T cell receptor specific for both RAC1-derived and RAC2-derived mutant epitopes

Abstract

The present invention is based on antigen binding proteins (ABPs), such as T cell receptors (TCRs), expressed on T cells, which have specificity for binding to MHC-presented Rac2 and Rac1 derived neoepitopes. Thus, such MHC-presented peptides may be derived from mutant forms of Rac1 and Rac2, e.g., preferably RAC2. P29L and / or RAC1 P29S Provided are isolated ABPs, as well as genetic constructs expressing the ABPs, recombinant host cells harboring the ABPs of the invention, and methods of producing such ABPs and host cells. Also provided are medical uses involving the TCRs of the invention, for example in the context of adoptive T cell therapy.

Description

[Technical field]

[0001] The present invention is based on antigen binding proteins (ABPs), such as T cell receptors (TCRs), expressed on T cells, which have specificity for binding to MHC-presented Rac2 and Rac1 derived neoepitopes. Thus, such MHC-presented peptides may be derived from mutant forms of Rac1 and Rac2, e.g., preferably RAC2. P29L and / or RAC1 P29S Provided are isolated ABPs, as well as genetic constructs expressing the ABPs, recombinant host cells harboring the ABPs of the invention, and methods of producing such ABPs and host cells. Also provided are medical uses involving the TCRs of the invention, for example in the context of adoptive T cell therapy. [Background technology]

[0002] Human T cells are lymphocytes that arise from bone marrow precursors and mature in the thymus. Several T cell subtypes have been identified, which are classified as CD4+ helper T cells and CD8+ killer T cells. In general, various types of CD4+ T cells mediate immune functions: for example, Th1 cells are associated with immune responses against intracellular bacteria, fungi, and protozoa, and can secrete IFN-gamma and enhance the microbial activity of macrophages. On the other hand, Th2 cells play a role in humoral immune responses. Through cytokine secretion and surface protein interactions, Th2 plays a key role in stimulating B cells to secrete antibodies. CD8+ T cells are specialized for the recognition of infected or mutated cells. Upon recognition of diseased cells, CD8+ T cells release a mixture of perforin, granzymes, and chemokines, which ultimately leads to apoptosis of the infected cells. (Non-Patent Document 1).

[0003] T cell activation depends, inter alia, on the interaction of their unique T cell receptor (TCR) with specific antigen-bearing MHC receptors of antigen-presenting cells. TCRs are surface-anchored protein complexes composed of two reactive antigen recognition chains and adjacent CD3 sequence elements. In TCRs, the transmembrane CD3 protein complex mediates signal transduction through cytoplasmic ITAM segments, whereas the central antigen recognition chains interact primarily with antigen-presenting cells with their distal reactive loops.

[0004] Most commonly, the antigen-interacting sequence element of the TCR is assembled as a dimer from two coupled TCRα and TCRβ chains. Each of these TCRα or TCRβ chains is assembled from a constant segment with a structural role and a variable segment that is responsible for antigen interaction. This interaction is governed by hypervariable regions located in the loops of each amino acid chain that forms the variable segment. Taken together, the TCRα and TCRβ variable segments form three antigen-interacting complementarity determining regions (CDRs). When they are complementary to the MHC / antigen complex of the presenting cell, a signal cascade is triggered that, depending on other costimulatory influences, can lead to various intracellular processes or T cell effector functions, such as apoptosis of the antigen-presenting cell.

[0005] A wide range of T cell specificities are acquired during their development in the thymus, where T cells undergo different maturation stages. During this process, T cells generate specific TCRs through somatic VJ or VDJ recombination of TCR alpha and beta coding gene segments. In addition, recombined TCR genes are further modified at their VDJ and VJ gene junctions through exonuclease activity and through the addition of random non-template-dependent nucleotides (N-nucleotides). This diversity creates highly variable complementarity determining regions that define the delicate specificity of the TCR chains.

[0006] TCRs recognize antigens in the form of short peptide fragments presented by cell surface glycoproteins termed major histocompatibility complexes (MHC), which in humans are encoded by human leukocyte antigen (HLA) genes and in mice by H-2 genes. In humans, two classes of MHC molecules have been identified. MHC class I molecules are expressed on all nucleated cells and are recognized by CD8+ T cells. MHC class II molecules are expressed on professional antigen presenting cells, B cells, and activated T cells and are recognized by CD4+ T cells. In MHC class I antigen presentation, intracellular proteins are endogenously processed in the proteasome into short peptide fragments, typically 8-10 amino acids in length. These antigens are translocated across the endoplasmic reticulum via transporters associated with antigen processing (TAPs) and loaded onto MHC class I molecules, which are then presented for CD8+ T cell recognition. Upon engagement of the TCRαβ dimer with the cognate antigen / MHC complex, the associated CD3 complex initiates a signal transduction cascade required for T cell activation (Non-Patent Document 2).

[0007] Adoptive T cell therapy is a general term for a therapeutic approach that relies on the in vitro expansion and administration of T cells with specificity for a patient's tumor or virus-infected cells. There are many variations of ATT. For example, expanded T cells can be generated by autologous harvesting and subsequent expansion / activation of a T cell fraction with the appropriate binding affinity. Other ATT therapies utilize T cells that are genetically engineered to express optimized naturally occurring TCRs or, as in the case of CAR-T cell therapy, artificially designed chimeric antigen-binding receptors. Naturally occurring TCRs offer the advantage of recognizing endogenously processed extracellular and intracellular antigens at the cell surface in an MHC-restricted manner, and therefore can target a different array of antigens other than CARs. The targets of T cell-based immunotherapy are peptide epitopes derived from pathogen-associated or tumor-associated (or tumor-specific) proteins, which are presented by molecules of the major histocompatibility complex (MHC). These disease-associated antigens can be peptides derived from all protein classes, e.g., enzymes, receptors, transcription factors, etc., that are expressed in the cells of the respective tumor and are usually upregulated when compared to unmodified cells of the same origin. In cases where the tumor is associated with a viral infection, e.g., MCV or human papillomavirus (HPV) infection, immunotherapy can be developed based on viral antigens expressed by tumor cells derived from the virus-infected host cells.

[0008] So far, ATT using TCR-modified T cells has been tested in clinical trials mainly in patients with solid tumors expressing tumor-associated antigens or cancer-testis antigens. The first published clinical trials using TCR-modified T cells targeted the tumor-associated antigens MART-1 and gp100 in patients with metastatic melanoma, leading to objective tumor regression rates of 19% or 30%, respectively. However, the infused T cells also recognized melanocytes in the patient's eyes, ears, and skin, thus leading to severe extratumoral on-target toxicity (Non-Patent Document 3). In another approach, sarcoma and melanoma patients were treated with TCR-modified T cells targeting the cancer-testis antigen NY-ESO-1, leading to objective response rates of 55% to 80%, while no severe toxicity was observed (Non-Patent Document 4 and Non-Patent Document 5). Finally, in another study using TCR-modified T cells targeting the cancer-testis antigen MAGE-A3, tumor regression was evident in five of seven patients, although two patients died of severe neurotoxicity (Non-Patent Document 6).

[0009] Rho GTPases of the Ras superfamily are involved in the regulation of multiple cellular functions and have been implicated in the pathology of various human diseases. Such diseases include cancer (Non-Patent Document 7, Non-Patent Document 8, Non-Patent Document 9), pathological angiogenesis, such as in diabetic retinopathy, tumor angiogenesis, glaucoma, and age-related macular degeneration (Non-Patent Document 10, Non-Patent Document 11, Non-Patent Document 12), asthma, Alzheimer's disease (Non-Patent Document 13), and cardiac left ventricular hypertrophy (Non-Patent Document 14, Non-Patent Document 15). Rho GTPases are attractive drug targets for future targeted therapy (Non-Patent Document 16).

[0010] High RhoA levels have been associated with human liver, skin, colon, ovarian, bladder, gastric, esophageal squamous cell, testicular, and breast cancer. High RhoB, C, or H levels have been associated with breast, squamous cell, pancreatic, breast, liver, ovarian, head and neck, prostate, non-small cell lung, and gastric cancer, as well as melanoma metastasis. High Rac1 levels have been associated with human testicular, gastric, breast, and squamous cell cancer. High Rac2 or Rac3 have been associated with breast, colon, head and neck, and squamous cell cancer (Non-Patent Document 17). Gain-of-function mutations, e.g., of Rac1, P29S It has been detected in melanoma, breast cancer, and head and neck cancer (Non-Patent Document 18).

[0011] Rac proteins (Rac1, 1b, 2, 3) belong to the RhoGTP-binding proteins (or GTPases) of the Ras superfamily and therefore act as molecular switches that cycle between active GTP-bound and inactive GDP-bound forms through nucleotide exchange and hydrolysis. Like most other GTPases, these proteins adopt various conformations depending on the bound nucleotide, with the main difference being the conformation of two short, flexible loop structures, named switch I and switch II regions. Three different mammalian Rac isoforms, Rac1, Rac2, and Rac3, share very high sequence identity (up to 90%), and Rac1b is an alternative splice variant of Rac1 with a 19 amino acid insertion near the switch II region. Rac1b has accelerated GEF-independent GDP / GTP exchange and impaired GTP hydrolysis, and is the main cause of autoactivating GTPases (Non-Patent Document 19).

[0012] RAC2 is a small GTPase that belongs to the Rho family of GTPases. P29L The mutation is another so-called driver mutation that promotes tumor growth and metastasis and is therefore a potential target in ATT.

[0013] Therefore, the object of the present invention is to provide a novel adoptive cell-based immunotherapy for treating patients suffering from cancer disease.In particular, the present invention seeks to provide a novel T cell receptor molecule that is related to mutant RAC1 and RAC2 and specific for MHC-presented peptides. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Nurieva R., Chung Y., Cellular & Molecular Immunology, 2010, 7, 190-197 [Non-Patent Document 2] RathJ. A., Arber C., Cells. 2020, 1485 [Non-Patent Document 3] Johnson et al. 2009 [Non-Patent Document 4] Rapoport et al. 2015 [Non-Patent Document 5] Robbins et al. 2015 [Non-Patent Document 6] Morgan et al. 2013 [Non-Patent Document 7] Fritz G., Just I., and Kaina B., Int. J. Cancer, 1999, 81, 682-687 [Non-Patent Document 8] Fritz G., Kaina B. Curr. Cancer Drug Targets, 2006, 6, 1-14 [Non-Patent Document 9] SahaiE., Marshall CJ, Nat. Rev. Cancer., 2002, 2, 133-42 [Non-Patent Document 10] ErikssonA., Cao R., Roy J., Tritsaris K., Wahlestedt C., Dissing S., Thyberg J., CaoY., Circulation, 2003, 107, 1532-8

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[0015] Generally, and by way of brief description, the main aspects of the present invention can be described as follows.

[0016] In a first aspect, the present invention relates to an antigen binding protein (ABP) or a variant thereof, which ABP optionally binds RAC2. P29L and / or RAC1 P29S An isolated ABP that specifically binds to a related antigenic peptide comprises at least one complementarity determining region (CDR), which is a CDR3 having an amino acid sequence with at least 80% sequence identity to a sequence selected from SEQ ID NOs: 3, 7, 11, and 15, or having no more than three or no more than two, preferably no more than one amino acid substitution(s), deletion(s), or insertion(s).

[0017] In a second aspect, the present invention relates to a nucleic acid or a nucleic acid construct (NAC) comprising a nucleic acid sequence encoding an ABP of the present invention.

[0018] In a third aspect, the present invention relates to a recombinant vector comprising a nucleic acid of the present invention.

[0019] In a fourth aspect, the present invention relates to a recombinant host cell, which contains or expresses an ABP of the invention, or which comprises a nucleic acid or vector of the invention.

[0020] In a fifth aspect, the present invention relates to a pharmaceutical composition comprising one or more, preferably in combination, of the ABP of the invention or the nucleic acid or cell of the invention.

[0021] In a sixth aspect, the present invention relates to a method for the detection of RAC2 in a subject, for use in medicine, in particular P29L and / or RAC1 P29S It also relates to a composition or composition comprising any of the products of the invention, or a combination thereof, for use in the treatment of a disease associated with expression, such as cancer.

[0022] In a seventh aspect, the present invention relates to a method for producing a cell line expressing a RAC1 and / or RAC2 specific antigen recognition construct, comprising the steps of: Providing a suitable host cell; Providing a genetic construct comprising a coding sequence encoding an ABP of the present invention; introducing said genetic construct into said suitable host cell; expressing said genetic construct by said suitable host cell; The present invention relates to a method comprising the steps of:

[0023] Detailed Description of the Invention The elements of the present invention are described below. Although these elements are listed with specific embodiments, it is understood that these elements can be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments are not to be construed as limiting the invention to only the exemplary described embodiments. It is to be understood that the specification supports and encompasses embodiments combining two or more exemplary described embodiments or embodiments combining one or more exemplary described embodiments with any number of the disclosed and / or preferred elements. Furthermore, unless the context indicates otherwise, all permutations and combinations of all elements described in this application are deemed to be disclosed by the specification of this application.

[0024] In a first aspect, the present invention relates to an antigen binding protein (ABP) or a variant thereof, which optionally specifically binds to RAC1 and / or RAC2, preferably mutated RAC1 and / or RAC2, and the isolated ABP comprises at least one complementarity determining region (CDR), which CDR is a CDR3 having an amino acid sequence with at least 60%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from SEQ ID NOs: 3, 7, 11, and 15, or having an amino acid sequence with no more than three or no more than two, preferably no more than one amino acid substitution(s), deletion(s), or insertion(s).

[0025] The TCRs and their individual variants of the present invention are specific for RAC2 P29L Although the peptide (SEQ ID NO: 17) was used, the TCR of the present invention is RAC1 P29S This provides the surprising advantage of being able to further recognize peptides derived from RAC2. P29L and / or RAC1 P29SThis offers a surprising advantage over prior art TCRs for such neoepitopes derived from Rac2, since when used in ATT, the constructs of the present invention have the potential to treat a broader patient population. The present invention relates to two primarily novel HLA-A2 restricted human TCRs (RAC2-T001 and RAC2-T002), which are derived from the mutant protein variant Rac2 P29L specifically recognizes Rac1, but not the corresponding Rac variant Rac1. P29S We also recognize that both mutations play a role in resistance and metastatic cancer, and therefore the present invention provides novel treatment options for patients suffering from such diseases, which have a poor prognosis.

[0026] The preferred ABP or its antigen-binding variant of the present invention is produced using RAC2-derived antigenic peptide and has high affinity to the corresponding RAC1 antigenic peptide covering the same epitope region.Preferably, the antigenic peptide derived from RAC2 used for immunization is the peptide of SEQ ID NO: 17, while the ABP has high affinity to the peptide of SEQ ID NO: 18 derived from RAC1.

[0027] In some embodiments, the ABP of the invention comprises a mutated RAC1 and / or RAC2 (e.g., RAC2 P29L and / or RAC1 P29S ) specifically binds to the antigenic peptide HLA molecule complex, and the RAC1 / RAC2 antigenic peptide is a sequence of a sequence containing a mutation, preferably RAC2 P29L and / or RAC1 P29S Such peptides are preferably 8-12, more preferably 10±1 amino acids in length and induce T cells that bind to HLA class I (preferably) or class II molecules and / or cross-react with said peptides, or a pharma- ceutically acceptable salt thereof, said peptides being not the basal full length polypeptide. Preferably, the ABPs of the present invention, when complexed with an antigenic peptide and HLA, induce / activate a T cell immune response, optionally in the presence of one or more co-activating stimuli.

[0028] The terms "antigen recognition construct" or "antigen binding protein" are used interchangeably herein and refer to a protein having the ability to specifically bind to another protein, preferably an immunoglobulin-like protein, such as a T-cell receptor or an antibody, or any known variant of this class of protein.

[0029] The term "isolated" as used herein in the context of a polypeptide, for example an antigen recognition construct (an example of which could be an antibody), refers to a polypeptide that has been purified from proteins or polypeptides, or from other contaminants that may interfere with therapeutic, diagnostic, preventive, research, or other uses. The antigen recognition construct according to the present invention can be a recombinant, synthetic, or modified (non-natural) antigen-binding construct. The term "isolated" as used herein in the context of a nucleic acid or cell refers to a nucleic acid or cell that has been purified from DNA, RNA, proteins, or polypeptides, or from other contaminants (e.g., other cells) that may interfere with therapeutic, diagnostic, preventive, research, or other uses, or the term refers to a recombinant, synthetic, or modified (non-natural) nucleic acid. In this context, a "recombinant" protein / polypeptide or nucleic acid is one that has been made using recombinant techniques. Methods and techniques for producing recombinant nucleic acids and proteins are known in the art.

[0030] As used herein, the term "identical" or percent "identity" wherever used herein when used in the context of two or more nucleic acid or protein / polypeptide sequences, refers to two or more sequences or subsequences that are the same or have (or at least have) the same specified percentage of amino acid residues or nucleotides (i.e., about 60% identity or at least about 60% identity over a designated region (preferably their full length sequences) when aligned and compared for maximum correspondence over a comparison window or designated region) when measured using a sequence comparison algorithm or when manually aligned and visually inspected (see, e.g., the NCBI website). In particular, for amino acid identity, BLASTP 2.2.28+ is used with the following parameters: matrix: BLOSUM62; gap penalty: presence: 11, extension: 1; neighbor word threshold: 11; multiple hit window: 40.

[0031] In another additional or alternative embodiment, where the ABP is preferably a TCR, the ABP may further comprise a CDR1 domain sequence and / or a CDR2 domain sequence. Within the variable domain, CDR1 and CDR2 are found in the variable (V) region of the polypeptide chain, and CDR3 comprises part of the V region, the diversity (D) region and all of the joining (J) region. CDR3 is the main CDR that is the most variable and responsible for the specificity and selectivity of antigen recognition. The CDR1 and CDR2 sequences may be selected from the CDR sequences of human variable chain alleles.

[0032] Native alpha-beta heterodimeric TCRs have an alpha chain and a beta chain. Each chain has a variable region, a binding region, and a constant region; the beta chain usually also has a short diversity region between the variable and binding regions, but this diversity region is often considered part of the binding region. Each variable region has three CDRs (complementarity determining regions) embedded in framework sequences, one of which is a hypervariable region named CDR3. There are multiple types of alpha chain variable (Vα) regions and multiple types of beta chain variable (Vβ) regions, identified by their framework, CDR1 and CDR2 sequences, and by partially defined CDR3 sequences. The Vα types are referred to by unique TRAV numbers in the IMGT nomenclature, and the Vβ types by unique TRBV numbers. For further information on immunoglobulin antibodies and TCR genes, see the International ImMunoGeneTics Information System™, Lefranc MP et al., (Nucleic Acids Res. 2015 Jan;43(Database issue):D413-22; and http: / / www.imgt.org / ).

[0033] Thus, in one further or alternative embodiment, the ABP of the invention comprises the CDR1, CDR2 and CDR3 sequences in combination as presented hereinbelow in Table 1, with each variable chain allele also indicated together with the CDR3 sequence. Thus, preferred is an ABP of the invention comprising the CDR1, CDR2 and CDR3 of at least one, and preferably all three CDR sequences. Preferably, the antigen recognition construct of the invention comprises each CDR1 to CDR3 of one individual herein disclosed TCR variable region of the invention (see Table 1 hereinbelow and the Examples section).

[0034] The term "specificity" or "antigen specificity" or "specific for" a given antigen, as used herein, means that the ABP is able to specifically bind to said antigen, preferably to RAC1 and / or RAC2 derived antigens, more preferably with high avidity, when said antigen is presented by HLA, preferably by HLA-A*02. For example, T cells expressing a TCR or an antigen-binding variant or derivative thereof can be incubated at low concentrations (e.g., about 10 -11 mol / l, 10 -10 mol / l, 10 -9 mol / l, 10 -8 mol / l, 10 -7 mol / l, 10 -6 mol / l, 10 -5 mol / l) of an antigenic peptide as provided herein below, e.g., RAC2 P29L and / or RAC1 P29S After co-culture with target cells pulsed with the epitope and antigen (containing the P29 mutant amino acid position), RAC2 P29L and / or RAC1 P29S If the T cells secrete at least about 200 pg / ml or more (e.g., 250 pg / ml or more, 300 pg / ml or more, 400 pg / ml or more, 500 pg / ml or more, 600 pg / ml or more, 700 pg / ml or more, 1000 pg / ml or more, 2000 pg / ml or more, 2500 pg / ml or more, 5000 pg / ml or more) of interferon gamma (IFN-γ) when contacted with a derived antigen peptide-presenting HLA, the TCR is selected as an antigen recognition construct that recognizes RAC2. P29L and / or RAC1 P29S Alternatively or additionally, T cells expressing a TCR may be considered to have "antigen specificity" for an antigenic peptide. P29L and / or RAC1 P29SA TCR can be considered to have "antigen specificity" for an antigenic peptide if, upon co-culture with target cells pulsed with the antigenic peptide, it secretes at least twice as much IFN-γ as the untransduced background level of IFN-γ. Such "specificity" as described above can be analyzed, for example, by ELISA.

[0035] In an alternative or additional embodiment of the invention, the antigen recognition construct selectively binds to an antigenic peptide, preferably a protein epitope or peptide having the amino acid sequence as set forth in SEQ ID NO: 17 or 18, preferably a peptide consisting of the amino acid sequence as set forth in SEQ ID NO: 17 or 18 or a variant thereof, which variant is an amino acid deletion, addition, insertion or substitution at not more than three, preferably at not more than two, particularly preferably at not more than one amino acid position. Such detection is achieved in the context of MHC presentation of said peptide.

[0036] The term "selectivity" or "selectively recognize / bind" is understood to refer to the property of the ABP, e.g., TCR or antibody, of the present invention to selectively recognize or bind to preferably only one specific epitope, and preferably to show no or substantially no cross-reactivity to another epitope. Preferably, "selectivity" or "selectively recognize / bind" means that the antigen recognition construct (e.g., TCR) selectively recognizes or binds to preferably only one specific epitope, and preferably to show no or substantially no cross-reactivity to another epitope, said epitope being unique to one protein, such that the antigen recognition construct shows no or substantially no cross-reactivity to another epitope and another protein. Surprisingly, the preferred TCR of the present invention shows an unexpected range of selectivity compared to the prior art TCRs by recognizing both the RAC1 neoepitope and the RAC2 neoepitope, and otherwise has reduced off-target binding to other epitope sequences, and thus the TCR of the present invention selectively recognizes or binds to mutant RAC proteins, preferably RAC2.P29L and / or RAC1 P29S It has been recognized that the present invention is particularly useful in the treatment of diseases associated with the expression of

[0037] The ABP according to the invention is preferably selected from an antibody, or a derivative or fragment thereof, or a T cell receptor (TCR), or a derivative or fragment thereof. The antibody or TCR derivative or fragment of the invention should preferably maintain the antigen binding / recognition ability of the parent molecule, in particular its specificity and / or selectivity as explained above. Such binding functionality can be maintained by the presence of the CDR3 region as defined herein. Such antigen binding specificity of the TCR usually depends on the HLA (MHC) presented antigen peptide, such that the TCR forms a complex together with the peptide and the HLA molecule presenting the peptide.

[0038] In an embodiment of the invention, the TCR of the invention binds RAC2 in a major histocompatibility complex (MHC) class I dependent manner. P29L Antigen and / or RAC1 P29S "MHC class I dependent manner" as used herein refers to the ability of a TCR to recognize an antigen within the context of an MHC class I molecule. P29L Antigen and / or RAC1 P29S It means that it induces an immune response when it binds to an antigen. The MHC class I molecule can be any MHC class I molecule known in the art, for example, an HLA-A molecule. In a preferred embodiment of the present invention, the MHC class I molecule is an HLA-A*02 molecule.

[0039] The present invention provides both single-chain and two-chain ABPs. The ABPs of the present invention can be soluble or membrane-bound constructs.

[0040] In some embodiments, the TCR alpha variable domain has at least one mutation compared to the TCR alpha domain shown in Table 1, and / or the TCR beta variable domain has at least one mutation compared to the TCR alpha domain shown in Table 1. In some embodiments, the TCR having at least one mutation in the TCR alpha variable domain and / or the TCR beta variable domain is RAC2. P29L and / or RAC1 P29S It has a binding affinity and / or binding half-life for a peptide-HLA molecule complex that is at least twice that of a TCR having an unmutated TCR alpha domain and / or an unmutated TCR beta variable domain.

[0041] The TCR alpha chain herein may further comprise a TCR alpha transmembrane domain and / or a TCR alpha intracellular domain. The TCR beta chain herein may further comprise a TCR beta transmembrane domain and / or a TCR beta intracellular domain.

[0042] The present invention particularly provides a TCR or a fragment or derivative thereof as an antigen recognition construct. The TCR is preferably human, which is understood to mean that it is generated from a human TCR locus and therefore contains a human TCR sequence. Furthermore, the TCR of the present invention is of human origin and is preferably selected from the group consisting of RAC2 and RAC3 of the present invention. P29L and / or RAC1 P29S It can be characterized by specific recognition of an antigen.

[0043] Another embodiment of the present invention additionally or alternatively provides an antigen recognition construct as described above which induces an immune response, preferably characterized by an increase in interferon (IFN) gamma levels.

[0044] The TCRs of the present invention may be provided as single chain α or β, or γ and δ molecules, or alternatively as two-chain constructs composed of both α and β, or γ and δ chains.

[0045] The antigen recognition construct of the present invention may comprise a TCR alpha or gamma chain, and / or a TCR beta or delta chain, wherein the TCR alpha or gamma chain comprises a CDR3 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 3 or 11, and / or the TCR beta or delta chain comprises a CDR3 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 7 or 15. Also preferred are ABPs having a TCR alpha or gamma chain, and / or a TCR beta or delta chain, wherein the TCR alpha or gamma chain comprises a CDR3 having an amino acid sequence that has at least 80% sequence identity or has no more than three or no more than two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to a sequence selected from SEQ ID NOs: 3 and 11, and / or the TCR beta or delta chain comprises a CDR3 having an amino acid sequence that has at least 80% sequence identity or has no more than three or no more than two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to a sequence selected from SEQ ID NOs: 7 and 15.

[0046] Most preferably, in some additional embodiments where the disclosure refers to an antigen recognition construct comprising any one, two or all of the CDR1-CDR3 regions of the TCR chains disclosed herein (see Table 1), such an antigen recognition construct may be preferred that comprises each CDR sequence of the invention with no more than three, no more than two, preferably only one modified amino acid residue. The modified amino acid residues may be selected from amino acid insertions, deletions, or substitutions. Most preferably, the three, two, preferably only one modified amino acid residues are the first or last amino acid residues of each CDR sequence. When the modifications are substitutions, in some embodiments, it is preferred that the substitutions are conservative amino acid substitutions. Furthermore, in some embodiments, it is preferred that any mutations in the CDRs are either at the most C-terminal and / or most N-terminal of the CDR.

[0047] When the antigen recognition construct of the invention is composed of at least two amino acid chains, for example a two-chain TCR or antigen-binding fragment thereof, the antigen recognition construct may comprise an amino acid sequence according to SEQ ID NO:3 in a first polypeptide chain and an amino acid sequence according to SEQ ID NO:7 in a second polypeptide chain, or an amino acid sequence according to SEQ ID NO:11 in a first polypeptide chain and an amino acid sequence according to SEQ ID NO:15 in a second polypeptide chain.

[0048] Any one of the above dual chain TCRs or antigen binding fragments thereof is a preferred TCR of the present invention. In some embodiments, the CDR3 of the dual chain TCR of the present invention may be mutated. The mutations provided as above in the CDR3 sequences as shown in Table 1 preferably comprise substitutions, deletions, additions or insertions of no more than three, preferably no more than two, most preferably no more than one amino acid residue. In some embodiments, the first polypeptide chain may be a TCR alpha or gamma chain and the second polypeptide chain may be a TCR beta or delta chain. Preferred are alpha beta or gamma delta TCR combinations. As stated, such mutated CDR3s may in preferred embodiments comprise only conservative amino acid exchanges or deletions and / or may be introduced at the most C-terminal and / or most N-terminal of such CDR3.

[0049] In some embodiments, the TCR or antigen-binding fragment thereof is composed of a TCR alpha and a TCR beta chain, or a gamma and a delta chain. Such dual chain TCRs comprise a variable region within each chain, each of which comprises one CDR1, one CDR2 and one CDR3 sequence. The TCR comprises the CDR1 through CDR3 sequences as contained in the variable chain amino acid sequences of SEQ ID NO:4 and SEQ ID NO:8, or SEQ ID NO:12 and SEQ ID NO:16.

[0050] Some embodiments of the present invention relate to a TCR or fragment thereof consisting of a TCR alpha chain and a TCR beta chain, said TCR comprising a variable region sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or preferably 100% sequence identity to an amino acid sequence selected from the alpha and beta chains according to SEQ ID NOs: 4 and 8, respectively, or according to SEQ ID NOs: 12 and 16, respectively.

[0051] The TCRs of the invention may further comprise a constant region derived from any suitable species, e.g., any mammal, e.g., human, rat, monkey, rabbit, donkey, or mouse. In certain embodiments of the invention, the TCRs of the invention further comprise a human constant region. In some preferred embodiments, the constant region of the TCRs of the invention may be slightly modified, e.g., by introduction of heterologous sequences, preferably mouse sequences, such modifications may increase TCR expression and stability.

[0052] The TCR alpha or gamma chain of the present invention may further comprise a CDR1 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity, or having no more than three or no more than two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s), compared to a sequence selected from SEQ ID NOs: 1 and 9, and / or a CDR2 having an amino acid sequence having at least 80% sequence identity, or having no more than three or no more than two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s), compared to a sequence selected from SEQ ID NOs: 2 and 10.

[0053] According to the present invention, the TCR β or δ chain may further comprise a CDR1 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity or having no more than three or no more than two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to a sequence selected from SEQ ID NOs: 5 and 13, and / or a CDR2 having an amino acid sequence having at least 80% sequence identity or having no more than three or no more than two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to a sequence selected from SEQ ID NOs: 6 and 14.

[0054] In a further embodiment, the antigen recognition construct may comprise a binding fragment of a TCR, said binding fragment comprising CDR1 to CDR3, optionally selected from CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NO: 1, 2, 3, or 5, 6, 7, or 9, 10, 11, or 13, 14, 15, each CDR independently having no more than three or no more than two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to these sequences.

[0055] In a further embodiment of the invention, the antigen recognition construct as described anywhere herein is a TCR or a fragment thereof composed of at least one TCR alpha chain sequence and one TCR beta chain sequence, wherein the TCR alpha chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 1 to 3, and the TCR beta chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 5 to 7, or the TCR alpha chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 9 to 11, and the TCR beta chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 13 to 15, and each CDR independently has no more than three or no more than two, preferably no more than one amino acid substitution(s), deletion(s), or insertion(s) compared to these sequences.

[0056] In a further embodiment of the invention, the antigen recognition construct as described herein above is a TCR or fragment thereof comprising at least one TCR alpha chain sequence and one TCR beta chain sequence, wherein the TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 4 and the TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 8, or wherein the TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 12 and the TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 16, and each variable domain sequence has not more than 20, preferably not more than 15, more preferably not more than 10, 5, 3 or 2, preferably not more than 1 amino acid substitution(s), deletion(s) or insertion(s) compared to these sequences.

[0057] In a further embodiment of the invention, the antigen recognition construct as hereinbefore described is a TCR or a fragment thereof further comprising a TCR constant region having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from a human TCR constant domain.

[0058] As used herein, the terms "mouse" or "human" when referring to an antigen recognition construct described herein, or a TCR, or any component of a TCR (e.g., the complementarity determining regions (CDRs), variable region, constant region, α chain, and / or β chain), refer to a TCR (or component thereof) derived from a mouse or human unrearranged TCR locus, respectively.

[0059] In one embodiment of the invention, chimeric TCRs are provided, in which the TCR chains comprise sequences from more than one species. Preferably, the TCRs of the invention may have an alpha chain comprising a human variable region of the alpha chain and a mouse constant region, for example a mouse TCR alpha chain.

[0060] In one embodiment, the TCR of the present invention is a human TCR comprising a human variable region according to the above embodiments and a human constant region.

[0061] In some embodiments, the antigen recognition construct is murine or humanized, these terms being used when amino acid sequences of heterologous origin are introduced into the construct of the invention.

[0062] The TCR of the present invention may be provided as a single chain TCR (scTCR). The scTCR may comprise a polypeptide of the variable region of a first TCR chain (e.g., an alpha chain) and a polypeptide of the entire (full length) second TCR chain (e.g., a beta chain), or vice versa. Furthermore, the scTCR may optionally comprise one or more linkers linking two or more polypeptides together. The linker may be, for example, a peptide, as described herein, linking two single chains together. Also provided are such scTCRs of the present invention, which are fused to a human cytokine, e.g., IL-2, IL-7, or IL-15.

[0063] The ABP according to the present invention may also be provided in the form of a multimeric complex comprising at least two TCR or scTCR molecules, each of which is fused to at least one biotin moiety or other interconnecting molecule / linker, and the TCRs or scTCRs are interconnected by biotin-streptavidin interactions to form the multimeric complex. Similar approaches to generate multimeric TCRs known in the art are also possible and encompassed by the present disclosure. Also provided are higher order multimeric complexes comprising three or more scTCRs of the present invention.

[0064] For the purposes of the present invention, a TCR is a moiety having at least one TCR alpha or gamma and / or TCR beta or delta variable domain. Generally, they contain both TCR alpha and TCR beta variable domains, or alternatively, they contain both TCR gamma and TCR delta variable domains. They can be αβ / γδ heterodimers or in a single chain format. For use in adoptive therapy, αβ or γδ heterodimeric TCRs can be transfected as full-length chains, for example, with both cytoplasmic and transmembrane domains. If desired, there can be disulfide bonds introduced between the residues of the respective constant domains.

[0065] In a preferred embodiment, the ABP of the invention is a human TCR or a fragment or derivative thereof. A human TCR or a fragment or derivative thereof is a TCR that contains more than 50% of the corresponding human TCR sequence. Preferably, only a small part of the TCR sequence is of artificial origin or originates from another species. However, chimeric TCRs, for example those of human origin but with mouse sequences in the constant domains, are known to be advantageous. Thus, particularly preferred are TCRs according to the invention that contain mouse sequences in the extracellular part of their constant domains.

[0066] It is therefore also preferred that the ABP of the present invention is capable of recognizing the respective antigen of said ABP in a human leukocyte antigen (HLA)-dependent manner, preferably in an HLA-A02-dependent manner. The term "HLA-dependent manner" in the context of the present invention means that the antigen recognition construct binds to an antigen only if the antigen peptide is presented by said HLA.

[0067] In one embodiment, the antigen recognition construct according to the present invention preferably induces an immune response, preferably characterized by an increase in interferon (IFN) gamma levels.

[0068] Also provided by the present invention is a polypeptide comprising a functional portion of any of the TCRs (or functional variants thereof) described herein, for example any one of the TCRs selected from the TCRs as provided in the Examples section and Table 1. The term "polypeptide" as used herein includes oligopeptides, which term refers to a single chain of amino acids linked by one or more peptide bonds. In relation to the polypeptides of the present invention, the functional portion can be any portion comprising consecutive amino acids of the TCR (or functional variants thereof), provided that the functional portion specifically binds to the RAC1 / RAC2 neo-antigens, preferably as disclosed herein as SEQ ID NOs: 17 and 18. The term "functional portion", when used in relation to a TCR (or functional variants thereof), refers to any portion or fragment of the TCR (or functional variants thereof) of the present invention, which portion or fragment retains the biological activity of the TCR (or functional variants thereof) and is a part of it (parent TCR or parent functional variants thereof). Functional portions include, for example, those portions of a TCR (or a functional variant thereof) that retain the ability to specifically bind (in an HLA-dependent manner) to a RAC1 / RAC2 antigen, or to detect, treat, or prevent a RAC1 / RAC2-associated disorder, such as cancer, to a similar, equal, or greater extent than the parent TCR (or a functional variant thereof). With respect to the parent TCR (or a functional variant thereof), the functional portion can include, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent TCR variable sequence (or a functional variant thereof).

[0069] A functional portion may comprise additional amino acids at the amino or carboxy terminus, or at both termini, of the portion, which additional amino acids are not found in the amino acid sequence of the parent TCR or a functional variant thereof. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, such as specifically binding to the RAC1 / RAC2 antigen and / or being capable of cancer detection, cancer treatment, or cancer prevention. More desirably, the additional amino acids improve the biological activity when compared to the biological activity of the parent TCR or a functional variant thereof.

[0070] A polypeptide may comprise a functional portion of one or both of the α and β chains of a TCR or a functional variant thereof of the invention, for example a functional portion comprising one or more of the CDR1, CDR2 and (preferably) CDR3 of the variable region(s) of the α and / or β chain of a TCR or a functional variant thereof of the invention. In certain embodiments of the invention, a polypeptide may comprise a functional portion comprising the amino acid sequence of SEQ ID NO: 3, 7, 11 or 15 (CDR3 of the variable region of a TCR of the invention), or a combination thereof. In certain embodiments of the invention, a polypeptide of the invention may comprise a variable region of a TCR or a functional variant thereof of the invention, for example comprising a combination of the above CDR regions. In this regard, a polypeptide may comprise the amino acid sequence of any of SEQ ID NO: 4, 8, 12 and 16 (the variable region of the α or β chain of a TCR of the invention).

[0071] In some examples, the construct of the present invention may comprise one or two polypeptide chains comprising a sequence according to any one of SEQ ID NOs: 1-16 (CDR sequences and variable region sequences), or a sequence encoded by a nucleic acid sequence, or a functional fragment thereof, and may further comprise other amino acid sequences, such as an amino acid sequence encoding an immunoglobulin or a portion thereof, in which case the protein of the present invention may be a fusion protein. In this regard, the present invention also provides fusion proteins comprising at least one of the polypeptides of the present invention described herein in combination with at least one other polypeptide. The other polypeptide may be present as a separate polypeptide of a fusion protein or as a polypeptide expressed in frame (in tandem) with one of the polypeptides of the present invention described herein. The other polypeptide may comprise any peptidic or proteinaceous molecule or a portion thereof, including, but not limited to, immunoglobulins, CD3, CD4, CD8, MHC molecules, CD1 molecules, such as CD1a, CD1b, CD1c, CD1d, etc.

[0072] A fusion protein may comprise one or more copies of a polypeptide of the invention and / or one or more copies of another polypeptide. For example, a fusion protein may comprise one, two, three, four, five or more copies of a polypeptide of the invention and / or another polypeptide. Suitable methods for making fusion proteins are known in the art and include, for example, recombinant methods. In some embodiments of the invention, the TCRs (and functional portions and variants thereof), polypeptides and proteins of the invention may be represented as a single protein comprising a linker peptide linking the alpha and beta chains and linking the gamma and delta chains. In this regard, the TCRs (and functional variants and variants thereof), polypeptides and proteins of the invention may comprise the amino acid sequence of the variable region of a TCR of the invention and may further comprise a linker peptide. The linker peptide may advantageously facilitate expression of the recombinant TCRs (including functional portions and variants thereof), polypeptides and / or proteins in a host cell. The linker peptide may comprise any suitable amino acid sequence. Linker sequences for single chain TCR constructs are known in the art. Such single chain constructs can further comprise one or two constant domain sequences. When constructs containing linker peptides are expressed by a host cell, the linker peptides can also be cleaved, resulting in separate α and β chains, and separate γ and δ chains.

[0073] As already mentioned above, the binding functions of the TCRs of the invention can be provided in the framework of an antibody. For example, the CDR sequences of the TCRs of the invention, possibly including three, two or one additional N- and / or c-terminal framework residues, can be directly grafted into an antibody variable heavy / light chain sequence. The term "antibody", in its various grammatical variations, is used herein to refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules comprising an antigen-binding site or paratope. Such molecules are also referred to as "antigen-binding fragments" of immunoglobulin molecules. The invention further provides antibodies or antigen-binding portions thereof, which specifically bind to the antigens described herein. The antibodies can be any type of immunoglobulin known in the art. For example, the antibodies can be of any isotype, e.g. IgA, IgD, IgE, IgG, IgM, etc. The antibodies can be monoclonal or polyclonal. The antibody can be a naturally occurring antibody, e.g., an antibody isolated and / or purified from a mammal, e.g., mouse, rabbit, goat, horse, chicken, hamster, human, etc. Alternatively, the antibody can be a genetically engineered antibody, e.g., a humanized antibody or a chimeric antibody. The antibody can be in monomeric or multimeric form.

[0074] The term "antibody" includes, but is not limited to, engineered or otherwise modified forms of immunoglobulins, such as intrabodies, chimeric antibodies, fully human antibodies, humanized antibodies (e.g., produced by "CDR grafting"), antibody fragments, and heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, etc.). The term "antibody" includes cis-diabodies and minibodies. Thus, any and all embodiments provided herein, with respect to "antibody" or "antibody-like construct", are also contemplated as embodiments of bispecific antibodies, diabodies, scFv fragments, chimeric antibody receptor (CAR) constructs, diabodies and / or minibodies, unless otherwise stated. The term "antibody" includes polypeptides comprising immunoglobulin family polypeptides or immunoglobulin fragments, as disclosed herein, capable of binding in a non-covalent, reversible and specific manner to the corresponding antigen, preferably the RAC1 / RAC2 mutant antigen peptide of the present invention. An exemplary antibody structural unit constitutes a tetramer. In some embodiments, a full-length antibody can be composed of two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain (linked through disulfide bonds). Antibody structures and isotypes are known to those skilled in the art (e.g., from Janeway's Immuno-biology, 9th edition, 2016).

[0075] Recognized mammalian immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes (for further information on immunoglobulin genes, see the International ImMunoGeneTics Information System™, Lefranc MP et al, Nucleic Acids Res. 2015 Jan;43(Database issue):D413-22, and http: / / www.imgt.org / ). For full-length chains, light chains are classified as either kappa or lambda. For full-length chains, heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. The N-terminus of each chain defines a variable region of about 100-110 or more amino acids that are primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these regions of the light and heavy chains, respectively. As used in the present invention, "antibody" encompasses all variants of antibodies and fragments thereof. Thus, full length antibodies, chimeric antibodies, humanized antibodies, single chain antibodies (scFv), Fab, Fab', and multimeric variants of these fragments (e.g., F(ab')2) with the same, essentially the same, or similar binding specificity are within the scope of this concept. In some embodiments, the antibody specifically binds to the mutated RAC1 / RAC2 peptide of the present invention. A suitable antigen recognition construct according to the present invention comprises an antibody heavy chain, preferably a variable domain thereof, or an antigen-binding fragment thereof, and / or an antibody light chain, preferably a variable domain thereof, or an antigen-binding fragment thereof. Similarly, disulfide-stabilized variable region fragments (dsFv) can be prepared by recombinant DNA technology, but the antibody fragments of the present invention are not limited to these exemplary types of antibody fragments. Similarly, the antibodies or antigen-binding portions thereof can be modified to contain detectable labels, such as radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).In some cases the CDR3 sequences of the TCRs may be slightly modified, but preferably at no more than three amino acid residues, preferably only two, most preferably only one amino acid position, when compared to the CDR3 sequences presented in SEQ ID NOs: 3, 7, 11 and 15. Preferably the antibody comprises a CDR3, preferably comprising all of the CDR1 to CDR3 regions in combination as shown for the TCRs of the invention in Table 1, optionally with in each case independently no more than three or no more than two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to these sequences.

[0076] Suitable methods for producing antibodies are known in the art, for example, standard hybridoma methods are described, for example, in Kohler and Milstein, Eur. J. Immunol, 5, 51 1-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA Janeway et al. (eds.), Immunobiology, 8 Ed., Garland Publishing, New York, NY (201 1)). Alternatively, other methods are known in the art, such as the EBV hybridoma method (Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), and Roder et al, Methods Enzymol, 121, 140-67 (1986)), and bacteriophage vector expression systems (see, e.g., Huse et al., Science, 246, 1275-81 (1989)). Additionally, methods for producing antibodies in non-human animals are described, for example, in U.S. Pat. Nos. 5,545,806, 5,569,825, and 5,714,352, and U.S. Patent Application Publication No. 2002 / 0197266.

[0077] Some embodiments of the invention also relate to TCRs, or functional fragments thereof, and polypeptides that are soluble TCRs. As used herein, the term "soluble T cell receptor" refers to heterodimeric truncated variants of native TCRs, which include the extracellular portions of the TCR α and β chains, which are linked, for example, by disulfide bonds, but lack the transmembrane and cytoplasmic domains of the native protein. The terms "soluble T cell receptor α and β chain sequences" refer to TCR α and β chain sequences that lack the transmembrane and cytoplasmic domains. The soluble TCR α and β chain sequences (amino acid or nucleic acid) can be identical to the corresponding sequences of a native TCR, or can include variant soluble TCR α and β chain sequences compared to the corresponding native TCR sequences. The term "soluble T cell receptor" as used herein encompasses soluble TCRs with variant or non-variant soluble TCR α and β chain sequences. The variations can be in the variable or constant regions of the soluble TCR α and β chain sequences and can include, but are not limited to, amino acid deletions, insertions, substitution mutations, and modifications of the nucleic acid sequence (which do not alter the amino acid sequence). The soluble TCRs of the invention, in any case, retain the binding functionality of their parent molecules.

[0078] In a second aspect, the present invention relates to a nucleic acid or a nucleic acid construct (NAC) comprising a nucleic acid sequence encoding an ABP of the present invention.

[0079] The above problem is further solved by a nucleic acid encoding an antigen recognition construct or ABP of the present invention, or by any of the protein or polypeptide constructs described above. The nucleic acid preferably has (a) a strand encoding an antigen recognition construct according to the present invention, (b) a strand complementary to the strand of (a), or (c) a strand that hybridizes under stringent conditions with a molecule as described in (a) or (b). Stringent conditions are known to the skilled artisan, in particular from Sambrook et al., "Molecular Cloning". In addition to the above, the nucleic acid optionally has further sequences necessary to express the nucleic acid sequence corresponding to the protein, in particular necessary for expression in mammalian / human cells. The nucleic acid used can be contained in a vector suitable for allowing the expression of the nucleic acid sequence corresponding to the peptide in the cell. However, the nucleic acid can also be used to transform an antigen presenting cell in such a way that the antigen presenting cell produces the corresponding protein on its own cell surface, and the antigen presenting cell is not limited to classical antigen presenting cells, e.g. dendritic cells.

[0080] In some embodiments, the antigen recognition construct or ABP polypeptide is encoded by a nucleic acid and can be expressed in vivo or in vitro. Thus, in some embodiments, a nucleic acid encoding an antigen recognition construct or ABP is provided. In some embodiments, a nucleic acid encodes one portion or monomer of an antigen recognition construct or ABP of the invention (e.g., one of the two chains of a TCR of the invention) and / or another nucleic acid encodes another portion or monomer of an antigen recognizing construct or ABP of the invention (e.g., the other of the two chains of a TCR). In some embodiments, a nucleic acid encodes more than one antigen recognition construct or ABP polypeptide chain, e.g., at least two TCR chains. A nucleic acid encoding multiple antigen recognition constructs or ABP chains can include a nucleic acid cleavage site between at least two chain sequences, can encode a transcription or translation initiation site between two or more chain sequences, and / or can encode a proteolytic target site between two or more antigen recognition constructs or ABP chains.

[0081] "Nucleic acid," as used herein, includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules," and generally refers to polymers of DNA or RNA, which can be single- or double-stranded, can be synthetic or obtained from natural sources (e.g., isolated and / or purified), and can have natural, non-natural, or modified nucleotides and can have natural, non-natural, or modified internucleotide linkages, e.g., phosphoramidate or phosphorothioate linkages in place of the phosphodiesters found between nucleotides in unmodified oligonucleotides.

[0082] Preferably, the nucleic acid of the invention is a recombinant nucleic acid. As used herein, the term "recombinant" refers to (i) a molecule constructed outside a living cell by joining a natural or synthetic nucleic acid segment to a nucleic acid molecule capable of replicating in a living cell, or (ii) a molecule resulting from the replication of a molecule as described in (i) above. For purposes herein, the replication can be in vitro or in vivo replication. The nucleic acid can comprise any nucleotide sequence encoding a TCR as described herein, or any of its polypeptides, or proteins, or functional portions, or functional variants.

[0083] In a third aspect, the present invention relates to a recombinant vector comprising a nucleic acid of the present invention.

[0084] Still further, the present invention provides a vector comprising a nucleic acid according to the present invention as described above. Desirably, the vector is an expression vector or a recombinant expression vector. The term "recombinant expression vector" in the context of the present invention refers to a nucleic acid construct that allows expression of mRNA, protein or polypeptide in a suitable host cell. The recombinant expression vector of the present invention can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and multiplication or for expression or both, e.g., plasmids and viruses. Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo. Preferably, the recombinant expression vector is a viral vector, e.g., a retroviral vector. The recombinant expression vector comprises control sequences, e.g., initiation and termination codons for transcription and translation, which are specific for the type of host cell (e.g., bacteria, fungi, plants or animals) into which the vector is to be introduced and in which expression of the nucleic acid of the present invention may take place. Still further, the vector of the present invention can comprise one or more marker genes, which allow for the selection of transformed or transfected hosts. The recombinant expression vector may comprise a native or exemplary promoter, which is operably linked to a nucleotide sequence encoding the construct of the present invention, or to a nucleotide sequence that is complementary to or hybridizes to the nucleotide sequence encoding the construct of the present invention. The selection of promoters may include, for example, strong promoters, weak promoters, inducible promoters, tissue-specific promoters, and development-specific promoters. The promoters may be non-viral or viral promoters. The recombinant expression vectors of the present invention may be designed for either transient expression, stable expression, or both. Similarly, the recombinant expression vectors may be made for constitutive or inducible expression.

[0085] Such vectors may contain nucleic acid encoding either one or both of the TCR chains, and may therefore include sequences encoding the alpha and / or beta chains of the ABP of the invention.

[0086] In a fourth aspect, the present invention relates to a recombinant host cell comprising or expressing an ABP of the invention or comprising a nucleic acid or vector of the invention.

[0087] The present invention also relates to a host cell comprising an antigen recognition construct or ABP according to the invention. In particular, the host cell of the present invention comprises a nucleic acid or vector as described herein above. The host cell can be a eukaryotic cell, such as a plant, animal, fungus or algae, or a prokaryotic cell, such as a bacterium or a protozoan. The host cell can be a cultured cell or a primary cell, i.e. directly isolated from an organism, such as a human. The host cell can be an adherent cell or a suspension cell, i.e. a cell growing in suspension. For the purpose of producing recombinant TCRs, polypeptides or proteins, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. Although the host cell can be of any cell type, can be derived from any kind of tissue and can be at any developmental stage, the host cell is preferably a peripheral blood leukocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell. The T cells can be any T cells, e.g., cultured T cells, e.g., primary T cells, or T cells from cultured T cell lines, e.g., Jurkat, SupT1, etc., or T cells obtained from a mammal, preferably T cells or T cell precursors from a human patient. If obtained from a mammal, the T cells can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cells can also be enriched or purified. Preferably, the T cells are human T cells. More preferably, the T cells are T cells isolated from a human. The T cells can be any type of T cell and can be at any stage of development, including, but not limited to, CD4-positive and / or CD8-positive cells, 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, etc. Preferably, the T cells are CD8 positive T cells or CD4 positive T cells.

[0088] Preferably, the host cell of the present invention is a lymphocyte, preferably a T lymphocyte, such as a CD4 positive or CD8 positive T cell. The host cell is even more preferably RAC2 P29L and / or RAC1 P29S RAC2, specific for antigen-presenting cells P29L and / or RAC1 P29S These are antigen-reactive T cells.

[0089] In a fifth aspect, the present invention relates to a pharmaceutical composition comprising one or more, preferably in combination, ABPs of the invention, or nucleic acids or cells of the invention.

[0090] Thus, also provided is a pharmaceutical composition comprising any of the products of the invention and TCR materials of the invention described herein, in particular any protein, nucleic acid, or host cell. In a preferred embodiment, the pharmaceutical composition is for use in immunotherapy, preferably adoptive cell therapy.

[0091] Preferably, the TCR material of the invention is administered by injection, for example, intravenous injection. When the TCR material of the invention is a host cell expressing a TCR of the invention (or a functional variant thereof), the pharma- ceutical acceptable carrier for injection for the cells can include any isotonic carrier, such as saline (about 0.90% w / v NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, Ill.), PLASMA-LYTE A (Baxter, Deerfield, Ill.), about 5% dextrose in water, lactated Ringer's solution, and the like. In certain embodiments, the pharma- ceutical acceptable carrier is supplemented with human serum albumin.

[0092] For purposes of the present invention, the amount or dose (e.g., number of cells, if the TCR material of the present invention is one or more cells) of the TCR material of the present invention administered can be sufficient to affect, e.g., produce a therapeutic or prophylactic response in the subject or animal over a reasonable time frame. For example, a dose of the TCR material of the present invention should be sufficient to bind a cancer antigen or detect, treat, or prevent cancer for a period of about 2 hours or more from the time of administration, e.g., 12 to 24 hours or more. In certain embodiments, the length of this period can be even longer. The dose will be determined by the efficacy of the particular TCR material of the present invention and the condition of the animal (e.g., human) being treated, as well as the body weight of the animal (e.g., human).

[0093] The pharmaceutical compositions, antigen recognition constructs, TCRs (including functional variants thereof), polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or cell populations of the present invention are useful in treating cancer, i.e. RAC2 P29L and / or RAC1 P29S It is contemplated that the TCRs of the present invention (and functional variants thereof) can be used in methods of treating or preventing pathologies associated with positive cells. The TCRs of the present invention (and functional variants thereof) can be used in methods of treating or preventing pathologies associated with positive cells, such as RAC2 of the present invention when the TCRs (or the associated polypeptides or proteins of the present invention and functional variants thereof) are expressed by or in cells, such as T cells. P29L and / or RAC1 P29S A target cell expressing an antigen, preferably RAC2, is expressed on the surface of said target cell via MHC I. P29L and / or RAC1 P29S The RAC2 of the present invention is capable of mediating an immune response against target cells presenting an antigenic peptide. P29L and / or RAC1 P29SIt is believed to specifically bind to antigen.In this regard, the present invention provides a method for treating or preventing a pathology, particularly cancer disease, in a mammalian subject, comprising administering to the mammal any of the pharmaceutical compositions of the present invention, isolated ABPs, or combinations of ABPs, in particular any nucleic acid or recombinant expression vector comprising the nucleotide sequence encoding any of the TCRs (and their functional variants), polypeptides, or proteins described herein, any of the TCRs (and their functional variants), polypeptides, or proteins described herein, or any host cell or cell population comprising a nucleic acid or recombinant vector (which encodes any of the constructs of the present invention (and their functional variants), polypeptides, or proteins described herein) in an amount effective for treating or preventing the pathology in the mammal, and the pathology is preferably cancer that is positive for mutant RAC1 and / or RAC2, or any disease or pathology associated with the expression of such mutant proteins.

[0094] Examples of pharma- ceutically acceptable carriers or diluents useful in the present invention include stabilizers, such as SPGA, carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins, such as albumin or casein, protein-containing agents, such as bovine serum or skim milk, and buffers (e.g., phosphate buffers).

[0095] In a sixth aspect, the present invention relates to a combination or composition comprising any of the products of the present invention, or a combination thereof, for use in medicine, in particular for use in the treatment of a disease associated with a mutated RAC1 / RAC2 antigen in a subject.

[0096] The constructs, proteins, TCR antibodies, polypeptides and nucleic acids of the invention are particularly intended for use in immunotherapy, preferably adoptive T cell therapy. Administration of the complexes of the invention can include, for example, injecting T cells of the invention into said patient. Preferably, such T cells are autologous T cells of the patient and have been transduced in vitro with a nucleic acid or antigen recognition construct of the invention. Thus, as used herein, the term "adoptive immunotherapy" or "adoptive cell therapy" (ACT) refers to the process of introducing autologous or allogeneic cells of various hematopoietic lineages (e.g., lymphocytes or T cells) into a patient or subject to treat a disease. The term "adoptive T cell therapy" refers to the process of introducing autologous or allogeneic T cells into a patient or subject to treat a disease, preferably such T cells recombinantly expressing the TCR (ABP) of the invention.

[0097] The conjugates and compositions of the present invention are useful for the prevention or treatment of proliferative disorders such as cancer. The term "cancer" or "tumor" or "proliferative disorder" refers to the presence of cells with characteristics typical of cancer-initiating cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of tumors, although such cells can exist alone in an animal or can be non-tumorigenic cancer cells, such as leukemia cells. Cancers include, but are not limited to, B cell cancers such as multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain diseases such as alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immune cell amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, cancer of the blood tissue, and the like.Other examples, but not limitations, of the types of cancers that are applicable to the methods encompassed by the present invention include human sarcomas and cell carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic lung carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, liver cancer, choriocarcinoma, seminoma, Embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, such as acute lymphocytic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia); chronic leukemias (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, the cancer phenotyped by the methods of the invention is an epithelial cancer, such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, renal cancer, pharyngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In yet other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial cancers can be characterized in a variety of other ways, including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or anaplastic.In some embodiments, the present invention finds use in the treatment, diagnosis, and / or prognosis of lymphomas or subtypes thereof, including but not limited to lymphocyte-rich classical Hodgkin lymphoma, mixed cellularity classical Hodgkin lymphoma, lymphopenic classical Hodgkin lymphoma, nodular sclerosis classical Hodgkin lymphoma, anaplastic large cell lymphoma, diffuse large B-cell lymphoma, MLL+ pre-B-cell ALL, based on the analysis of the markers described herein.

[0098] Preferred cancers of the present invention are those characterized by the expression of RAC1 and / or RAC2, and in preferred embodiments, mutated variants of RAC1 and / or RAC2, i.e. preferably RAC1 P29S and / or RAC2 P29L and characterized by the expression of at least a peptide derived from RAC1 / RAC2, preferably a mutated amino acid P29S or P29L and most preferably, such cancers are characterized by direct or immune cell-mediated presentation of a peptide comprising SEQ ID NO: 17 or 18.

[0099] Most preferably, the ABP of the present invention is for use in the treatment of a cancer disease, which cancer disease is characterized by a mutant RAC1 protein, preferably RAC1 P29S In the examples, it is surprisingly found that the RAC2-derived mutant peptide (RAC2 P29L It was shown that the TCR of the present invention generated against RAC1 has a significantly better therapeutic effect in vivo against RAC1-driven cancers than the TCR generated using RAC1 peptide. Thus, the TCR of the present invention is P29S The present invention is particularly useful for treating or preventing diseases characterized by the expression of

[0100] The antigen recognition constructs, ABPs, e.g., TCRs, polypeptides, proteins (including functional variants thereof), nucleic acids, recombinant expression vectors, host cells (including populations thereof), and antibodies (including antigen-binding portions thereof) of the invention (all of which are hereinafter collectively referred to as "TCR materials of the invention") can be formulated into compositions, such as pharmaceutical compositions. In this regard, the invention provides pharmaceutical compositions comprising any of the antigen recognition constructs, TCRs, polypeptides, proteins, functional portions, functional variants, nucleic acids, expression vectors, host cells (including populations thereof), and antibodies (including antigen-binding portions thereof) described herein, together with a pharma- ceutically acceptable carrier, excipient, and / or stabilizer. Pharmaceutical compositions of the invention containing any of the TCR materials of the invention can include two or more TCR materials of the invention, e.g., polypeptides and nucleic acids, or two or more different TCRs (including functional portions and functional variants thereof). Alternatively, the pharmaceutical composition may comprise the TCR material of the invention in combination with another pharma- ceutical active agent(s) or drug(s), such as, for example, chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like. Preferably, the carrier is a pharma- ceutical acceptable carrier. For pharmaceutical compositions, the carrier can be any of those commonly used for the particular TCR material of the invention under consideration. Such pharma- ceutical acceptable carriers are known to those skilled in the art and readily available to the general consumer. Preferably, the pharma- ceutical acceptable carrier is one that has no adverse side effects or toxicity under the conditions of use.

[0101] As used herein, the term "subject" refers to a human or another mammal (e.g., primate, dog, cat, goat, horse, pig, mouse, rat, rabbit, etc.), preferably suffering from a disease treatable or preventable according to the invention described herein. Such a subject is preferably a subject that is positive for peptide presentation of RAC peptide as disclosed herein.

[0102] The term "treatment" is used herein to characterize a method or process that aims to (1) delay or prevent the onset of a disease or condition (e.g., cancer); (2) slow or halt the progression, worsening, or progression of the symptoms of a disease or condition; (3) bring about the remission of the symptoms of a disease or condition; or (4) cure a disease or condition. Treatment can be administered prior to the onset of a disease or condition for prophylactic or preventative measures. Alternatively, or additionally, treatment can be administered after the onset of a disease for therapeutic measures.

[0103] In a seventh aspect, the present invention relates to a method for producing a cell line expressing a RAC1 and / or RAC2 neo-antigen specific antigen recognition construct, comprising the steps of: Providing a suitable host cell; Providing a genetic construct comprising a coding sequence encoding an ABP of the present invention; introducing said genetic construct into said suitable host cell; expressing said genetic construct by said suitable host cell; The present invention relates to a method comprising the steps of:

[0104] This method may further comprise the step of displaying the antigen recognition construct on the cell surface of the suitable host cell.

[0105] In another preferred embodiment, the genetic construct is an expression construct comprising a promoter sequence operably linked to said coding sequence.

[0106] Preferably, said antigen recognition construct is of mammalian origin, preferably of human origin.Suitable host cells for use in the method of the present invention are mammalian cells, such as human cells, particularly human T lymphocytes.The T cells used in the present invention are described in detail herein above.

[0107] Also included in the invention are embodiments in which the antigen recognition construct is a modified TCR, the modification being the addition of a functional domain, such as a label or a therapeutically active agent. Still further included are TCRs with alternative domains, such as alternative membrane anchoring domains in place of the integral transmembrane region.

[0108] Desirably, the transfection system for introducing the gene construct into the suitable host cell is a retroviral vector system. Such systems are known to those skilled in the art.

[0109] In one embodiment, also encompassed by the invention is the additional method step of isolating and purifying the antigen recognition construct from the cell, and, optionally, reconstituting the translated antigen recognition construct fragment in a T cell.

[0110] In an alternative embodiment of the present invention, a T cell receptor (TCR) producing cell line as described (wherein the TCR is specific for RAC1 / RAC2 positive cells), preferably RAC2 as described herein above, is used. P29L and / or RAC1 P29S T cells are provided that are obtained or obtainable by the method for producing positive, high avidity cells, such T cells depending on the host cell used in the method of the invention may be, for example, human or non-human T cells, preferably human TCR.

[0111] Another aspect of the invention further relates to detection of RAC2 in a (biological) sample, such as one obtained from a subject or patient. P29L and / or RAC1 P29S Antigen, or MHC and RAC2 P29Land / or RAC1 P29S Antigen peptide (RAC2 P29L and / or RAC1 P29S The present invention relates to a method for detecting a complex between a RAC2 protein and a protein epitope of a related protein, the method comprising: P29L and / or RAC1 P29S peptide or RAC2 P29L and / or RAC1 P29S contacting said peptide / MHC complex with an ABP that specifically binds to said ABP; P29L and / or RAC1 P29S With peptides or RAC2 P29L and / or RAC1 P29S and detecting binding to the peptide / MHC complex. In some embodiments, the antigen recognition construct or ABP is a TCR or an antibody, or a similar construct, or preferably an antigen recognition construct according to the invention as described herein. In some embodiments, the (biological) sample is a liver sample (e.g., one of those described anywhere herein), such as a RAC2 antibody of the subject. P29L and / or RAC1 P29S The sample contains potentially positive cells, for example melanoma cells.

[0112] Also provided is a method of treating cancer in a subject in need of cancer treatment, the method comprising: isolating cells from the subject; and transforming a cell with at least one vector encoding an antigen recognition construct or ABP of the invention to generate a transformed cell; expanding the transformed cells to generate a plurality of transformed cells; administering a plurality of transformed cells to the subject; Includes.

[0113] Also provided is a method of treating cancer in a subject in need of cancer treatment, the method comprising: Isolating cells from a healthy donor; transforming a cell with a vector encoding an antigen recognition construct of the present invention to generate a transformed cell; expanding the transformed cells to generate a plurality of transformed cells; administering a plurality of transformed cells to the subject; Includes.

[0114] Also provided is a method for detecting RAC2 in a biological sample. P29L and / or RAC1 P29S A method for detecting positive cancer cells, the method comprising: Contacting a biological sample with the ABP of the present invention, the biological sample preferably containing RAC2 P29L and / or RAC1 P29S Contains positive host cells; detecting binding of the ABP to the biological sample; Includes.

[0115] In some embodiments, RAC2 P29L and / or RAC1 P29S Methods for detecting positive cells include in vitro, in vivo, or in situ detection of, for example, RAC2 P29L and / or RAC1 P29S The method is performed using a subject's sample containing disease-associated cells that may contain

[0116] Also provided is a method of detecting the presence of a pathology in a mammal, the method comprising: (i) contacting a sample comprising one or more cells from the mammal with any of the TCRs (and functional variants thereof), polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, antibodies or antigen-binding portions thereof, or pharmaceutical compositions of the invention described herein, thereby forming a complex, and detecting the complex, wherein detection of the complex indicates the presence of a pathology in the mammal, the pathology being cancer, e.g., RAC2. P29L and / or RAC1 P29S It's positive for cancer.

[0117] For the methods of the present invention for detecting a pathology in a mammal, the cellular sample can be a sample comprising whole cells, a lysate thereof, or a fraction of a whole cell lysate, such as a nuclear or cytoplasmic fraction, a total protein fraction, or a nucleic acid fraction.

[0118] For purposes of the detection methods of the present invention, the contacting can occur in vitro or in vivo with respect to a mammal. Preferably, the contacting is in vitro.

[0119] Similarly, detection of the complex can occur through a number of ways known in the art. For example, the antigen recognition constructs (and their functional variants), polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, or antibodies or TCRs of the present invention described herein, or antigen-binding portions thereof, can be labeled with a detectable label, such as a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and an elemental particle (e.g., gold particle).

[0120] For purposes of the methods of the invention, when a host cell or cell population is administered, the cells can be allogeneic or autologous cells to the mammal. Preferably, the cells are autologous to the mammal.

[0121] Generally, the present invention provides a method for the preparation of a RAC2 P29L and / or RAC1 P29S The present invention provides a method for treating a subject suffering from a RAC2 positive cancer, the method comprising administering an antigen recognition construct, an ABP, a nucleic acid, a vector, a pharmaceutical composition, and / or a host cell as disclosed by the present invention. Preferably, the subject is in need of such treatment. In a preferred embodiment, the subject is a subject having a RAC2 positive cancer. P29L and / or RAC1 P29S The subject is a mammalian subject, preferably a human patient, suffering from a positive disease.

[0122] In addition to the above, the present invention further relates to the following specific embodiments:

[0123] Item 1: An isolated antigen binding protein (ABP) that specifically binds to an antigenic peptide derived from RAC1 and / or RAC2, preferably mutated RAC1 and / or RAC2, or a variant thereof, wherein the isolated ABP comprises at least one complementarity determining region (CDR), the CDR having an amino acid sequence with at least 80% sequence identity compared to a sequence selected from SEQ ID NOs: 3, 7, 11, and 15, or a CDR3 having no more than three or no more than two, preferably no more than one amino acid substitution(s), deletion(s), or insertion(s).

[0124] Item 2: The above ABP is RAC2 P29L and / or RAC1 P29S and preferably such derived peptides are capable of specifically and / or selectively binding to antigenic peptides derived from RAC2. P29L and / or RAC1 P29S Contains mutations and therefore P29L and / or P29S The isolated ABP of item 1, comprising a mutation. The position of the mutation is not relevant. Most preferably, the isolated ABP of item 1 binds to the peptides of SEQ ID NO: 17 and / or 18.

[0125] Item 3: The isolated ABP of items 1 or 2, wherein the ABP is an antibody, or an antigen-binding derivative or fragment thereof, or preferably a T cell receptor (TCR), or an antigen-binding derivative or fragment thereof.

[0126] Item 4: The isolated ABP according to any one of items 1 to 3, wherein the ABP binds to a human leukocyte antigen (HLA)-presented antigen peptide, the HLA being preferably of type A2, and preferably the ABP forms a specific complex with an MHC and an antigen peptide.

[0127] Item 5: ABP is RAC2 P29Land / or RAC1 P29S 5. The isolated ABP according to any one of items 1 to 4, which specifically and / or selectively binds to an epitope having an amino acid sequence selected from an MHC-presented peptide of the protein, preferably the peptide comprises such a variant amino acid position, most preferably the peptide consists of SEQ ID NO: 17 or 18.

[0128] Item 6: The isolated ABP of any one of items 1 to 5, wherein the ABP is an α / β-TCR, or an antigen-binding derivative or fragment thereof, or the construct is a γ / δ-TCR, or an antigen-binding derivative or fragment thereof.

[0129] Item 7: ABP is of human origin and RAC2 P29L and / or RAC1 P29S 7. The isolated ABP according to any one of items 1 to 6, characterized in that it specifically and / or selectively recognizes an antigen peptide.

[0130] Item 8: The isolated ABP of any one of items 1 to 7, wherein the ABP is capable of inducing an immune response in a subject, and optionally, the immune response is characterized by an increase in interferon (IFN) gamma levels.

[0131] Item 9: The isolated ABP of any one of items 1 to 8, comprising a TCR alpha or gamma chain, and / or a TCR beta or delta chain, wherein the TCR alpha or gamma chain has an amino acid sequence with at least 80% sequence identity with a sequence selected from SEQ ID NOs: 3 and 11, or has no more than three or no more than two, preferably no more than one amino acid substitution(s), deletion(s), or insertion(s), and / or the TCR beta or delta chain has an amino acid sequence with at least 80% sequence identity with a sequence selected from SEQ ID NOs: 7 and 15, or has no more than three or no more than two, preferably no more than one amino acid substitution(s), deletion(s), or insertion(s).

[0132] Item 10: The isolated ABP of item 9, wherein the TCR alpha or gamma chain further comprises a CDR1 having an amino acid sequence with at least 80% sequence identity or having no more than three or no more than two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to a sequence selected from SEQ ID NO: 1 or 9, and / or a CDR2 having an amino acid sequence with at least 80% sequence identity or having no more than three or no more than two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to a sequence selected from SEQ ID NO: 2 or 10.

[0133] Item 11: The isolated ABP of item 9 or 10, wherein the TCR β or δ chain further comprises a CDR1 having an amino acid sequence with at least 80% sequence identity or having no more than 3 or no more than 2, preferably no more than 1 amino acid substitution(s), deletion(s) or insertion(s) compared to a sequence selected from SEQ ID NOs: 5 and 13, and / or a CDR2 having an amino acid sequence with at least 80% sequence identity or having no more than 3 or no more than 2, preferably no more than 1 amino acid substitution(s), deletion(s) or insertion(s) compared to a sequence selected from SEQ ID NOs: 6 and 14.

[0134] Item 12: The isolated ABP of any one of items 1 to 11, comprising a TCR variable chain region having at least 80% sequence identity or having no more than 20, preferably no more than 15, more preferably no more than 10, no more than 5, no more than 3, or no more than 2, preferably no more than 1 amino acid substitution(s), deletion(s), or insertion(s) compared to an amino acid sequence selected from SEQ ID NOs: 4, 8, 12, and 16.

[0135] Item 13: The isolated ABP of any one of items 1 to 12, wherein the construct is humanized, chimeric, and / or murine.

[0136] Item 14: The isolated ABP according to any one of items 1 to 13, comprising a binding fragment of a TCR, said binding fragment comprising CDR1 to CDR3, optionally selected from CDR1 sequences to CDR3 sequences having the amino acid sequences of SEQ ID NO: 1, 2, 3, or 5, 6, 7, or 9, 10, 11, or 13, 14, 15, and each CDR independently has no more than three or no more than two, preferably no more than one amino acid substitution(s), deletion(s), or insertion(s) compared to these sequences.

[0137] Item 15: The isolated ABP according to any one of Items 1 to 14, wherein the construct is a TCR or a fragment thereof composed of at least one TCR α chain sequence and one TCR β chain sequence, the TCR α chain sequence comprising CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 1 to 3, the TCR β chain sequence comprising CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 5 to 7, or the TCR α chain sequence comprising CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 9 to 11, the TCR β chain sequence comprising CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 13 to 15, and each CDR independently has three or less or two or less, preferably one or less amino acid substitution(s), deletion(s), or insertion(s) compared to these sequences.

[0138] Item 16: The isolated ABP of any one of items 1 to 15, wherein the ABP is a TCR or an antigen-binding fragment or derivative thereof comprising at least one TCR alpha chain sequence and one TCR beta chain sequence, wherein the TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 4, and the TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 8, or wherein the TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 12, and the TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 16, and each variable domain sequence has 20 or less, preferably 15 or less, more preferably 10 or less, 5 or less, 3 or less, or 2 or less, preferably 1 or less amino acid substitution(s), deletion(s), or insertion(s) compared to these sequences.

[0139] Item 17: The isolated ABP of any one of items 1 to 16, wherein the ABP is a TCR or an antigen-binding fragment or derivative thereof and further comprises a TCR constant region, preferably a human TCR constant region sequence.

[0140] Item 18: An isolated nucleic acid encoding the ABP of any one of items 1 to 17.

[0141] Item 19: The isolated nucleic acid of Item 18, comprising a nucleic acid sequence having at least 80% sequence identity or having no more than 20, preferably no more than 15, more preferably no more than 10, no more than 5, no more than 3, or no more than 2, preferably no more than 1 nucleic acid substitution(s), deletion(s), or insertion(s) compared to a nucleic acid sequence encoding a polypeptide selected from SEQ ID NOs: 4, 8, 12, and 16.

[0142] Item 20: A recombinant vector comprising the nucleic acid of Item 18 or 19.

[0143] Item 21: A recombinant host cell comprising the ABP of any one of items 1 to 17, or the nucleic acid of item 18 or 19, or the vector of item 20.

[0144] Item 22: The recombinant host cell of item 21, wherein the cell is a lymphocyte, preferably a T lymphocyte or a precursor T lymphocyte, more preferably a CD4 positive or, most preferably, a CD8 positive T cell.

[0145] Item 23: A pharmaceutical composition comprising an ABP according to any one of items 1 to 17, or a nucleic acid according to item 18 or 19, or a vector according to item 20, or a recombinant host cell according to item 21 or 22, and a pharma- ceutically acceptable carrier, stabilizer, and / or excipient, preferably the pharmaceutical composition comprising at least two different ABPs according to any one of items 1 to 17.

[0146] Item 24: A compound or composition used in medicine, selected from the ABP of any one of items 1 to 17, or the nucleic acid of item 18 or 19, or the vector of item 20, or the recombinant host cell of item 21 or 22, or the pharmaceutical composition of item 23.

[0147] Item 25: The use is directed to the treatment of proliferative disorders, preferably cancer, e.g. RAC2 P29L and / or RAC1 P29S 25. The composition or composition for use according to item 24, for the treatment of cancer in which expression of

[0148] Item 26: The combination or composition for use of item 25, wherein the treatment comprises immunotherapy, preferably adoptive autologous or xenogeneic T cell therapy.

[0149] Item 27: The composition or composition for use according to item 26, wherein the T cell therapy involves the use of cells containing the ABP or nucleic acid according to any one of the above items.

[0150] Item 28: RAC2 P29L and / or RAC1 P29S 1. A method for producing a cell line expressing a specific antigen recognition construct, comprising: (a) providing a suitable host cell; (b) providing a genetic construct comprising a coding sequence encoding the ABP according to any one of items 1 to 17; (c) introducing said genetic construct into said suitable host cell; and (d) expressing said genetic construct by said suitable host cell. A method comprising:

[0151] Item 29: The method of item 28, further comprising cell surface display of the antigen recognition construct.

[0152] Item 30: The method of items 28 or 29, wherein the genetic construct is an expression construct comprising a promoter sequence operably linked to the coding sequence.

[0153] Item 31: The method according to any one of Items 28 to 30, wherein the antigen recognition construct is of mammalian origin, preferably of human origin.

[0154] Item 32: The method of any one of items 28 to 31, wherein the suitable host cell is a mammalian cell, optionally selected from a human cell or a human T lymphocyte.

[0155] Item 33: The method of any one of items 28 to 32, wherein the antigen recognition construct is a modified TCR, and the modification comprises the addition of a functional domain comprising a label, or an alternative domain comprising a membrane anchor domain.

[0156] Item 34: The method of item 33, wherein the antigen recognition construct is an ABP, preferably an alpha / beta TCR, a gamma / delta TCR, or a TCR variant, such as a single chain TCR (scTCR).

[0157] Item 35: The method of any one of items 28 to 34, wherein the genetic construct is introduced into the suitable host cell by retroviral transfection.

[0158] Item 36: The method of any one of items 28 to 35, further comprising isolating and purifying the antigen recognition construct from a suitable host cell, and optionally reconstituting the antigen recognition construct in a T cell.

[0159] Item 37: A suitable host cell is RAC2 P29L and / or RAC1 P29S 36. The method of any one of items 28 to 35, wherein the cell, e.g. a T cell, is obtained from a patient suffering from a disorder associated with the expression, e.g., preferably a cancer.

[0160] Hereinafter, further preferred embodiments of the present invention will be described with respect to the ABP of the present invention described herein above, in particular the most preferred TCRs as described herein and their fragments and variants (RAC2-T001 and RAC2-T002). The above description of the ABP of the present invention and the antigen-binding peptides recognized by the ABP shall equally apply.

[0161] In one such preferred embodiment, the mutant RAC1 and / or RAC2 derived antigenic peptides according to the present invention disclosed herein are selected from peptides having an amino acid sequence selected from SEQ ID NOs: 17 to 21, preferably SEQ ID NOs: 19, 20, 21 (in some embodiments, being at least 90%, preferably 100% identical to the sequence).

[0162] In another such embodiment, the isolated ABP of the present invention or an antigen-binding fragment thereof disclosed herein can specifically and / or selectively bind to an antigen peptide having an amino acid sequence represented by a consensus sequence (or consensus motif) set forth in one of SEQ ID NOs: 19 to 21, preferably MHC-presented. For such ABPs, the above description of the ABP of the present invention applies equally. For such ABPs, the above description of the ABP of the present invention applies equally.

[0163] In particular, in such an embodiment, the above description of the ABP with reference to any of the sequences of RAC1-T001 according to Table 1 herein below specifically and / or selectively binds to the peptide epitope of the consensus sequence shown in SEQ ID NO: 19 or 20 (of which SEQ ID NO: 20 is preferred). On the other hand, in a further form of such an embodiment, the above description of the ABP with reference to any of the sequences of RAC1-T002 according to Table 1 herein below specifically and / or selectively binds to the peptide epitope of the consensus sequence shown in SEQ ID NO: 21 (of which SEQ ID NO: 20 is preferred).

[0164] Preferably, in such an embodiment, the isolated ABP of the invention is capable of specifically and / or selectively binding to an antigenic peptide as described herein and does not show non-self-recognition to non-specific peptides, such non-specific peptides being preferably understood as peptides not derived from a mutant RAC1 or RAC2 protein.

[0165] As used herein, the terms "of the invention," "in accordance with the invention," "according to the invention," and the like are intended to refer to all aspects and embodiments of the invention described and / or claimed herein.

[0166] As used herein, the term "comprising" should be interpreted as embracing both "including" and "consisting of," both of which are specifically contemplated and thus mean the embodiments according to the invention that are separately disclosed. As used herein, "and / or" should be considered as a specific disclosure of each of the two specified features or components, with or without the other. For example, "A and / or B" should be considered as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were separately presented herein. In the context of the present invention, the terms "about" and "approximately" represent an interval of precision that is understood by the skilled artisan to still ensure the technical effect of the feature in question. This term typically indicates a deviation of ±20%, ±15%, ±10%, e.g. ±5% from the specified numerical value. As will be understood by the skilled artisan, the deviation of such a specific numerical value for a given technical effect depends on the nature of the technical effect. For example, natural or biological technical effects may generally have a greater variation than man-made or engineered technical effects. As will be understood by one of ordinary skill in the art, such specific numerical variation for a given technical effect will depend on the nature of the technical effect. For example, natural or biological technical effects may generally have a greater variation than man-made or engineered technical effects. When an indefinite or definite article is used in reference to a singular noun, such as "a," "an," or "the," the plural of that noun is also included unless something specifically specified otherwise.

[0167] It should be understood that application of the teachings of the present invention to a particular problem or environment, and the inclusion of variations of the present invention or additional features thereof (such as further aspects and embodiments), are within the capabilities of one of ordinary skill in the art in light of the teachings contained herein.

[0168] Unless the context dictates otherwise, the feature descriptions and definitions presented above are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0169] All references, patents and publications cited herein are hereby incorporated by reference in their entirety.

[0170] The drawings show: [Brief description of the drawings]

[0171] [Figure 1] FIG. 1 shows the identification and isolation of RAC2P29L-specific CTLs in ABabDII mice. [Figure 1-1] Same as above [Diagram 2] FIG. 1 shows that TCR gene transfer confers specificity for mutant RAC2P29L peptides. [Figure 2-1] Same as above [Diagram 3] FIG. 1 shows that human melanoma cells harboring RAC1P29S are recognized by RAC2P29L TCR-redirected T cells. [Figure 4] FIG. 1 shows that heterologous RAC2P29L-specific TCR induces efficient tumor rejection upon ATT. [Diagram 5] Figure 1 shows the analysis of the consensus epitope of RAC2-T001 TCR and RAC2-T002 TCR. IFNγ production by T cells transduced with T001 TCR or T002 TCR either when co-cultured with RAC2P29L-loaded T2 cells or when co-cultured with RAC2P29L-loaded T2 cells with the respective amino acid substitutions. The respective T001 / T002 TCR recognition motifs are shown. "X" can be any naturally occurring amino acid. [Figure 6]Figure 1 shows the cross-reactivity analysis of potential human HLA-A2 restricted epitopes containing the T001 / T002 TCR recognition motif. Y-axis: IFNγ production by T001 TCR transduced T cells when co-cultured with peptide-loaded T2 cells (dark grey). (A) Analysis of recognition of potential human HLA-A2 restricted GNAZ peptides. X-axis: peptide concentration during loading (in mol / L). (B) Recognition of endogenously expressed GNAZ by T001 TCR according to SEQ ID NOs: 1-16: the TCR sequences of the present invention disclosed herein are shown in Table 1 below. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0172] SEQ ID NO: 17 is the peptide RAC2 P29L :F L Show GEYIPTV.

[0173] SEQ ID NO: 18 is peptide RAC1 P29S :F S Show GEYIPTV.

[0174] SEQ ID NOs: 19-21 show the T001 / T002 TCR consensus binding epitopes.

[0175] SEQ ID NO:22 shows the HLA-A2 restricted GNAZ peptide. EXAMPLES

[0176] Certain aspects and embodiments of the invention will now be described, by way of example, with reference to the description, figures and tables provided herein. Such examples of methods, uses and other aspects of the invention are merely representative and should not be considered as limiting the scope of the invention solely to such representative examples.

[0177] The examples show the following:

[0178] Example 1: Generation of TCRs of the invention Figure 1: RAC2 in ABabDII mice P29LIdentification and isolation of specific CTLs. P29L Immunization with peptides induces CTL responses. Representative example of ex vivo ICS analysis of RAC2 mutant peptide immunized ABabDII mice 7 days after the last immunization. Cells are gated on lymphocytes and CD3+ cells. Numbers in brackets represent the percentage of IFNg+ CD8+ T cells. (B) Identification of IFNγ+ CD8+ T cells using IFNγ capture assay. 10 -8 The mutant RAC2 peptide was cultured in spleen cells for 10 days in the presence of the mutant RAC2 peptide of M. P29L Representative IFNγ capture ab / CD8+ staining of specific CD8+ T cells. Cells were gated on lymphocytes and CD3+ cells, and unstimulated splenocytes were used as negative controls. Numbers in brackets represent the percentage of IFNγ++ CD8+ T cells. Sorted cells are shown in Figure 1. Table 1 shows the TCR amino acid sequences as isolated.

[0179] Table 1: Isolated TCR sequences of the present invention [Table 1]

[0180] Example 2: TCR gene transfer using mutant RAC2 P29L Provides specificity for peptides Two RAC2s P29L The corresponding TCR alpha and beta chains (RAC2-T001 and RAC2-T001) isolated from peptide-immunized ABabDII mice were cloned into the retroviral vector pMP71 and re-expressed in human PBMCs. (A) Transduction efficiency was measured by staining the mouse TCRb chain on CD8+ T cells. The number of positive CD8+ T cells is indicated in brackets. (B-D) Transduction efficiency of RAC2 when co-cultured with peptide-loaded T2 cells. P29L IFNγ production of T cells transduced with TCR (RAC2-T002, black bars; RAC2-T001 TCR, red bars). P29SEither peptide (D) was used for loading; as a negative control, T2 cells were not loaded (T2), and for maximal stimulation, PMA and ionomycin (P+I) were added to the co-cultures.

[0181] Example 3: RAC2 P29L TCR-redirected T cells, RAC1 P29S Recognition of human melanoma cells harboring Human PBMCs were transfected with RAC2 P29L MB231 carrying the RAC1 / 2wt gene and RAC1 P29S When co-cultured with Mel55 carrying the gene, RAC2 P29L IFNγ production of −TCR-transduced T cells is shown. As controls, mutant peptide-loaded cells (+RAC1 or +RAC2) or no cells (oe) were used in the co-cultures. For maximum stimulation, PMA and ionomycin (P+I) were added to the co-cultures.

[0182] Example 4: Heterologous RAC2 P29L Specific TCR induces efficient tumor rejection upon ATT FIG. 4(A): To analyze the in vivo efficacy of RAC1 / 2-specific TCRs, RAC1 P29S HHD+ MC703 tumor cells expressing a minigene encoding three copies of the epitope (MC703-FSG) were generated by retroviral transduction and subsequent FAC sorting of gfp+ cells. The amount of HLA-A2+gfp+ tumor cells used in subsequent experiments is shown. (B) Mouse T cells obtained from HHD transgenic mice were retrovirally transduced with RAC1- and RAC2-specific TCRs.

[0183] Each CD8+ T cell provided efficient recognition of epitope-expressing tumor cells; 14 / 35 TCR-transduced HHD+ T cells and non-transduced HHD+ T cells (oe) served as negative controls. (C) HHD Rag- / - mice bearing established MC703-FSG tumors were treated with either 5934RAC1-specific T cells or RAC2-T001 RAC2-specific T cells (1 × 10 6 Tumor-bearing HHD Rag- / - mice receiving T cells expressing CDK4R24C-specific TCR (14 / 35) are shown as controls. One representative experiment is shown.

[0184] Example 5: Identification of the consensus epitope of RAC2-T001 and RAC2-T002 RAC2 P29L Each amino acid in the peptide was replaced by alanine in turn. -5 mol / l and 10 -9 The T001 and T002 TCR-transduced T cells were loaded with 100 μg / l of TCR-transduced T cells (black and grey bars, respectively) and IFNγ production by the T001 and T002 TCR-transduced T cells was measured. The results of these experiments are shown in Figure 5 and Table 2. IFNγ production in response to the unmodified peptide was set to 100%. Furthermore, RAC1 P29S The reactivity against RAC1 (bars marked with #) and wtRAC1 / 2 (open bars) is shown. Both T001 and T002 TCR-transduced T cells showed reactivity against RAC1 P29S In the case of T001 TCR, the transduced T cells hardly produced any IFNγ upon co-culture with wtRAC1 / 2-loaded T2 cells. In the case of T002 TCR, the transduced T cells showed a marked decrease in production upon co-culture with wtRAC1 / 2 (unmodified RAC2 P29LAnalysis of IFNγ production monitored for each of the amino acid substitutions yielded the respective T001 / T002 recognition motifs. For T001 TCR transduced cells, these motifs are XXXXYIPTV (SEQ ID NO: 19) and XXXEYIPTV (SEQ ID NO: 20). For T002 TCR transduced cells, the motif XXXEYXPTV (SEQ ID NO: 21) was identified. "X" can be any naturally occurring amino acid.

[0185] Table 2: Antigenic peptides and T001 / T002 recognition motifs [Table 2] *The underlined amino acids indicate the position of the neoantigenic mutation; "X" indicates a variable amino acid side chain, which can be any naturally occurring amino acid.

[0186] Example 6: Cross-reactivity and non-self recognition of RAC2-T001 RAC2 when co-cultured with peptide-loaded T2 cells P29L IFNγ production of TCR-transduced T cells was monitored.

[0187] Human genes containing peptide motifs similar to the T001 / T002 recognition motif were included if they contained the T001 / T002 recognition motif, were not present in any mouse protein, and had a predicted HLA-A2 affinity of IC50<5000 nM. IC50, i.e. peptide-HLA-A2 binding affinity, was predicted by NetMHCPan. AAADYIPTV was the only peptide that met these criteria. Therefore, recognition of the potential human HLA-A2 restricted GNAZ (GTP-binding protein Gz subunit alpha) peptide AAADYIPTV, which contains the T001 recognition motif XXXXYIPTV, was analyzed. In these experiments, T2 cells were not loaded (T2) as a negative control, and PMA and ionomycin (P+I) were added to the co-culture for maximum stimulation. The results are shown in Figure 6A. This figure includes IFNγ production data from RAC2-T001 TCR-transduced T cells (dark bars) and mock experiments (light grey bars). T001 TCR-transduced T cells (peptide loading concentration was 10 -6 M) produced the most IFNγ, at approximately 30% of maximum stimulation.

[0188] Furthermore, recognition of endogenously expressed GNAZ by the T001 TCR was tested by co-culture of human HLA-A2+ HepG2 and SH-SY5Y cells with T001-transduced T cells as targets (dark bars, effector cells). Results are shown in Figure 6B, which includes IFNγ production data from RAC2-T001 TCR-transduced T cells (dark bars) and mock experiments (light grey bars). Peptides were delivered to T2 cells at 10 -6 GNAZ expressing cell lines are not recognized by the TCR. GNAZ peptide loaded T cells were used as a positive control. PMA and ionomycin (P+I) were added to the co-cultures for maximum stimulation.

[0189] We additionally performed LCL scans, which did not provide any evidence of allo-recognition by T001 TCR-modified T cells. In these experiments, T001 TCR-transduced T cells were co-cultured with B-LCL expressing various HLA allotypes. Positive control HLA-A2+ LCL expressed RAC2 P29L The peptide was loaded.

[0190] Drawing translation Figure 1A Restimulation with: RAC2 mutant 9mer peptide Figure 1B Stimulated stimulation Unstimulated Figure 2B Peptide Figure 2C Peptide Figure 2D Peptide Figure 4C Tumor volume Time after ATTATT Figure 5 % of RAC2 P29L RAC2 P29L of% T2 cellsT2 cells

Claims

1. 1. An isolated antigen binding protein (ABP) that specifically binds to a mutant RAC1 and / or RAC2 derived antigenic peptide or a variant thereof, the isolated ABP comprising a T cell receptor (TCR) alpha or gamma chain, and / or a TCR beta or delta chain, wherein the TCR alpha or gamma chain has a complementarity determining region (CDR) 3 that has an amino acid sequence with at least 80% sequence identity or has no more than three amino acid substitution(s), deletion(s), or insertion(s) compared to a sequence selected from SEQ ID NOs: 3 and 11, and / or the TCR beta or delta chain has a CDR3 that has an amino acid sequence with at least 80% sequence identity or has no more than three amino acid substitution(s), deletion(s), or insertion(s) compared to a sequence selected from SEQ ID NOs: 7 and 15.

2. The isolated ABP of claim 1, wherein the ABP is capable of specifically and / or selectively binding to an antigenic peptide comprising the P29 variant amino acid position of RAC1 and / or RAC2, or an antigenic peptide of SEQ ID NO: 17 and / or 18.

3. 2. The isolated ABP of claim 1, wherein the ABP is a TCR or an antigen-binding derivative or fragment thereof.

4. The isolated ABP of claim 1, wherein the TCR alpha or gamma chain further comprises a CDR1 having an amino acid sequence with at least 80% sequence identity or having no more than three amino acid substitution(s), deletion(s) or insertion(s) compared to a sequence selected from SEQ ID NO: 1 or 9, and / or a CDR2 having an amino acid sequence with at least 80% sequence identity or having no more than three amino acid substitution(s), deletion(s) or insertion(s) compared to a sequence selected from SEQ ID NO: 2 or 10.

5. The isolated ABP of claim 1, wherein the TCR beta or delta chain further comprises a CDR1 having an amino acid sequence with at least 80% sequence identity or having no more than three amino acid substitution(s), deletion(s) or insertion(s) compared to a sequence selected from SEQ ID NOs: 5 and 13, and / or a CDR2 having an amino acid sequence with at least 80% sequence identity or having no more than three amino acid substitution(s), deletion(s) or insertion(s) compared to a sequence selected from SEQ ID NOs: 6 and 14.

6. The isolated ABP of claim 1, comprising a TCR variable chain region having at least 80% sequence identity or having no more than 20 amino acid substitution(s), deletion(s), or insertion(s) compared to a sequence selected from SEQ ID NOs: 4, 8, 12, and 16.

7. 2. The isolated ABP of claim 1, wherein the ABP is a TCR or an antigen-binding fragment or derivative thereof and further comprises a TCR constant region or a human TCR constant region sequence.

8. 2. An isolated nucleic acid encoding the ABP of claim 1.

9. A recombinant vector comprising the nucleic acid of claim 8.

10. A recombinant host cell comprising the ABP of claim 1, a nucleic acid encoding said ABP, or a vector comprising said nucleic acid.

11. The recombinant host cell of claim 10, wherein the cell is a lymphocyte, a T lymphocyte or a precursor T lymphocyte, a CD4 positive or a CD8 positive T cell.

12. A pharmaceutical composition comprising the ABP according to claim 1, a nucleic acid encoding said ABP, a vector comprising said nucleic acid, or a recombinant host cell comprising said ABP, said nucleic acid or said vector, together with a pharma- ceutically acceptable carrier, stabilizer and / or excipient; or 13. A pharmaceutical composition comprising the ABP of claim 1, a nucleic acid encoding said ABP, a vector comprising said nucleic acid, or a recombinant host cell comprising said ABP, said nucleic acid or said vector, and a pharma- ceutical acceptable carrier, stabilizer, and / or excipient, wherein said pharmaceutical composition comprises at least two different ABPs of claim 1.

13. A medicament for the treatment of a proliferative disorder comprising an ABP according to any one of claims 1 to 7, or a nucleic acid according to claim 8, or a vector according to claim 9, or a recombinant host cell according to claim 10 or 11, or a pharmaceutical composition according to claim 12.

14. The pharmaceutical described in claim 13, wherein the treatment includes immunotherapy, adoptive T cell therapy, autologous T cell therapy, or xenogeneic T cell therapy using the cells described in claim 10 or 11.

15. RAC2 P29L and / or RAC1 P29S 1. A method for producing a cell line expressing a specific antigen recognition construct, comprising: (a) providing a suitable host cell; (b) providing a genetic construct comprising a coding sequence encoding the ABP according to any one of claims 1 to 7; (c) introducing the genetic construct into the suitable host cell; and (d) expressing the genetic construct by the suitable host cell to obtain a cell line expressing the specific antigen recognition construct; A method comprising:

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