Regions (CDRs) CDRa1, CDRa2 and CDRa3, and a second polypeptide comprising or consisting of the variable domain V _B comprising CDRa1, CDRa2 and CDRa3, wherein CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO:2, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:4, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:8, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:10, wherein the CDRa1, CDRa3 and/or two or three amino acids may comprise mutations. The antigen binding protein comprising the CDR sequences is also referred to hereinafter as "antigen binding protein 5". In a preferred embodiment of the antigen binding protein 5, CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 3 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 9, wherein the CDRa2 and/or CDRb2 sequences may comprise one, two, three or four amino acid mutations. Antigen binding protein 5 specifically binds to a functional epitope comprising or consisting of 2,3 or 4 amino acid positions selected from positions 3, 6, 7 and 8 of SEQ ID No. 138. The EC ₅₀ of cells expressing the antigen binding protein 5 (e.g., T2 cells loaded with CT 45-IP) is measured (e.g., in a luciferase release assay) to be less than about 25nM, less than about 15nM, less than about 10nM, less than about 5nM, less than about 2.5nM, less than about 1.5nM, or less than about 1nM. Antigen binding protein 5 did not bind significantly to: at least 1, at least 2, at least 3, at least 4, at least 5 or all analogous peptides selected from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO 155 (SP-05-0010). In a preferred embodiment of antigen binding protein 5, V _A comprises or consists of: the amino acid sequence of SEQ ID No. 1 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 1 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 7 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 7; wherein V _A and V _B comprise a CDR sequence as described above for antigen binding protein 5, wherein the CDR sequence may comprise one, two or three amino acid mutations, preferably no amino acid mutations. Preferably, the alpha chain variable region of antigen binding protein 5 is encoded by TRAV35 and/or the beta chain variable region of antigen binding protein 5 is encoded by TRBV 9. In preferred embodiments, the antigen binding protein 5 is capable of activating CD8+ T cells (particularly CD8+ CD4-T cells), and/or CD4+ T cells (particularly CD4+ CD8-T cells).

Antigen binding protein 6

In some embodiments, the invention relates to an antigen binding protein that specifically binds to a CT45 antigen peptide, the CT45 antigen peptide being in a complex with a Major Histocompatibility Complex (MHC) protein, wherein the CT45 antigen peptide comprises or consists of the amino acid sequence of SEQ ID NO:138 (KIFEMLEGV) and wherein the antigen binding protein comprises a first polypeptide comprising or consisting of the variable domain V _A comprising Complementarity Determining Regions (CDRs) CDRa1, CDRa2 and CDRa3, and a second polypeptide comprising or consisting of the variable domain V _B comprising CDRa1, CDRa2 and CDRa3, wherein CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO:53, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:58, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:60, wherein the CDRa1, CDRa3 and/or two or three amino acid sequences may be mutated. The antigen binding protein comprising the CDR sequences is also referred to hereinafter as "antigen binding protein 6". In a preferred embodiment of antigen binding protein 6, CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 54 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 59, wherein CDRa2 and/or CDRb2 sequences may comprise one, two, three or four amino acid mutations. Antigen binding protein 6 specifically binds to a functional epitope comprising or consisting of 2, 3 or 4 amino acid positions selected from positions 1, 3, 4 and 6 of SEQ ID No. 138. The EC ₅₀ of cells expressing the antigen binding protein 6 (e.g., T2 cells loaded with CT 45-IP) that are tested (e.g., in a luciferase release assay) to kill cells presenting the CT45-IP MHC complex is less than about 30nM, less than about 25nM, less than about 20nM, less than about 15nM, less than about 10nM, or less than about 5nM. Antigen binding protein 6 did not bind significantly to: at least 1, at least 2, at least 3, at least 4, at least 5 or all like peptides selected from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO:155 (SP-05-0010), preferably from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO:155 (SP-05-0010), more preferably from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007) and SEQ ID NO:155 (SP-05-0010). In a preferred embodiment of antigen binding protein 6, V _A comprises or consists of: the amino acid sequence of SEQ ID No. 52 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 52 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 57 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 57; wherein V _A and V _B comprise a CDR sequence as described above for antigen binding protein 6, wherein the CDR sequence may comprise one, two or three amino acid mutations, preferably no amino acid mutations. Preferably, the alpha chain variable region of antigen binding protein 6 is encoded by TRAV12-3 and/or the beta chain variable region of antigen binding protein 6 is encoded by TRBV 4-1. In preferred embodiments, the antigen binding protein 6 is capable of activating CD8+ T cells (particularly CD8+ CD4-T cells), and/or CD4+ T cells (particularly CD4+ CD8-T cells).

Antigen binding protein 7

In some embodiments, the invention relates to an antigen binding protein that specifically binds to a CT45 antigen peptide, the CT45 antigen peptide being in a complex with a Major Histocompatibility Complex (MHC) protein, wherein the CT45 antigen peptide comprises or consists of the amino acid sequence of SEQ ID NO:138 (KIFEMLEGV) and wherein the antigen binding protein comprises a first polypeptide comprising or consisting of the variable domain V _A comprising Complementarity Determining Regions (CDRs) CDRa1, CDRa2 and CDRa3, and a second polypeptide comprising or consisting of the variable domain V _B comprising CDRa1, CDRa2 and CDRa3, wherein CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO:71, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:75, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:77, wherein the CDRa1, CDRa3 and/or two or three amino acids may comprise mutations. The antigen binding protein comprising the CDR sequences is also referred to hereinafter as "antigen binding protein 7". In a preferred embodiment of the antigen binding protein 7, CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 15 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 76, wherein the CDRa2 and/or CDRb2 sequences may comprise one, two, three or four amino acid mutations. Antigen binding protein 7 specifically binds to a functional epitope comprising or consisting of: positions 1,4, 6 and 7 of SEQ ID NO. 138. The EC ₅₀ of cells expressing the antigen binding protein 7 (e.g., in a luciferase release assay) that are induced to kill cells presenting the CT45-IP: MHC complex (e.g., CT45-IP loaded T2 cells) is measured to be less than about 5nM, less than about 2.5nM, less than about 1.5nM, or less than about 1nM. Antigen binding protein 7 did not bind significantly to: at least 1, at least 2, at least 3, at least 4, at least 5 or all analogous peptides selected from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO 155 (SP-05-0010). In a preferred embodiment of antigen binding protein 7, V _A comprises or consists of: the amino acid sequence of SEQ ID No. 70 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 70 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 74 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 74; wherein V _A and V _B comprise a CDR sequence as described above for antigen binding protein 7, wherein the CDR sequence may comprise one, two or three amino acid mutations, preferably no amino acid mutations. Preferably, the alpha chain variable region of antigen binding protein 7 is encoded by TRAV38-2/DV8 and/or the beta chain variable region of antigen binding protein 7 is encoded by TRBV 13. In preferred embodiments, the antigen binding protein 7 is capable of activating CD8+ T cells (particularly CD8+ CD4-T cells), and/or CD4+ T cells (particularly CD4+ CD8-T cells).

Antigen binding protein 8

In some embodiments, the invention relates to an antigen binding protein that specifically binds to a CT45 antigen peptide, the CT45 antigen peptide being in a complex with a Major Histocompatibility Complex (MHC) protein, wherein the CT45 antigen peptide comprises or consists of the amino acid sequence of SEQ ID NO:138 (KIFEMLEGV) and wherein the antigen binding protein comprises a first polypeptide comprising or consisting of the variable domain V _A comprising Complementarity Determining Regions (CDRs) CDRa1, CDRa2 and CDRa3, and a second polypeptide comprising or consisting of the variable domain V _B comprising CDRa1, CDRa2 and CDRa3, wherein CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO:99, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:75, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:104, wherein the CDRa1, CDRa3 and/or two or three amino acids may comprise mutations. The antigen binding protein comprising the CDR sequences is also referred to hereinafter as "antigen binding protein 8". In a preferred embodiment of antigen binding protein 8, CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 100 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 76, wherein CDRa2 and/or CDRb2 sequences may comprise one, two, three or four amino acid mutations. Antigen binding protein 8 specifically binds to a functional epitope comprising or consisting of: 1 or 2 amino acid positions selected from positions 5 and 7 of SEQ ID NO. 138. The EC ₅₀ of cells expressing the antigen binding protein 8 (e.g., T2 cells loaded with CT 45-IP) that are tested (e.g., in a luciferase release assay) to kill cells presenting the CT45-IP, MHC complex, is less than about 30nM, less than about 25nM, less than about 20nM, less than about 15nM, or less than about 10nM. Antigen binding protein 8 did not bind significantly to: at least 1, at least 2, at least 3, at least 4, at least 5 or all of the like peptides selected from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO:155 (SP-05-0010), preferably from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO:155 (SP-05-0010). In a preferred embodiment of antigen binding protein 8, V _A comprises or consists of: the amino acid sequence of SEQ ID No. 98 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 98 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 103 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 103; wherein V _A and V _B comprise a CDR sequence as described above for antigen binding protein 8, wherein the CDR sequence may comprise one, two or three amino acid mutations, preferably no amino acid mutations. Preferably, the alpha chain variable region of antigen binding protein 8 is encoded by TRAV19 and/or the beta chain variable region of antigen binding protein 8 is encoded by TRBV 13. In preferred embodiments, antigen binding protein 8 is capable of activating CD8+ T cells (particularly CD8+ CD4-T cells), and/or CD4+ T cells (particularly CD4+ CD8-T cells).

Antigen binding protein 9

In some embodiments, the invention relates to an antigen binding protein that specifically binds to a CT45 antigen peptide, the CT45 antigen peptide being in a complex with a Major Histocompatibility Complex (MHC) protein, wherein the CT45 antigen peptide comprises or consists of the amino acid sequence of SEQ ID NO:138 (KIFEMLEGV) and wherein the antigen binding protein comprises or consists of a first polypeptide comprising or consisting of the variable domain V _A comprising Complementarity Determining Regions (CDRs) CDRa1, CDRa2 and CDRa3, and the second polypeptide comprising or consisting of the variable domain V _B comprising CDRa1, CDRa2 and CDRa3, wherein CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO:80, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:85, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:87, wherein the CDRa1, CDRa3 and/or two or three amino acids may be mutated. The antigen binding protein comprising the CDR sequences is also referred to hereinafter as "antigen binding protein 9". In a preferred embodiment of antigen binding protein 9, CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 81 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 86, wherein CDRa2 and/or CDRb2 sequences may comprise one, two, three or four amino acid mutations. Antigen binding protein 9 specifically binds to a functional epitope comprising or consisting of: positions 1, 3, 4, 5, 6, 7 and 8 of SEQ ID NO. 138. The EC ₅₀ of cells expressing the antigen binding protein 9 (e.g., T2 cells loaded with CT 45-IP) that are tested (e.g., in a luciferase release assay) to kill cells presenting the CT45-IP, MHC complex, is less than about 5nM, less than about 2.5nM, less than about 1.5nM, or less than about 1nM. Antigen binding protein 9 did not bind significantly to: at least 1, at least 2, at least 3, at least 4, at least 5 or all of the like peptides selected from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO:155 (SP-05-0010), preferably from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO:155 (SP-05-0010). In a preferred embodiment of antigen binding protein 9, V _A comprises or consists of: the amino acid sequence of SEQ ID No. 79 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 79 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 84 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 84; wherein V _A and V _B comprise a CDR sequence as described above for antigen binding protein 9, wherein the CDR sequence may comprise one, two or three amino acid mutations, preferably no amino acid mutations. Preferably, the alpha chain variable region of antigen binding protein 9 is encoded by TRAV5 and/or the beta chain variable region of antigen binding protein 9 is encoded by TRBV 2. In preferred embodiments, antigen binding protein 9 is capable of activating CD8+ T cells (particularly CD8+ CD4-T cells), and/or CD4+ T cells (particularly CD4+ CD8-T cells).

Antigen binding protein 10

In some embodiments, the invention relates to an antigen binding protein that specifically binds to a CT45 antigen peptide, the CT45 antigen peptide being in a complex with a Major Histocompatibility Complex (MHC) protein, wherein the CT45 antigen peptide comprises or consists of the amino acid sequence of SEQ ID NO:138 (KIFEMLEGV) and wherein the antigen binding protein comprises or consists of a first polypeptide comprising or consisting of the variable domain V _A comprising Complementarity Determining Regions (CDRs) CDRa1, CDRa2 and CDRa3, and the second polypeptide comprising or consisting of the variable domain V _B comprising CDRa1, CDRa2 and CDRa3, wherein CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO:107, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:112, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:114, wherein the CDRa1, CDRa3 and/or two or three amino acids may comprise mutations. The antigen binding protein comprising the CDR sequences is also referred to hereinafter as "antigen binding protein 10". In a preferred embodiment of the antigen binding protein 10, CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 108 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 113, wherein the CDRa2 and/or CDRb2 sequences may comprise one, two, three or four amino acid mutations. Antigen binding protein 10 specifically binds to a functional epitope comprising or consisting of: positions 5, 6 and 7 of SEQ ID NO. 138. The measured (e.g., in a luciferase release assay) induction of antigen binding protein 10-expressing T cell killing presents CT45-IP MHC complex cells (e.g., CT45-IP loaded T2 cells) with an EC ₅₀ of less than about 30nM, less than about 25nM, less than about 20nM, less than about 15nM, or less than about 10nM. Antigen binding protein 10 did not bind significantly to: at least 1, at least 2, at least 3, at least 4, at least 5 or all analogous peptides selected from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO 155 (SP-05-0010). In a preferred embodiment of the antigen binding protein 10, V _A comprises or consists of: the amino acid sequence of SEQ ID No. 106 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 106 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 111 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 111; wherein V _A and V _B comprise a CDR sequence as described above for antigen binding protein 10, wherein the CDR sequence may comprise one, two or three amino acid mutations, preferably no amino acid mutations. Preferably, the alpha chain variable region of antigen binding protein 10 is encoded by TRAV1-2 and/or the beta chain variable region of antigen binding protein 10 is encoded by TRBV 6-1. In preferred embodiments, antigen binding protein 10 is capable of activating CD8+ T cells (particularly CD8+ CD4-T cells), and/or CD4+ T cells (particularly CD4+ CD8-T cells).

Antigen binding protein 11

In some embodiments, the invention relates to an antigen binding protein that specifically binds to a CT45 antigen peptide, the CT45 antigen peptide being in a complex with a Major Histocompatibility Complex (MHC) protein, wherein the CT45 antigen peptide comprises or consists of the amino acid sequence of SEQ ID NO:138 (KIFEMLEGV) and wherein the antigen binding protein comprises or consists of a first polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:112 and a second polypeptide comprising or consisting of the variable domain V _A comprising Complementarity Determining Regions (CDRs) CDRa1, CDRa2 and CDRa3, and the second polypeptide comprising or consisting of the variable domain V _B comprising CDRa1, CDRa2 and CDRa3, wherein CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO:125, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:112 and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:130, wherein the CDRa1, CDRa3 and/or two or three amino acid sequences may be mutated. The antigen binding protein comprising the CDR sequences is also referred to hereinafter as "antigen binding protein 11". In a preferred embodiment of antigen binding protein 11, CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 126 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 113, wherein CDRa2 and/or CDRb2 sequences may comprise one, two, three or four amino acid mutations. Antigen binding protein 11 specifically binds to a functional epitope comprising or consisting of: position 1,3, 4, 5, 6 and 8 of SEQ ID NO. 138. The EC ₅₀ of cells that induce the killing of cells presenting the CT45-IP: MHC complex (e.g., CT45-IP loaded T2 cells) that express antigen binding protein 11 is measured (e.g., in a luciferase release assay) to be less than about 60nM. Antigen binding protein 11 did not bind significantly to: at least 1, at least 2, at least 3, at least 4, at least 5 or all analogous peptides selected from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO 155 (SP-05-0010). In a preferred embodiment of antigen binding protein 11, V _A comprises or consists of: the amino acid sequence of SEQ ID No. 124 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 124 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 129 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 129; wherein V _A and V _B comprise a CDR sequence as described above for antigen binding protein 11, wherein the CDR sequence may comprise one, two or three amino acid mutations, preferably no amino acid mutations. Preferably, the alpha chain variable region of antigen binding protein 11 is encoded by TRAV22 and/or the beta chain variable region of antigen binding protein 11 is encoded by TRBV 6-1. In preferred embodiments, antigen binding protein 11 is capable of activating CD8+ T cells (particularly CD8+ CD4-T cells), and/or CD4+ T cells (particularly CD4+ CD8-T cells).

Antigen binding protein 12

In some embodiments, the invention relates to an antigen binding protein that specifically binds to a CT45 antigen peptide, the CT45 antigen peptide being in a complex with a Major Histocompatibility Complex (MHC) protein, wherein the CT45 antigen peptide comprises or consists of the amino acid sequence of SEQ ID NO:138 (KIFEMLEGV) and wherein the antigen binding protein comprises or consists of a first polypeptide comprising or consisting of the variable domain V _A comprising Complementarity Determining Regions (CDRs) CDRa1, CDRa2 and CDRa3, and the second polypeptide comprising or consisting of the variable domain V _B comprising CDRa1, CDRa2 and CDRa3, wherein CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO:117, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:119, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:58, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:122, wherein the CDRa1, CDRa3 and/or two or three amino acids may comprise mutations. The antigen binding protein comprising the CDR sequences is also referred to hereinafter as "antigen binding protein 12". In a preferred embodiment of antigen binding protein 12, CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 118 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 59, wherein CDRa2 and/or CDRb2 sequences may comprise one, two, three or four amino acid mutations. Antigen binding protein 12 specifically binds to a functional epitope comprising or consisting of 2,3 or 4 amino acid positions selected from positions 3,4, 6 and 7 of SEQ ID No. 138. The measured (e.g., in a luciferase release assay) EC ₅₀ of cells that express the antigen binding protein 12 that kill cells presenting the CT45-IP: MHC complex (e.g., CT45-IP loaded T2 cells) is less than about 60nM or less than about 50nM. Antigen binding protein 12 did not bind significantly to: at least 1, at least 2, at least 3, at least 4, at least 5 or all analogous peptides selected from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO 155 (SP-05-0010). In a preferred embodiment of antigen binding protein 12, V _A comprises or consists of: the amino acid sequence of SEQ ID NO. 116 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 116, and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 121 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 121; wherein V _A and V _B comprise a CDR sequence as described above for antigen binding protein 12, wherein the CDR sequence may comprise one, two or three amino acid mutations, preferably no amino acid mutations. Preferably, the alpha chain variable region of antigen binding protein 12 is encoded by TRAV27 and/or the beta chain variable region of antigen binding protein 12 is encoded by TRBV 4-1. In preferred embodiments, the antigen binding protein 12 is capable of activating CD8+ T cells (particularly CD8+ CD4-T cells), and/or CD4+ T cells (particularly CD4+ CD8-T cells).

Antigen binding protein 13

In some embodiments, the invention relates to an antigen binding protein that specifically binds to a CT45 antigen peptide, the CT45 antigen peptide being in a complex with a Major Histocompatibility Complex (MHC) protein, wherein the CT45 antigen peptide comprises or consists of the amino acid sequence of SEQ ID NO:138 (KIFEMLEGV) and wherein the antigen binding protein comprises a first polypeptide comprising or consisting of the variable domain V _A comprising Complementarity Determining Regions (CDRs) CDRa1, CDRa2 and CDRa3, and a second polypeptide comprising or consisting of the variable domain V _B comprising CDRa1, CDRa2 and CDRa3, wherein CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO:24, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:38, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:40, wherein the CDRa1, CDRa3 and/or two or three amino acids may comprise mutations. The antigen binding protein comprising the CDR sequences is also referred to hereinafter as "antigen binding protein 13". In a preferred embodiment of the antigen binding protein 13, CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 25 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 39, wherein the CDRa2 and/or CDRb2 sequences may comprise one, two, three or four amino acid mutations. Antigen binding protein 13 specifically binds to a functional epitope comprising or consisting of: position 1,3, 4, 6 and 7 of SEQ ID NO. 138. The EC ₅₀ of cells that express the antigen binding protein 13 (e.g., in a luciferase release assay) that are induced to kill cells presenting the CT45-IP: MHC complex (e.g., CT45-IP loaded T2 cells) is measured to be less than about 50nM, less than about 30nM, less than about 25nM, less than about 20nM, or less than about 15nM. Antigen binding protein 13 did not bind significantly to: at least 1, at least 2, at least 3, at least 4, at least 5 or all analogous peptides selected from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO 155 (SP-05-0010). In a preferred embodiment of antigen binding protein 13, V _A comprises or consists of: the amino acid sequence of SEQ ID No. 34 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 34 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 37 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 37; wherein V _A and V _B comprise a CDR sequence as described above for antigen binding protein 13, wherein the CDR sequence may comprise one, two or three amino acid mutations, preferably no amino acid mutations. Preferably, the alpha chain variable region of antigen binding protein 13 is encoded by TRAV14/DV4 and/or the beta chain variable region of antigen binding protein 13 is encoded by TRBV 19. In preferred embodiments, the antigen binding protein 13 is capable of activating CD8+ T cells (particularly CD8+ CD4-T cells), and/or CD4+ T cells (particularly CD4+ CD8-T cells).

Antigen binding protein 14

In some embodiments, the invention relates to an antigen binding protein that specifically binds to a CT45 antigen peptide, the CT45 antigen peptide being in a complex with a Major Histocompatibility Complex (MHC) protein, wherein the CT45 antigen peptide comprises or consists of the amino acid sequence of SEQ ID NO:138 (KIFEMLEGV) and wherein the antigen binding protein comprises a first polypeptide comprising or consisting of the variable domain V _A comprising Complementarity Determining Regions (CDRs) CDRa1, CDRa2 and CDRa3, and a second polypeptide comprising or consisting of the variable domain V _B comprising CDRa1, CDRa2 and CDRa3, wherein CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO:24, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:29, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:31, wherein the CDRa1, CDRa3 and/or two or three amino acids may comprise mutations. The antigen binding protein comprising the CDR sequences is also referred to hereinafter as "antigen binding protein 14". In a preferred embodiment of the antigen binding protein 14, CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 25 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 30, wherein the CDRa2 and/or CDRb2 sequences may comprise one, two, three or four amino acid mutations. Antigen binding protein 14 specifically binds to a functional epitope comprising or consisting of 2,3 or 4 amino acid positions selected from positions 1,3, 4 and 5 of SEQ ID No. 138. The measured (e.g., in a luciferase release assay) EC ₅₀ of cells expressing the antigen binding protein 14 that kill cells presenting the CT45-IP: MHC complex (e.g., T2 cells loaded with CT 45-IP) is less than about 50nM, less than about 30nM, less than about 25nM, or less than about 20nM. Antigen binding protein 14 did not bind significantly to: at least 1, at least 2, at least 3, at least 4, at least 5 or all analogous peptides selected from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO 155 (SP-05-0010). In a preferred embodiment of the antigen binding protein 14, V _A comprises or consists of: the amino acid sequence of SEQ ID No. 23 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 23 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 28 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 28; wherein V _A and V _B comprise a CDR sequence as described above for antigen binding protein 14, wherein the CDR sequence may comprise one, two or three amino acid mutations, preferably no amino acid mutations. Preferably, the alpha chain variable region of antigen binding protein 14 is encoded by TRAV14/DV4 and/or the beta chain variable region of antigen binding protein 14 is encoded by TRBV 11-2. In preferred embodiments, the antigen binding protein 14 is capable of activating CD8+ T cells (particularly CD8+ CD4-T cells), and/or CD4+ T cells (particularly CD4+ CD8-T cells).

Antigen binding protein 15

In some embodiments, the invention relates to an antigen binding protein that specifically binds to a CT45 antigen peptide, the CT45 antigen peptide being in a complex with a Major Histocompatibility Complex (MHC) protein, wherein the CT45 antigen peptide comprises or consists of the amino acid sequence of SEQ ID NO:138 (KIFEMLEGV) and wherein the antigen binding protein comprises a first polypeptide comprising or consisting of the variable domain V _A comprising Complementarity Determining Regions (CDRs) CDRa1, CDRa2 and CDRa3, and a second polypeptide comprising or consisting of the variable domain V _B comprising CDRa1, CDRa2 and CDRa3, wherein CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO:43, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:45, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:50, wherein the CDRa1, CDRa3 and/or two or three amino acids may comprise mutations. The antigen binding protein comprising the CDR sequences is also referred to hereinafter as "antigen binding protein 15". In a preferred embodiment of the antigen binding protein 15, CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 44 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 49, wherein the CDRa2 and/or CDRb2 sequences may comprise one, two, three or four amino acid mutations. Antigen binding protein 15 specifically binds to a functional epitope comprising or consisting of: position 1, 3, 4,5, 6 and 7 of SEQ ID NO. 138. The EC ₅₀ of cells that express the antigen binding protein 15 (e.g., in a luciferase release assay) that are induced to kill cells presenting the CT45-IP: MHC complex (e.g., CT45-IP loaded T2 cells) is measured to be less than about 50nM, less than about 30nM, less than about 25nM, or less than about 20nM. Antigen binding protein 15 did not bind significantly to: at least 1, at least 2, at least 3, at least 4, at least 5 or all of the like peptides selected from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO:155 (SP-05-0010), preferably from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007) and SEQ ID NO:155 (SP-05-0010). In a preferred embodiment of antigen binding protein 15, V _A comprises or consists of: the amino acid sequence of SEQ ID No. 42 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID No. 42 and V _B comprises or consists of: the amino acid sequence of SEQ ID NO. 27 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 27; wherein V _A and V _B comprise a CDR sequence as described above for antigen binding protein 15, wherein the CDR sequence may comprise one, two or three amino acid mutations, preferably no amino acid mutations. Preferably, the alpha chain variable region of antigen binding protein 15 is encoded by TRAV21 and/or the beta chain variable region of antigen binding protein 15 is encoded by TRBV 5-1. In preferred embodiments, antigen binding protein 15 is capable of activating CD8+ T cells (particularly CD8+ CD4-T cells), and/or CD4+ T cells (particularly CD4+ CD8-T cells).

The invention also includes particles displaying antigen binding proteins, particularly TCRs, and including the particles in a library of particles. Such particles include, but are not limited to, phage, yeast, ribosomes, or mammalian cells. Methods of generating such particles and libraries are known in the art (see, e.g., WO2004/044004; WO01/48145, chervin et al (2008) J.Immuno. Methods 339.2:175-184).

Nucleic acids, vectors and recombinant host cells

The polypeptides of the antigen binding proteins of the invention may be encoded by nucleic acids and expressed in vivo, ex vivo or in vitro. Thus, in a second aspect, the present invention relates to one or more nucleic acids comprising or consisting of one or more sequences encoding the antigen binding proteins of the first aspect of the invention.

The nucleic acid may be comprised in one nucleic acid molecule or may be split into two or more nucleic acid molecules, wherein each nucleic acid molecule comprises at least one of the one or more sequences encoding the antigen binding protein of the first aspect of the invention. In some embodiments, one nucleic acid molecule encodes one portion or monomer of an antigen binding protein of the invention (e.g., one of the two chains of a TCR of the invention) and another nucleic acid molecule encodes another portion or monomer of an antigen binding protein of the invention (e.g., the other of the two chains of a TCR). In some embodiments, the nucleic acid encodes two or more antigen binding protein polypeptide chains, e.g., at least two TCR chains. The nucleic acid encoding the plurality of antigen binding protein polypeptide chains may include a nucleic acid cleavage site between at least two chain sequences, may encode a transcription or translation initiation site (e.g., an Internal Ribosome Entry Site (IRES) between two or more chain sequences), and/or may encode a proteolytic target site between two or more antigen binding protein chains. If two or more antigen binding protein polypeptide chains are encoded on one nucleic acid molecule, the two or more antigen binding protein polypeptide chains may be under the control of the same promoter or under the control of separate promoters.

In the context of the present invention, the term "nucleic acid" refers to a single-or double-stranded oligomer or polymer of deoxyribonucleotide or ribonucleotide bases, or both. The nucleotide monomers consist of nucleobases, five carbon sugars (such as, but not limited to, ribose or 2' -deoxyribose) and one to three phosphate groups. Typically, nucleic acids are formed via phosphodiester bonds between individual nucleotide monomers, and in the context of the present invention, the term nucleic acid includes, but is not limited to, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) molecules, but also includes synthetic forms that include other linked nucleic acids (e.g., peptide nucleic acids, as described in Nielsen et al (Science 254:1497-1500, 1991). Typically, a nucleic acid is a single-or double-stranded molecule and is composed of naturally occurring nucleotides. The description of a single strand of nucleic acid also defines, at least in part, the sequence of the complementary strand. The nucleic acid may be single-stranded or double-stranded, or may contain both double-stranded and single-stranded sequences. The illustrated double stranded nucleic acid molecule may have a single stranded overhang of 3 'or 5' and thus need not be or is assumed to be fully double stranded throughout its length. The term nucleic acid includes a chromosome or a segment of a chromosome, a vector (e.g., an expression vector), an expression cassette, naked DNA or RNA polymers, primers, probes, cDNA, genomic DNA, recombinant DNA, cRNA, mRNA, tRNA, micrornas (mirnas), or small interfering RNAs (sirnas). The nucleic acid may be, for example, single-stranded, double-stranded or triple-stranded, and is not limited to any particular length. Unless otherwise indicated, a particular nucleic acid sequence comprises or encodes a complementary sequence in addition to any explicitly indicated sequence.

In a preferred embodiment, the nucleic acid is an isolated nucleic acid. In a preferred embodiment, the nucleic acid is a recombinant nucleic acid.

The nucleic acid may be present in whole cells, in cell lysates, or may be in partially purified or substantially pure form. Nucleic acids are "isolated" or "become substantially pure" when other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) are removed by standard techniques for purification.

The nucleic acid molecules of the present disclosure can be obtained using standard molecular biology techniques, including, but not limited to, methods of amplification and reverse transcription of RNA. Once the DNA fragments encoding, for example, variable strands are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length genes. In these manipulations, a DNA fragment encoding a variant is operably linked to another DNA molecule or to a fragment encoding another protein (e.g., a constant region or flexible linker). The term "operably linked" as used in this context is intended to mean that the two DNA fragments are joined in a functional manner, e.g. such that the amino acid sequences encoded by the two DNA fragments remain in frame or such that the protein is expressed under the control of a desired promoter. The isolated DNA encoding a variable region (e.g., a variable alpha region and/or a variable beta region) can be converted to a full-length gene by operably linking the DNA encoding the variable region to another DNA molecule encoding a constant region. The sequences of human constant region genes (e.g., for TCRs or antibodies) are known in the art, and DNA fragments encompassing these regions can be obtained by standard PCR amplification.

Typically, the nucleic acid comprises one or more DNA or RNA molecules, which may be included in one or more suitable vectors.

In a third aspect, the invention relates to a vector or collection of vectors comprising one or more nucleic acids of the second aspect of the invention. Preferably, the sequence encoding the antigen binding protein is operably linked to a promoter sequence. Herein, "vector collection" refers to two or more vectors. If two or more antigen binding protein polypeptide chains are encoded on one vector, the two or more antigen binding protein polypeptide chains may be under the control of the same promoter or under the control of separate promoters.

The terms "vector," "cloning vector," and "expression vector" refer to a vector by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell to transform the host and facilitate expression (e.g., transcription and translation) of the introduced sequence.

A variety of expression vectors may be employed to express polynucleotides encoding antigen binding proteins or functional fragments thereof. Both viral-based and non-viral expression vectors can be used to produce the antigen binding proteins described herein, or functional fragments thereof, in mammalian host cells. Non-viral vectors and systems include a variety of plasmids, cosmids, episomes, artificial chromosomes, phage, or viral vectors.

Such vectors may comprise regulatory elements (e.g., promoters, enhancers, terminators, etc.) to cause or direct expression of the polypeptide upon administration to a subject. Examples of promoters and enhancers used in expression vectors of animal cells include the early promoter and enhancer of SV40 (Mizukami T. Et al 1987), the LTR promoter and enhancer of Moloney murine leukemia virus (Kuwana Y et al 1987), the promoter of antibody heavy chain (Mason JO et al 1985) and enhancer (GILLIES SD et al 1983), and the like.

For example, non-viral vectors useful for expressing the polynucleotides and polypeptides described herein in mammalian (e.g., human or non-human) cells include all suitable vectors known in the art for expressing proteins. Other examples of plasmids and include replicative plasmids that include an origin of replication, or integrative plasmids (such as pUC, pcDNA, pBR, for example).

The term "viral vector" refers to a nucleic acid vector construct comprising at least one element of viral origin and having the ability to be packaged into viral vector particles and encoding at least an exogenous nucleic acid. The vector and/or particle may be used for the purpose of transferring the nucleic acid of interest into a cell in vitro or in vivo. Various forms of viral vectors are known in the art. Useful viral vectors include vectors based on: retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, SV 40-based vectors, papilloma virus, EB virus, vaccinia virus vectors, and Semliki Forest Virus (SFV). Recombinant viruses can be produced by techniques known in the art, such as by transfection of packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, psiCRIP cells, GPenv + cells, 293 cells, and the like. Detailed protocols for the generation of such replication-defective recombinant viruses can be found, for example, in the following documents: WO 95/14785, WO 96/22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94/19478.

The first polypeptide and the second polypeptide described herein may be present in the same vector or in separate vectors (i.e., a collection of vectors).

In a fourth aspect, the invention relates to a host cell comprising an antigen binding protein of the first aspect of the invention, a nucleic acid of the second aspect of the invention, or a vector of the third aspect of the invention. The host cell may be transfected, infected or transduced or transformed, in particular with a nucleic acid and/or vector according to the invention.

The host cell may be a eukaryotic cell (e.g., plant, animal, fungus, or algae) or a prokaryotic cell (e.g., bacterial or protozoa). The host cell may be a cultured cell or a primary cell (i.e., isolated directly from an organism (e.g., a human). The host cell may be an adherent cell or a cell in suspension (i.e., a cell grown in suspension). For the purpose of producing a recombinant antigen binding protein (e.g., a TCR, polypeptide, or protein), the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. Although the host cell may be any cell type, may be derived from any type of tissue, and may be at any stage of development, the host cell is preferably Peripheral Blood Leukocytes (PBLs) or Peripheral Blood Mononuclear Cells (PBMCs). More preferably, the host cell is a lymphocyte, such as a T cell, T cell progenitor cell or NK cell. NK cells are naturally occurring lymphoid non-T cells that can rapidly kill virus-infected cells and tumor cells. NK cells can be engineered to express tumor-specific TCRs for use as cell therapy products in cancer therapy (SHIMASAKI et al, nat Rev Drug discovery, 3 months 2020; 19 (3): 200-218). In a preferred embodiment, the host cell is a T cell, such as a CD4 or CD8 positive T cell or γδ T cell. The T cell may be any T cell, such as a cultured T cell, e.g. a primary T cell or a T cell from a cultured T cell line (e.g. Jurkat, supT1, etc.), or a T cell obtained from a mammal, preferably a T cell or a precursor T cell from a human patient. If obtained from a mammal, T cells may be obtained from a number of sources including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells may also be enriched or purified. Preferably, the T cell is a human T cell. More preferably, the T cell is a T cell isolated from a human. T cells may be any type of T cell and may be at any stage of development, including but not limited to CD4 positive helper T cells (e.g., th1 and Th2 cells), CD8 positive T cells (e.g., cytotoxic T cells), tumor infiltrating cells (TILs), memory T cells, naive T cells, γδ T cells, and the like.

In other preferred embodiments, the host cell is or is a cell for recombinant expression, such as a Chinese Hamster Ovary (CHO) cell or a yeast cell.

The term "transformation" means the introduction of a "foreign" (i.e., extrinsic) gene, DNA or RNA sequence into a host cell such that the host cell will express the introduced gene or sequence to produce the desired substance, typically an antigen binding protein or functional fragment thereof as described herein. Host cells that receive and express the introduced DNA or RNA have been "transformed".

The nucleic acids of the invention may be used to produce the recombinant antigen-binding proteins of the invention in a suitable expression system. The term "expression system" means a host cell and a compatible vector under suitable conditions, e.g., for expressing a protein encoded by foreign DNA carried by and introduced into the host cell.

Common expression systems include E.coli (E.coli) host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E.coli, kluyveromyces (Kluyveromyces) or Saccharomyces (Saccharomyces) yeast, mammalian cell lines (e.g., green monkey kidney cells (Vero cells), CHO cells, 3T3 cells, COS cells, HEK cells, etc.), primary or established mammalian cell cultures (e.g., produced from primordial lymphocytes, fibroblasts, embryonic cells, epithelial cells, neural cells, adipocytes, etc.). Examples also include mouse SP2/0-Ag14 cells (ATCC CRL 1581), mouse P3X63-Ag8.653 cells (ATCC CRL 1580), CHO cells in which the dihydrofolate reductase gene is deficient (Urlaub G et al; 1980), rat YB2/3HL.P2.G11.16Ag.20 cells (ATCC CRL 1662), and the like. In some embodiments, YB2/0 cells may be preferred because the ADCC activity of the chimeric or humanized antibody is enhanced when expressed in the cells. In a preferred embodiment, the above-described host cell is used as an expression system.

In particular, for the expression of some antigen binding proteins of the invention, particularly antigen binding proteins comprising two polypeptides that are not linked, the expression vector may be of the type in which the gene encoding the first polypeptide (e.g., a TCR alpha chain) and the gene encoding the second polypeptide (e.g., a TCR beta chain) are present on separate vectors, or of the type in which both genes are present on the same vector (tandem type). As described in the context of humanized antibodies, tandem expression vectors are preferred with respect to ease of construction of antigen binding protein expression vectors, ease of introduction into animal cells, and balance between expression levels of the two polypeptides (e.g., TCR alpha and beta chains) in animal cells (Shitara K et al J Immunol methods.1994, 3. 1/3; 167 (1-2): 271-8). Examples of tandem expression vectors as described in the context of humanized antibodies include pKANTEX93 (WO 97/10354), pEE18, and the like.

In one embodiment, such recombinant host cells may be used to produce at least one antigen binding protein of the invention.

Pharmaceutical composition

The antigen binding proteins of the invention have been shown to be capable of achieving cytotoxicity against cells presenting CT45-IP antigenic peptides. Because the peptide is specifically presented by tumor cells, the antigen binding proteins of the invention can be used to destroy tumor cells in a patient. The immune response in the patient may be induced by direct administration of the antigen binding protein to the patient, desirably in combination with an agent (i.e., adjuvant) that enhances immunogenicity. Immune responses derived from such therapeutic vaccination can be expected to be highly specific for tumor cells, since peptide KIFEMLEGV (SEQ ID NO: 138) is not presented or over-presented on normal tissue in comparable copy numbers, thereby preventing the risk of an undesired autoimmune response against normal tissue cells in the patient.

The invention also relates to an antigen binding protein according to the invention for use as a medicament. The invention also relates to a pharmaceutical composition of the invention for use as a medicament.

The term "pharmaceutical composition" or "therapeutic composition" as used herein refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a subject.

In some embodiments, the subject may also be referred to as a patient.

Such therapeutic or pharmaceutical compositions may comprise a therapeutically effective amount of an antigen binding protein of the invention or an antigen binding protein further comprising a therapeutic agent, admixed with a pharmaceutically or physiologically acceptable formulation selected for suitability to the mode of administration.

In some embodiments, the antigen binding proteins of the invention will be supplied as part of a sterile pharmaceutical composition, which will typically include a pharmaceutically acceptable carrier.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to a mammal, particularly a human, as the case may be. Pharmaceutically acceptable carrier means any type of nontoxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation aid.

"Pharmaceutically acceptable carrier" and may include physiologically compatible solvents, compatibilizers, stabilizers, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In one embodiment, the carrier is an aqueous carrier. In some embodiments, the aqueous carrier, when combined with the antigen binding proteins described herein, is capable of imparting improved properties, e.g., improved solubility, efficacy, and/or improved immunotherapy.

Additional examples of pharmaceutically acceptable carriers or diluents that may be used in the present invention include stabilizers (e.g., SPGA), carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins (e.g., albumin or casein), protein-containing agents (e.g., bovine serum or skim milk), and buffers (e.g., phosphate buffers).

The form, route of administration, dosage and regimen of the pharmaceutical composition will naturally depend on the condition to be treated, the severity of the condition, the age, weight and sex of the patient, etc. The pharmaceutical composition may be in any suitable form (depending on the desired method of administration to the patient). It may be provided in unit dosage form, will typically be provided in a sealed container, and may be provided as part of a kit. Such a kit will typically (but not necessarily) include instructions for use. Which may comprise a plurality of said unit dosage forms.

Preferably, the pharmaceutical composition is administered by injection (e.g., intravenously). When the pharmaceutical composition comprises a host cell expressing an antigen binding protein of the invention (preferably a TCR), the pharmaceutically acceptable carrier for the injected cell may comprise any isotonic carrier, such as, for example, physiological saline (about 0.90% w/v aqueous NaCl, about 300mOsm/L aqueous NaCl, or about 9.0g NaCl/liter water), NORMOSOL R electrolyte solution (Abbott, chicago, illinois), PLASMALYTE A (Baxter, dilfield, illinois), about 5% aqueous dextrose, or lactated ringer. In one embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

Empirical considerations (such as biological half-life) will generally aid in the determination of the dosage. The frequency of administration can be determined and adjusted during the course of treatment and is based on reducing the number of cancer cells, maintaining a reduction in cancer cells, reducing proliferation of cancer cells, or killing cancer cells. Alternatively, a sustained continuous release formulation of the antigen binding protein may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

In one embodiment, the dose of antigen binding protein in an individual that has been administered one or more administrations can be determined empirically. Increasing doses of antigen binding protein are administered to an individual. To assess the efficacy of antigen binding proteins, markers of cancer cell status can be tracked. These include direct measurement of cancer cell proliferation and cell death by FACS, other imaging techniques; improvement in health as assessed by such measurements, or improvement in quality of life or prolongation of survival as measured by accepted tests. It will be apparent to those skilled in the art that the dosage will vary depending on the individual, the disease state, and the past and concurrent treatments used.

The dosage for administration may be adjusted according to a number of parameters, in particular according to the mode of administration used, the relevant pathology, or alternatively the desired duration of treatment.

The pharmaceutical compositions, vectors, nucleic acids and cells of the invention may be provided in substantially pure form, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure.

Method for producing antigen binding proteins

In one embodiment, the method further comprises isolating and purifying the antigen binding protein from the host cell, and optionally, reconstituting the antigen binding protein in a host cell (preferably a lymphocyte, more preferably a T cell or NK cell, most preferably a T cell). The person skilled in the art is fully capable of selecting suitable host cells for expression of the antigen binding protein.

The antigen binding proteins of the invention may be produced by any technique known in the art, such as, but not limited to, any chemical, biological, genetic or enzymatic technique, alone or in combination.

The antigen binding proteins of the invention are suitably isolated from the culture medium by antibody purification procedures such as, for example, protein a-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

In one embodiment, recovering the expressed antigen binding protein or polypeptide herein refers to performing protein a chromatography, kappa selection chromatography and/or size exclusion chromatography, preferably protein a chromatography and/or size exclusion chromatography, more preferably protein a chromatography and size exclusion chromatography.

Knowing the amino acid sequence of the desired sequence, one skilled in the art can produce the antigen binding proteins of the invention by standard techniques for producing polypeptides. For example, the antigen binding proteins may be synthesized using well known solid phase methods, particularly using commercially available peptide synthesis equipment (such as manufactured by Applied Biosystems of foster, california) and following the manufacturer's instructions. Alternatively, the antibodies and antigen binding proteins of the invention may be produced by recombinant DNA and gene transfection techniques well known in the art (see Morrison SL. et al (1984) and patent documents US5,202,238 and US5,204,244). For example, fragments may be obtained as DNA expression products after the following process: the DNA sequence encoding the desired (poly) peptide is incorporated into an expression vector, and such vector is introduced into a suitable eukaryotic or prokaryotic host that will express the desired polypeptide, from which the polypeptide can then be isolated using well-known techniques.

Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, e.g., riechmann l. 1988;Neuberger MS et al 1985) and can be readily applied to the production of antigen binding proteins of the present invention.

In one example, the vector used to express the recombinant antigen-binding proteins of the invention is designed as a monocistronic pUC19 derivative, for example, under the control of a HCMV-derived promoter element. Plasmid DNA is amplified, for example, in E.coli according to standard culture methods, followed by purification using commercially available kits (Macherey & Nagel). Purified plasmid DNA was used for transient transfection of, for example, CHO-S cells according to the manufacturer' S instructions (ExpiCHO ^TM systems; thermo FISHER SCIENTIFIC). Transfected CHO cells are cultured, for example, at, for example, 32 ℃ to 37 ℃ for 6-14 days and receive one to two feeds of ExpiCHO ^TM feed solutions.

By, for example, filtration (0.22 μm), using, for example SartoclearLaboratory filter aids (Sartorius) to clarify conditioned cell supernatants. Using, for example/>Pure 25L FPLC system (GE LIFESCIENCES) for purification of bispecific antigen binding proteins, the system was equipped for tandem affinity chromatography and size exclusion chromatography. Affinity chromatography is performed according to standard affinity chromatography protocols on, for example, a protein a or protein L column (GE LIFESCIENCES). For example, size exclusion chromatography is performed directly after elution from the affinity column (pH 2.8) using, for example, a Superdex 200pg 16/600 column (GE LIFESCIENCES) according to standard protocols to obtain high purity monomeric proteins. The calculated extinction coefficient is used on, for example, a NanoDrop system (Thermo Scientific) to determine protein concentration from the predicted protein sequence. The concentration was adjusted by using a Vivaspin device (Sartorius), if necessary. Finally, the purified molecules are stored at a concentration of about 1mg/mL in, for example, phosphate buffered saline at a temperature of 2 ℃ to 8 ℃.

The mass of the purified bispecific antigen binding protein is determined by e.g. HPLC-SEC on a MabPac SEC-1 column (5 μm,7.8X300 mM) which is run in a Vanquish UHPLC system in e.g. 50mM sodium phosphate pH 6.8 containing 300mM NaCl.

Therapeutic methods and uses

The antigen binding proteins of the invention are particularly useful in immunotherapy (preferably adoptive cell therapy, more preferably adoptive T cell therapy) for the prevention and/or treatment of proliferative diseases. Administration of the compounds of the invention may for example involve infusion of lymphocytes, preferably NK cells or T cells, more preferably T cells of the invention, into the patient. Preferably, such lymphocytes are autologous lymphocytes of the patient and are transduced with the nucleic acids or antigen binding proteins of the invention in vitro.

In a preferred embodiment, the proliferative disease is cancer, in particular a cancer expressing CT 45.

In the context of the present invention, a cancer is considered to be a "CT 45 expressing cancer" (also referred to as CT45 "positive" cancer) if, according to the guidelines of NCI, the relevant peptide such as, for example, CT45-IP peptide is presented in >98% of all cancers. In all other indications mentioned herein, a biopsy may be performed, as it is standard in the treatment of these cancers and may be based onAnd related methods for identifying the peptides (according to WO 03/100432; WO 2005/076009; WO 2011/128448; WO 2016/107740, US 7,811,828, US 9,791,444 and US 2016/0187351, the contents of each of which are hereby incorporated by reference in their entirety). In one embodiment, the cancer is readily determined (i.e., diagnosed), for example, by using the antigen binding proteins of the invention. Methods for using antigen binding proteins to identify cancers that express antigens are known to those of skill in the art. It should be understood that the terms "cancer" and "cancer" are not used interchangeably herein, as cancer (cancer) is a special type of cancer that is newly developed in the skin or in tissues lining or covering body organs.

In one embodiment, the cancer expressing CT45 is selected from lung cancer, NSCLC, gall bladder cancer, bile duct cancer, lymph node cancer, ovarian cancer, esophageal cancer, liver cancer, uterine cancer, and melanoma.

In one embodiment, the cancer is a cancer in which CT45 antigen is overexpressed, mutated, and/or presented with CT45 antigen peptides. Such cancers are readily determined (i.e., diagnosed), for example, by using the antigen binding proteins of the invention. Methods for using antigen binding proteins to identify cancers that express antigens are known to those of skill in the art.

In another aspect, the present invention relates to a method of treating a proliferative disease, said method comprising administering to a subject in need thereof a therapeutically effective amount of an antigen binding protein, nucleic acid or vector, host cell or pharmaceutical composition according to the invention as defined above.

In a particular embodiment, the invention relates to a method of treating a subject suffering from a proliferative disease, said method comprising administering to said subject lymphocytes (preferably NK cells or T cells, more preferably T cells) expressing an antigen binding protein of the invention on the cell surface.

The terms "subject" or "individual" are used interchangeably and may be, for example, a human or a non-human mammal, preferably a human.

In the context of the present invention, the term "treatment" includes therapeutic treatment (i.e. for a subject suffering from a given disease) and/or prophylactic treatment (i.e. for a subject susceptible to a given disease). Therapeutic treatment and means reversing, alleviating and/or inhibiting the progression of one or more symptoms of a disorder or condition. Prophylactic treatment means preventing the occurrence of one or more symptoms of a disorder or condition. Thus, treatment refers not only to treatment that results in complete cure of the disease, but also to treatment that slows disease progression, prevents or delays onset of the disease, and/or prolongs survival of the subject.

In one embodiment, a "disease" or "disorder" is any condition that would benefit from treatment with an antigen binding protein of the invention. In one embodiment, this includes chronic and acute disorders or diseases, including those pathological conditions that predispose a subject to the disorder. The term "in need of treatment" refers to those subjects who have had a disorder and are to be prevented from the disorder.

"Proliferative disorders", such as cancer, involve deregulated and/or inappropriate proliferation of cells.

In one embodiment, the method of treatment comprises immunotherapy, in particular adoptive autologous or heterologous cell therapy (preferably T cell therapy).

In a preferred embodiment, the antigen binding protein is or comprises a TCR or a functional fragment thereof.

Preferably, the antigen binding protein is expressed on the surface of the host cell.

In one embodiment, the method of treatment comprises administering a host cell expressing an antigen binding protein, wherein the host cell is a T cell, a T cell progenitor cell, or an NK cell (preferably a T cell).

In one embodiment, the host cell (preferably a T cell, T cell progenitor or NK cell, more preferably a T cell) is autologous.

In one embodiment, the host cell (preferably a T cell, a T cell group cell or an NK cell, more preferably a T cell) is allogeneic.

In one embodiment, the antigen binding protein is conjugated to a therapeutically active agent (preferably a therapeutically active agent selected from the group consisting of radionuclides, chemotherapeutic agents, and toxins).

In one embodiment, the method of treatment further comprises administering at least one chemotherapeutic agent to a subject in need of treatment.

In one embodiment, the method of treatment further comprises administering radiation therapy to a subject in need of treatment.

In a related aspect, the invention relates to a method of eliciting an immune response in a patient suffering from a proliferative disease, in particular a cancer presenting a peptide in complex with an MHC protein, said peptide comprising or consisting of the amino acid sequence of KIFEMLEGV (SEQ ID NO: 138), said method comprising administering to said patient an antigen binding protein of the disclosure, wherein said cancer is selected from the group of cancers consisting of: lung cancer, NSCLC, gall bladder cancer, bile duct cancer, lymph node cancer, ovarian cancer, esophageal cancer, liver cancer, uterine cancer and melanoma. In one embodiment, the immune response mentioned in the method is a cytotoxic T cell response.

In yet another aspect, the invention relates to the use of an antigen binding protein, nucleic acid or vector, host cell or pharmaceutical composition according to the invention for the manufacture of a medicament for the treatment of a proliferative disease in a subject.

In yet another aspect, the invention relates to the use of an antigen binding protein, nucleic acid or vector, host cell or pharmaceutical composition according to the invention for treating a disease in a subject.

Documents providing guidelines for Cancer therapy include Cancer, PRINCIPLES AND PRACTICE of Oncology, 4 th edition, deVita et al, editors J.B. Lippincott Co., philadelphia, pa. (1993). The appropriate treatment is selected based on the particular type of cancer and other factors such as the general health of the patient, as is well known in the relevant arts. In treating cancer patients, the antigen binding proteins of the invention may be used alone or may be added to treatment regimens with other antineoplastic agents.

Thus, in some embodiments, the antigen binding protein may be administered concurrently with, before or after, a variety of drugs and treatments (such as, for example, chemotherapeutic agents, non-chemotherapeutic agents, antineoplastic agents, and/or radiation) that are widely used in cancer treatment.

"Diagnosis" herein refers to medical diagnosis, and refers to determining which disease or condition explains the symptoms and signs of an individual.

By "therapeutically effective amount" of an antigen binding protein or pharmaceutical composition thereof is meant an amount of antigen binding protein sufficient to treat the proliferative disease at a reasonable benefit/risk ratio applicable to any medical treatment. However, it will be appreciated that the total daily dose of the antigen binding protein, nucleic acid or vector, host cell or pharmaceutical composition of the invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the particular antigen binding protein employed; the particular composition employed, the age, weight, general health, sex and diet of the patient; the time of administration, route of administration and rate of excretion of the particular polypeptide employed; duration of treatment; a medicament for use in combination or simultaneously with the particular polypeptide employed; and similar factors well known in the medical arts. For example, it is well known in the art to start doses of the compound at levels below those required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

In one embodiment, the efficacy of treatment with an antigen binding protein of the invention is determined in vivo, e.g., in a mouse cancer model, and by measuring, e.g., the change in tumor volume between the treatment group and the control group.

The antigen binding protein of the invention, the nucleic acid of the invention or the vector of the invention, the host cell of the invention or the pharmaceutical composition of the invention may be administered by any feasible method.

As disclosed herein, in some embodiments, a host cell as defined above is used in a medical use or method of treatment as described herein. In such embodiments, the host cell is preferably a lymphocyte, such as an NK cell, a T cell or a T cell progenitor cell, preferably a CD4 and/or CD8 positive T cell or γδ T cell, most preferably a CD4 and/or CD8 positive T cell.

Thus, the host cells of the invention, preferably T cells, may be used as the active ingredient of a therapeutic composition. Accordingly, the present invention also provides a method of killing target cells of a patient that abnormally express a polypeptide comprising peptide KIFEMLEGV (SEQ ID NO: 138), comprising administering to the patient an effective amount of a host cell, preferably a T cell. In the case of this method, the host cell preferably elicits an immune response once administered to the subject.

In one aspect, a TCR-elicited immune response or T cell response may refer to proliferation and activation of effector function induced by a peptide such as KIFEMLEGV (SEQ ID NO: 138) in vitro, ex vivo or in vivo. For MHC class I restricted cytotoxic T cells, for example, effector function may be peptide pulsed, peptide precursor pulsed or lysis of naturally peptide-presenting target cells; secretion of cytokines (preferably interferon-gamma, TNF-alpha or IL-2) induced by the peptide; secretion of effector molecules (e.g., granzymes or perforins) induced by peptides; or degranulation.

In the context of the present invention, T cells are typically collected from a subject by apheresis when T cells are used as a medicament. The T cells are then genetically engineered to express the antigen binding proteins of the invention on their cell surfaces, and then the genetically engineered T cells are expanded and then reinfused into a subject. In this example, the antigen binding protein is preferably a membrane-bound antigen binding protein, more preferably a TCR.

Thus, a host cell has been transfected, infected or transformed with a nucleic acid and/or vector according to the invention as described above in the section "nucleic acid, vector and recombinant host cell".

When transfecting a host cell to express an antigen binding protein of the invention, preferably the cell comprises an expression vector capable of expressing the antigen binding protein. The host cell may be referred to as an activated host cell.

Protocols for this so-called adoptive transfer of T cells are well known in the art. An overview can be found in: gattioni et al and Morgan et al (Gattinoni, L. Et al, nat. Rev. Immunol.6 (2006): 383-393; morgan, R.A. et al, science 314 (2006): 126-129).

For the purposes of the present invention, the amount or dose of the antigen binding protein of the first aspect of the invention, the nucleic acid of the second aspect of the invention, the vector of the third aspect of the invention, the host cell of the fourth aspect of the invention or the pharmaceutical composition of the fifth aspect of the invention administered may be sufficient to achieve, for example, a therapeutic or prophylactic response in a subject or animal within a reasonable time frame. For example, the dose of an antigen binding protein, nucleic acid, vector, host cell, or pharmaceutical composition according to the invention should be sufficient to bind to a cancer antigen, or detect, treat, or prevent cancer, over a period of time of about 2 hours or more (e.g., 12 to 24 hours or more) from the time of administration. In certain embodiments, the period of time may be even longer. The dosage will be determined according to the efficacy of the antigen binding protein, nucleic acid, vector, host cell or pharmaceutical composition according to the invention and the condition of the animal (e.g., human) and the weight of the animal (e.g., human) to be treated.

Various other methods can be used to generate T cells in vitro. For example, autologous tumor infiltrating lymphocytes may be used in the generation of CTLs. Plabanski et al (Plabanski, M. Et al, eur. J Immunol 25 (1995): 1783-1787) utilized autologous peripheral blood lymphocytes (PLB) in the preparation of T cells. Likewise, B cells can be used in the production of autologous T cells.

Allogeneic cells may also be used in the preparation of T cells, and the methods are described in detail in US6805861, incorporated herein by reference.

Host cells expressing the antigen binding proteins of the invention against peptide KIFEMLEGV (SEQ ID NO: 138) can be used in therapy. Thus, a further aspect of the invention provides an activated host cell obtainable by the foregoing method of the invention.

Activated host cells produced by the above method can specifically recognize cells that abnormally express a polypeptide comprising peptide KIFEMLEGV (SEQ ID NO: 138).

By "aberrant expression", the inventors also mean that the polypeptide is overexpressed compared to the level of expression in normal (healthy) tissue, or that the gene is silenced in the tissue from which the tumor is derived, but expressed in the tumor. By "over-expression", the inventors mean that the polypeptide is present at a level that is at least 1.2 times the level present in normal tissue; preferably at least 2 times, and more preferably at least 5 times or 10 times the level present in normal tissue.

In one aspect, a host cell (particularly a T cell) recognizes the cell by interaction (e.g., binding) with the CT45-IP: MHC complex via its antigen binding protein (particularly its TCR). Host cells can be used in a method of killing target cells of a patient that abnormally express a polypeptide comprising peptide KIFEMLEGV (SEQ ID NO: 138), wherein an effective amount of the activated host cells is administered to the patient. T cells administered to a patient may originate from the patient and be activated as described above (i.e., the T cells are autologous T cells). Alternatively, the T cells are not from the patient, but from another individual (i.e., the T cells are heterologous T cells). In this case, it is preferable if the individual is a healthy individual. By "healthy individual" the inventors mean that the individual is generally in good health, preferably has an active immune system, and more preferably is not suffering from any disease that can be easily tested and detected.

In vivo, the target cells of CD8 positive T cells according to the invention may be tumor cells (which sometimes express MHC class II) and/or stromal cells surrounding a tumor (tumor cells) (Dengjel, J. Et al, CLIN CANCER RES 12 (2006): 4163-4170).

Diagnostic use

CT45 is expressed on the surface of the cancers defined hereinabove. CT45 antigenic peptides constitute cancer markers and thus have the potential to be used to indicate the effectiveness of anti-cancer therapies or to detect disease recurrence.

Thus, in a further aspect, the present invention provides an antigen binding protein of the first aspect, a nucleic acid of the second aspect, a vector of the third aspect, a host cell of the fourth aspect or a pharmaceutical composition of the fifth aspect for use as a diagnostic agent, in particular as an in vivo diagnostic agent. In a preferred embodiment, the diagnostic agent is for diagnosing a proliferative disease. In a more preferred embodiment, the diagnostic agent is used for diagnosing a cancer presenting a peptide comprising or consisting of the amino acid sequence KIFEMLEGV (SEQ ID NO: 138) in complex with an MHC protein, preferably wherein the cancer is selected from the group of cancers consisting of: lung cancer, NSCLC, gall bladder cancer, bile duct cancer, lymph node cancer, ovarian cancer, esophageal cancer, liver cancer, uterine cancer and melanoma.

In one embodiment, the antigen binding proteins of the invention are used as components of an assay to determine the sensitivity of a patient to a therapeutic agent, to monitor the effectiveness of an anti-cancer therapy or to detect recurrence of a disease after treatment in the context of a therapy targeting a tumor expressing CT 45.

Thus, another object of the present invention relates to an antigen binding protein according to the present invention for use in vivo detection of CT45 expression in a subject, or for use in ex vivo or in vitro detection of CT45 expression in a biological sample of a subject. The detection may be intended in particular for

A) Diagnosing the presence of cancer in a subject, or

B) Determining the sensitivity of a patient suffering from cancer to a therapeutic agent targeting CT45, or

C) Monitoring the effectiveness of anti-CT 45 cancer therapy or detecting cancer recurrence following anti-CT 45 cancer therapy, particularly for therapies using antigen binding proteins according to the invention; by detecting the presentation of CT45 antigen peptide on tumor cells.

In one embodiment, the antigen binding protein is intended for in vitro or ex vivo use.

In yet another aspect, the invention relates to an in vitro method of detecting cancer in a biological sample, the in vitro method comprising the steps of: (a) Contacting the biological sample with an antigen binding protein of the first aspect of the invention, and (b) detecting binding of the antigen binding protein to the biological sample.

Kit for detecting a substance in a sample

Finally, the invention also provides a kit comprising at least one antigen binding protein of the invention.

In one embodiment, the kit comprises

A) At least one antigen binding protein of the invention as defined above in the section "antigen binding protein", a nucleic acid encoding said antigen binding protein, a vector comprising said nucleic acid, or a host cell comprising said antigen binding protein, nucleic acid and/or vector,

B) Optionally packaging material, and

C) Optionally a label or package insert contained within the packaging material that indicates that the antigen binding protein is effective for treating cancer or for use in cancer treatment.

In a preferred embodiment, the kit comprises a nucleic acid encoding an antigen binding protein of the invention, or a vector comprising the nucleic acid. The kit may further comprise instructions for the regulatable expression of the antigen-binding protein on the surface of a cell (preferably a T cell, a T cell progenitor cell or an NK cell, more preferably a T cell).

The kits of the present disclosure may further include any other agent useful for the regulatable expression of antigen binding proteins on the surface of cells (preferably T cells, T cell progenitors or NK cells, more preferably T cells), such as transfection/transduction agents useful for introducing nucleic acids or expression vectors into cells.

It may also be preferred that the kit comprises a host cell comprising a nucleic acid encoding an antigen binding protein of the invention or a vector comprising the nucleic acid, i.e. a host cell capable of expressing an antigen binding protein of the invention.

The components of the kit may be present in separate containers, or the components may be present in a single container. Suitable containers include single tubes, one or more wells of a plate (e.g., 96-well plate, 384-well plate, etc.), and the like.

In related embodiments, at least one antigen binding protein of the invention is contained in single-and/or multi-chamber pre-filled syringes (e.g., liquid syringes and freeze syringes).

In one embodiment, the invention encompasses a kit for producing a single dose administration unit.

Thus, in one embodiment, at least one antigen binding protein of the invention as mentioned in a) of the kit of the invention is a dried antigen binding protein of the invention contained in a first container. The kit also contains a second container with an aqueous formulation.

Thus, in one embodiment, the kit comprises

A) A first container comprising at least one dried antigen binding protein of the invention as defined above in section "antigen binding protein",

B) A second container comprising an aqueous formulation;

c) Optionally packaging material, and

D) Optionally a label or package insert contained within the packaging material that indicates that the antigen binding protein is effective for treating cancer or for use in cancer treatment.

The aqueous formulation is typically a solution comprising a pharmaceutically acceptable carrier as defined above in section "pharmaceutical composition".

In related embodiments, the "first container" and "second" container refer to the chambers of a multi-chamber prefilled syringe (e.g., a freeze syringe).

Throughout this disclosure, the term "and/or" is a grammatical conjunctive term that should be construed to cover one or more situations in which it is connected. For example, the phrase "such native sequence proteins can be prepared using standard recombinant and/or synthetic methods" indicates that native sequence proteins can be prepared using standard recombinant and synthetic methods, or native sequence proteins can be prepared using standard recombinant methods, or native sequence proteins can be prepared using synthetic methods.

Furthermore, throughout the present application, the term "comprising" should be interpreted to cover all the explicitly mentioned features as well as any optional, additional, unspecified features. As used herein, the use of the term "comprising" also discloses embodiments in which no other features (i.e., "consisting of … …") are present other than the explicitly mentioned features.

Furthermore, the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

The invention will now be described in more detail with reference to the following figures and examples. All documents and patent documents cited herein are hereby incorporated by reference. While the invention has been illustrated and described in detail in the foregoing description, the embodiments are to be considered as illustrative or exemplary and not restrictive.

Drawings

Fig. 1: functional avidity (EC ₅₀) was measured according to the killing efficiency of TCR transfected T cells against CT45-IP peptide loaded T2 cells. T2 cells constitutively expressing luciferase were loaded with a drop-wise amount of CT45-IP peptide, which was then co-cultured with cd8+ T cells transfected with the specific TCR. Killing was analyzed by measuring luciferase activity released by dying T2 cells in the supernatant. The assay was repeated twice with cells from two different donors (indicated by filled circles and diamond symbols). Functional avidity was assessed by calculating the half maximum killing efficiency of the tested TCRs.

Fig. 2: cross-reactivity check of sequence-like peptides. CT45-IP peptide, 10 different sequence-like peptides, unrelated peptide controls NYESO-001 or no loading were loaded at a concentration of 10. Mu.M for each peptide, respectively, into T2 cells constitutively expressing luciferase. Those T2 cells were then co-incubated with cd8+ T cells transfected with the specific TCRs. Killing was analyzed by measuring luciferase activity released by dying T2 cells in the supernatant. The assay was repeated twice with cells from two different donors (black bar = assay 1, donor 1; red bar = assay 2, donor 2).

Fig. 3: TCR surface staining. Assessed by flow cytometry of pHLA-Dextramer in combination with measured TCR expression. The histograms show T cells electroporated with TCR-mRNA after staining with CT45-IP-HLA-A 2X 02dextramer (black line) and simulated TCR control (light grey dashed line). The percentage of positive events is indicated in the figure. The simulated TCR control was used as a reference for the dextramer negative region.

Fig. 4: tumor cell line efficacy. Live cell monitoring of RFP expressing tumor cell lines co-cultured with or without T cells expressing our TCR of interest. Red counts representing tumor cells were quantified over a 48h period and normalized to time point 0 h. The left panel shows tumor cell line NCIH, 1703 and the right panel shows proliferation of cell line a 375. Cd8+ T cells electroporated with mock TCRs (negative control, top), TCR-9 (middle) and TCR-7 (bottom) are shown. Target cells additionally loaded with 10. Mu.M CT45-IP peptide were used as positive control (dotted circles in the middle) as well as target cells without effector cells (asterisks) and effector cells expressing the desired TCR in different E: T ratios (gray circles of different shades).

Examples

Materials and methods

TCR identification

The alpha and beta TCR chain sequences were isolated from T cells of healthy donors. To ensure enrichment of peptide-specific T cells, cells were repeatedly stimulated with CT45-IP-MHC and CD28 (priming) -coated artificial antigen presenting cells (as described in Walter et al, 2003J Immunol.,11 months 15 days; 171 (10): 4974-8) and subsequently single cell sorted using CT 45-IP-HLA-A.times.02 tetramers, or alternatively stimulated with CT45-IP loaded T2 cells. After sufficient expansion, cells were sorted using CT 45-IP-HLA-A.times.02 tetramer.

TCR nucleotide sequences are obtained via standard methods, such as 5' race and sanger sequencing, as described, for example, in Green and Sambrook Molecular Cloning, laboratory Manual, fourth edition. Genes encoding the V and J regions of the TCR are listed in table 2. The annotation is made by GeneData 11.0.1 using IMGT/GENE-DB (version: 28/11/2019) as a reference database. The TCR amino acid sequences are listed in table 3.

TABLE 2 identified TCRs

ID	Vα	Jα	Vβ	Jβ
					TCR-1	TRAV38-1	TRAJ22	TRBV7-9	TRBJ2-7
TCR-2	TRAV14/DV4	TRAJ52	TRBV13	TRBJ1-1
					TCR-3	TRAV14/DV4	TRAJ33	TRBV27	TRBJ1-5
TCR-4	TRAV3	TRAJ30	TRBV6-2	TRBJ2-1
					TCR-5	TRAV35	TRAJ26	TRBV9	TRBJ2-7
TCR-6	TRAV12-3	TRAJ7	TRBV4-1	TRBJ2-1
					TCR-7	TRAV38-2/DV8	TRAJ32	TRBV13	TRBJ2-1
TCR-8	TRAV19	TRAJ40	TRBV13	TRBJ2-1
					TCR-9	TRAV5	TRAJ17	TRBV2	TRBJ2-1
TCR-10	TRAV1-2	TRAJ44	TRBV6-1	TRBJ2-1
					TCR-11	TRAV22	TRAJ44	TRBV6-1	TRBJ2-7
TCR-12	TRAV27	TRAJ50	TRBV4-1	TRBJ1-2
					TCR-13	TRAV14/DV4	TRAJ5	TRBV19	TRBJ2-1
TCR-14	TRAV14/DV4	TRAJ22	TRBV11-2	TRBJ1-6
					TCR-15	TRAV21	TRAJ37	TRBV5-1	TRBJ2-7

TABLE 3 TCR amino acid sequence

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Re-expression of TCR

The human constant chain domain is exchanged with its murine counterpart and has additional mutations in the transmembrane domain to enhance hydrophobicity. Both modifications are in Jin et al, JCI insight.2018;3 (8): e 99488.

To assess their functional characteristics, TCR mRNA electroporation techniques were used to re-express the identified TCRs in primary T cells of healthy donors. Briefly, T7 transcription was performed on DNA templates of TCR a and β chain nucleotide sequences. The resulting TCR mRNA was used for electroporation of pre-activated T cells for transient expression of exogenous TCRs verified in co-culture experiments.

Co-culture assays (evaluation of functional avidity, specificity and TCR motif)

The functional characteristics of TCRs were evaluated in co-culture experiments with T2 cells. Cd8+ T cells were pre-stimulated and electroporated with TCR mRNA. On the day of co-culture, luciferase-transduced T2 cells were loaded with: peptide CT45-IP (SEQ ID NO: 138) at different concentrations (evaluation of functional affinity, EC ₅₀), sequence-like peptide at a concentration of 10. Mu.M (SEQ ID NO: 146-155), or alanine-substituted variants of CT45-IP peptide (SEQ ID NO: 139-145) at a concentration ranging from 10-30 fold higher than the average EC ₅₀ of the corresponding TCR (TCR motif determination). As a control, irrelevant peptide NYESO-001 (SEQ ID NO: 188) and unloaded T2 cells were used at a concentration of 10. Mu.M. Briefly, T2 cells were incubated with the corresponding amount of peptide for 2 hours and subsequently washed and harvested.

Cells electroporated with model TCR 1G4-a95Ly and mock electroporated T cells without exogenous TCR served as controls. T cells and peptide-loaded T2 cells were seeded at a 1:1 ratio and incubated for 24h until the supernatant was harvested. The supernatant was subjected to an analysis for the presence of luciferase released by apoptotic/necrotic T2 cells killed by peptide-specific T cells. The amount of luciferase present in the supernatant was determined by adding specific substrate and measuring chemiluminescent signal in a microplate reader.

Testing of functionality in CD4+ T cells

Cd3+ T cells were stimulated and electroporated with TCR mRNA. After overnight incubation, TCR transfected T cells were co-incubated with T2 cells loaded with either 10. Mu.M CT45-IP (SEQ ID NO: 138) or 10. Mu.M unrelated NYESO-001 (SEQ ID NO: 188) peptide at a 1:1 ratio. After the start of co-culture, cytokine secretion blocking reagent was directly added and incubated at 37 ℃ for 5h. Thereafter, T cells were stained with fluorescently labeled antibodies against different surface markers (e.g., CD4 and CD 8). After fixation and permeabilization, cells were stained for intracellular cytokines (TNF- α and IFN- γ) and analyzed on a flow cytometer (data not shown).

Lentiviral co-transduction of co-stimulatory molecule CD8

To engage CD 4T cells in an immune response following transduction with MHC class I restricted TCRs, co-transduction with co-stimulatory molecule CD8 was performed. For this purpose, pre-stimulated cd3+ T cells were transduced with lentiviral vectors encoding TCR chains and CD8 molecules. The T cells were activated for 24h using coated plates of CD3 and anti-CD 28 together with added IL-2. The pre-titrated concentrated lentiviral supernatant is combined with an adjuvant to enhance transduction of lentiviral particles (e.g.,Reagent (Sirion Biotech)) is added to the cells together. T cells were expanded over the course of 10 days using increasing volumes of medium and decreasing concentrations of IL-2, while cells were continuously transferred to larger cell culture flasks. Prior to freezing the cells, the transduction efficiency and resting state of T cells were examined via flow cytometry. Subsequently, the effect of CD8 co-transfection on killing efficiency against CT45-IP presenting tumor cells was analyzed in a live cell monitoring killing assay (data not shown).

TCR surface staining

Surface marker staining was performed with electroporated and pre-activated cd8+ T cells. For this purpose, anti-CD 8, CD3 and/or mTCRB antibodies were used as well as TCR staining with fluorescent labeled Dextramer (dextramer scaffold with conjugated CT 45-IP-HLA-A-02 or unrelated NYESO 1-001-HLA-A-02). After 30 minutes, the cells were washed, fixed and then analyzed by flow cytometry. Gating was performed on the signal of cells stained with irrelevant peptide-MHCdextramer to define dextramer positive cells.

Live cell monitoring killing assay

Proliferation of tumor cell lines expressing Red Fluorescent Protein (RFP) was monitored by quantifying red object counts over time using a living cell imaging system. Cell lines NCIH and A375 were co-cultured with T cells expressing TCRs of the invention at E:T ratios of 9:1, 3:1 or 1:1, or were not co-cultured with T cells and monitored over a period of 48 h. As a positive control, target cells were loaded with 10. Mu.M of peptide CT45-IP. The decline in proliferation of tumor cell lines over time is an indicator of tumor cell killing.

Example 1: functional affinity

As measured by peptide titration experiments, all fifteen identified CT45-IP specific TCRs showed high functional avidity, expressed as half maximal killing capacity EC ₅₀ shown in fig. 1. The EC ₅₀ values measured ranged from 0.15nM to 59.5nM. Specifically, EC ₅₀ value is 7.78nM(TCR-1);4.69nM(TCR-2);1.02nM(TCR-3);1.31nM(TCR-4);1.32nM(TCR-5);3.25nM(TCR-6);0.48nM(TCR-7);6.52nM(TCR-8);0.15nM(TCR-9);8.75nM(TCR-10);59.50nM(TCR-11);47.47nM(TCR-12);11.38nM(TCR-13);17.69nM(TCR-14);18.60nM(TCR-15).

Example 2: specificity and TCR motifs

The TCRs described herein were tested for their specific characteristics by testing their ability to recognize 10 CT45-IP sequence-like peptides (SEQ ID NOS: 146-155). The loading with CT45-IP and CT45-IP sequence-like peptides was performed at extremely high concentrations of 10. Mu.M to detect low signals. TCRs were shown to not bind to another peptide than CT 45-IP. A small amount of binding signal of TCR-9 and TCR-6 to peptide SP-05-0004 (SEQ ID NO: 149) was detected as depicted in FIG. 2. TCR characterization also included determining TCR binding motifs for CT45-IP peptides, as listed in table 4. For this purpose, CT45-IP alanine-exchange peptide variants (SEQ ID NOS: 139-145) were tested in a co-culture experiment using all fifteen TCRs. Alanine exchange peptides were tested at a concentration range of 10-30 fold higher than the average EC ₅₀ of the corresponding TCR. Relevant positions are indicated by numerals (referring to the positions of amino acids in the peptide), irrelevant positions are indicated by hyphens, and untested positions are marked by x.

TABLE 4 consensus motifs

Example 3: CD4 functionality

Functional assays in cd4+ T cells were performed as described above. Of the fifteen tested TCRs, the inventors identified TCRs that showed functionality in cd8+ T cells as well as cd4+ T cells (data not shown).

Example 4: surface expression

The surface expression of TCRs described herein was measured by CT45-IP-HLA-A2 x 02dextramer staining and is shown in figure 3. After electroporation of TCR mRNA, surface expression varied from 0.53% (TCR-11) to 55.5% (TCR-5) positive events after gating on cd3+ T cells. Specifically, the surface is expressed as 8.9％(TCR-1);39.1％(TCR-2);27.2％(TCR-3);39.1％(TCR-4);61.1％(TCR-5);2.9％(TCR-6);53.3％(TCR-7);60.7％(TCR-8);44.0％(TCR-9);52.8％(TCR-10);6.6％(TCR-11);20.1％(TCR-12);53.1％(TCR-13);11.6％(TCR-14);22.6％(TCR-15).

Example 5: efficacy against tumor cell lines

Two tumor cell lines (i.e., A375 with CT45-IP of about 30 copies/cell and NCIH1703 with CT45-IP of about 150 copies/cell) were co-cultured with CD8+ T cells expressing TCR-9, TCR-7 or mimicking TCR (FIG. 4). The efficacy of those TCRs to kill both cell lines was assessed in live cell monitoring experiments. As measured by the normalized red subject count of <2, the mock TCRs did not result in a significant reduction in tumor cell proliferation, whereas at E: T ratios of 6:1 (TCR-9) or 1.8:1 (TCR-7) (NCIH 1703 and a 375), at E: T ratios of 6:1 (TCR-9) or 1.8:1 (TCR-7) (NCIH 1703 and a 375), and at E: T ratios of 2:1 (TCR-9) or 0.6:1 (TCR-7) and 0.6:1 (TCR-9) or 0.2:1 (TCR-7) (NICIH 1703), TCR-9 and TCR-7 effectively killed tumor cell lines loaded with CT45-IP peptide.

Example 6: security window

Co-culture assays were performed using T2 cells loaded with the peptides CT45-IP and SP-05-0004, respectively, of the titration series. The safety window is determined as EC ₅₀ SP-05-0004 divided by EC ₅₀ CT45-IP (data not shown).

Example 7: CT45-IP rendering

The frequency of detection of CT45-IP antigen peptides was analyzed in primary and cultured tumor samples. A summary of the results is shown in table 5. In the table, expression >0% is expressed as +, expression. Gtoreq.10% is expressed as ++, and expression. Gtoreq.30% is expressed as ++. The tumor entities that were detected as presented were cholangiocarcinoma (CCC), liver cancer (HCC), skin cancer (MEL, identified by cell lines), lymph node cancer (NHL), non-small cell lung cancer (NSCLC), ovarian Cancer (OC), esophageal cancer (OSCAR), and uterine cancer (UEC).

TABLE 5 target presentation

Example 8: efficacy of CD4+ T cells co-transduced with CT45-IP specific TCR and CD8 against tumor cells

Tumor cells presenting CT45-IP were co-cultured with CD4+ T cells or CD3+ T cells co-transduced with CT45-IP specific TCR and CD8 as described herein. The killing efficacy of T cells was evaluated in live cell monitoring experiments. After co-transduction with CT45-IP specific TCRs and CD8, effective killing of tumor cell lines as measured by normalized red object count <2 was observed (data not shown).

Clause of (b)

1. An antigen binding protein that specifically binds to a CT45 antigen peptide, the CT45 antigen peptide being in a complex with a Major Histocompatibility Complex (MHC) protein, wherein the CT45 antigen peptide comprises or consists of the amino acid sequence of SEQ ID No. 138 (KIFEMLEGV) and wherein the antigen binding protein comprises a first polypeptide comprising a variable domain V _A comprising Complementarity Determining Regions (CDRs) CDRa1, CDRa2 and CDRa3 and a second polypeptide comprising a variable domain V _B comprising CDRb1, CDRb2 and CDRb3, wherein

1) CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 14, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 16, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 19, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 21,

2) CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 24, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 133, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 75, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 136,

3) CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 24, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 63, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 66, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 68,

4) CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 90, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 92, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 66, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 96,

5) CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 2, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 4, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 8, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 10,

6) CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 53, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 55, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 58, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 60,

7) CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 71, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 72, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 75, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 77,

8) CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 99, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 101, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 75, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 104,

9) CDRA1 comprises or consists of the amino acid sequence of SEQ ID NO. 80, CDRA3 comprises or consists of the amino acid sequence of SEQ ID NO. 82, CDRB1 comprises or consists of the amino acid sequence of SEQ ID NO. 85, and CDRB3 comprises or consists of the amino acid sequence of SEQ ID NO. 87,

10 CDRa1 comprises or consists of the amino acid sequence of SEQ ID No. 107, CDRa3 comprises or consists of the amino acid sequence of SEQ ID No. 109, CDRb1 comprises or consists of the amino acid sequence of SEQ ID No. 112, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID No. 114,

11 CDRa1 comprises or consists of the amino acid sequence of SEQ ID No. 125, CDRa3 comprises or consists of the amino acid sequence of SEQ ID No. 127, CDRb1 comprises or consists of the amino acid sequence of SEQ ID No. 112, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID No. 130, or

12 CDRa1 comprises or consists of the amino acid sequence of SEQ ID No. 117, CDRa3 comprises or consists of the amino acid sequence of SEQ ID No. 119, CDRb1 comprises or consists of the amino acid sequence of SEQ ID No. 58, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID No. 122,

13 CDRa1 comprises or consists of the amino acid sequence of SEQ ID No. 24, CDRa3 comprises or consists of the amino acid sequence of SEQ ID No. 35, CDRb1 comprises or consists of the amino acid sequence of SEQ ID No. 38, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID No. 40,

14 CDRa1 comprises or consists of the amino acid sequence of SEQ ID No. 24, CDRa3 comprises or consists of the amino acid sequence of SEQ ID No. 26, CDRb1 comprises or consists of the amino acid sequence of SEQ ID No. 29, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID No. 31, or

15 CDRa1 comprises or consists of the amino acid sequence of SEQ ID No. 43, CDRa3 comprises or consists of the amino acid sequence of SEQ ID No. 45, CDRb1 comprises or consists of the amino acid sequence of SEQ ID No. 48, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID No. 50,

Wherein the antigen binding protein comprises the CDRa1, CDRa3, CDRb1 and CDRb3 sequence(s) having no more than one, two or three amino acid mutations, wherein each of CDRa1, CDRa3, CDRb1 and/or CDRb3 can comprise one, two or three amino acid mutations.

2. The antigen binding protein of clause 1, wherein

5) CDRA2 comprises or consists of the amino acid sequence of SEQ ID NO.3 and CDRB2 comprises or consists of the amino acid sequence of SEQ ID NO. 9,

11 CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 126 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 113, or

12 CDRa2 comprises or consists of the amino acid sequence of SEQ ID No. 118 and CDRb2 comprises or consists of the amino acid sequence of SEQ ID No. 59,

Wherein the antigen binding protein comprises the CDRa2 and CDRb2 sequence(s) having no more than one, two, three or four amino acid mutations, wherein each of CDRa2 and/or CDRb2 is capable of comprising one, two, three or four amino acid mutations.

3. The antigen binding protein of clause 1 or 2, wherein the antigen binding protein specifically binds to a complex of the CT45 antigen peptide and an MHC protein.

4. The antigen binding protein of any one of clauses 1 to 3, wherein the MHC protein is an HLA protein, more particularly HLA-a, even more particularly HLA-a x 02.

5. The antigen binding protein of any one of clauses 1-4, wherein EC ₅₀ of CT45-IP for inducing killing of cells presenting the CT45-IP, MHC complex, by T cells expressing the antigen binding protein is less than about 60nM, less than about 50nM, less than about 30nM, less than about 25nM, less than about 20nM, less than about 15nM, less than about 10nM, less than about 5nM, less than about 2.5nM, less than about 1.5nM, or less than about 1nM.

6. The antigen binding protein of any one of clauses 1 to 5, wherein the antigen binding protein specifically binds to a functional epitope comprising or consisting of 2, 3 or 4 amino acid positions selected from positions 3, 4, 5, 6 and 7 of SEQ ID No. 138.

7. The antigen binding protein of any one of clauses 1 to 6, wherein the antigen binding protein does not bind significantly to: at least 1, at least 2, at least 3, at least 4, at least 5 or all of the like peptides selected from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO:155 (SP-05-0010), preferably from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO:155 (SP-05-0010), more preferably from SEQ ID NO:147 (SP-05-0002), SEQ ID NO:151 (SP-05-0006), SEQ ID NO:152 (SP-05-0007) and SEQ ID NO:155 (SP-05-0010).

8. The antigen binding protein of any one of clauses 1 to 7, wherein the first polypeptide and the second polypeptide are comprised on two polypeptide chains, preferably wherein V _A is comprised in a first polypeptide chain and V _B is comprised in a second polypeptide chain.

9. The antigen binding protein of any one of clauses 1 to 7, wherein the antigen binding protein is a single chain antigen binding protein, preferably a single chain TCR; or a single chain bispecific antigen binding protein, preferably a single chain bispecific TCR.

10. The antigen binding protein of any one of clauses 1 to 9, wherein the antigen binding protein is monovalent or multivalent, e.g., tetravalent, trivalent, or divalent.

11. The antigen binding protein of any one of clauses 1 to 10, wherein the antigen binding protein is monospecific or multispecific, e.g., tetraspecific, trispecific, or bispecific.

12. The antigen binding protein of any one of clauses 1 to 11, wherein the antigen binding protein is a soluble protein.

13. The antigen binding protein of any one of clauses 1 to 12, wherein the antigen binding protein is a TCR.

14. The antigen binding protein of clause 13, wherein the TCR is selected from the group consisting of an alpha/beta TCR, a gamma/delta TCR, a single chain TCR, a membrane bound TCR, a soluble TCR, a monovalent, divalent or multivalent TCR, a monospecific, bispecific or multispecific TCR, a functional fragment of a TCR, and a fusion protein or chimeric protein comprising a functional fragment of a TCR.

15. The antigen binding protein of clause 13, wherein the TCR is an alpha/beta TCR or a gamma/delta TCR, preferably an alpha/beta TCR.

16. The antigen binding protein of any one of clauses 1 to 15, further comprising one or more of the following:

(i) One or more additional antigen binding sites;

(ii) A transmembrane domain, optionally comprising a cytoplasmic signaling region;

(iii) A diagnostic agent;

(iv) A therapeutic agent.

17. The antigen binding protein of clause 16, wherein the one or more additional antigen binding sites comprise or consist of an antibody-derived antigen binding site, preferably comprising or consisting of V _L and V _H.

18. The antigen binding protein of any one of clauses 1 to 17, wherein V _A comprises or consists of: an amino acid sequence selected from SEQ ID NOs 13, 132, 62, 89, 1, 52, 70, 98, 79, 106, 124, 116, 34, 23 and 42 or having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NOs 13, 132, 62, 89, 1, 52, 70, 98, 79, 106, 124, 116, 34, 23 or 42 and comprising an amino acid sequence of CDRa1, CDRa2 and CDRa3 according to clause 1 or 2; and wherein V _B comprises or consists of: an amino acid sequence selected from SEQ ID NOs 18, 135, 65, 94, 7, 57, 74, 103, 84, 111, 129, 121, 37, 28 and 47, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NOs 18, 135, 65, 94, 7, 57, 74, 103, 84, 111, 129, 121, 37, 28 or 47 and comprising CDRb1, CDRb2 and CDRb3 according to clause 1 or 2, wherein said CDRa1, CDRa2, CDRa3, CDRb1, CDRb2 and/or CDRb3 sequence can comprise one, two or three amino acid mutations, preferably amino acid substitutions.

19. The antigen binding protein of any one of clauses 1 to 18, wherein V _A and V _B are TCR variable domains, preferably TCR α, β, γ or δ variable domains, more preferably wherein V _A is a TCR α or γ variable domain, preferably an α variable domain, and V _B is a TCR β or δ variable domain, preferably a β variable domain.

20. The antigen binding protein of any one of clauses 1 to 19, further comprising a constant domain, wherein the constant domain comprises or consists of: an amino acid sequence selected from the group consisting of SEQ ID NO. 5, 750, 751, 156, 11, 32 and 157, preferably selected from the group consisting of SEQ ID NO. 5, 750, 751, 11 and 32, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 5, 750, 751, 156, 11, 32 or 157.

21. The antigen binding protein of any one of clauses 1 to 20, wherein the first polypeptide is a TCR a chain and the second polypeptide is a TCR β chain, or the first polypeptide is a TCR γ chain and the second polypeptide is a TCR δ chain, preferably wherein the first polypeptide is a TCR a chain and the second polypeptide is a TCR β chain.

22. The antigen binding protein of any one of clauses 1 to 21, wherein the first polypeptide comprises or consists of: an amino acid sequence selected from the group consisting of SEQ ID NOs 17, 134, 64, 93, 6, 56, 73, 102, 83, 110, 128, 120, 36, 27, 46 and 158-172, preferably selected from the group consisting of SEQ ID NOs 17, 134, 64, 93, 6, 56, 73, 102, 83, 110, 128, 120, 36, 27, 46, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NOs 17, 134, 64, 93, 6, 56, 73, 102, 83, 110, 128, 120, 36, 27, 46 or 158-172, and said second polypeptide comprises or consists of: an amino acid sequence selected from the group consisting of SEQ ID NO:22, 137, 69, 97, 12, 61, 78, 105, 88, 115, 131, 123, 41, 33, 51 and 173-187, preferably selected from the group consisting of SEQ ID NO:22, 137, 69, 97, 12, 61, 78, 105, 88, 115, 131, 123, 41, 33, 51, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO:22, 137, 69, 97, 12, 61, 78, 105, 88, 115, 131, 123, 41, 33, 51 or 173-187.

23. The antigen binding protein of any one of clauses 1 to 22, wherein the antigen binding protein specifically binds to a functional epitope comprising or consisting of:

Amino acid positions 1, 3 and 4 of SEQ ID NO. 138, preferably amino acid positions 1, 3, 4 and 5, or 1, 3, 4 and 6 or 1, 3, 4, 5, 6 and 7 of SEQ ID NO. 138;

Amino acid positions 4, 6 and 7 of SEQ ID NO. 138, preferably amino acid positions 1, 4, 6 and 7, or 3, 4, 6 and 7 or 1, 3, 4, 6 and 7 of SEQ ID NO. 138; or (b)

Amino acid positions 5 and 7 of SEQ ID NO. 138, preferably amino acid positions 5, 6 and 7, or 3, 4,5, 6 and 7 of SEQ ID NO. 138.

24. The antigen binding protein of any one of clauses 1 to 23, wherein the antigen binding protein does not significantly bind to a similar peptide of the group consisting of:

i.SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) And SEQ ID NO 155 (SP-05-0010); or alternatively

ii.SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) And SEQ ID NO. 155 (SP-05-0010).

25. The antigen binding protein of any one of clauses 1 to 24, wherein the antigen binding protein has an average expression of at least 5%, at least 10%, at least 20%, at least 30%, or at least 40%.

26. The antigen binding protein of any one of clauses 1 to 25, wherein V _A comprises a V segment encoded by TRAV14, particularly TRAV14/DV4, and CDRa1 according to SEQ ID No. 24 and CDRa2 according to SEQ ID No. 25.

27. The antigen binding protein of any one of clauses 1 to 26, wherein V _B comprises

I. a V segment encoded by TRBV13, and CDRb1 according to SEQ ID No. 75 and CDRb2 according to SEQ ID No. 76;

v region encoded by TRBV4-1, and CDRb1 according to SEQ ID No. 58 and CDRb2 according to SEQ ID No. 59, or

The V region encoded by TRBV6-1, and CDRB1 according to SEQ ID NO. 112 and CDRB2 according to SEQ ID NO. 113.

28. The antigen binding protein of any one of clauses 1 to 27, wherein the antigen binding protein is capable of activating

-Cd4+ T cells, in particular cd4+ CD8-T cells, and/or

CD8+ T cells, in particular CD8+ CD4-T cells,

And wherein the antigen binding protein is preferably a TCR, more preferably an alpha/beta TCR or a gamma/delta TCR.

29. One or more nucleic acids comprising one or more sequences encoding the antigen binding protein of any one of clauses 1-28.

30. A vector or collection of vectors comprising one or more nucleic acids according to clause 29.

31. A host cell comprising the antigen binding protein of any one of clauses 1-28, or the one or more nucleic acids of clause 29, or the vector or collection of vectors of clause 30.

32. The host cell of clause 31, wherein the host cell is

-Lymphocytes, preferably T cells, T cell progenitors or NK cells, more preferably CD4 or CD8 positive T cells; or alternatively

Cells for recombinant expression, such as Chinese Hamster Ovary (CHO) cells or yeast cells.

33. A pharmaceutical composition comprising the antigen binding protein of any one of clauses 1-28, one or more nucleic acids of clause 29, the vector or collection of vectors of clause 30, or the host cell of clause 31 or 32, and optionally a pharmaceutically acceptable carrier.

34. The antigen binding protein of any one of clauses 1 to 28, the one or more nucleic acids of clauses 29, the vector or collection of vectors of clauses 30, the host cell of clauses 31 or 32, or the pharmaceutical composition of clause 33, for use in medicine.

35. The antigen binding protein according to any one of clauses 1 to 28, the one or more nucleic acids according to clause 29, the vector or collection of vectors according to clause 30, the host cell according to clause 31 or 32, or the pharmaceutical composition according to clause 33, for use in a method of treating and/or diagnosing a proliferative disease, in particular cancer.

36. A method of treating a proliferative disease, in particular cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antigen binding protein according to any one of clauses 1 to 28, one or more nucleic acids according to clause 29, a vector or collection of vectors according to clause 30, a host cell according to clause 31 or 32, or a pharmaceutical composition according to clause 33.

37. The antigen binding protein, one or more nucleic acids, vector or collection of vectors, host cell or pharmaceutical composition for use according to clause 35 or the method of treatment according to clause 36, wherein the cancer is a CT45 expressing cancer, more particularly selected from the group of cancers consisting of: lung cancer, NSCLC, gall bladder cancer, bile duct cancer, lymph node cancer, ovarian cancer, esophageal cancer, liver cancer, uterine cancer and melanoma.

38. The antigen binding protein, one or more nucleic acids, vector or collection of vectors, host cell or pharmaceutical composition for use according to clause 35 or 37 or the method of treatment according to clause 36 or 37, wherein the method of treatment comprises immunotherapy, in particular adoptive autologous or heterologous T cell therapy.

39. The antigen binding protein, one or more nucleic acids, vector or collection of vectors, host cell or pharmaceutical composition for use according to any one of clauses 35, 37 or 38 or the method of treatment according to any one of clauses 36 to 38, wherein the antigen binding protein is a TCR.

40. The antigen binding protein, one or more nucleic acids, vector or collection of vectors, host cell or pharmaceutical composition for use according to any one of clauses 35 or 37-39 or the method of treatment according to any one of clauses 36-39, wherein the antigen binding protein is expressed on the surface of a host cell.

41. The antigen binding protein, one or more nucleic acids, vector or collection of vectors, host cell or pharmaceutical composition for use according to any one of clauses 35 or 37 to 40 or the method of treatment according to any one of clauses 36 to 40, wherein the method of treatment comprises administering a host cell expressing the antigen binding protein, wherein the host cell is a T cell, a T cell group cell or an NK cell, preferably a T cell.

42. The antigen binding protein, one or more nucleic acids, vector or collection of vectors, host cell or pharmaceutical composition for use according to clause 40 or the method of treatment according to clause 40, wherein the host cell, preferably T cell, T cell group cell or NK cell, more preferably T cell is autologous.

43. The antigen binding protein, one or more nucleic acids, vector or collection of vectors, host cell or pharmaceutical composition for use according to clause 40 or the method of treatment according to clause 40, wherein the host cell, preferably T cell, T cell group cell or NK cell, more preferably T cell is allogeneic.

44. The antigen binding protein, one or more nucleic acids, vector or collection of vectors, host cell or pharmaceutical composition for use according to any one of clauses 35 or 37-43 or the method of treatment according to any one of clauses 36-43, wherein the antigen binding protein is conjugated to a therapeutically active agent.

45. The antigen binding protein, one or more nucleic acids, vector or collection of vectors, host cell or pharmaceutical composition for use according to any one of clauses 44 or the method of treatment according to any one of clauses 44, wherein the therapeutically active agent is selected from the group consisting of a radionuclide, a chemotherapeutic agent, and a toxin.

46. The antigen binding protein, one or more nucleic acids, vector or collection of vectors, host cell or pharmaceutical composition for use according to any one of clauses 35 or 37-45 or the method of treatment according to any one of clauses 36-45, wherein the method of treatment further comprises administering at least one chemotherapeutic agent to the subject in need of treatment.

47. The antigen binding protein, one or more nucleic acids, vector or collection of vectors, host cell or pharmaceutical composition for use according to any one of clauses 35 or 37-46 or the method of treatment according to any one of clauses 36-46, wherein the method of treatment further comprises administering radiation therapy to the subject in need of treatment.

48. A method of treating cancer in a subject in need thereof, the method comprising:

a) Isolating cells from the subject;

b) Transforming the cell with a vector or collection of vectors encoding the antigen binding protein of any one of clauses 1-28 to produce a transformed cell;

c) Amplifying the transformed cells to produce a plurality of transformed cells; and

D) Administering the plurality of transformed cells to the subject.

49. A method of treating cancer in a subject in need thereof, the method comprising:

a) Isolating cells from a healthy donor;

D) Administering the plurality of transformed cells to the subject.

50. The method of clause 48 or 49, wherein the transformed cell is a lymphocyte, preferably an NK cell or a T cell progenitor cell, more preferably a T cell.

51. Use of the antigen binding protein of any one of clauses 1-28 for the manufacture of a medicament for the treatment of a proliferative disease.

52. An in vitro method for detecting cancer, in particular a CT45 expressing cancer, in a biological sample, the in vitro method comprising:

a) Contacting the biological sample with the antigen binding protein according to any one of clauses 1 to 28, and

B) Detecting binding of the antigen binding protein to the biological sample.

53. A method of producing an antigen binding protein according to any one of clauses 1 to 28, the method comprising

A) Providing a host cell, wherein the host cell,

B) Providing a genetic construct comprising one or more nucleic acids encoding an antigen binding protein according to any one of clauses 1 to 28,

C) Introducing the genetic construct into the host cell, and

D) Expressing the genetic construct by the host cell.

54. The method of clause 53, further comprising isolating and purifying the antigen binding protein from the host cell, and optionally, reconstituting the antigen binding protein in a T cell.

55. The method of clause 53 or 54, further comprising cell surface presentation of the antigen binding protein.

56. The method of any one of clauses 53-55, wherein the gene construct is an expression construct comprising a promoter sequence operably linked to the nucleic acid encoding the antigen binding protein.

57. The method of any one of clauses 53 to 56, wherein the gene construct is introduced into the host cell by retroviral transfection.

Claims

2. The antigen binding protein of claim 1, wherein

1) CDRA2 comprises or consists of the amino acid sequence of SEQ ID NO. 15 and CDRB2 comprises or consists of the amino acid sequence of SEQ ID NO. 20

3. The antigen binding protein of claim 1 or 2, wherein the antigen binding protein is a TCR, preferably wherein the TCR is selected from the group consisting of an alpha/beta TCR, a gamma/delta TCR, a single chain TCR, a membrane bound TCR, a soluble TCR, a monovalent, divalent or multivalent TCR, a monospecific, bispecific or multispecific TCR, a fusion protein or chimeric protein of a functional fragment of a TCR and a functional fragment comprising a TCR, more preferably wherein the TCR is an alpha/beta TCR or a gamma/delta TCR, most preferably an alpha/beta TCR.

4. The antigen binding protein of any one of claims 1 to 3, wherein V _A comprises or consists of: an amino acid sequence selected from SEQ ID NOs 13, 132, 62, 89, 1, 52, 70, 98, 79, 106, 124, 116, 34, 23 and 42 or having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NOs 13, 132, 62, 89, 1, 52, 70, 98, 79, 106, 124, 116, 34, 23 and 42 and comprising the amino acid sequences of CDRa1, CDRa2 and CDRa3 according to claim 1 or 2; and wherein V _B comprises or consists of: an amino acid sequence selected from SEQ ID NOs 18, 135, 65, 94, 7, 57, 74, 103, 84, 111, 129, 121, 37, 28 and 47, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NOs 18, 135, 65, 94, 7, 57, 74, 103, 84, 111, 129, 121, 37, 28 and 47 and comprising said CDRb1, CDRb2 and CDRb3 according to claim 1 or 2, wherein said CDRa1, CDRa2, CDRa3, CDRb1, CDRb2 and/or CDRb3 sequence is capable of comprising one, two or three amino acid mutations, preferably amino acid substitutions.

5. The antigen binding protein of any one of claims 1 to 4, further comprising a constant domain, wherein the constant domain comprises or consists of: an amino acid sequence selected from the group consisting of SEQ ID NO. 5, 750, 751, 156, 11, 32 and 157, preferably selected from the group consisting of SEQ ID NO. 5, 750, 751, 11 and 32, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 5, 750, 751, 156, 11, 32 or 157.

6. The antigen binding protein of any one of claims 1 to 5, wherein the first polypeptide comprises or consists of: an amino acid sequence selected from the group consisting of SEQ ID NOs 17, 134, 64, 93, 6, 56, 73, 102, 83, 110, 128, 120, 36, 27, 46 and 158-172, preferably selected from the group consisting of SEQ ID NOs 17, 134, 64, 93, 6, 56, 73, 102, 83, 110, 128, 120, 36, 27 and 46, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NOs 17, 134, 64, 93, 6, 56, 73, 102, 83, 110, 128, 120, 36, 27, 46 or 158-172, and said second polypeptide comprises or consists of: an amino acid sequence selected from the group consisting of SEQ ID NO. 22, 137, 69, 97, 12, 61, 78, 105, 88, 115, 131, 123, 41, 33, 51 and 173-187, preferably selected from the group consisting of SEQ ID NO. 22, 137, 69, 97, 12, 61, 78, 105, 88, 115, 131, 123, 41, 33 and 51, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO. 22, 137, 69, 97, 12, 61, 78, 105, 88, 115, 131, 123, 41, 33, 51 or 173-187.

7. The antigen binding protein of any one of claims 1 to 6, wherein the antigen binding protein does not bind significantly to: at least 1, at least 2, at least 3, at least 4, at least 5 or all of the like peptides selected from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:149(SP-05-0004)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO:155 (SP-05-0010), preferably from SEQ ID NO:146(SP-05-0001)、SEQ ID NO:147(SP-05-0002)、SEQ ID NO:148(SP-05-0003)、SEQ ID NO:150(SP-05-0005)、SEQ ID NO:151(SP-05-0006)、SEQ ID NO:152(SP-05-0007)、SEQ ID NO:153(SP-05-0008)、SEQ ID NO:154(SP-05-0009) and SEQ ID NO:155 (SP-05-0010), more preferably from SEQ ID NO:147 (SP-05-0002), SEQ ID NO:151 (SP-05-0006), SEQ ID NO:152 (SP-05-0007) and SEQ ID NO:155 (SP-05-0010).

8. The antigen binding protein of any one of claims 1 to 7, wherein the antigen binding protein is capable of activating

-Cd4+ T cells, in particular cd4+ CD8-T cells, and/or

CD8+ T cells, in particular CD8+ CD4-T cells,

9. A nucleic acid comprising a sequence encoding the antigen binding protein of any one of claims 1 to 8.

10. A vector comprising the nucleic acid of claim 9.

11. A host cell comprising the antigen binding protein of any one of claims 1 to 8, or the nucleic acid of claim 9, or the vector of claim 10.

12. A pharmaceutical composition comprising the antigen binding protein of any one of claims 1 to 8, the nucleic acid of claim 9, the vector of claim 10, or the host cell of claim 11 and optionally a pharmaceutically acceptable carrier.

13. The antigen binding protein according to any one of claims 1 to 8, the nucleic acid according to claim 9, the vector according to claim 10, the host cell according to claim 11, or the pharmaceutical composition according to claim 12, for use in medicine, preferably for use in a method of treating and/or diagnosing a proliferative disease, in particular cancer.

14. An in vitro method for detecting cancer, in particular a CT45 expressing cancer, in a biological sample, the in vitro method comprising:

a) Contacting the biological sample with the antigen binding protein of any one of claims 1 to 8, and

B) Detecting binding of the antigen binding protein to the biological sample.

15. A method of producing the antigen binding protein of any one of claims 1 to 8, the method comprising

A) Providing a host cell, wherein the host cell,

B) Providing a genetic construct comprising a nucleic acid encoding an antigen binding protein according to any one of claims 1 to 8,

C) Introducing the genetic construct into the host cell, and

D) Expressing the genetic construct by the host cell.