HLA class II-restricted T cell receptors against RAS with the G12R mutation

Abstract

The present invention discloses an isolated or purified T cell receptor (TCR), wherein the TCR has antigenic specificity for a mutant human RAS amino acid sequence in which glycine at position 12 is substituted with arginine. Also provided are related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, cell populations, and pharmaceutical compositions. Also disclosed are methods for detecting the presence of cancer in a mammal, and methods for treating or preventing cancer in a mammal.

Description

[0001] Cross - Reference to Related Applications

[0002] This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 795,203, filed on January 22, 2019, which is incorporated herein by reference in its entirety.

[0003] Statement Regarding Federally Sponsored Research or Development

[0004] This invention was made with government support under NIH, National Cancer Institute grant number ZIABC010984. The government has certain rights in the invention.

[0005] Incorporation by Reference of Materials Submitted Electronically

[0006] A computer - readable nucleotide / amino acid sequence listing, submitted herewith and identified as follows, is incorporated herein by reference in its entirety: a 74,093 - byte ASCII (text) file named "746666_ST25.txt", dated January 21, 2020. BACKGROUND OF THE INVENTION

[0008] Some cancers can have extremely limited treatment options, especially when the cancer becomes metastatic and unresectable. Despite some progress in treatments such as surgery, chemotherapy, and radiation therapy, the prognosis for many cancers (e.g., pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, and prostate cancer) can be poor. Thus, there is an unmet need for additional cancer treatments. SUMMARY OF THE INVENTION

[0009] One embodiment of the invention provides an isolated or purified T - cell receptor (TCR), wherein the TCR has antigenic specificity for a mutant human RAS amino acid sequence in which glycine at position 12 is replaced by arginine, wherein the mutant human RAS amino acid sequence is a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence, and wherein position 12 is defined with reference to the wild - type (WT) human KRAS protein, WT human HRAS protein, or WT human NRAS protein, respectively.

[0010] Another embodiment of the present invention provides an isolated or purified polypeptide comprising the functional portion of the TCR of the present invention, wherein the functional portion comprises the following amino acid sequences: (a) all of SEQ ID NO: 1-3, (b) all of SEQ ID NO: 4-6, (c) all of SEQ ID NO: 7-9, (d) all of SEQ ID NO: 10-12, (e) all of SEQ ID NO: 1-6 or (f) all of SEQ ID NO: 7-12.

[0011] Another embodiment of the present invention provides an isolated or purified protein comprising at least one polypeptide of the present invention.

[0012] Embodiments of the present invention further provide nucleic acids, recombinant expression vectors, host cells, cell populations and pharmaceutical compositions related to the TCR, polypeptides and proteins of the present invention.

[0013] One embodiment of the present invention provides an isolated or purified nucleic acid comprising a first nucleic acid sequence and a second nucleotide sequence from 5' to 3', wherein the first and second nucleotide sequences encode the amino acid sequences of the following SEQ ID NOs: 13 and 14; 14 and 13; 15 and 16; 16 and 15; 30 and 31; 31 and 30; 32 and 33; 33 and 32; 34 and 35; 35 and 34; 36 and 37; 37 and 36; 41 and 42; 42 and 41; 43 and 44; 44 and 43; 45 and 46; 46 and 45; 47 and 48; 48 and 47; 49 and 50; 50 and 49; 51 and 52; or 52 and 51.

[0014] Embodiments of the present invention further provide methods for detecting the presence of cancer in a mammal, methods for treating or preventing cancer in a mammal, methods for inducing an immune response against cancer in a mammal, methods for generating host cells expressing the TCR that is antigen-specific for the peptide of SEQ ID NO: 39, and methods for generating the TCR, polypeptides and proteins of the present invention

[0015] Brief Description of the Drawings

[0016] Figure 1 is a graph showing the number of interferon γ (IFNγ)-positive spots detected after co-culturing 4270 TCR-transduced cells with autologous DCs / 2 × 10 4 (2E4) cells. Prior to co-culture, autologous DCs were pulsed with serial dilutions (ng / mL) of WT RAS peptide (squares) or G12R RAS peptide (circles).

[0017] Figure 2It is a graph showing the IFNγ concentration (pg / mL) detected after co-culturing 4270 TCR-transduced cells with COS7 cells. Before co-culturing, COS7 cells were transfected with one of the indicated HLA molecules and then pulsed with the G12R RAS peptide. HLA-DRA01:01 is the second member of the heterodimer of each of DRB1*11:01, DRB1*15:01, DRB3*02:02, DRB4*01:01, and DRB5*01.

[0018] Figure 3 It is a graph showing the number of IFNγ-positive spots / 2E4 cells detected after co-culturing 4268 TCR-transduced cells with autologous DCs. Before co-culturing, autologous DCs were pulsed with serial dilutions (ng / mL) of the WT RAS peptide (squares) or the G12R RAS peptide (circles).

[0019] Figure 4 It is a graph showing the IFNγ concentration (pg / mL) detected after co-culturing 4268 TCR-transduced cells with COS7 cells. Before co-culturing, COS7 cells were transfected with one of the indicated HLA molecules and then pulsed with the G12R RAS peptide. HLA-DRA01:01 is the second member of the heterodimer of each of DRB1*11:01, DRB1*12:01, DRB3*02, DRB4*01, and DRB5*01.

[0020] Detailed Description of the Invention

[0021] RAS family proteins belong to a large family of small GTPases. Without being limited to a particular theory or mechanism, it is believed that upon mutation, RAS proteins can be involved in signal transduction in the early stages of tumorigenesis of many human cancers. A single amino acid substitution can activate the protein. The mutant RAS protein product can be constitutively activated. Mutant RAS proteins can be expressed in any of a variety of human cancers such as pancreatic cancer (e.g., pancreatic carcinoma), colorectal cancer, lung cancer (e.g., lung adenocarcinoma), endometrial cancer, ovarian cancer (e.g., epithelial ovarian cancer), and prostate cancer. Human RAS family proteins include KRAS, HRAS, and NRAS.

[0022] KRAS is also known as GTPase KRas, V-Ki-Ras2 Kirsten rat sarcoma viral oncogene, or KRAS2. There are two transcript variants of KRAS: KRAS variant A and KRAS variant B. WT KRAS variant A has the amino acid sequence of SEQ ID NO:17. WT KRAS variant B has the amino acid sequence of SEQ ID NO:18. Hereinafter, unless otherwise specified, "KRAS" (mutated or unmutated (WT)) refers to both variant A and variant B. Upon activation, mutant KRAS binds to guanosine 5'-triphosphate (GTP) and converts GTP into guanosine 5'-diphosphate (GDP).

[0023] HRAS is another member of the RAS protein family. HRAS is also known as Harvey rat sarcoma viral oncoprotein, V-Ha-Ras Harvey rat sarcoma viral oncogene homolog, or Ras family small GTP-binding protein H-Ras. WT HRAS has the amino acid sequence of SEQ ID NO:19.

[0024] NRAS is yet another member of the RAS protein family. NRAS is also known as GTPase NRas, V-Ras neuroblastoma RAS viral oncogene homolog, or NRAS1. WT NRAS has the amino acid sequence of SEQ ID NO:20.

[0025] One embodiment of the present invention provides an isolated or purified TCR, wherein the TCR has antigenic specificity for a mutant human RAS amino acid sequence in which glycine at position 12 is replaced by arginine, wherein the mutant human RAS amino acid sequence is the amino acid sequence of mutant human KRAS, mutant human HRAS, or mutant human NRAS, and wherein position 12 is defined with reference to the WT human KRAS protein, the WT human HRAS protein, or the WT human NRAS protein, respectively. Hereinafter, unless otherwise specified, "TCR" also refers to the functional portion and functional variants of the TCR.

[0026] The mutant human RAS amino acid sequence can be a mutant human KRAS amino acid sequence, a mutant human HRAS amino acid sequence, or a mutant human NRAS amino acid sequence. The amino acid sequences of WT human KRAS, NRAS, and HRAS proteins each have a length of 188 or 189 amino acid residues and are highly identical to each other. For example, the amino acid sequence of the WT human NRAS protein is 86.8% identical to the amino acid sequence of the WT human KRAS protein. The amino acid residues 1-86 of the WT human NRAS protein and the WT human KRAS protein are 100% identical. The amino acid sequence of the WT human HRAS protein is 86.3% identical to the amino acid sequence of the WT human KRAS protein. The amino acid residues 1-94 of the WT human HRAS protein and the WT human KRAS protein are 100% identical. Hereinafter, unless otherwise specified, the "RAS" (mutant or unmutated (WT)) mentioned commonly refers to KRAS, HRAS, and NRAS.

[0027] In one embodiment of the present invention, the mutant human RAS amino acid sequence includes a human RAS amino acid sequence in which glycine at position 12 is replaced by arginine, where position 12 is defined with reference to the corresponding WT RAS protein. The WT RAS protein can be any one of the WT KRAS protein (SEQ ID NO:17 or 18), the WT HRAS protein (SEQ ID NO:19), or the WT NRAS protein (SEQ ID NO:20), because as explained above, the amino acid residues 1-86 of the WT human NRAS protein and the WT human KRAS protein are 100% identical, and the amino acid residues 1-94 of the WT human HRAS protein and the WT human KRAS protein are 100% identical. Therefore, the amino acid residue at position 12 of each of the WT KRAS, WT HRAS, and WT NRAS proteins is the same, i.e., glycine.

[0028] The mutant human RAS amino acid sequence has glycine at position 12 replaced by arginine. In this regard, the embodiments of the present invention provide TCRs that are antigen-specific for any human RAS protein, polypeptide, or peptide amino acid sequence containing the G12R mutation.

[0029] Mutations and substitutions of RAS are defined herein with reference to the amino acid sequences of the corresponding WT RAS proteins. Thus, mutations and substitutions of RAS are described herein in the following format: the amino acid residue present at a specific position (i.e., position 12) in the WT RAS protein, followed by the position number, followed by the amino acid residue that replaces that residue in the specific mutation or substitution being discussed. The RAS amino acid sequence (e.g., RAS peptide) may include amino acid residues that are fewer than all of the amino acid residues of the full-length WT RAS protein. Thus, position 12 is defined herein with reference to the WT full-length RAS protein (i.e., any one of SEQ ID NOs: 17-20), and it should be understood that the actual position of the corresponding residue in a specific instance of the RAS amino acid sequence may vary. When the position is defined according to any one of SEQ ID NOs: 17-20, the term "G12" refers to the glycine that is normally present at position 12 of any one of SEQ ID NOs: 17-20, and "G12R" indicates that the glycine that is normally present at position 12 of any one of SEQ ID NOs: 17-20 is replaced by arginine. For example, in a specific instance where the RAS amino acid sequence is (e.g.) TEYKLVVVGA G GVGKSALTIQLI (SEQ ID NO:25) (an exemplary WT KRAS peptide corresponding to contiguous amino acid residues 2 to 24 of SEQ ID NO:17), "G12R" refers to the underlined glycine in SEQ ID NO:25 being replaced by valine, but the actual position of the underlined glycine in SEQ ID NO:25 is 11. The human RAS amino acid sequence containing the G12R mutation is hereinafter referred to as "G12R RAS".

[0030] Examples of full-length RAS proteins containing the G12R mutation are set forth in Table 1 below.

[0031] Table 1

[0032] Mutant full-length RAS protein SEQ ID NO: G12R KRAS variant A 21 G12R KRAS variant B 22 G12R HRAS 23 G12R NRAS 24

[0033] In one embodiment of the present invention, the TCR has antigenic specificity for the RAS peptide containing the G12R mutation as set forth above, wherein the G12R RAS peptide has any length. In one embodiment of the present invention, the G12R RAS peptide has any length suitable for binding to any of the HLA class II molecules set forth herein. For example, the TCR may have antigenic specificity for a RAS peptide containing the G12R mutation, the RAS peptide having a length of about 11 to about 30 amino acid residues, about 12 to about 24 amino acid residues, or about 18 to about 20 amino acid residues. The G12R RAS peptide may include any contiguous amino acid residues in the mutant RAS protein that contain the G12R mutation. In one embodiment of the present invention, the TCR may have antigenic specificity for a RAS peptide containing the G12R mutation, the mutant RAS peptide having the following lengths: about 30 amino acid residues, about 29 amino acid residues, about 28 amino acid residues, about 27 amino acid residues, about 26 amino acid residues, about 25 amino acid residues, about 24 amino acid residues, about 23 amino acid residues, about 22 amino acid residues, about 21 amino acid residues, about 20 amino acid residues, about 19 amino acid residues, about 18 amino acid residues, about 17 amino acid residues, about 16 amino acid residues, about 15 amino acid residues, about 14 amino acid residues, about 13 amino acid residues, about 12 amino acid residues, about 11 amino acid residues, or a range of any two of the foregoing values. An example of a specific peptide containing the G12R mutation that can be recognized by the TCR of the present invention is MTEYKLVVVGA R GVGKSALTIQLI (SEQ ID NO:39). In one embodiment of the present invention, the TCR has antigenic specificity for the mutant human RAS amino acid sequence of SEQ ID NO:39. In one embodiment of the present invention, the TCR does not have antigenic specificity for the wild-type human RAS amino acid sequence of SEQ ID NO:40.

[0034] In one embodiment of the present invention, the TCR of the present invention is capable of recognizing G12R RAS presented by an HLA class II molecule. In this regard, the TCR may induce an immune response upon binding to G12R RAS within the context of an HLA class II molecule. The TCR of the present invention is capable of recognizing G12R RAS presented by an HLA class II molecule and may bind to HLA class II molecules other than G12R RAS.

[0035] In one embodiment of the present invention, the HLA class II molecule is an HLA-DR heterodimer. The HLA-DR heterodimer is a cell surface receptor comprising an α chain and a β chain. The HLA-DR α chain is encoded by the HLA-DRA gene. The HLA-DR β chain is encoded by the HLA-DRB1 gene, the HLA-DRB3 gene, the HLA-DRB4 gene, or the HLA-DRB5 gene. Examples of molecules encoded by the HLA-DRB1 gene may include (but are not limited to) HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, HLA-DR5, HLA-DR6, HLA-DR7, HLA-DR8, HLA-DR9, HLA-DR10, HLA-DR11, HLA-DR12, HLA-DR13, HLA-DR14, HLA-DR15, HLA-DR16, and HLA-DR17. The HLA-DRB3 gene encodes HLA-DR52. The HLA-DRB4 gene encodes HLA-DR53. The HLA-DRB5 gene encodes HLA-DR51. In one embodiment of the present invention, the HLA class II molecule is an HLA-DRB5:HLA-DRA heterodimer. In a particularly preferred embodiment, the HLA class II molecule is expressed by the HLA-DRB5*01:HLA-DRA*01:01 allele (i.e., the HLA-DRB5*01:HLA-DRA*01:01 heterodimer).

[0036] In another embodiment of the present invention, the HLA class II molecule is an HLA-DQ heterodimer. The HLA-DQ heterodimer is a cell surface receptor comprising an α-chain and a β-chain. The HLA-DQ α-chain is encoded by the HLA-DQA1 gene. HLA-DQA1 alleles may include DQA1*01:01, DQA1*01:02, DQA1*01:03, DQA1*01:04, DQA1*02:01, DQA1*03:01, DQA1*03:02, DQA1*03:03, DQA1*04:01, DQA1*05:01, DQA1*05:05, and DQA1*06:01. The HLA-DQ β-chain is encoded by the HLA-DQB1 gene. HLA-DQB1 alleles may include HLA-DQB1*02:01, HLA-DQB1*02:02, HLA-DQB1*02:03, HLA-DQB1*03:01, HLA-DQB1*03:02, HLA-DQB1*03:03, HLA-DQB1*03:04, HLA-DQB1*03:05, HLA-DQB1*04:01, HLA-DQB1*04:02, HLA-DQB1*05:01, HLA-DQB1*05:02, HLA-DQB1*05:03, HLA-DQB1*05:04, HLA-DQB1*06:01, HLA-DQB1*06:02, HLA-DQB1*06:03, HLA-DQB1*06:04, HLA-DQB1*06:05, and HLA-DQB1*06:09. In one embodiment of the present invention, the HLA class II molecule is an HLA-DQA1:HLA-DQB1 heterodimer. In a particularly preferred embodiment, the HLA class II molecule is expressed by the HLA-DQA1*05:05:HLA-DQB1*03:01 alleles (i.e., the HLA-DQA1*05:05:HLA-DQB1*03:01 heterodimer).

[0037] The TCRs of the present invention can provide any one or more of a variety of advantages, including for adoptive cell transfer when expressed by cells. G12R RAS is expressed by cancer cells and not by normal, non-cancerous cells. Without being limited to a particular theory or mechanism, it is believed that the TCRs of the present invention advantageously destroy cancer cells and at the same time minimize or eliminate the destruction of normal, non-cancerous cells, thereby reducing (e.g., by minimizing or eliminating) toxicity. In addition, because the G12R mutation can occur early in tumor formation, the G12R RAS mutation can be expressed on substantially all patient cancer cells. The TCRs of the present invention can advantageously and successfully treat or prevent G12R RAS-positive cancers that do not respond to other types of treatment (e.g., chemotherapy, surgery, or radiation). Additionally, the TCRs of the present invention can recognize G12R RAS with high affinity, which can enable the recognition of unmanipulated tumor cells (e.g., tumor cells that have not been treated with interferon (IFN)-γ, transfected with a vector encoding one or both of G12R RAS and HLA-DRB5*01:HLA-DRA*01:01, one or both of G12R RAS and HLA-DQA1*05:05:HLA-DQB1*03:01, pulsed with G12R RAS peptides, or a combination thereof). Furthermore, the HLA-DRB5*01 allele is expressed in the human population at a frequency of approximately 18%, and HLA-DQA1*05:05:HLA-DQB1*03:01 is expressed at a frequency of approximately 11% in the Caucasian race in the United States. Thus, the TCRs of the present invention can increase the number of cancer patients eligible for immunotherapy to include patients who express one or both of the HLA-DRB5*01:HLA-DRA*01:01 allele and the HLA-DQA1*05:05:HLA-DQB1*03:01 allele and who may not be eligible for immunotherapy using TCRs that recognize G12R RAS presented by other MHC molecules. Based on the frequencies of the HLA-DRB5*01:HLA-DRA*01:01 and HLA-DQA1*05:05:HLA-DQB1*03:01 alleles and the estimated G12R RAS expression in various cancers in the United States, it is estimated that greater than approximately 1,000 pancreatic cancer patients are potentially eligible for treatment with a TCR of the present invention each year. For example, the KRAS G12R mutation is expressed in greater than approximately 8% of pancreatic cancer patients.

[0038] As used herein, the phrase "antigen specific" means that the TCR can specifically bind with high avidity and immunologically recognize G12R RAS. For example, after co-culture with (a) antigen-negative, HLA class II molecule-positive target cells pulsed with low concentrations of G12R RAS peptide (e.g., from about 0.05 ng / mL to about 10 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 8 ng / mL, 10 ng / mL, or a range defined by any two of the foregoing values) or (b) antigen-negative, HLA class II molecule-positive target cells into which a nucleotide sequence encoding G12R RAS has been introduced for expression of G12R RAS, if about 1×10 4 to about 1×10 5 T cells expressing the TCR secrete at least about 200 pg / mL or more (e.g., 200 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, 5,000 pg / mL or more, 7,000 pg / mL or more, 10,000 pg / mL or more, 20,000 pg / mL or more, or a range defined by any two of the foregoing values) of IFN-γ, then the TCR can be considered to have "antigen specificity" for G12R RAS. After co-culture with antigen-negative, HLA class II molecule-positive target cells pulsed with higher concentrations of G12R RAS peptide, cells expressing the TCR of the present invention can also secrete IFN-γ. The HLA class II molecule can be any HLA class II molecule described herein (e.g., the HLA-DRB5*01:HLA-DRA*01:01 heterodimer or the HLA-DQA1*05:05:HLA-DQB1*03:01 heterodimer).

[0039] Alternatively or additionally, after co-culture with (a) antigen-negative, HLA class II molecule-positive target cells pulsed with low concentration G12R RAS peptide or (b) antigen-negative, HLA class II molecule-positive target cells into which a nucleotide sequence encoding G12R RAS has been introduced such that the target cells express G12R RAS, a TCR can be considered to have "antigen specificity" for G12R RAS if T cells expressing the TCR secrete at least twice as much IFN-γ as compared to the amount of IFN-γ expressed by a negative control. The negative control can be, for example, (i) T cells expressing a TCR and co-cultured with (a) antigen-negative, HLA class II molecule-positive target cells pulsed with the same concentration of an irrelevant peptide (e.g., some other peptide having a different sequence from the G12R RAS peptide) or (b) antigen-negative, HLA class II molecule-positive target cells into which a nucleotide sequence encoding an irrelevant peptide has been introduced such that the target cells express the irrelevant peptide; or (ii) untransduced T cells (e.g., derived from PBMCs that do not express a TCR) co-cultured with (a) antigen-negative, HLA class II molecule-positive target cells pulsed with the same concentration of G12R RAS peptide or (b) antigen-negative, HLA class II molecule-positive target cells into which a nucleotide sequence encoding G12R RAS has been introduced such that the target cells express G12R RAS. The HLA class II molecules expressed by the target cells of the negative control are the same as the HLA class II molecules expressed by the target cells co-cultured with the T cells being tested. The HLA class II molecules can be any of the HLA class II molecules described herein (e.g., the HLA-DRB5*01:HLA-DRA*01:01 heterodimer or the HLA-DQA1*05:05:HLA-DQB1*03:01 heterodimer). The IFN-γ secretion can be measured by methods known in the art (e.g., enzyme-linked immunosorbent assay (ELISA)).

[0040] Alternatively or additionally, after co-culture with (a) antigen-negative, HLA class II molecule-positive target cells pulsed with low concentration G12R RAS peptide or (b) antigen-negative, HLA class II molecule-positive target cells into which a nucleotide sequence encoding G12R RAS has been introduced such that the target cells express G12R RAS antigen, a TCR can be considered to have "antigen specificity" for G12R RAS if at least twice as many T cells expressing the TCR secrete IFN-γ as compared to the number of negative control T cells that secrete IFN-γ. The HLA class II molecules, peptide concentration, and negative control can be as described herein for other aspects of the invention. The number of cells secreting IFN-γ can be measured by methods known in the art (e.g., ELISPOT).

[0041] Alternatively or additionally, after stimulation of target cells expressing G12R RAS, if a TCR-expressing T cell upregulates the expression of one or more T cell activation markers (such as, for example, as measured by flow cytometry), the TCR can be considered to have "antigen specificity" for G12R RAS. Examples of T cell activation markers include 4-1BB, OX40, CD107a, CD69, and cytokines (such as tumor necrosis factor (TNF), interleukin (IL)-2, etc.) that are upregulated upon antigen stimulation.

[0042] One embodiment of the invention provides a TCR comprising two polypeptides (i.e., polypeptide chains), which are, for example, the alpha (α) chain of a TCR, the beta (β) chain of a TCR, the gamma (γ) chain of a TCR, the delta (δ) chain of a TCR, or a combination thereof. The polypeptides of the TCR of the invention can comprise any amino acid sequence, provided that the TCR has antigen specificity for G12R RAS. In some embodiments, the TCR is non-natural.

[0043] In one embodiment of the invention, the TCR comprises two polypeptide chains, each of which comprises a variable region containing the complementarity determining regions (CDRs) 1, 2, and 3 of the TCR. In one embodiment of the invention, the TCR comprises: a first polypeptide chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:1 (CDR1 of the α chain of 4268TCR), a CDR2 having the amino acid sequence of SEQ ID NO:2 (CDR2 of the α chain of 4268TCR), and a CDR3 having the amino acid sequence of SEQ ID NO:3 (CDR3 of the α chain of 4268TCR); and a second polypeptide chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:4 (CDR1 of the β chain of 4268TCR), a CDR2 having the amino acid sequence of SEQ ID NO:5 (CDR2 of the β chain of 4268TCR), and a CDR3 having the amino acid sequence of SEQ ID NO:6 (CDR3 of the β chain of 4268TCR).

[0044] In another embodiment of the present invention, the TCR comprises: a first polypeptide chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:7 (CDR1 of the α-chain of 4270TCR), a CDR2 having the amino acid sequence of SEQ ID NO:8 (CDR2 of the α-chain of 4270TCR), and a CDR3 having the amino acid sequence of SEQ ID NO:9 (CDR3 of the α-chain of 4270TCR); and a second polypeptide chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:10 (CDR1 of the β-chain of 4270TCR), a CDR2 having the amino acid sequence of SEQ ID NO:11 (CDR2 of the β-chain of 4270TCR), and a CDR3 having the amino acid sequence of SEQ ID NO:12 (CDR3 of the β-chain of 4270TCR).

[0045] In this regard, the TCR of the present invention may comprise any one or more of the amino acid sequences selected from SEQ ID NO:1-12. In one embodiment of the present invention, the TCR comprises the following amino acid sequences: (a) all of SEQ ID NO:1-3, (b) all of SEQ ID NO:4-6, (c) all of SEQ ID NO:7-9, (d) all of SEQ ID NO:10-12, (e) all of SEQ ID NO:1-6, or (f) all of SEQ ID NO:7-12. In a particularly preferred embodiment, the TCR comprises the following amino acid sequences: (i) all of SEQ ID NO:1-6 or (ii) all of SEQ ID NO:7-12.

[0046] In one embodiment of the present invention, the TCR comprises the amino acid sequence of the variable region of the TCR containing the above CDRs. In this regard, the TCR may comprise the following amino acid sequences: SEQ ID NO:13 (variable region of the α-chain of 4268TCR with an N-terminal signal peptide); SEQ ID NO:14 (variable region of the β-chain of 4268TCR with an N-terminal signal peptide); SEQ ID NO:15 (variable region of the α-chain of 4270TCR with an N-terminal signal peptide); SEQ ID NO:16 (variable region of the β-chain of 4270TCR with an N-terminal signal peptide); SEQ ID NO:43 (variable region of the α-chain of 4268TCR without an N-terminal signal peptide); SEQ ID NO:44 (variable region of the β-chain of 4268TCR without an N-terminal signal peptide); SEQ ID NO:41 (variable region of the α-chain of 4270TCR without an N-terminal signal peptide); SEQ ID NO:42 (variable region of the β-chain of 4270TCR without an N-terminal signal peptide); both SEQ ID NO:13 and 14; both SEQ ID NO:15 and 16; both SEQ ID NO:41 and 42; or both SEQ ID NO:43 and 44. Preferably, the TCR comprises the following amino acid sequences: (i) both SEQ ID NO:13 and 14; (ii) both SEQ ID NO:15 and 16; (iii) both SEQ ID NO:41 and 42; or (iv) both SEQ ID NO:43 and 44.

[0047] The TCR of the present invention may further comprise an α-chain constant region and a β-chain constant region. The constant regions may be derived from any suitable species, such as human or mouse. In one embodiment of the present invention, the TCR further comprises murine α- and β-chain constant regions or human α- and β-chain constant regions. As used herein, when referring to the TCR or any component of the TCR described herein (such as CDRs, variable regions, constant regions, α-chain and / or β-chain), the terms "murine" or "human" mean TCRs (or their components) derived from mice or humans, respectively, i.e., TCRs (or their components) originating from murine T cells or human T cells or expressed therefrom once.

[0048] One embodiment of the invention provides a chimeric TCR comprising a human variable region and a murine constant region, wherein the TCR has antigen specificity for a mutant human RAS amino acid sequence in which glycine at position 12 is replaced by arginine. The murine constant region can provide any one or more advantages. For example, the murine constant region can reduce the mismatch between the TCR of the invention and the endogenous TCR of the host cell into which the TCR of the invention is introduced. Alternatively or additionally, the murine constant region can increase the expression of the TCR of the invention compared to the same TCR having a human constant region. The chimeric TCR can comprise the amino acid sequence of SEQ ID NO:28 (WT murine α-chain constant region), SEQ ID NO:29 (WT murine β-chain constant region), or both SEQ ID NO:28 and 29. Preferably, the TCR of the invention comprises the amino acid sequences of both SEQ ID NO:28 and 29. The chimeric TCR can comprise a combination of any murine constant region described herein and any CDR region described herein for other aspects of the invention. In this regard, the TCR can comprise the following amino acid sequences: (a) all of SEQ ID NO:1-3 and 28; (b) all of SEQ ID NO:4-6 and 29; (c) all of SEQ ID NO:7-9 and 28; (d) all of SEQ ID NO:10-12 and 29; (e) all of SEQ ID NO:1-6 and 28-29; or (f) all of SEQ ID NO:7-12 and 28-29. In another embodiment of the invention, the chimeric TCR can comprise a combination of any murine constant region described herein and any variable region described herein for other aspects of the invention. In this regard, the TCR can comprise the following amino acid sequences: (i) both SEQ ID NO:13 and 28; (ii) both SEQ ID NO:14 and 29; (iii) both SEQ ID NO:15 and 28; (iv) both SEQ ID NO:16 and 29; (v) all of SEQ ID NO:13-14 and 28-29; or (vi) all of SEQ ID NO:15-16 and 28-29.

[0049] In another embodiment of the present invention, the TCR comprises the following amino acid sequences: SEQ ID NO:34 (4268 TCR α-chain with WT murine constant region and N-terminal signal peptide), SEQ ID NO:35 (4268 TCR β-chain with WT murine constant region and N-terminal signal peptide), SEQ ID NO:36 (4270 TCR α-chain with WT murine constant region and N-terminal signal peptide), SEQ ID NO:37 (4270 TCR β-chain with WT murine constant region and N-terminal signal peptide), SEQ ID NO:49 (4268 TCR α-chain with WT murine constant region and without N-terminal signal peptide), SEQ ID NO:50 (4268 TCR β-chain with WT murine constant region and without N-terminal signal peptide), SEQ ID NO:51 (4270 TCR α-chain with WT murine constant region and without N-terminal signal peptide), SEQ ID NO:52 (4270 TCR β-chain with WT murine constant region and without N-terminal signal peptide), both of SEQ ID NO:34-35, both of SEQ ID NO:36-37, both of SEQ ID NO:49-50 or both of SEQ ID NO:51-52.

[0050] In one embodiment of the present invention, the TCR comprises an α chain containing a variable region and a constant region and a β chain containing a variable region and a constant region. In this regard, the TCR can comprise (a) an α chain comprising the amino acid sequence of SEQ ID NO: 30 (α chain of 4268 TCR with an N-terminal signal peptide), wherein: (i) X at position 180 of SEQ ID NO: 30 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 246 of SEQ ID NO: 30 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 247 of SEQ ID NO: 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) a β chain comprising the amino acid sequence of SEQ ID NO: 31 (β chain of 4268 TCR with an N-terminal signal peptide), wherein X at position 198 of SEQ ID NO: 31 is Ser or Cys; (c) an α (α chain of 4270 TCR with an N-terminal signal peptide) comprising the amino acid sequence of SEQ ID NO: 32, wherein: (i) X at position 188 of SEQ ID NO: 32 is Thr or Cys; (ii) X at position 252 of SEQ ID NO: 32 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 254 of SEQ ID NO: 32 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 255 of SEQ ID NO: 32 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) a β chain comprising the amino acid sequence of SEQ ID NO: 33 (β chain of 4270 TCR with an N-terminal signal peptide), wherein X at position 191 of SEQ ID NO: 33 is Ser or Cys; (e) both (a) and (b); (f) both (c) and (d); (g) an α chain comprising the amino acid sequence of SEQ ID NO: 45 (α chain of 4268 TCR without an N-terminal signal peptide), wherein: (i) X at position 161 of SEQ ID NO: 45 is Thr or Cys; (ii) X at position 225 of SEQ ID NO: 45 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 227 of SEQ ID NO: 45 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp;and (iv) X at position 228 of SEQ ID NO:45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (h) a β-chain comprising the amino acid sequence of SEQ ID NO:46 (β-chain of 4268 TCR without an N-terminal signal peptide), wherein X at position 178 of SEQ ID NO:46 is Ser or Cys; (i) an α-chain comprising the amino acid sequence of SEQ ID NO:47 (α-chain of 4270 TCR without an N-terminal signal peptide), wherein: (i) X at position 168 of SEQ ID NO:47 is Thr or Cys; (ii) X at position 232 of SEQ ID NO:47 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 234 of SEQ ID NO:47 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 235 of SEQ ID NO:47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (j) a β-chain comprising the amino acid sequence of SEQ ID NO:48 (β-chain of 4270 TCR without an N-terminal signal peptide), wherein X at position 171 of SEQ ID NO:48 is Ser or Cys; (k) both (g) and (h); or (l) both (i) and (j).;

[0051] In one embodiment of the invention, the TCR comprises a substituted constant region. In this regard, the TCR can comprise the amino acid sequence of any of the TCRs set forth herein, having one, two, three or four amino acid substitutions in the constant region of one or both of the α and β chains. Preferably, the TCR comprises a murine constant region having one, two, three or four amino acid substitutions in the murine constant region of one or both of the α and β chains. In a particularly preferred embodiment, the TCR comprises a murine constant region having one, two, three or four amino acid substitutions in the murine constant region of the α chain and one amino acid substitution in the murine constant region of the β chain. In some embodiments, compared to a parental TCR comprising an unsubstituted (wild-type) constant region, the TCR comprising a substituted constant region advantageously provides one or more of the following advantages: increased G12R RAS +Target recognition, increased host cell expression, reduced endogenous TCR mismatch, and increased anti-tumor activity. Generally, the substituted amino acid sequences SEQ ID NO: 26 and 27 of the murine constant regions of the TCR α and β chains respectively correspond to all or a part of the unsubstituted murine constant region amino acid sequences SEQ ID NO: 28 and 29, wherein SEQ ID NO: 26 has one, two, three, or four amino acid substitutions compared to SEQ ID NO: 28 and SEQ ID NO: 27 has one amino acid substitution compared to SEQ ID NO: 29. In this regard, one embodiment of the present invention provides a TCR comprising the following amino acid sequences: (a) SEQ ID NO: 26 (constant region of the α chain), wherein (i) X at position 48 is Thr or Cys; (ii) X at position 112 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 114 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) SEQ ID NO: 27 (constant region of the β chain), wherein X at position 57 is Ser or Cys; or (c) both SEQ ID NO: 26 and 27. In one embodiment of the present invention, the TCR comprising SEQ ID NO: 26 does not include SEQ ID NO: 28 (unsubstituted murine constant region of the α chain). In one embodiment of the present invention, the TCR comprising SEQ ID NO: 27 does not include SEQ ID NO: 29 (unsubstituted murine constant region of the β chain).

[0052] In one embodiment of the invention, the substituted constant region comprises a cysteine substitution in the constant region of one or both of the α and β chains to provide a cysteine-substituted TCR. The opposing cysteines in the α and β chains provide a disulfide bond that links the constant regions of the α and β chains of the substituted TCR to each other and that is not present in TCRs comprising unsubstituted murine constant regions. In this regard, the TCR can be a cysteine-substituted TCR in which one or both of the native Thr (Thr48) at position 48 of SEQ ID NO:28 and the native Ser (Ser57) at position 57 of SEQ ID NO:29 can be substituted with Cys. Preferably, both the native Thr48 of SEQ ID NO:28 and the native Ser57 of SEQ ID NO:29 are substituted with Cys. Examples of cysteine-substituted TCR constant region sequences are set forth in Table 2. In one embodiment of the invention, the cysteine-substituted TCR comprises (i) SEQ ID NO:26, (ii) SEQ ID NO:27, or (iii) both SEQ ID NO:26 and 27, where both SEQ ID NO:26 and 27 are as defined in Table 2. In addition to any CDR or variable region set forth herein, the cysteine-substituted TCRs of the invention can also comprise a substituted constant region.

[0053] In one embodiment of the invention, the cysteine-substituted chimeric TCR comprises a full-length α chain and a full-length β chain. Examples of cysteine-substituted chimeric TCR α and β chain sequences are set forth in Table 2. In one embodiment of the invention, the TCR comprises (i) SEQ ID NO:30, (ii) SEQ ID NO:31, (iii) SEQ ID NO:32, (iv) SEQ ID NO:33, (v) both SEQ ID NO:30 and 31, (vi) both SEQ ID NO:32 and 33, (vii) SEQ ID NO:45, (viii) SEQ ID NO:46, (ix) SEQ ID NO:47, (x) SEQ ID NO:48, (xi) both SEQ ID NO:45 and 46, or (xii) both SEQ ID NO:47 and 48, where SEQ ID NO:30-33 and 45-48 are all as defined in Table 2.

[0054] Table 2

[0055]

[0056]

[0057] In one embodiment of the present invention, one, two, or three amino acids in the transmembrane (TM) domain of the constant region of one or both of the α and β chains of the substituted amino acid sequence are substituted with hydrophobic amino acids to provide a TCR substituted with hydrophobic amino acids (also referred to herein as "LVL-modified TCR"). Compared to a TCR without hydrophobic amino acid substitution in the TM domain, the hydrophobic amino acid substitution in the TM domain of the TCR can increase the hydrophobicity of the TM domain of the TCR. In this regard, the TCR is an LVL-modified TCR, wherein one, two, or three of the natural Ser112, Met114, and Gly115 of SEQ ID NO: 28 can be independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, preferably Leu, Ile, or Val. Preferably, all three of the natural Ser112, Met114, and Gly115 of SEQ ID NO: 28 can be independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, preferably Leu, Ile, or Val. In one embodiment of the present invention, the LVL-modified TCR comprises (i) SEQ ID NO: 26, (ii) SEQ ID NO: 27, or (iii) both SEQ ID NO: 26 and 27, wherein both SEQ ID NO: 26 and 27 are as defined in Table 3. In addition to any CDR or variable region described herein, the LVL-modified TCR of the present invention may comprise a substituted constant region.

[0058] In one embodiment of the present invention, the LVL-modified TCR comprises a full-length α chain and a full-length β chain. Examples of the LVL-modified TCR α and β chain sequences are set forth in Table 3. In one embodiment of the present invention, the LVL-modified TCR comprises (i) SEQ ID NO: 30, (ii) SEQ ID NO: 31, (iii) SEQ ID NO: 32, (iv) SEQ ID NO: 33, (v) both SEQ ID NO: 30 and 31, (vi) both SEQ ID NO: 32 and 33, (vii) SEQ ID NO: 45, (viii) SEQ ID NO: 46, (ix) SEQ ID NO: 47, (x) SEQ ID NO: 48, (xi) both SEQ ID NO: 45 and 46, or (xii) both SEQ ID NO: 47 and 48, wherein SEQ ID NO: 30-33 and 45-48 are all as defined in Table 3.

[0059] Table 3

[0060]

[0061]

[0062] In one embodiment of the present invention, the substituted amino acid sequence comprises the following combination of substitutions: a cysteine substitution in the constant region of one or both of the α and β chains; and substitution of one, two or three amino acids in the transmembrane (TM) domain of one or both of the constant region α and β chains with hydrophobic amino acids (also referred to herein as "cysteine-substituted, LVL-modified TCR"). In this regard, the TCR is a cysteine-substituted, LVL-modified chimeric TCR, wherein the native Thr48 of SEQ ID NO:28 is substituted with Cys; one, two or three of the native Ser112, Met114 and Gly115 of SEQ ID NO:28 are independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met or Trp, preferably Leu, Ile or Val; and the native Ser57 of SEQ ID NO:29 is substituted with Cys. Preferably, all three of the native Ser112, Met114 and Gly115 of SEQ ID NO:28 can be independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met or Trp, preferably Leu, Ile or Val. In one embodiment of the present invention, the cysteine-substituted, LVL-modified TCR comprises (i) SEQ ID NO:26, (ii) SEQ ID NO:27 or (iii) both SEQ ID NO:26 and 27, wherein both SEQ ID NO:26 and 27 are defined as in Table 4. In addition to any CDR or variable region described herein, the cysteine-substituted, LVL-modified TCR of the present invention may comprise a substituted constant region.

[0063] In one embodiment, the cysteine-substituted, LVL-modified TCR comprises a full-length α chain and a full-length β chain. In one embodiment of the present invention, the cysteine-substituted, LVL-modified TCR comprises (i) SEQ ID NO:30, (ii) SEQ ID NO:31, (iii) SEQ ID NO:32, (iv) SEQ ID NO:33, (v) both SEQ ID NO:30 and 31, (vi) both SEQ ID NO:32 and 33, (vii) SEQ ID NO:45, (viii) SEQ ID NO:46, (ix) SEQ ID NO:47, (x) SEQ ID NO:48, (xi) both SEQ ID NO:45 and 46 or (xii) both SEQ ID NO:47 and 48, wherein SEQ ID NO:30-33 and 45-48 are all defined as in Table 4.

[0064] Table 4

[0065]

[0066]

[0067]

[0068] The present invention also provides a polypeptide comprising a functional portion of any of the TCRs set forth herein. As used herein, the term "polypeptide" includes oligopeptides and refers to a single chain of amino acids linked by one or more peptide bonds.

[0069] For the polypeptides of the present invention, the functional portion can be any portion that includes contiguous amino acids of a TCR as part thereof, provided that the functional portion specifically binds to G12R RAS. The term "functional portion", when referring to the use of a TCR, refers to any portion or fragment of a TCR of the present invention that retains the biological activity of the TCR (parent TCR) of which it is a part. Functional portions encompass, for example, those portions of a TCR that are still able to specifically bind to G12R RAS to a similar, the same or a higher degree compared to the parent TCR (e.g., within the context of an HLA-DRB5*01:HLA-DRA*01:01 heterodimer or an HLA-DQA1*05:05:HLA-DQB1*03:01 heterodimer) or are able to detect, treat or prevent cancer. With reference to the parent TCR, the functional portion can include, for example, about 10%, about 25%, about 30%, about 50%, about 68%, about 80%, about 90%, about 95% or more of the parent TCR.

[0070] The functional portion can include other amino acids at the amino or carboxyl terminus or both termini of the portion, which other amino acids are not found in the amino acid sequence of the parent TCR. Desirably, the other amino acids do not interfere with the biological function of the functional portion (e.g., specifically binding to G12R RAS and / or being able to detect cancer, treat or prevent cancer, etc.). More desirably, the other amino acids can enhance the biological activity compared to the biological activity of the parent TCR.

[0071] The polypeptide may include a functional portion of either or both of the α and β chains of the TCR of the present invention, for example, a functional portion including one or more of CDR1, CDR2, and CDR3 of the variable region of the α chain and / or β chain in the TCR of the present invention. In one embodiment of the present invention, the polypeptide may include the amino acid sequence of SEQ ID NO:1 (CDR1 of the α chain), SEQ ID NO:2 (CDR2 of the α chain), SEQ ID NO:3 (CDR3 of the α chain), SEQ ID NO:4 (CDR1 of the β chain), SEQ ID NO:5 (CDR2 of the β chain), SEQ ID NO:6 (CDR3 of the β chain), or a combination thereof. In another embodiment of the present invention, the polypeptide may include the amino acid sequence of SEQ ID NO:7 (CDR1 of the α chain), SEQ ID NO:8 (CDR2 of the α chain), SEQ ID NO:9 (CDR3 of the α chain), SEQ ID NO:10 (CDR1 of the β chain), SEQ ID NO:11 (CDR2 of the β chain), SEQ ID NO:12 (CDR3 of the β chain), or a combination thereof.

[0072] In this regard, the polypeptide of the present invention may include any one or more of the amino acid sequences selected from SEQ ID NO:1-12. In one embodiment of the present invention, the TCR includes the following amino acid sequences: (a) all of SEQ ID NO:1-3, (b) all of SEQ ID NO:4-6, (c) all of SEQ ID NO:7-9, (d) all of SEQ ID NO:10-12, (e) all of SEQ ID NO:1-6, or (f) all of SEQ ID NO:7-12. In a preferred embodiment, the polypeptide includes the following amino acid sequences: (i) all of SEQ ID NO:1-6 or (ii) all of SEQ ID NO:7-12.

[0073] In one embodiment of the present invention, the polypeptide of the present invention may include, for example, the variable region of the TCR of the present invention comprising a combination of the above CDR regions. In this regard, the polypeptide may include the following amino acid sequences: (i) SEQ ID NO:13 (variable region of the α chain), (ii) SEQ ID NO:14 (variable region of the β chain), (iii) both SEQ ID NO:13 and 14, (iv) SEQ ID NO:15 (variable region of the α chain), (v) SEQ ID NO:16 (variable region of the β chain), (vi) both SEQ ID NO:15 and 16, (vii) SEQ ID NO:41 (variable region of the α chain), (viii) SEQ ID NO:42 (variable region of the β chain), (ix) both SEQ ID NO:41 and 42, (x) SEQ ID NO:43 (variable region of the α chain), (xi) SEQ ID NO:44 (variable region of the β chain), or (xii) both SEQ ID NO:43 and 44. Preferably, the polypeptide includes the following amino acid sequences: (i) both SEQ ID NO:13 and 14, (ii) both SEQ ID NO:15 and 16, (iii) both SEQ ID NO:41 and 42, or (iv) SEQ ID NO:43 and 44.

[0074] In one embodiment of the present invention, the polypeptide of the present invention may further include the constant region of the TCR of the present invention as described above. In this regard, the polypeptide may further include the following amino acid sequences: SEQ ID NO:28 (WT murine constant region of the α chain), SEQ ID NO:29 (WT murine constant region of the β chain), SEQ ID NO:26 (substituted murine constant region of the α chain), SEQ ID NO:27 (substituted murine constant region of the β chain), both SEQ ID NO:28 and 29, or both SEQ ID NO:26 and 27. Preferably, the polypeptide further includes a combination of the amino acid sequences of both SEQ ID NO:26 and 27 or both SEQ ID NO:28 and 29 with any of the CDR regions or variable regions described herein for other aspects of the present invention. In one embodiment of the present invention, one or both of SEQ ID NO:26 and 27 of the polypeptide are defined as any of those in Table 2-4.

[0075] In one embodiment of the present invention, the polypeptide of the present invention may comprise the entire length of the α or β chain of the TCR as set forth herein. In this regard, the polypeptide of the present invention may comprise the following amino acid sequences: SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, both SEQ ID NO:30-31, both SEQ ID NO:32-33, SEQ ID NO:34-35, both SEQ ID NO:36-37, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, both SEQ ID NO:45 and 46, both SEQ ID NO:47 and 48, both SEQ ID NO:49 and 50, or both SEQ ID NO:51 and 52. Alternatively, the polypeptide of the present invention may comprise both chains of the TCR as set forth herein.

[0076] For example, the polypeptides of the present invention may include (a) the amino acid sequence of SEQ ID NO: 30, wherein: (i) X at position 180 of SEQ ID NO: 30 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 246 of SEQ ID NO: 30 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 247 of SEQ ID NO: 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) the amino acid sequence of SEQ ID NO: 31, wherein X at position 198 of SEQ ID NO: 31 is Ser or Cys; (c) the amino acid sequence of SEQ ID NO: 32, wherein: (i) X at position 188 of SEQ ID NO: 32 is Thr or Cys; (ii) X at position 252 of SEQ ID NO: 32 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 254 of SEQ ID NO: 32 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 255 of SEQ ID NO: 32 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) the amino acid sequence of SEQ ID NO: 33, wherein X at position 191 of SEQ ID NO: 33 is Ser or Cys; (e) both (a) and (b); (f) both (c) and (d); (g) the amino acid sequence of SEQ ID NO: 45, wherein: (i) X at position 161 of SEQ ID NO: 45 is Thr or Cys; (ii) X at position 225 of SEQ ID NO: 45 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 227 of SEQ ID NO: 45 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 228 of SEQ ID NO: 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (h) the amino acid sequence of SEQ ID NO: 46, wherein X at position 178 of SEQ ID NO: 46 is Ser or Cys; (i) the amino acid sequence of SEQ ID NO: 47, wherein: (i) X at position 168 of SEQ ID NO: 47 is Thr or Cys;(ii) X at position 232 of SEQ ID NO:47 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 234 of SEQ ID NO:47 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 235 of SEQ ID NO:47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (j) the amino acid sequence of SEQ ID NO:48, wherein X at position 171 of SEQ ID NO:48 is Ser or Cys; (k) both (g) and (h); or (l) both (i) and (j). In one embodiment of the present invention, any one or more of SEQ ID NOs: 30-33 and 45-48 of the polypeptide is defined as in any one of Tables 2-4.;

[0077] The present invention further provides a protein comprising at least one polypeptide as set forth herein. "Protein" means a molecule comprising one or more polypeptide chains.

[0078] In one embodiment, the protein of the present invention may comprise (a) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:1-3 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:4-6; or (b) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:7-9 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:10-12.

[0079] In another embodiment of the present invention, the protein may comprise (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:13 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:14; (ii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:15 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:16; (iii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:41 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:42; or (iv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:43 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:44.

[0080] The protein of the present invention may further comprise any constant region as set forth herein with respect to other aspects of the present invention. In this regard, in one embodiment of the present invention, (i) the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 26 and the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 27; or (ii) the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 28 and the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 29. In one embodiment of the present invention, one or both of SEQ ID NOs: 26 and 27 of the protein are defined as in any one of Tables 2-4.

[0081] Alternatively or additionally, the protein of an embodiment of the present invention may comprise (a) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 30, wherein: (i) X at position 180 of SEQ ID NO: 30 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 246 of SEQ ID NO: 30 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 247 of SEQ ID NO: 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 31, wherein X at position 198 of SEQ ID NO: 31 is Ser or Cys; (c) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 32, wherein: (i) X at position 188 of SEQ ID NO: 32 is Thr or Cys; (ii) X at position 252 of SEQ ID NO: 32 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 254 of SEQ ID NO: 32 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 255 of SEQ ID NO: 32 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 33, wherein X at position 191 of SEQ ID NO: 33 is Ser or Cys; (e) both (a) and (b); (f) both (c) and (d); (g) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 45, wherein: (i) X at position 161 of SEQ ID NO: 45 is Thr or Cys; (ii) X at position 225 of SEQ ID NO: 45 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 227 of SEQ ID NO: 45 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 228 of SEQ ID NO: 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (h) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 46, wherein X at position 178 of SEQ ID NO: 46 is Ser or Cys;(i) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 47, wherein: (i) X at position 168 of SEQ ID NO: 47 is Thr or Cys; (ii) X at position 232 of SEQ ID NO: 47 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 234 of SEQ ID NO: 47 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 235 of SEQ ID NO: 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (j) A second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 48, wherein X at position 171 of SEQ ID NO: 48 is Ser or Cys; (k) Both (g) and (h); or (l) Both (i) and (j). In one embodiment of the present invention, one or more of SEQ ID NOs: 30-33 and 45-48 are defined as in any of those defined in Tables 2-4.;

[0082] The protein of the present invention may be a TCR. Alternatively, for example, if the protein comprises a single polypeptide chain containing the amino acid sequences of both SEQ ID NO: 30 and 31, both SEQ ID NO: 32 and 33, or if the first and / or second polypeptide chain of the protein further comprises other amino acid sequences (such as amino acid sequences encoding an immunoglobulin or a portion thereof), then the protein of the present invention may be a fusion protein. In this regard, the present invention also provides a fusion protein comprising at least one polypeptide of the present invention as described herein and at least one other polypeptide. The other polypeptide may exist as a separate polypeptide of the fusion protein or may exist as a polypeptide expressed in-frame (in tandem) with one of the polypeptides of the present invention as described herein. The other polypeptide may encode any peptide or protein 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.).

[0083] The fusion protein may comprise one or more copies of the polypeptide of the present invention and / or one or more copies of the other polypeptide. For example, the fusion protein may comprise 1, 2, 3, 4, 5 or more copies of the polypeptide of the present invention and / or the other polypeptide. Suitable methods for preparing the fusion protein are known in the art and include, for example, recombinant methods.

[0084] In some embodiments of the present invention, the TCRs, polypeptides, and proteins of the present invention can be expressed as a single protein comprising a linker peptide that links the α-chain and the β-chain. In this regard, the TCRs, polypeptides, and proteins of the present invention can further comprise a linker peptide. The linker peptide can advantageously facilitate the expression of the recombinant TCRs, polypeptides, and / or proteins in host cells. The linker peptide can comprise any suitable amino acid sequence. The linker peptide can be a cleavable linker peptide. For example, the linker peptide can be a furin-SGSG-P2A linker comprising the amino acid sequence of SEQ ID NO:38. When a construct comprising the linker peptide is expressed by a host cell, the linker peptide can be cleaved, generating separate α- and β-chains. In one embodiment of the present invention, the TCR, polypeptide, or protein can comprise an amino acid sequence containing a full-length α-chain, a full-length β-chain, and a linker peptide located between the α-chain and the β-chain.

[0085] The protein of the present invention can be a recombinant antibody or an antigen-binding portion thereof comprising at least one polypeptide of the present invention as described herein. As used herein, "recombinant antibody" refers to a recombinant (e.g., genetically engineered) protein comprising at least one polypeptide of the present invention and a polypeptide chain of an antibody or an antigen-binding portion thereof. The polypeptide of the antibody or an antigen-binding portion thereof can be a heavy chain, a light chain, a variable or constant region of a heavy chain or a light chain, a single-chain variable fragment (scFv), or an Fc, Fab, or F(ab')2 fragment, etc. The polypeptide chain of the antibody or an antigen-binding portion thereof can exist as an isolated polypeptide of the recombinant antibody. Alternatively, the polypeptide chain of the antibody or an antigen-binding portion thereof can exist as a polypeptide expressed in-frame (in tandem) with a polypeptide of the present invention. The polypeptide of the antibody or an antigen-binding portion thereof can be a polypeptide of any antibody or any antibody fragment (including any antibody and antibody fragment described herein).

[0086] The scope of the present invention encompasses functional variants of the TCRs, polypeptides, or proteins of the present invention as set forth herein. As used herein, the term "functional variant" refers to a TCR, polypeptide, or protein that has substantial or significant sequence identity or similarity to a parental TCR, polypeptide, or protein, and the functional variant retains the biological activity of the TCR, polypeptide, or protein of which it is a variant. Functional variants encompass, for example, those variants of the TCRs, polypeptides, or proteins (parental TCRs, polypeptides, or proteins) as set forth herein that are still capable of binding to G12R RAS with a similar degree, the same degree, or a higher degree of specificity compared to the parental TCR, polypeptide, or protein (the parental TCR has antigen specificity thereto or the parental polypeptide or protein binds specifically thereto). With reference to the parental TCR, polypeptide, or protein, the functional variant can, for example, be at least about 30%, about 50%, about 75%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or a higher degree identical to the amino acid sequence of the parental TCR, polypeptide, or protein.

[0087] Functional variants can (for example) include the amino acid sequence of a parental TCR, polypeptide or protein having at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acid having the same chemical or physical properties. For example, a conservative amino acid substitution can be an acidic amino acid substituted for another acidic amino acid (e.g., Asp or Glu), an amino acid having a nonpolar side chain substituted for another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid substituted for another basic amino acid (Lys, Arg, etc.), an amino acid having a polar side chain substituted for another amino acid having a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

[0088] Alternatively or additionally, functional variants can include the amino acid sequence of a parental TCR, polypeptide or protein having at least one non-conservative amino acid substitution. In this case, preferably, the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitution enhances the biological activity of the functional variant such that the biological activity of the functional variant is greater than that of the parental TCR, polypeptide or protein.

[0089] A TCR, polypeptide or protein may consist essentially of one or more of the specified amino acid sequences set forth herein, such that other components of the TCR, polypeptide or protein (such as other amino acids) do not substantially alter the biological activity of the TCR, polypeptide or protein. In this regard, the TCRs, polypeptides or proteins of the invention may, for example, consist essentially of the following amino acid sequences: SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, both of SEQ ID NO:30-31, both of SEQ ID NO:32-33, both of SEQ ID NO:34-35 or both of SEQ ID NO:36-37. Similarly, by way of example, the TCRs, polypeptides or proteins of the invention may consist essentially of the following amino acid sequences: (i) SEQ ID NO:13, (ii) SEQ ID NO:14, (iii) SEQ ID NO:15, (iv) SEQ ID NO:16, (v) both of SEQ ID NO:13 and 14 or (vi) both of SEQ ID NO:15 and 16. Additionally, the TCRs, polypeptides or proteins of the invention may consist essentially of the following amino acid sequences: (a) any one or more of SEQ ID NO:1-12; (b) all of SEQ ID NO:1-3; (c) all of SEQ ID NO:4-6; (d) all of SEQ ID NO:7-9; (e) all of SEQ ID NO:10-12; (f) all of SEQ ID NO:1-6; or (g) all of SEQ ID NO:7-12.

[0090] The TCRs, polypeptides and proteins of the invention can have any length, i.e., can comprise any number of amino acids, provided that the TCR, polypeptide or protein retains its biological activity (such as being able to specifically bind to G12R RAS; detecting cancer in a mammal; or treating or preventing cancer in a mammal, etc.). By way of example, the length of the polypeptide can range from about 50 to about 5000 amino acids, such as about 50, about 70, about 75, about 100, about 125, about 150, about 175, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000 or more amino acids. In this regard, the polypeptides of the invention also include oligopeptides.

[0091] The TCRs, polypeptides, and proteins of the present invention may include synthetic amino acids in place of one or more natural amino acids. The synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-aminodecanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0092] The TCRs, polypeptides, and proteins of the present invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, or converted to acid addition salts and / or dimerized or polymerized, or conjugated, for example, via disulfide bridges.

[0093] The TCRs, polypeptides, and / or proteins of the present invention may be obtained by methods known in the art, such as resynthesis. Additionally, the nucleic acids described herein may be used recombinantly and standard recombinant methods may be used to produce polypeptides and proteins. See, for example, Green and Sambrook, Molecular Cloning:A Laboratory Manual, 4th Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Alternatively, the TCRs, polypeptides, and / or proteins described herein can be synthesized commercially by companies such as Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), and Multiple Peptide Systems (San Diego, CA). In this regard, the TCRs, polypeptides, and proteins of the present invention can be synthetic, recombinant, isolated, and / or purified. One embodiment of the present invention provides an isolated or purified TCR, polypeptide, or protein encoded by any of the nucleic acids or vectors described herein for other aspects of the present invention. Another embodiment of the present invention provides an isolated or purified TCR, polypeptide, or protein that is produced by expressing any of the nucleic acids or vectors described herein for other aspects of the present invention in a cell. Yet another embodiment of the present invention provides a method for producing any of the TCRs, polypeptides, or proteins described herein, the method comprising culturing any of the host cells or host cell populations described herein to produce the TCR, polypeptide, or protein.

[0094] The scope of the present invention includes conjugates (e.g., bioconjugates) that include any one of the TCRs, polypeptides, or proteins of the present invention (including any functional portion or variant thereof), nucleic acids, recombinant expression vectors, host cells, host cell populations, or antibodies or antigen-binding portions thereof. In general, conjugates and methods for synthesizing conjugates are known in the art.

[0095] One embodiment of the present invention provides a nucleic acid comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. As used herein, "nucleic acid" includes "polynucleotide", "oligonucleotide", and "nucleic acid molecule", and generally refers to a polymer of DNA or RNA, which can be single-stranded or double-stranded, can contain natural, unnatural, or modified nucleotides, and can contain natural, unnatural, or modified internucleotide linkages (e.g., phosphoramidate linkages or phosphorothioate linkages) instead of the phosphodiester between nucleotides found in unmodified oligonucleotides. In one embodiment, the nucleic acid comprises complementary DNA (cDNA). Generally preferably, the nucleic acid does not include any insertions, deletions, inversions, and / or substitutions. However, in some cases, as discussed herein, the nucleic acid may suitably include one or more insertions, deletions, inversions, and / or substitutions.

[0096] Preferably, the nucleic acid of the present invention is recombinant. As used herein, the term "recombinant" means (i) a molecule is constructed outside of a living cell by joining natural or synthetic nucleic acid segments to a nucleic acid molecule that can replicate in a living cell, or (ii) a molecule is derived from the replication of that described in (i) above. For the purposes herein, replication can be in vitro replication or in vivo replication.

[0097] Nucleic acids can be constructed using procedures known in the art based on chemical synthesis and / or enzymatic ligation reactions. See, for example, the above references of Green and Sambrook et al. For example, nucleic acids can be synthesized chemically using natural nucleotides or various modified nucleotides (e.g., phosphorothioate derivatives and acridine substituted nucleotides) designed to increase the biological stability of the molecule or increase the physical stability of the duplex formed upon hybridization. Examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N 6 -isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouridine, β-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -isopentenyladenine, uridine-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine, uridine-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more nucleic acids of the present invention can be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0098] The nucleic acid may include any nucleotide sequence encoding any TCR, polypeptide, or protein described herein. In one embodiment of the invention, the nucleic acid includes a codon-optimized nucleotide sequence encoding any TCR, polypeptide, or protein described herein. Without being limited to any particular theory or mechanism, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcript. Codon optimization of the nucleotide sequence may involve replacing the native codon with another codon encoding the same amino acid but which can be translated by a tRNA more readily available in the cell, thereby increasing translation efficiency. Optimization of the nucleotide sequence may also reduce secondary mRNA structures that interfere with translation, thereby increasing translation efficiency.

[0099] The invention also provides a nucleic acid comprising: a nucleotide sequence complementary to the nucleotide sequence of any nucleic acid described herein; or a nucleotide sequence that hybridizes to the nucleotide sequence of any nucleic acid described herein under stringent conditions.

[0100] Nucleotide sequences that hybridize under stringent conditions preferably hybridize under high-stringency conditions. "High-stringency conditions" means that the nucleotide sequence specifically hybridizes to the target sequence (the nucleotide sequence of any nucleic acid described herein) in an amount detectably stronger than non-specific hybridization. High-stringency conditions include conditions that distinguish polynucleotides having exact complementary sequences or only a few scattered mismatches from random sequences that have exactly a few smaller regions (e.g., 3 - 10 bases) of matching nucleotide sequences. Such smaller regions of complementarity are more readily melted than full-length complements of 14 - 17 or more bases, and high-stringency hybridization allows them to be easily distinguished. Relatively high-stringency conditions include (e.g.) low salt and / or high temperature conditions, such as provided by about 0.02 - 0.1 M NaCl or equivalent at a temperature of about 50 - 70 °C. Such high-stringency conditions tolerate fewer (if any) mismatches between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting the expression of any TCR of the present invention. It should generally be understood that the conditions can be made more stringent by adding increasing amounts of formamide.

[0101] One embodiment of the invention also provides a nucleic acid comprising a nucleotide sequence that is at least about 70% or identical (e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical) to any nucleic acid described herein. In this regard, the nucleic acid may consist essentially of any nucleotide sequence described herein.

[0102] One embodiment of the present invention provides an isolated or purified nucleic acid comprising, from 5' to 3', a first nucleic acid sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence encode the following amino acid sequences: SEQ ID NO: 13 and 14; 14 and 13; 15 and 16; 16 and 15; 30 and 31; 31 and 30; 32 and 33; 33 and 32; 34 and 35; 35 and 34; 36 and 37; 37 and 36; 41 and 42; 42 and 41; 43 and 44; 44 and 43; 45 and 46; 46 and 45; 47 and 48; 48 and 47; 49 and 50; 50 and 49; 51 and 52; or 52 and 51.

[0103] In one embodiment of the present invention, the isolated or purified nucleic acid further comprises a third nucleotide sequence inserted between the first nucleotide sequence and the second nucleotide sequence, wherein the third nucleotide sequence encodes a cleavable linker peptide. In one embodiment of the present invention, the cleavable linker peptide comprises the amino acid sequence of SEQ ID NO: 38.

[0104] The nucleic acid of the present invention can be incorporated into a recombinant expression vector. In this regard, the present invention provides a recombinant expression vector comprising any nucleic acid of the present invention. In one embodiment of the present invention, the recombinant expression vector comprises nucleotide sequences encoding an α-chain, a β-chain, and a linker peptide.

[0105] For the purposes herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that, when the construct comprises a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and the vector is contacted with a cell under conditions sufficient to permit expression of the mRNA, protein, polypeptide, or peptide within the host cell, allows expression of the mRNA, protein, polypeptide, or peptide by the cell. The vector as a whole of the present invention is not natural. However, a part of the vector can be natural. The recombinant expression vector of the present invention can comprise any type of nucleotide, which includes (but is not limited to) DNA and RNA, which can be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and can contain natural, non-natural, or altered nucleotides. The recombinant expression vector can comprise natural, non-natural inter-nucleotide linkages or both types of linkages. Preferably, the non-natural or altered nucleotides or inter-nucleotide linkages do not impede transcription or replication of the vector.

[0106] 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 cell. Suitable vectors include those designed for propagation and amplification or for expression or for both, such as plasmids and viruses. The vectors can be selected from: pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA). Phage vectors can also be used, such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector, such as a retroviral vector. In a particularly preferred embodiment, the recombinant expression vector is the MSGV1 vector. In one embodiment of the present invention, the recombinant expression vector is a transposon or lentiviral vector.

[0107] The recombinant expression vectors of the present invention can be prepared using standard recombinant DNA techniques described, for example, in the above references by Greene and Sambrook et al. Constructs of circular or linear expression vectors containing replication systems functional in prokaryotic or eukaryotic host cells can be prepared. The replication systems can be derived from, for example, ColE1, 2μ plasmid, λ, SV40, bovine papillomavirus, etc.

[0108] Desirably, and where appropriate and considering whether the vector is DNA- or RNA-based, the recombinant expression vector includes regulatory sequences specific to the type of host cell into which the vector is to be introduced (such as bacteria, fungi, plants, or animals), such as transcription and translation start and stop codons.

[0109] The recombinant expression vector may contain one or more marker genes that allow selection of transformed or transfected host cells. Marker genes include biocide resistance (e.g., resistance to antibiotics, heavy metals, etc.), complementation in auxotrophic host cells (to provide prototrophy), etc. Suitable marker genes for the expression vectors of the present invention include, for example, the neomycin / G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0110] The recombinant expression vector may include a natural or non-natural promoter operably linked to a nucleotide sequence encoding a TCR, polypeptide, or protein, or a nucleotide sequence complementary to or hybridizing to a nucleotide sequence encoding a TCR, polypeptide, or protein. Those skilled in the art are familiar with the selection of promoters (e.g., strong, weak, inducible, tissue-specific, and development-specific). Similarly, those skilled in the art are also familiar with the combination of nucleotide sequences and promoters. The promoter can be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and the promoter found in the long terminal repeat of murine stem cell virus.

[0111] The recombinant expression vector of the present invention can be designed for transient expression, stable expression, or both. Similarly, the recombinant expression vector can be prepared for constitutive expression or inducible expression.

[0112] In addition, a recombinant expression vector containing a suicide gene can be prepared. As used herein, the term "suicide gene" refers to a gene that causes the death of cells expressing the suicide gene. The suicide gene can be a gene that confers sensitivity of the expressing cells to a pharmaceutical agent (e.g., a drug) and causes cell death when the cells are contacted or exposed to the agent. Suicide genes are known in the art and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and the inducible caspase 9 gene system.

[0113] Another embodiment of the present invention further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain the recombinant expression vector of the present invention. The host cell can be a eukaryotic cell (e.g., a plant, animal, fungal, or algal cell), or can be a prokaryotic cell (e.g., a bacterial or protozoal cell). 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 that grows in suspension). Suitable host cells are known in the art and include, for example, DH5α Escherichia coli (E. coli) cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For the purpose of amplifying or replicating the recombinant expression vector, the host cell is preferably a prokaryotic cell, such as a DH5α cell. For the purpose of producing a recombinant TCR, polypeptide, or protein, the host cell is preferably a mammalian cell. Most preferably, the host cell line is a human cell. Although the host cell can be any cell type, can be derived from any type of tissue, and can be at any developmental stage, the host cell is preferably a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell line is a T cell. In one embodiment of the present invention, the host cell line is a human lymphocyte. In another embodiment of the present invention, the host cell is selected from: T cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, and natural killer (NK) cells. Yet another embodiment of the present invention provides a method for producing a host cell that expresses a TCR that is antigen-specific for the peptide of SEQ ID NO: 39, the method comprising contacting the cell with any of the vectors described herein under conditions that permit the introduction of the vector into the cell.

[0114] For the purposes herein, the T cell can be any T cell, such as a cultured T cell (e.g., a primary T cell, or a T cell from a cultured T cell line (e.g., Jurkat, SupT1, etc.), or a T cell obtained from a mammal). If obtained from a mammal, the T cell can be obtained from a variety of sources, including (but not limited to) blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cell can also be enriched or purified. Preferably, the T cell is a human T cell. The T cell can be any type of T cell and can be at any developmental stage, including (but not limited to) CD4 + / CD8 + double-positive T cells, CD4 + helper T cells (e.g., Th1 and Th2 cells), CD4 + T cells, CD8 +T cells (such as cytotoxic T cells), tumor-infiltrating lymphocytes (TIL), memory T cells (such as central memory T cells and effector memory T cells), naive T cells, etc.

[0115] The present invention also provides a cell population comprising at least one host cell described herein. The cell population can be a heterogeneous population, which includes host cells containing any of the described recombinant expression vectors and at least one other cell (such as a host cell that does not include any recombinant expression vector (such as a T cell) or a cell other than a T cell (such as a B cell, macrophage, neutrophil, red blood cell, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc.)). Alternatively, the cell population can be a substantially homogeneous population, wherein the population mainly comprises host cells containing the recombinant expression vector (such as consisting essentially of the same). The population can also be a clonal cell population, wherein all the cells of the population are clones of a single host cell including the recombinant expression vector, so that all the cells of the population include the recombinant expression vector. In one embodiment of the present invention, the cell population is a clonal population comprising host cells containing the recombinant expression vector as described herein.

[0116] In one embodiment of the present invention, the number of cells in the population can be rapidly expanded. The number of T cells can be expanded by any of a number of methods known in the art, such as those described in, for example: U.S. Patent No. 8,034,334; U.S. Patent No. 8,383,099; U.S. Patent Application Publication No. 2012 / 0244133; Dudley et al., J. Immunol., 26:332-42 (2003); and Riddell et al., J. Immunol. Methods, 128:189-201 (1990). In one embodiment, the number of T cells is expanded by co-culturing T cells with OKT3 antibody, IL-2, and feeder PBMC (such as irradiated allogeneic PBMC).

[0117] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) of the present invention can be isolated and / or purified. As used herein, the term "isolated" means removed from its natural environment. As used herein, the term "purified" means having increased purity, where "purity" is a relative term and need not be construed as absolute purity. For example, the purity can be at least about 50%, can be greater than about 60%, about 70%, about 80%, about 90%, about 95%, or can be about 100%.

[0118] The TCR, polypeptide, protein, nucleic acid, recombinant expression vector, and host cell of the present invention (including populations thereof, hereinafter collectively referred to as "the TCR materials of the present invention") can be formulated into a composition (such as a pharmaceutical composition). In this regard, the present invention provides a pharmaceutical composition comprising any one of the TCR, polypeptide, protein, nucleic acid, expression vector, and host cell (including populations thereof) described herein and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention containing any of the TCR materials of the present invention may include more than one of the TCR materials of the present invention (such as a polypeptide and a nucleic acid) or two or more different TCRs. Alternatively, the pharmaceutical composition may include a combination of the TCR materials of the present invention and another pharmaceutically active agent or drug (such as a chemotherapeutic agent), and the chemotherapeutic agent is (for example) asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

[0119] Preferably, the carrier is a pharmaceutically acceptable carrier. For a pharmaceutical composition, the carrier can generally be used for any of the specific TCR materials of the present invention being considered. Methods for preparing a pharmaceutically administrable composition are known or apparent to those skilled in the art and are described in more detail in (for example) Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press (2012). Preferably, a pharmaceutically acceptable carrier is one that is free of adverse side effects or toxicity under the conditions of use.

[0120] The choice of carrier depends in part on the specific TCR material of the present invention and the specific method for administering the TCR material of the present invention. Accordingly, there are various suitable formulations for the pharmaceutical composition of the present invention. Suitable formulations can include any for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, or intraperitoneal administration. More than one route can be used to administer the TCR material of the present invention, and in some cases, a particular route may provide a more rapid and effective response than another route.

[0121] Preferably, the TCR material of the present invention is administered by injection (e.g., intravenously). When the TCR material of the present invention is a host cell (or a population thereof) expressing the TCR of the present invention, a pharmaceutically acceptable carrier for the cells for injection may comprise any isotonic carrier (e.g., physiological saline (about 0.90% w / v NaCl in water, about 300 mOsm / L NaCl in water or about 9.0 g NaCl / liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water or Ringer's lactate). In one embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

[0122] For the purposes of the present invention, the amount or dose of the TCR material of the present invention administered (e.g., the number of cells when the TCR material of the present invention is one or more cells) should be sufficient to produce a certain effect (e.g., a therapeutic or prophylactic response) in an individual or animal over a suitable time period. For example, the dose of the TCR material of the present invention should be sufficient to bind to a cancer antigen (e.g., G12R RAS) or detect, treat or prevent cancer within a period of about 2 hours or more (e.g., 12 to 24 or more hours) after administration. In certain embodiments, this time period can even be longer. The dose depends on the potency of the particular TCR material of the present invention and the condition of the animal (e.g., human) as well as the weight of the animal (e.g., human) to be treated.

[0123] Many assays are known in the art for determining the administered dose. For the purposes of the present invention, an assay comprising the following procedure can be used to determine the starting dose to be administered to a mammal: in a group of mammals each given a different dose of T cells, compare the extent of target cell lysis or IFN-γ secretion by T cells expressing the TCR, polypeptide or protein of the present invention after administration of a given dose of such T cells to the mammals. The extent of target cell lysis or IFN-γ secretion after administration of a certain dose can be analyzed by methods known in the art.

[0124] The dose of the TCR material of the present invention also depends on the presence, nature and extent of any adverse side effects that may accompany the administration of a particular TCR material of the present invention. Generally, after considering various factors (such as age, weight, general health, diet, sex, the TCR material of the present invention to be administered, the route of administration and the severity of the cancer to be treated), the attending physician determines the dose of the TCR material of the present invention for each individual patient to be treated. In an embodiment where the TCR material of the present invention is a cell population, the number of cells administered per infusion can vary, e.g., from about 1×10 6 to about 1×10 12one cell or more. In certain embodiments, fewer than 1×10 6 cells may be administered.

[0125] Those skilled in the art will readily appreciate that the inventive TCR materials of the present invention can be modified in any number of ways to increase the therapeutic or prophylactic efficacy of the TCR materials of the present invention via the modification. For example, the TCR materials of the present invention can be directly conjugated or indirectly conjugated via a bridge to a chemotherapeutic agent. The practice of conjugating compounds to chemotherapeutic agents is known in the art. Those skilled in the art recognize that sites on the TCR materials of the present invention that are not required for the function of the TCR materials of the present invention are suitable sites for attaching the bridge and / or the chemotherapeutic agent, provided that the bridge and / or the chemotherapeutic agent do not interfere with the function of the TCR materials of the present invention after attachment to the TCR materials of the present invention (i.e., capable of binding to G12R RAS or detecting, treating, or preventing cancer).

[0126] The pharmaceutical compositions, TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, and cell populations of the present invention are expected to be useful in methods for treating or preventing cancer. Without being limited to a particular theory, it is believed that the TCRs of the present invention specifically bind to G12R RAS, such that the TCR (or related polypeptides or proteins of the present invention) can mediate an immune response against target cells expressing G12R RAS when expressed by a cell. In this regard, one embodiment of the present invention provides a method for treating or preventing cancer in a mammal, which comprises administering to the mammal an amount of any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein, or any host cell or cell population comprising a recombinant vector encoding any of the TCRs, polypeptides, or proteins described herein, in an amount effective to treat or prevent cancer in the mammal.

[0127] One embodiment of the present invention provides a method for inducing an immune response against cancer in a mammal, which comprises administering to the mammal an amount of any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein, or any host cell or cell population comprising a recombinant vector encoding any of the TCRs, polypeptides, or proteins described herein, in an amount effective to induce an immune response against cancer in the mammal.

[0128] One embodiment of the invention provides any of the pharmaceutical compositions, TCRs, polypeptides or proteins, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, proteins described herein, or any host cell or cell population comprising a recombinant vector encoding any of the TCRs, polypeptides or proteins described herein, for treating or preventing cancer in a mammal.

[0129] One embodiment of the invention provides any of the pharmaceutical compositions, TCRs, polypeptides or proteins, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides or proteins described herein, or any host cell or cell population comprising a recombinant vector encoding any of the TCRs, polypeptides or proteins described herein, for inducing an immune response against cancer in a mammal.

[0130] As used herein, the terms "treatment" and "prevention" and words derived therefrom do not necessarily mean 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that those skilled in the art would consider to have a potential benefit or therapeutic effect. In this regard, the methods of the invention can provide any level of treatment or prevention of cancer in a mammal in any amount. Additionally, the treatment or prevention provided by the methods of the invention can include treating or preventing one or more conditions or symptoms of the cancer being treated or prevented. For example, the treatment or prevention can include promoting tumor regression. Similarly, for the purposes herein, "prevention" can encompass delaying the onset of cancer or its symptoms or conditions. Alternatively or additionally, "prevention" can encompass preventing or delaying the onset of cancer or its symptoms or conditions.

[0131] Also provided is a method of detecting the presence of cancer in a mammal. The method includes (i) contacting a sample comprising one or more cells from the mammal with any of the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations 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 cancer in the mammal.

[0132] For the method of the invention for detecting cancer in a mammal, the cell sample can be a sample comprising whole cells, lysates thereof, or a portion of a whole cell lysate (such as a nuclear or cytoplasmic portion, a whole protein portion or a nucleic acid portion).

[0133] For the purposes of the method of the invention for detecting cancer, the contacting can occur in vitro or in vivo in the mammal. Preferably, the contacting is performed in vitro.

[0134] Similarly, the complex can be detected by any number of ways known in the art. For example, the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells or cell populations of the invention described herein can be labeled with a detectable label (such as a radioisotope, fluorophore (such as fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzyme (such as alkaline phosphatase, horseradish peroxidase) and elemental particles (such as gold particles)).

[0135] For the purposes of the methods of the invention for administering host cells or cell populations, the cells can be allogeneic or autologous cells of a mammal. Preferably, the cells are autologous to the mammal.

[0136] For the methods of the invention, the cancer can be any cancer, including any of the following: acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer or rectoanal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, uterine cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal cancer, omental cancer and diaphragmatic cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer and bladder cancer. Preferred cancers are pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer or prostate cancer. Preferably, the lung cancer is lung adenocarcinoma, the ovarian cancer is epithelial ovarian cancer, and the pancreatic cancer is pancreatic adenocarcinoma. In one embodiment of the invention, the cancer expresses a mutant human RAS amino acid sequence in which glycine at position 12 is replaced by arginine, wherein the mutant human RAS amino acid sequence is a mutant human KRAS, mutant human HRAS or mutant human NRAS amino acid sequence, and wherein position 12 is defined with reference to the WT human KRAS, WT human HRAS or WT human NRAS protein, respectively. The mutant human KRAS, mutant human HRAS and mutant human NRAS expressed by the cancer can be as described herein for other aspects of the invention.

[0137] The mammalian species referred to in the method of the present invention can be any mammalian species. As used herein, the term "mammalian species" refers to any mammalian species, including (but not limited to) mammalian species of the order Rodentia (e.g., mice and hamsters) and mammalian species of the order Lagomorpha (e.g., rabbits). Preferably, the mammalian species is from the order Carnivora, including the families Felidae (cats) and Canidae (dogs). More preferably, the mammalian species is from the order Artiodactyla (including the families Bovidae (cows) and Suidae (pigs)) or the order Perissodactyla (including the family Equidae (horses)). Most preferably, the mammalian species is from the order Primates, the suborder Anthropoidea or the infraorder Simiiformes (monkeys) or the parvorder Catarrhini (humans and apes). A particularly preferred mammalian species is the human species.

[0138] The following examples further illustrate the present invention, but should of course not be construed as limiting its scope in any way.

[0139] Example 1

[0140] This example shows the isolation of a TCR that is antigen-specific for human KRAS containing the G12R mutation and presented by the HLA-DRB5*01:HLA-DRA*01:01 heterodimer.

[0141] A TCR that is antigen-specific for human KRAS containing the G12R mutation and presented by the HLA-DRB5*01:HLA-DRA*01:01 heterodimer was isolated from TILs of a patient with metastatic pancreatic cancer (Patient 4270). The TCR was isolated from TILs that showed specific recognition of KRAS G12R and did not show specific recognition of WT KRAS.

[0142] To sequence the reactive 4270 TCR, reactive TILs were sorted by fluorescence-activated cell sorting (FACS) based on upregulation of the T cell activation marker 4-1BB. Subsequently, the cells were lysed and the TCR transcripts were Sanger sequenced. The amino acid sequences of the variable regions of the 4270 TCR α and β chains are shown in Table 5. The CDRs are underlined.

[0143] Table 5

[0144]

[0145] Example 2

[0146] This example demonstrates that cells transduced with the 4270 TCR isolated in Example 1 specifically recognize the G12R RAS peptide.

[0147] The nucleic acid sequence encoding the G12R RAS-reactive 4270 TCR of Example 1 and containing a murine constant region substituted with cysteine and modified with LVL was cloned into a retroviral expression vector. The murine constant region of the α-chain includes the amino acid sequence of SEQ ID NO:26, where X at position 48 is Cys, X at position 112 is Leu, X at position 114 is Ile, and X at position 115 is Val. The constant region of the β-chain includes the amino acid sequence of SEQ ID NO:27, where X at position 57 is Cys. A linker comprising the amino acid sequence RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO:38) is located between the α-chain constant region and the β-chain variable region. Allogeneic PBMCs were transduced with the retroviral expression vector.

[0148] The transduced cells were co-cultured overnight with autologous DCs pulsed with serial dilutions of the WT RAS peptide MTEYKLVVVGA G GVGKSALTIQLI (SEQ ID NO:40) or the G12R RAS peptide MTEYKLVVVGA R GVGKSALTIQLI (SEQ ID NO:39). IFNγ secretion was evaluated by enzyme-linked immunospot (ELISpot). The results are shown in Figure 1 .

[0149] As Figure 1 shown, cells transduced with the 4270 TCR specifically recognize the G12R RAS peptide.

[0150] Example 3

[0151] This example demonstrated that cells transduced with the 4270 TCR isolated in Example 1 recognize the G12R RAS peptide presented in the context of the HLA-DRB5*01:HLA-DRA*01:01 heterodimer.

[0152] Allogeneic PBMCs were transduced virally using the retroviral expression vector of Example 2. Target COS7 cells were transfected with one of the HLA molecules expressed by patient 4270 and shown in Figure 2 . The transfected COS7 cells were then pulsed with the G12R RAS peptide MTEYKLVVVGA R GVGKSALTIQLI (SEQ ID NO:39). The 4270 TCR-transduced cells were co-cultured overnight with the pulsed, transfected COS7 cells. IFNγ secretion was evaluated by ELISA. The results are shown in Figure 2 .

[0153] As Figure 2As shown, the cells transduced with the 4270 TCR isolated in Example 1 specifically recognize the G12R RAS peptide presented in the context of the HLA-DRB5*01:HLA-DRA*01:01 heterodimer.

[0154] Example 4

[0155] This example shows the isolation of a TCR that is antigen-specific for human KRAS containing the G12R mutation and presented by the HLA-DQA1*05:05:HLA-DQB1*03:01 heterodimer.

[0156] TCRs that are antigen-specific for human KRAS containing the G12R mutation and presented by the HLA-DQA1*05:05:HLA-DQB1*03:01 heterodimer were isolated from the TILs of a patient with metastatic colon cancer (Patient 4268). TCRs were isolated from TILs that showed specific recognition of KRAS G12R and did not show specific recognition of WT KRAS.

[0157] To sequence the reactive 4268 TCR, reactive TILs were FACS sorted based on upregulation of the T cell activation marker 4-1BB. Subsequently, the cells were lysed and Sanger sequencing of the TCR transcripts was performed. The amino acid sequences of the variable regions of the 4268 TCR α and β chains are shown in Table 6. The CDRs are underlined.

[0158] Table 6

[0159]

[0160] Example 5

[0161] This example confirms that cells transduced with the 4268 TCR isolated in Example 4 specifically recognize the G12R RAS peptide.

[0162] The nucleic acid sequence encoding the G12R RAS-reactive 4268 TCR of Example 4 and containing a murine constant region modified with cysteine substitutions and LVL was cloned into a retroviral expression vector. The murine constant region of the α chain includes the amino acid sequence of SEQ ID NO:26, where X at position 48 is Cys, X at position 112 is Leu, X at position 114 is Ile, and X at position 115 is Val. The constant region of the β chain includes the amino acid sequence of SEQ ID NO:27, where X at position 57 is Cys. A linker including the amino acid sequence RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO:38) is located between the constant region of the α chain and the variable region of the β chain. Allogeneic PBMCs were transduced with the retroviral expression vector.

[0163] Co-culture the transduced cells overnight with autologous DC pulsed with serial dilutions of the WT RAS peptide MTEYKLVVVGA G GVGKSALTIQLI (SEQ ID NO:40) or the G12R RAS peptide MTEYKLVVVGA R GVGKSALTIQLI (SEQ ID NO:39). Evaluate IFNγ secretion by ELIspot. The results are shown in Figure 3 .

[0164] As Figure 3 shown, cells transduced with 4268 TCR specifically recognize the G12R RAS peptide.

[0165] Example 6

[0166] This example demonstrates that cells transduced with the 4268 TCR isolated in Example 4 recognize the G12R RAS peptide presented in the context of the HLA-DQA1*05:05:HLA-DQB1*03:01 heterodimer.

[0167] Virally transduce allogeneic PBMCs using the retroviral expression vector of Example 5. Transfect target COS7 cells with one of the HLA molecules expressed by patient 4268 and shown in Figure 4 . Then pulse the transfected COS7 cells with the G12R RAS peptide MTEYKLVVVGA R GVGKSALTIQLI (SEQ ID NO:39). Co-culture the 4268 TCR-transduced cells overnight with the pulsed, transfected COS7 cells. Evaluate IFNγ secretion by ELISA. The results are shown in Figure 4 .

[0168] As Figure 4 shown, cells transduced with the 4268 TCR isolated in Example 4 specifically recognize the G12R RAS peptide presented in the context of the HLA-DQA1*05:05:HLA-DQB1*03:01 heterodimer.

[0169] All references cited herein (including publications, patent applications, and patents) are incorporated herein by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0170] In the description of the background of the present invention (especially in the background of the following claims), unless otherwise indicated herein or clearly contradicted by the context, the use of the terms "a", "an", "the", "at least one", and similar indicatives are intended to cover the singular and the plural. Unless otherwise indicated herein or clearly contradicted by the context, the term "at least one" (e.g., "at least one of A and B") followed by a list of one or more items is intended to mean one item selected from the listed items (A or B) or a combination of two or more of the listed items (A and B). Unless otherwise stated, the terms "comprising", "having", "including", and "containing" are intended to be open-ended terms (i.e., meaning "including but not limited to"). Unless otherwise indicated herein, the recitation of a range of values herein is merely intended as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise indicated herein or otherwise clearly contradicted by the context, all methods described herein may be performed in any suitable order. Unless otherwise asserted, the use of any and all examples or exemplary language (e.g., "such as") provided herein is merely intended to better illustrate the invention and does not impose a limitation on the scope of the invention. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0171] The preferred embodiments of the present invention are described herein, including the best mode known to the inventors for practicing the present invention. Variations of those preferred embodiments will be apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to appropriately employ such variations, and the inventors expect the invention to be practiced otherwise than as specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter recited in the appended claims as permitted by applicable law. In addition, unless otherwise indicated herein or otherwise clearly contradicted by the context, the present invention covers any combination of the above elements in all possible variations thereof. Sequence Listing <110> U.S. Department of Health and Human Services <120> HLA Class II-Restricted T Cell Receptors Against RAS Containing the G12R Mutation <130> 746666 <150> US 62 / 795,203 <151> 2019-01-22 <160> 52 <170> PatentIn version 3.5 <210> 1 <211> 6 <212> PRT <213> Homo sapiens <400> 1 Asp Arg Gly Ser Gln Ser 1 5 <210> 2 <211> 6 <212> PRT <213> Homo sapiens <400> 2 Ile Tyr Ser Asn Gly Asp 1 5 <210> 3 <211> 13 <212> PRT <213> Homo sapiens <400> 3 Cys Ala Val Glu Gly Ala Gly Ser Tyr Gln Leu Thr Phe 1 5 10 <210> 4 <211> 6 <212> PRT <213> Homo sapiens <400> 4 Asp Phe Gln Ala Thr Thr 1 5 <210> 5 <211> 7 <212> PRT <213> Homo sapiens <400> 5 Ser Asn Glu Gly Ser Lys Ala 1 5 <210> 6 <211> 14 <212> PRT <213> Homo sapiens <400> 6 Cys Ser Ala Ser Asn Arg Leu Ala Val Asn Glu Gln Phe Phe 1 5 10 <210> 7 <211> 6 <212> PRT <213> Homo sapiens <400> 7 Asn Ser Met Phe Asp Tyr 1 5 <210> 8 <211> 9 <212> PRT <213> Homo sapiens <400> 8 Ile Ser Ile Ser Ser Ile Lys Asp Lys 1 5 <210> 9 <211> 15 <212> PRT <213> Homo sapiens <400> 9 Cys Ala Ala Ser Gly Asn Thr Gly Thr Ala Ser Lys Leu Thr Phe 1 5 10 15 <210> 10 <211> 5 <212> PRT <213> Homo sapiens <400> 10 Leu Asn His Asp Ala 1 5 <210> 11 <211> 6 <212> PRT <213> Homo sapiens <400> 11 Ser Gln Ile Val Asn Asp 1 5 <210> 12 <211> 16 <212> PRT <213> Homo sapiens <400> 12 Cys Ala Ser Ser Asn Gln Leu Val Thr Gly Gly Tyr Gly Tyr Thr Phe 1 5 10 15 <210> 13 <211> 132 <212> PRT <213> Homo sapiens <400> 13 Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser 1 5 10 15 Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu 20 25 30 Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp 35 40 45 Arg Gly Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser 50 55 60 Pro Glu Leu Ile Met Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly 65 70 75 80 Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu 85 90 95 Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val 100 105 110 Glu Gly Ala Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys Leu 115 120 125 Ser Val Ile Pro 130 <210> 14 <211> 141 <212> PRT <213> Homo sapiens <400> 14 Met Leu Leu Leu Leu Leu Leu Leu Gly Pro Gly Ile Ser Leu Leu Leu 1 5 10 15 Pro Gly Ser Leu Ala Gly Ser Gly Leu Gly Ala Val Val Ser Gln His 20 25 30 Pro Ser Trp Val Ile Cys Lys Ser Gly Thr Ser Val Lys Ile Glu Cys 35 40 45 Arg Ser Leu Asp Phe Gln Ala Thr Thr Met Phe Trp Tyr Arg Gln Phe 50 55 60 Pro Lys Gln Ser Leu Met Leu Met Ala Thr Ser Asn Glu Gly Ser Lys 65 70 75 80 Ala Thr Tyr Glu Gln Gly Val Glu Lys Asp Lys Phe Leu Ile Asn His 85 90 95 Ala Ser Leu Thr Leu Ser Thr Leu Thr Val Thr Ser Ala His Pro Glu 100 105 110 Asp Ser Ser Phe Tyr Ile Cys Ser Ala Ser Asn Arg Leu Ala Val Asn 115 120 125 Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 130 135 140 <210> 15 <211> 140 <212> PRT <213> Homo sapiens <400> 15 Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro 1 5 10 15 Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln 20 25 30 Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn 35 40 45 Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys 50 55 60 Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys 65 70 75 80 Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala 85 90 95 Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala 100 105 110 Val Tyr Phe Cys Ala Ala Ser Gly Asn Thr Gly Thr Ala Ser Lys Leu 115 120 125 Thr Phe Gly Thr Gly Thr Arg Leu Gln Val Thr Leu 130 135 140 <210> 16 <211> 134 <212> PRT <213> Homo sapiens <400> 16 Met Ser Asn Gln Val Leu Cys Cys Val Val Leu Cys Phe Leu Gly Ala 1 5 10 15 Asn Thr Val Asp Gly Gly Ile Thr Gln Ser Pro Lys Tyr Leu Phe Arg 20 25 30 Lys Glu Gly Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His 35 40 45 Asp Ala Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Gln Ile Val Asn Asp Phe Gln Lys Gly Asp Ile Ala 65 70 75 80 Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr 85 90 95 Val Thr Ser Ala Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser 100 105 110 Ser Asn Gln Leu Val Thr Gly Gly Tyr Gly Tyr Thr Phe Gly Ser Gly 115 120 125 Thr Arg Leu Thr Val Val 130 <210> 17 <211> 189 <212> PRT <213> Homo sapiens <400> 17 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His His Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Glu Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Ser Arg Thr Val Asp Thr Lys 115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Phe Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Arg Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln Tyr Arg Leu Lys Lys Ile Ser Lys Glu Glu Lys 165 170 175 Thr Pro Gly Cys Val Lys Ile Lys Lys Cys Ile Ile Met 180 185 <210> 18 <211> 188 <212> PRT <213> Homo sapiens <400> 18 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His His Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Glu Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Ser Arg Thr Val Asp Thr Lys 115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Phe Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Asp Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Lys His Lys Glu Lys Met Ser Lys Asp Gly Lys Lys 165 170 175 Lys Lys Lys Lys Ser Lys Thr Lys Cys Val Ile Met 180 185 <210> 19 <211> 189 <212> PRT <213> Homo sapiens <400> 19 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His Gln Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Ala Ala Arg Thr Val Glu Ser Arg 115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Tyr Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln His Lys Leu Arg Lys Leu Asn Pro Pro Asp Glu 165 170 175 Ser Gly Pro Gly Cys Met Ser Cys Lys Cys Val Leu Ser 180 185 <210> 20 <211> 189 <212> PRT <213> Homo sapiens <400> 20 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Ser Lys Ser Phe Ala Asp Ile Asn Leu Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Thr Arg Thr Val Asp Thr Lys 115 120 125 Gln Ala His Glu Leu Ala Lys Ser Tyr Gly Ile Pro Phe Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln Tyr Arg Met Lys Lys Leu Asn Ser Ser Asp Asp 165 170 175 Gly Thr Gln Gly Cys Met Gly Leu Pro Cys Val Val Met 180 185 <210> 21 <211> 189 <212> PRT <213> Homo sapiens <400> 21 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Arg Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His His Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Glu Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Ser Arg Thr Val Asp Thr Lys 115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Phe Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Arg Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln Tyr Arg Leu Lys Lys Ile Ser Lys Glu Glu Lys 165 170 175 Thr Pro Gly Cys Val Lys Ile Lys Lys Cys Ile Ile Met 180 185 <210> 22 <211> 188 <212> PRT <213> Homo sapiens <400> 22 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Arg Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His His Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Glu Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Ser Arg Thr Val Asp Thr Lys 115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Phe Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Asp Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Lys His Lys Glu Lys Met Ser Lys Asp Gly Lys Lys 165 170 175 Lys Lys Lys Lys Ser Lys Thr Lys Cys Val Ile Met 180 185 <210> 23 <211> 189 <212> PRT <213> Homo sapiens <400> 23 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Arg Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His Gln Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Ala Ala Arg Thr Val Glu Ser Arg 115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Tyr Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln His Lys Leu Arg Lys Leu Asn Pro Pro Asp Glu 165 170 175 Ser Gly Pro Gly Cys Met Ser Cys Lys Cys Val Leu Ser 180 185 <210> 24 <211> 189 <212> PRT <213> Homo sapiens <400> 24 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Arg Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Ser Lys Ser Phe Ala Asp Ile Asn Leu Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Thr Arg Thr Val Asp Thr Lys 115 120 125 Gln Ala His Glu Leu Ala Lys Ser Tyr Gly Ile Pro Phe Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln Tyr Arg Met Lys Lys Leu Asn Ser Ser Asp Asp 165 170 175 Gly Thr Gln Gly Cys Met Gly Leu Pro Cys Val Val Met 180 185 <210> 25 <211> 23 <212> PRT <213> Homo sapiens <400> 25 Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys Ser 1 5 10 15 Ala Leu Thr Ile Gln Leu Ile 20 <210> 26 <211> 137 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Synthetic <220> <221> SITE <222> (48)..(48) <223> X is Thr or Cys <220> <221> SITE <222> (112)..(112) <223> X is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp <220> <221> SITE <222> (114)..(114) <223> X is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp <220> <221> SITE <222> (115)..(115) <223> X is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp <400> 26 Asp Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg 1 5 10 15 Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile 20 25 30 Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Xaa 35 40 45 Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala 50 55 60 Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr 65 70 75 80 Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr 85 90 95 Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Xaa 100 105 110 Val Xaa Xaa Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu 115 120 125 Leu Met Thr Leu Arg Leu Trp Ser Ser 130 135 <210> 27 <211> 173 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Synthetic <220> <221> SITE <222> (57)..(57) <223> X is Ser or Cys <400> 27 Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro 1 5 10 15 Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val Cys Leu 20 25 30 Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn 35 40 45 Gly Lys Glu Val His Ser Gly Val Xaa Thr Asp Pro Gln Ala Tyr Lys 50 55 60 Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala 65 70 75 80 Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe 85 90 95 His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro Lys Pro 100 105 110 Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly 115 120 125 Ile Thr Ser Ala Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu 130 135 140 Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser 145 150 155 160 Thr Leu Val Val Met Ala Met Val Lys Arg Lys Asn Ser 165 170 <210> 28 <211> 137 <212> PRT <213> House mouse (Mus musculus) <400> 28 Asp Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg 1 5 10 15 Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile 20 25 30 Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Thr 35 40 45 Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala 50 55 60 Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr 65 70 75 80 Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr 85 90 95 Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Ser 100 105 110 Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu 115 120 125 Leu Met Thr Leu Arg Leu Trp Ser Ser 130 135 <210> 29 <211> 173 <212> PRT <213> Mus musculus <400> 29 Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro 1 5 10 15 Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val Cys Leu 20 25 30 Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn 35 40 45 Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Ala Tyr Lys 50 55 60 Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala 65 70 75 80 Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe 85 90 95 His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro Lys Pro 100 105 110 Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly 115 120 125 Ile Thr Ser Ala Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu 130 135 140 Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser 145 150 155 160 Thr Leu Val Val Met Ala Met Val Lys Arg Lys Asn Ser 165 170 <210> 30 <211> 269 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Synthetic <220> <221> SITE <222> (180)..(180) <223> X is Thr or Cys <220> <221> SITE <222> (244)..(244) <223> X is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp <220> <221> SITE <222> (246)..(246) <223> X is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp <220> <221> SITE <222> (247)..(247) <223> X is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp <400> 30 Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser 1 5 10 15 Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu 20 25 30 Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp 35 40 45 Arg Gly Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser 50 55 60 Pro Glu Leu Ile Met Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly 65 70 75 80 Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu 85 90 95 Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val 100 105 110 Glu Gly Ala Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys Leu 115 120 125 Ser Val Ile Pro Asp Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu 130 135 140 Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe 145 150 155 160 Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile 165 170 175 Thr Asp Lys Xaa Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn 180 185 190 Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile 195 200 205 Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp 210 215 220 Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe 225 230 235 240 Gln Asn Leu Xaa Val Xaa Xaa Leu Arg Ile Leu Leu Leu Lys Val Ala 245 250 255 Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 <210> 31 <211> 314 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Synthetic <220> <221> SITE <222> (198)..(198) <223> X is Ser or Cys <400> 31 Met Leu Leu Leu Leu Leu Leu Leu Gly Pro Gly Ile Ser Leu Leu Leu 1 5 10 15 Pro Gly Ser Leu Ala Gly Ser Gly Leu Gly Ala Val Val Ser Gln His 20 25 30 Pro Ser Trp Val Ile Cys Lys Ser Gly Thr Ser Val Lys Ile Glu Cys 35 40 45 Arg Ser Leu Asp Phe Gln Ala Thr Thr Met Phe Trp Tyr Arg Gln Phe 50 55 60 Pro Lys Gln Ser Leu Met Leu Met Ala Thr Ser Asn Glu Gly Ser Lys 65 70 75 80 Ala Thr Tyr Glu Gln Gly Val Glu Lys Asp Lys Phe Leu Ile Asn His 85 90 95 Ala Ser Leu Thr Leu Ser Thr Leu Thr Val Thr Ser Ala His Pro Glu 100 105 110 Asp Ser Ser Phe Tyr Ile Cys Ser Ala Ser Asn Arg Leu Ala Val Asn 115 120 125 Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp Leu 130 135 140 Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro Ser Lys Ala 145 150 155 160 Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val Cys Leu Ala Arg Gly 165 170 175 Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu 180 185 190 Val His Ser Gly Val Xaa Thr Asp Pro Gln Ala Tyr Lys Glu Ser Asn 195 200 205 Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp 210 215 220 His Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe His Gly Leu 225 230 235 240 Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro Lys Pro Val Thr Gln 245 250 255 Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser 260 265 270 Ala Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile 275 280 285 Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Thr Leu Val 290 295 300 Val Met Ala Met Val Lys Arg Lys Asn Ser 305 310 <210> 32 <211> 277 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Synthetic <220> <221> SITE <222> (188)..(188) <223> X is Thr or Cys <220> <221> SITE <222> (252)..(252) <223> X is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp <220> <221> SITE <222> (254)..(254) <223> X is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp <220> <221> SITE <222> (255)..(255) <223> X is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp <400> 32 Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro 1 5 10 15 Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln 20 25 30 Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn 35 40 45 Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys 50 55 60 Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys 65 70 75 80 Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala 85 90 95 Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala 100 105 110 Val Tyr Phe Cys Ala Ala Ser Gly Asn Thr Gly Thr Ala Ser Lys Leu 115 120 125 Thr Phe Gly Thr Gly Thr Arg Leu Gln Val Thr Leu Asp Ile Gln Asn 130 135 140 Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser 145 150 155 160 Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn Val Pro Lys 165 170 175 Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Xaa Val Leu Asp Met 180 185 190 Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln 195 200 205 Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr 210 215 220 Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe 225 230 235 240 Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Xaa Val Xaa Xaa Leu 245 250 255 Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu 260 265 270 Arg Leu Trp Ser Ser 275 <210> 33 <211> 307 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Synthetic <220> <221> SITE <222> (191)..(191) <223> X is Ser or Cys <400> 33 Met Ser Asn Gln Val Leu Cys Cys Val Val Leu Cys Phe Leu Gly Ala 1 5 10 15 Asn Thr Val Asp Gly Gly Ile Thr Gln Ser Pro Lys Tyr Leu Phe Arg 20 25 30 Lys Glu Gly Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His 35 40 45 Asp Ala Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Gln Ile Val Asn Asp Phe Gln Lys Gly Asp Ile Ala 65 70 75 80 Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr 85 90 95 Val Thr Ser Ala Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser 100 105 110 Ser Asn Gln Leu Val Thr Gly Gly Tyr Gly Tyr Thr Phe Gly Ser Gly 115 120 125 Thr Arg Leu Thr Val Val Glu Asp Leu Arg Asn Val Thr Pro Pro Lys 130 135 140 Val Ser Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys 145 150 155 160 Ala Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu 165 170 175 Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Xaa Thr 180 185 190 Asp Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser 195 200 205 Arg Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe 210 215 220 Arg Cys Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro 225 230 235 240 Glu Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp 245 250 255 Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr Gln Gln Gly Val 260 265 270 Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu 275 280 285 Tyr Ala Val Leu Val Ser Thr Leu Val Val Met Ala Met Val Lys Arg 290 295 300 Lys Asn Ser 305 <210> 34 <211> 269 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Synthetic <400> 34 Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser 1 5 10 15 Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu 20 25 30 Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp 35 40 45 Arg Gly Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser 50 55 60 Pro Glu Leu Ile Met Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly 65 70 75 80 Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu 85 90 95 Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val 100 105 110 Glu Gly Ala Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys Leu 115 120 125 Ser Val Ile Pro Asp Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu 130 135 140 Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe 145 150 155 160 Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile 165 170 175 Thr Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn 180 185 190 Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile 195 200 205 Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp 210 215 220 Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe 225 230 235 240 Gln Asn Leu Ser Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala 245 250 255 Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 <210> 35 <211> 314 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Synthetic <400> 35 Met Leu Leu Leu Leu Leu Leu Leu Gly Pro Gly Ile Ser Leu Leu Leu 1 5 10 15 Pro Gly Ser Leu Ala Gly Ser Gly Leu Gly Ala Val Val Ser Gln His 20 25 30 Pro Ser Trp Val Ile Cys Lys Ser Gly Thr Ser Val Lys Ile Glu Cys 35 40 45 Arg Ser Leu Asp Phe Gln Ala Thr Thr Met Phe Trp Tyr Arg Gln Phe 50 55 60 Pro Lys Gln Ser Leu Met Leu Met Ala Thr Ser Asn Glu Gly Ser Lys 65 70 75 80 Ala Thr Tyr Glu Gln Gly Val Glu Lys Asp Lys Phe Leu Ile Asn His 85 90 95 Ala Ser Leu Thr Leu Ser Thr Leu Thr Val Thr Ser Ala His Pro Glu 100 105 110 Asp Ser Ser Phe Tyr Ile Cys Ser Ala Ser Asn Arg Leu Ala Val Asn 115 120 125 Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp Leu 130 135 140 Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro Ser Lys Ala 145 150 155 160 Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val Cys Leu Ala Arg Gly 165 170 175 Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu 180 185 190 Val His Ser Gly Val Ser Thr Asp Pro Gln Ala Tyr Lys Glu Ser Asn 195 200 205 Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp 210 215 220 His Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe His Gly Leu 225 230 235 240 Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro Lys Pro Val Thr Gln 245 250 255 Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser 260 265 270 Ala Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile 275 280 285 Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Thr Leu Val 290 295 300 Val Met Ala Met Val Lys Arg Lys Asn Ser 305 310 <210> 36 <211> 277 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Synthetic <400> 36 Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro 1 5 10 15 Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln 20 25 30 Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn 35 40 45 Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys 50 55 60 Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys 65 70 75 80 Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala 85 90 95 Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala 100 105 110 Val Tyr Phe Cys Ala Ala Ser Gly Asn Thr Gly Thr Ala Ser Lys Leu 115 120 125 Thr Phe Gly Thr Gly Thr Arg Leu Gln Val Thr Leu Asp Ile Gln Asn 130 135 140 Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser 145 150 155 160 Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn Val Pro Lys 165 170 175 Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Thr Val Leu Asp Met 180 185 190 Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln 195 200 205 Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr 210 215 220 Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe 225 230 235 240 Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Ser Val Met Gly Leu 245 250 255 Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu 260 265 270 Arg Leu Trp Ser Ser 275 <210> 37 <211> 307 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Synthetic <400> 37 Met Ser Asn Gln Val Leu Cys Cys Val Val Leu Cys Phe Leu Gly Ala 1 5 10 15 Asn Thr Val Asp Gly Gly Ile Thr Gln Ser Pro Lys Tyr Leu Phe Arg 20 25 30 Lys Glu Gly Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His 35 40 45 Asp Ala Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Gln Ile Val Asn Asp Phe Gln Lys Gly Asp Ile Ala 65 70 75 80 Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr 85 90 95 Val Thr Ser Ala Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser 100 105 110 Ser Asn Gln Leu Val Thr Gly Gly Tyr Gly Tyr Thr Phe Gly Ser Gly 115 120 125 Thr Arg Leu Thr Val Val Glu Asp Leu Arg Asn Val Thr Pro Pro Lys 130 135 140 Val Ser Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys 145 150 155 160 Ala Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu 165 170 175 Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr 180 185 190 Asp Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser 195 200 205 Arg Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe 210 215 220 Arg Cys Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro 225 230 235 240 Glu Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp 245 250 255 Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr Gln Gln Gly Val 260 265 270 Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu 275 280 285 Tyr Ala Val Leu Val Ser Thr Leu Val Val Met Ala Met Val Lys Arg 290 295 300 Lys Asn Ser 305 <210> 38 <211> 27 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Synthetic <400> 38 Arg Ala Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys 1 5 10 15 Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 20 25 <210> 39 <211> 24 <212> PRT <213> Homo sapiens <400> 39 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Arg Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile 20 <210> 40 <211> 24 <212> PRT <213> Homo sapiens <400> 40 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile 20 <210> 41 <211> 120 <212> PRT <213> Homo sapiens <400> 41 Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln Asn Ser Pro Ser 1 5 10 15 Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn Cys Asp Tyr Thr 20 25 30 Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys Tyr Pro Ala Glu 35 40 45 Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys Asp Lys Asn Glu 50 55 60 Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala Lys His Leu Ser 65 70 75 80 Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Ala Ser Gly Asn Thr Gly Thr Ala Ser Lys Leu Thr Phe Gly Thr 100 105 110 Gly Thr Arg Leu Gln Val Thr Leu 115 120 <210> 42 <211> 114 <212> PRT <213> Homo sapiens <400> 42 Gly Gly Ile Thr Gln Ser Pro Lys Tyr Leu Phe Arg Lys Glu Gly Gln 1 5 10 15 Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His Asp Ala Met Tyr 20 25 30 Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu Ile Tyr Tyr Ser 35 40 45 Gln Ile Val Asn Asp Phe Gln Lys Gly Asp Ile Ala Glu Gly Tyr Ser 50 55 60 Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr Val Thr Ser Ala 65 70 75 80 Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser Ser Asn Gln Leu 85 90 95 Val Thr Gly Gly Tyr Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr 100 105 110 Val Val <210> 43 <211> 113 <212> PRT <213> Homo sapiens <400> 43 Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro 1 5 10 15 Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser 20 25 30 Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu 35 40 45 Ile Met Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr 50 55 60 Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp 65 70 75 80 Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Glu Gly Ala 85 90 95 Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys Leu Ser Val Ile 100 105 110 Pro <210> 44 <211> 121 <212> PRT <213> Homo sapiens <400> 44 Ala Gly Ser Gly Leu Gly Ala Val Val Ser Gln His Pro Ser Trp Val 1 5 10 15 Ile Cys Lys Ser Gly Thr Ser Val Lys Ile Glu Cys Arg Ser Leu Asp 20 25 30 Phe Gln Ala Thr Thr Met Phe Trp Tyr Arg Gln Phe Pro Lys Gln Ser 35 40 45 Leu Met Leu Met Ala Thr Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu 50 55 60 Gln Gly Val Glu Lys Asp Lys Phe Leu Ile Asn His Ala Ser Leu Thr 65 70 75 80 Leu Ser Thr Leu Thr Val Thr Ser Ala His Pro Glu Asp Ser Ser Phe 85 90 95 Tyr Ile Cys Ser Ala Ser Asn Arg Leu Ala Val Asn Glu Gln Phe Phe 100 105 110 Gly Pro Gly Thr Arg Leu Thr Val Leu 115 120 <210> 45 <211> 250 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Synthetic <220> <221> SITE <222> (161)..(161) <223> X is Thr or Cys <220> <221> SITE <222> (225)..(225) <223> X is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp <220> <221> SITE <222> (227)..(227) <223> X is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp <220> <221> SITE <222> (228)..(228) <223> X is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp <400> 45 Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro 1 5 10 15 Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser 20 25 30 Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu 35 40 45 Ile Met Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr 50 55 60 Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp 65 70 75 80 Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Glu Gly Ala 85 90 95 Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys Leu Ser Val Ile 100 105 110 Pro Asp Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro 115 120 125 Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln 130 135 140 Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys 145 150 155 160 Xaa Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile 165 170 175 Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu 180 185 190 Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu 195 200 205 Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu 210 215 220 Xaa Val Xaa Xaa Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn 225 230 235 240 Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 245 250 <210> 46 <211> 294 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Synthetic <220> <221> SITE <222> (178)..(178) <223> X is Ser or Cys <400> 46 Ala Gly Ser Gly Leu Gly Ala Val Val Ser Gln His Pro Ser Trp Val 1 5 10 15 Ile Cys Lys Ser Gly Thr Ser Val Lys Ile Glu Cys Arg Ser Leu Asp 20 25 30 Phe Gln Ala Thr Thr Met Phe Trp Tyr Arg Gln Phe Pro Lys Gln Ser 35 40 45 Leu Met Leu Met Ala Thr Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu 50 55 60 Gln Gly Val Glu Lys Asp Lys Phe Leu Ile Asn His Ala Ser Leu Thr 65 70 75 80 Leu Ser Thr Leu Thr Val Thr Ser Ala His Pro Glu Asp Ser Ser Phe 85 90 95 Tyr Ile Cys Ser Ala Ser Asn Arg Leu Ala Val Asn Glu Gln Phe Phe 100 105 110 Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Arg Asn Val Thr 115 120 125 Pro Pro Lys Val Ser Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn 130 135 140 Lys Gln Lys Ala Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp 145 150 155 160 His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly 165 170 175 Val Xaa Thr Asp Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys 180 185 190 Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg 195 200 205 Asn His Phe Arg Cys Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp 210 215 220 Lys Trp Pro Glu Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala 225 230 235 240 Glu Ala Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr Gln 245 250 255 Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys 260 265 270 Ala Thr Leu Tyr Ala Val Leu Val Ser Thr Leu Val Val Met Ala Met 275 280 285 Val Lys Arg Lys Asn Ser 290 <210> 47 <211> 257 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Synthetic <220> <221> SITE <222> (168)..(168) <223> X at position 168 is Thr or Cys <220> <221> SITE <222> (232)..(232) <223> X is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp <220> <221> SITE <222> (234)..(234) <223> X is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp <220> <221> SITE <222> (235)..(235) <223> X is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp <400> 47 Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln Asn Ser Pro Ser 1 5 10 15 Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn Cys Asp Tyr Thr 20 25 30 Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys Tyr Pro Ala Glu 35 40 45 Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys Asp Lys Asn Glu 50 55 60 Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala Lys His Leu Ser 65 70 75 80 Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Ala Ser Gly Asn Thr Gly Thr Ala Ser Lys Leu Thr Phe Gly Thr 100 105 110 Gly Thr Arg Leu Gln Val Thr Leu Asp Ile Gln Asn Pro Glu Pro Ala 115 120 125 Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu 130 135 140 Phe Thr Asp Phe Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser 145 150 155 160 Gly Thr Phe Ile Thr Asp Lys Xaa Val Leu Asp Met Lys Ala Met Asp 165 170 175 Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr 180 185 190 Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp 195 200 205 Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met 210 215 220 Asn Leu Asn Phe Gln Asn Leu Xaa Val Xaa Xaa Leu Arg Ile Leu Leu 225 230 235 240 Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser 245 250 255 Ser <210> 48 <211> 287 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Synthetic <220> <221> SITE <222> (171)..(171) <223> X is Ser or Cys <400> 48 Gly Gly Ile Thr Gln Ser Pro Lys Tyr Leu Phe Arg Lys Glu Gly Gln 1 5 10 15 Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His Asp Ala Met Tyr 20 25 30 Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu Ile Tyr Tyr Ser 35 40 45 Gln Ile Val Asn Asp Phe Gln Lys Gly Asp Ile Ala Glu Gly Tyr Ser 50 55 60 Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr Val Thr Ser Ala 65 70 75 80 Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser Ser Asn Gln Leu 85 90 95 Val Thr Gly Gly Tyr Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr 100 105 110 Val Val Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe 115 120 125 Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val 130 135 140 Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp 145 150 155 160 Val Asn Gly Lys Glu Val His Ser Gly Val Xaa Thr Asp Pro Gln Ala 165 170 175 Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val 180 185 190 Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val 195 200 205 Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro 210 215 220 Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp 225 230 235 240 Cys Gly Ile Thr Ser Ala Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr 245 250 255 Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu 260 265 270 Val Ser Thr Leu Val Val Met Ala Met Val Lys Arg Lys Asn Ser 275 280 285 <210> 49 <211> 250 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Synthetic <400> 49 Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro 1 5 10 15 Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser 20 25 30 Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu 35 40 45 Ile Met Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr 50 55 60 Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp 65 70 75 80 Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Glu Gly Ala 85 90 95 Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys Leu Ser Val Ile 100 105 110 Pro Asp Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro 115 120 125 Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln 130 135 140 Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys 145 150 155 160 Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile 165 170 175 Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu 180 185 190 Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu 195 200 205 Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu 210 215 220 Ser Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn 225 230 235 240 Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 245 250 <210> 50 <211> 294 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Synthetic <400> 50 Ala Gly Ser Gly Leu Gly Ala Val Val Ser Gln His Pro Ser Trp Val 1 5 10 15 Ile Cys Lys Ser Gly Thr Ser Val Lys Ile Glu Cys Arg Ser Leu Asp 20 25 30 Phe Gln Ala Thr Thr Met Phe Trp Tyr Arg Gln Phe Pro Lys Gln Ser 35 40 45 Leu Met Leu Met Ala Thr Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu 50 55 60 Gln Gly Val Glu Lys Asp Lys Phe Leu Ile Asn His Ala Ser Leu Thr 65 70 75 80 Leu Ser Thr Leu Thr Val Thr Ser Ala His Pro Glu Asp Ser Ser Phe 85 90 95 Tyr Ile Cys Ser Ala Ser Asn Arg Leu Ala Val Asn Glu Gln Phe Phe 100 105 110 Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Arg Asn Val Thr 115 120 125 Pro Pro Lys Val Ser Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn 130 135 140 Lys Gln Lys Ala Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp 145 150 155 160 His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly 165 170 175 Val Ser Thr Asp Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys 180 185 190 Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg 195 200 205 Asn His Phe Arg Cys Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp 210 215 220 Lys Trp Pro Glu Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala 225 230 235 240 Glu Ala Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr Gln 245 250 255 Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys 260 265 270 Ala Thr Leu Tyr Ala Val Leu Val Ser Thr Leu Val Val Met Ala Met 275 280 285 Val Lys Arg Lys Asn Ser 290 <210> 51 <211> 257 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Synthetic <400> 51 Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln Asn Ser Pro Ser 1 5 10 15 Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn Cys Asp Tyr Thr 20 25 30 Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys Tyr Pro Ala Glu 35 40 45 Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys Asp Lys Asn Glu 50 55 60 Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala Lys His Leu Ser 65 70 75 80 Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Ala Ser Gly Asn Thr Gly Thr Ala Ser Lys Leu Thr Phe Gly Thr 100 105 110 Gly Thr Arg Leu Gln Val Thr Leu Asp Ile Gln Asn Pro Glu Pro Ala 115 120 125 Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu 130 135 140 Phe Thr Asp Phe Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser 145 150 155 160 Gly Thr Phe Ile Thr Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp 165 170 175 Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr 180 185 190 Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp 195 200 205 Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met 210 215 220 Asn Leu Asn Phe Gln Asn Leu Ser Val Met Gly Leu Arg Ile Leu Leu 225 230 235 240 Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser 245 250 255 Ser <210> 52 <211> 287 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Synthetic <400> 52 Gly Gly Ile Thr Gln Ser Pro Lys Tyr Leu Phe Arg Lys Glu Gly Gln 1 5 10 15 Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His Asp Ala Met Tyr 20 25 30 Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu Ile Tyr Tyr Ser 35 40 45 Gln Ile Val Asn Asp Phe Gln Lys Gly Asp Ile Ala Glu Gly Tyr Ser 50 55 60 Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr Val Thr Ser Ala 65 70 75 80 Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser Ser Asn Gln Leu 85 90 95 Val Thr Gly Gly Tyr Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr 100 105 110 Val Val Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe 115 120 125 Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val 130 135 140 Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp 145 150 155 160 Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Ala 165 170 175 Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val 180 185 190 Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val 195 200 205 Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro 210 215 220 Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp 225 230 235 240 Cys Gly Ile Thr Ser Ala Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr 245 250 255 Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu 260 265 270 Val Ser Thr Leu Val Val Met Ala Met Val Lys Arg Lys Asn Ser 275 280 285

Claims

1. An isolated or purified T cell receptor (TCR), wherein the TCR is antigen - specific for the mutant human RAS amino acid sequence shown in SEQ ID NO:39, and the TCR comprises: (a) an α - chain CDR1 having the amino acid sequence shown in SEQ ID NO:1, an α - chain CDR2 having the amino acid sequence shown in SEQ ID NO:2, an α - chain CDR3 having the amino acid sequence shown in SEQ ID NO:3, a β - chain CDR1 having the amino acid sequence shown in SEQ ID NO:4, a β - chain CDR2 having the amino acid sequence shown in SEQ ID NO:5, and a β - chain CDR3 having the amino acid sequence shown in SEQ ID NO:6; or (b) an α - chain CDR1 having the amino acid sequence shown in SEQ ID NO:7, an α - chain CDR2 having the amino acid sequence shown in SEQ ID NO:8, an α - chain CDR3 having the amino acid sequence shown in SEQ ID NO:9, a β - chain CDR1 having the amino acid sequence shown in SEQ ID NO:10, a β - chain CDR2 having the amino acid sequence shown in SEQ ID NO:11, and a β - chain CDR3 having the amino acid sequence shown in SEQ ID NO:

12.

2. The TCR according to claim 1, wherein the TCR is not antigen - specific for the wild - type human RAS amino acid sequence of SEQ ID NO:

40.

3. The TCR according to claim 1 or 2, wherein the mutant human RAS amino acid sequence is presented by a human leukocyte antigen (HLA) class II molecule.

4. The TCR according to claim 3, wherein the HLA class II molecule is an HLA - DR heterodimer or an HLA - DQ heterodimer.

5. The TCR according to claim 3, wherein the HLA class II molecule is an HLA - DRB5:HLA - DRA heterodimer or an HLA - DQA1:HLA - DQB1 heterodimer.

6. The TCR according to claim 3, wherein the HLA class II molecule is an HLA - DRB5*01:HLA - DRA*01:01 heterodimer or an HLA - DQA1*05:05:HLA - DQB1*03:01 heterodimer.

7. The TCR according to claim 1 or 2, which comprises: (i) both the amino acid sequence of SEQ ID NO:13 and the amino acid sequence of SEQ ID NO:14, (ii) both the amino acid sequence of SEQ ID NO:15 and the amino acid sequence of SEQ ID NO:16, (iii) both the amino acid sequence of SEQ ID NO:41 and the amino acid sequence of SEQ ID NO:42, or (iv) both the amino acid sequence of SEQ ID NO:43 and the amino acid sequence of SEQ ID NO:

44.

8. The TCR according to claim 1 or 2, which further comprises: (a) an α - chain constant region comprising the amino acid sequence of SEQ ID NO:26, wherein: (i)X at position 48 of SEQ ID NO:26 is Cys; (ii)X at position 112 of SEQ ID NO:26 is Leu; (iii)X at position 114 of SEQ ID NO:26 is Ile; and (iv)X at position 115 of SEQ ID NO:26 is Val; (b) a beta chain constant region comprising the amino acid sequence of SEQ ID NO:27, wherein X at position 57 of SEQ ID NO:27 is Cys; or (c) both (a) and (b).

9. The isolated or purified TCR according to claim 1 or 2, comprising: (a) an alpha chain comprising the amino acid sequence of SEQ ID NO:30, wherein: (i)X at position 180 of SEQ ID NO:30 is Cys; (ii)X at position 244 of SEQ ID NO:30 is Leu; (iii)X at position 246 of SEQ ID NO:30 is Ile; and (iv)X at position 247 of SEQ ID NO:30 is Val; (b) a beta chain comprising the amino acid sequence of SEQ ID NO:31, wherein X at position 198 of SEQ ID NO:31 is Cys; (c) an alpha chain comprising the amino acid sequence of SEQ ID NO:32, wherein: (i)X at position 188 of SEQ ID NO:32 is Cys; (ii)X at position 252 of SEQ ID NO:32 is Leu; (iii)X at position 254 of SEQ ID NO:32 is Ile; and (iv)X at position 255 of SEQ ID NO:32 is Val; (d) a beta chain comprising the amino acid sequence of SEQ ID NO:33, wherein X at position 191 of SEQ ID NO:33 is Cys; (e) both (a) and (b); (f) both (c) and (d); (g) an alpha chain comprising the amino acid sequence of SEQ ID NO:45, wherein: (i)X at position 161 of SEQ ID NO:45 is Cys; (ii)X at position 225 of SEQ ID NO:45 is Leu; (iii)X at position 227 of SEQ ID NO:45 is Ile; and (iv)X at position 228 of SEQ ID NO:45 is Val; (h) a beta chain comprising the amino acid sequence of SEQ ID NO:46, wherein X at position 178 of SEQ ID NO:46 is Cys; (i) an alpha chain comprising the amino acid sequence of SEQ ID NO:47, wherein: (i)X at position 168 of SEQ ID NO:47 is Cys; (ii)X at position 232 of SEQ ID NO:47 is Leu; (iii)X at position 234 of SEQ ID NO:47 is Ile; and (iv) X at position 235 of SEQ ID NO:47 is Val; (j) a β-chain comprising the amino acid sequence of SEQ ID NO:48, wherein X at position 171 of SEQ ID NO:48 is Cys; (k) both (g) and (h); or (l) both (i) and (j).

10. An isolated or purified polypeptide comprising a functional portion of the TCR according to any one of claims 1 to 9, wherein the functional portion is antigen-specific for the mutant human RAS amino acid sequence shown in SEQ ID NO:39 and comprises the following amino acid sequences: (a) an α-chain CDR1 having the amino acid sequence of SEQ ID NO:1, an α-chain CDR2 having the amino acid sequence of SEQ ID NO:2, an α-chain CDR3 having the amino acid sequence of SEQ ID NO:3, a β-chain CDR1 having the amino acid sequence of SEQ ID NO:4, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:6; or (b) an α-chain CDR1 having the amino acid sequence of SEQ ID NO:7, an α-chain CDR2 having the amino acid sequence of SEQ ID NO:8, an α-chain CDR3 having the amino acid sequence of SEQ ID NO:9, a β-chain CDR1 having the amino acid sequence of SEQ ID NO:10, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:11, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:

12.

11. The isolated or purified polypeptide according to claim 10, wherein the functional portion comprises: (i) the amino acid sequence of SEQ ID NO:13, (ii) the amino acid sequence of SEQ ID NO:14, (iii) the amino acid sequence of SEQ ID NO:15, (iv) the amino acid sequence of SEQ ID NO:16, (v) both the amino acid sequence of SEQ ID NO:13 and the amino acid sequence of SEQ ID NO:14, (vi) both the amino acid sequence of SEQ ID NO:15 and the amino acid sequence of SEQ ID NO:16, (vii) the amino acid sequence of SEQ ID NO:41, (viii) the amino acid sequence of SEQ ID NO:42, (ix) the amino acid sequence of SEQ ID NO:43, (x) the amino acid sequence of SEQ ID NO:44, (xi) both the amino acid sequence of SEQ ID NO:41 and the amino acid sequence of SEQ ID NO:42, or (xii) both the amino acid sequence of SEQ ID NO:43 and the amino acid sequence of SEQ ID NO:

44.

12. The isolated or purified polypeptide according to claim 10 or 11, further comprising: (a) The amino acid sequence of SEQ ID NO:26, wherein: (i) X at position 48 of SEQ ID NO:26 is Cys; (ii) X at position 112 of SEQ ID NO:26 is Leu; (iii) X at position 114 of SEQ ID NO:26 is Ile; and (iv) X at position 115 of SEQ ID NO:26 is Val; (b) The amino acid sequence of SEQ ID NO:27, wherein X at position 57 of SEQ ID NO:27 is Cys; or (c) Both (a) and (b).

13. The isolated or purified polypeptide according to claim 10 or 11, comprising: (a) The amino acid sequence of SEQ ID NO:30, wherein: (i) X at position 180 of SEQ ID NO:30 is Cys; (ii) X at position 244 of SEQ ID NO:30 is Leu; (iii) X at position 246 of SEQ ID NO:30 is Ile; and (iv) X at position 247 of SEQ ID NO:30 is Val; (b) The amino acid sequence of SEQ ID NO:31, wherein X at position 198 of SEQ ID NO:31 is Cys; (c) The amino acid sequence of SEQ ID NO:32, wherein: (i) X at position 188 of SEQ ID NO:32 is Cys; (ii) X at position 252 of SEQ ID NO:32 is Leu; (iii) X at position 254 of SEQ ID NO:32 is Ile; and (iv) X at position 255 of SEQ ID NO:32 is Val; (d) The amino acid sequence of SEQ ID NO:33, wherein X at position 191 of SEQ ID NO:33 is Cys; (e) Both (a) and (b); (f) Both (c) and (d); (g) The amino acid sequence of SEQ ID NO:45, wherein: (i) X at position 161 of SEQ ID NO:45 is Cys; (ii) X at position 225 of SEQ ID NO:45 is Leu; (iii) X at position 227 of SEQ ID NO:45 is Ile; and (iv) X at position 228 of SEQ ID NO:45 is Val; (h) The amino acid sequence of SEQ ID NO:46, wherein X at position 178 of SEQ ID NO:46 is Cys; (i) The amino acid sequence of SEQ ID NO:47, wherein: (i) X at position 168 of SEQ ID NO:47 is Cys; (ii) X at position 232 of SEQ ID NO:47 is Leu; (iii) X at position 234 of SEQ ID NO:47 is Ile; and (iv) X at position 235 of SEQ ID NO:47 is Val; (j) The amino acid sequence of SEQ ID NO: 48, wherein X at position 171 of SEQ ID NO: 48 is Cys; (k) Both (g) and (h); or (l) Both (i) and (j).

14. An isolated or purified protein that has antigenic specificity for the mutant human RAS amino acid sequence shown in SEQ ID NO: 39 and comprises: (a) A first polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 1 to 3 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 4 to 6; or (b) A first polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 7 to 9 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 10 to 12.

15. The isolated or purified protein according to claim 14, wherein: (i) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 13 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 14; (ii) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 15 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16; (iii) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 41 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 42; or (iv) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 43 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

44.

16. The isolated or purified protein according to claim 14 or 15, wherein: (a) The first polypeptide chain further comprises the amino acid sequence of SEQ ID NO: 26, wherein: (i) X at position 48 of SEQ ID NO: 26 is Cys; (ii) X at position 112 of SEQ ID NO: 26 is Leu; (iii) X at position 114 of SEQ ID NO: 26 is Ile; and (iv) X at position 115 of SEQ ID NO: 26 is Val; (b) The second polypeptide chain further comprises the amino acid sequence of SEQ ID NO: 27, wherein X at position 57 of SEQ ID NO: 27 is Cys; or (c) Both (a) and (b).

17. The isolated or purified protein according to claim 14 or 15, wherein: (a) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 30, wherein: (i) X at position 180 of SEQ ID NO: 30 is Cys; (ii) X at position 244 of SEQ ID NO: 30 is Leu; (iii) X at position 246 of SEQ ID NO: 30 is Ile; and (iv) X at position 247 of SEQ ID NO: 30 is Val; (b) The second polypeptide chain comprises the amino acid sequence of SEQ ID NO:31, wherein X at position 198 of SEQ ID NO:31 is Cys; (c) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO:32, wherein: (i) X at position 188 of SEQ ID NO:32 is Cys; (ii) X at position 252 of SEQ ID NO:32 is Leu; (iii) X at position 254 of SEQ ID NO:32 is Ile; and (iv) X at position 255 of SEQ ID NO:32 is Val; (d) The second polypeptide chain comprises the amino acid sequence of SEQ ID NO:33, wherein X at position 191 of SEQ ID NO:33 is Cys; (e) Both (a) and (b); (f) Both (c) and (d); (g) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO:45, wherein: (i) X at position 161 of SEQ ID NO:45 is Cys; (ii) X at position 225 of SEQ ID NO:45 is Leu; (iii) X at position 227 of SEQ ID NO:45 is Ile; and (iv) X at position 228 of SEQ ID NO:45 is Val; (h) The second polypeptide chain comprises the amino acid sequence of SEQ ID NO:46, wherein X at position 178 of SEQ ID NO:46 is Cys; (i) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO:47, wherein: (i) X at position 168 of SEQ ID NO:47 is Cys; (ii) X at position 232 of SEQ ID NO:47 is Leu; (iii) X at position 234 of SEQ ID NO:47 is Ile; and (iv) X at position 235 of SEQ ID NO:47 is Val; (j) The second polypeptide chain comprises the amino acid sequence of SEQ ID NO:48, wherein X at position 171 of SEQ ID NO:48 is Cys; (k) Both (g) and (h); or (l) Both (i) and (j).

18. An isolated or purified nucleic acid comprising a nucleotide sequence encoding a TCR as described in any one of claims 1 to 9, a polypeptide as described in any one of claims 10 to 13, or a protein as described in any one of claims 14 to 17.

19. An isolated or purified nucleic acid comprising, from 5' to 3', a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence encode the following amino acid sequences respectively: SEQ ID NO: 13 and 14; 14 and 13; 15 and 16; 16 and 15; 30 and 31; 31 and 30; 32 and 33; 33 and 32; 41 and 42; 42 and 41; 43 and 44; 44 and 43; 45 and 46; 46 and 45; 47 and 48; or 48 and 47, wherein: (i) X at position 180 of SEQ ID NO: 30 is Cys, X at position 244 of SEQ ID NO: 30 is Leu, X at position 246 of SEQ ID NO: 30 is Ile, and X at position 247 of SEQ ID NO: 30 is Val; (ii) X at position 198 of SEQ ID NO: 31 is Cys; (iii) X at position 188 of SEQ ID NO: 32 is Cys, X at position 252 of SEQ ID NO: 32 is Leu, X at position 254 of SEQ ID NO: 32 is Ile, and X at position 255 of SEQ ID NO: 32 is Val; (iv) X at position 191 of SEQ ID NO: 33 is Cys; (v) X at position 161 of SEQ ID NO: 45 is Cys, X at position 225 of SEQ ID NO: 45 is Leu, X at position 227 of SEQ ID NO: 45 is Ile, and X at position 228 of SEQ ID NO: 45 is Val; (vi) X at position 178 of SEQ ID NO: 46 is Cys; (vii) X at position 168 of SEQ ID NO: 47 is Cys, X at position 232 of SEQ ID NO: 47 is Leu, X at position 234 of SEQ ID NO: 47 is Ile, and X at position 235 of SEQ ID NO: 47 is Val; and (viii) X at position 171 of SEQ ID NO: 48 is Cys.

20. The isolated or purified nucleic acid according to claim 19, further comprising a third nucleotide sequence inserted between the first nucleotide sequence and the second nucleotide sequence, wherein the third nucleotide sequence encodes a cleavable linker peptide.

21. The isolated or purified nucleic acid according to claim 20, wherein the cleavable linker peptide comprises the amino acid sequence of SEQ ID NO:

38.

22. A recombinant expression vector comprising the nucleic acid according to any one of claims 18 to 21.

23. The recombinant expression vector according to claim 22, which is a transposon or a lentiviral vector.

24. A method for producing a host cell expressing a TCR that is antigen - specific for the peptide of SEQ ID NO:39, the method comprising contacting an isolated or purified cell with the vector as recited in claim 22 or 23 under conditions that permit introduction of the vector into the cell.

25. An isolated or purified host cell comprising the nucleic acid as recited in any one of claims 18 to 21 or the recombinant expression vector as recited in claim 22 or 23.

26. The host cell as recited in claim 25, wherein the cell is a human lymphocyte.

27. The host cell as recited in claim 25 or 26, wherein the cell is selected from T cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, and natural killer (NK) cells.

28. An isolated or purified cell population comprising the host cell as recited in any one of claims 25 to 27.

29. A method for producing a TCR as recited in any one of claims 1 to 9, a polypeptide as recited in any one of claims 10 to 13, or a protein as recited in any one of claims 14 to 17, the method comprising culturing the host cell as recited in any one of claims 25 - 27 or the host cell population as recited in claim 28 to produce the TCR, the polypeptide, or the protein.

30. A pharmaceutical composition comprising (a) a TCR as recited in any one of claims 1 to 9, a polypeptide as recited in any one of claims 10 to 13, or a protein as recited in any one of claims 14 to 17, a nucleic acid as recited in any one of claims 18 to 21, a recombinant expression vector as recited in claim 22 or 23, a host cell as recited in any one of claims 25 to 27, or a cell population as recited in claim 28, and (b) a pharmaceutically acceptable carrier.

31. Use of a TCR as recited in any one of claims 1 to 9, a polypeptide as recited in any one of claims 10 to 13, or a protein as recited in any one of claims 14 to 17, a nucleic acid as recited in any one of claims 18 to 21, a recombinant expression vector as recited in claim 22 or 23, a host cell as recited in any one of claims 25 to 27, a cell population as recited in claim 28, or the pharmaceutical composition as recited in claim 30 in the preparation of a medicament for inducing an immune response against cancer in a mammal.

32. Use of a TCR as recited in any one of claims 1 to 9, a polypeptide as recited in any one of claims 10 to 13, or a protein as recited in any one of claims 14 to 17, a nucleic acid as recited in any one of claims 18 to 21, a recombinant expression vector as recited in claim 22 or 23, a host cell as recited in any one of claims 25 to 27, a cell population as recited in claim 28, or the pharmaceutical composition as recited in claim 30 in the preparation of a medicament for detecting, treating, or preventing cancer in a mammal.

33. The use according to claim 31 or 32, wherein the cancer expresses a mutant human RAS amino acid sequence in which glycine at position 12 is replaced by arginine, wherein the mutant human RAS amino acid sequence is a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), mutant human Harvey rat sarcoma viral oncogene homolog (HRAS) or mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence, and wherein position 12 is defined with reference to the wild-type human KRAS protein, wild-type human HRAS protein or wild-type human NRAS protein, respectively.

34. The use according to claim 33, wherein the mutant human RAS amino acid sequence is a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS) amino acid sequence.

35. The use according to claim 33, wherein the mutant human RAS amino acid sequence is a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence.

36. The use according to claim 33, wherein the mutant human RAS amino acid sequence is a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS) amino acid sequence.

37. The use according to claim 33, wherein the cancer is pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer or prostate cancer.

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