A high affinity t cell receptor recognizing an hpv antigen

Abstract

The present application provides a T cell receptor (TCR) having the property of binding to YMLDLQPET-HLA A0201 complex; and the binding affinity of the TCR to the YMLDLQPET-HLA A0201 complex is at least 2 times the binding affinity of the wild-type TCR to the YMLDLQPET-HLA A0201 complex. The present application also provides a fusion molecule of such TCR and a therapeutic agent. Such TCR can be used alone or in combination with a therapeutic agent to target tumor cells presenting YMLDLQPET-HLA A0201 complex.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of biotechnology, more specifically to a T cell receptor (TCR) capable of recognizing a polypeptide derived from HPV16 E7 protein. The present invention also relates to the preparation and use of said receptor. BACKGROUND

[0002] There are only two types of molecules that can recognize antigens in a specific manner. One is the immunoglobulin or antibody; the other is the T cell receptor (TCR), which is a glycoprotein on the cell membrane in the form of a heterodimer of a chain / β chain or a chain / δ chain. The composition of the TCR repertoire of the immune system is generated in the thymus by V(D)J recombination, followed by positive and negative selection. In the peripheral environment, the TCR mediates the specific recognition of the major histocompatibility complex-peptide complex (pMHC) by T cells, and thus is essential for the cellular immune function of the immune system.

[0003] The TCR is the only receptor for specific antigen peptides presented on the major histocompatibility complex (MHC), and such exogenous or endogenous peptides can be the only sign of cell abnormality. In the immune system, the binding of antigen-specific TCR to the pMHC complex initiates direct physical contact between T cells and antigen-presenting cells (APCs), and then other cell membrane surface molecules of both T cells and APCs interact, which leads to a series of subsequent cell signaling and other physiological responses, thus enabling different antigen-specific T cells to exert immune effects on their target cells.

[0004] The MHC class I and class II molecule ligands corresponding to the TCR are also proteins of the immunoglobulin superfamily but have specificity for antigen presentation, and different individuals have different MHCs, thus being able to present different short peptides in a protein antigen to the surface of their respective APC cells. The human MHC is commonly referred to as the HLA gene or HLA complex.

[0005] The HPV16 E7 gene is one of the early region genes of the human papillomavirus (HPV) genome, and the E7 protein encoded by the gene is a small acidic protein containing about 100 amino acids. The high-risk HPV16 E7 protein is an important cause of inducing tumors such as cervical cancer, head and neck tumors, anal cancer, etc. After HPV16 E7 is generated in cells, it is degraded into small polypeptides and binds to MHC (major histocompatibility complex) molecules to form a complex, which is presented to the cell surface. YMLDLQPET is a short peptide derived from the HPV16 E7 antigen and is a target for the treatment of HPV16 E7-related diseases.

[0006] Accordingly, the YMLDLQPET-HLA A0201 complex provides a marker against which a TCR can target tumor cells. A TCR capable of binding to the YMLDLQPET-HLA A0201 complex has high application value for the treatment of tumors. For example, a TCR capable of targeting this tumor cell marker can be used to deliver cytotoxic or immunostimulatory agents to target cells, or be transformed into T cells, enabling T cells expressing this TCR to destroy tumor cells, in order to be administered to a patient in a treatment process known as adoptive immunotherapy. For the former purpose, a TCR with a high affinity is desirable, enabling the TCR to reside on the targeted cell for a long period of time. For the latter purpose, it is preferred to use a TCR with a medium affinity. Therefore, the skilled person is committed to developing TCRs targeting tumor cell markers for different purposes. SUMMARY

[0007] The present application aims to provide a TCR with a high affinity for the YMLDLQPET-HLA A0201 complex.

[0008] A further object of the present application is to provide a method for preparing a TCR of the above type and the use of a TCR of the above type.

[0009] In a first aspect, the present application provides a T cell receptor (TCR) comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, having the activity of binding to a YMLDLQPET-HLA A0201 complex, and wherein the amino acid sequence of the TCR alpha chain variable domain has at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence of the TCR beta chain variable domain has at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 2.

[0010] In a preferred embodiment, the amino acid sequence of the TCR alpha chain variable domain and the amino acid sequence of the TCR beta chain variable domain are not simultaneously the amino acid sequence of a wild-type TCR alpha chain variable domain and the amino acid sequence of a wild-type TCR beta chain variable domain.

[0011] In a further preferred embodiment, the amino acid sequence of the TCR alpha chain variable domain is not the amino acid sequence shown in SEQ ID NO: 1 and / or

[0012] the amino acid sequence of the TCR beta chain variable domain is not the amino acid sequence shown in SEQ ID NO: 2.

[0013] In another preferred embodiment, the alpha chain variable domain of the TCR comprises an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology to the sequence set forth in SEQ ID NO: 1.

[0014] In another preferred embodiment, the beta chain variable domain of the TCR is an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to the sequence set forth in SEQ ID NO: 2.

[0015] In another preferred embodiment, the amino acid sequence of the TCR alpha chain variable domain has at least 95% sequence homology to the amino acid sequence set forth in SEQ ID NO: 1.

[0016] In another preferred embodiment, the amino acid sequence of the TCR beta chain variable domain has at least 95% sequence homology to the amino acid sequence set forth in SEQ ID NO: 2.

[0017] In another preferred embodiment, the TCR has at least 2-fold higher affinity for the YMLDLQPET-HLA A0201 complex than the wild-type TCR.

[0018] In another preferred embodiment, the three CDR regions (complementarity determining regions) of the TCR alpha chain variable domain have the following reference sequences,

[0019] CDR1a: DRVSQS

[0020] CDR2a: IYSNGD

[0021] CDR3a: AVNPRYGNKLV, and the TCR alpha chain variable domain contains at least one of the following mutations:

[0022]

[0023]

[0024] In another preferred embodiment, the TCR alpha chain variable domain has two or three or four amino acid mutations.

[0025] In another preferred embodiment, the amino acid sequence of CDR3a of the TCR alpha chain variable domain is: AVNPRYGNKLV.

[0026] In another preferred embodiment, the three CDRs of the TCR alpha chain variable domain are: CDR1a: DRVSQS;

[0027] CDR2a: IYSNGD; and CDR3a: AVNPRYGNKLV.

[0028] In another preferred embodiment, the TCR a chain variable domain has the amino acid sequence of SEQ ID NO: 1.

[0029] In another preferred embodiment, the three CDRs of the TCR β chain variable domain are:

[0030] CDR1 β: KGHDR;

[0031] CDR2 β: SFDVKD; and

[0032] CDR3 β: ATSDRGQGAFGEQY, and CDR3 β contains at least one of the following mutations:

[0033] residue before mutation residue after mutation G at position 6 of CDR3β Q Q at position 7 of CDR3β F or W or Y G at position 11 of CDR3β A Q at position 13 of CDR3β Y Y at position 14 of CDR3β F or H

[0034] In another preferred embodiment, the amino acid mutations in the CDR3 β include:

[0035] residue before mutation residue after mutation G at position 6 of CDR3β Q

[0036] In another preferred embodiment, the number of mutations in the CDR3 β is 1 or 2 or 3.

[0037] In another preferred embodiment, the TCR β chain variable domain has the amino acid sequence of SEQ ID NO: 1.

[0038] In another preferred embodiment, the TCR β chain variable domain has the amino acid sequence of SEQ ID NO: 1.

[0039] In another preferred embodiment, the TCR a chain variable domain comprises CDR1 a, CDR2 a, and CDR3 a, wherein the amino acid sequence of CDR3 a is: AVNPRYGNKLV, and the amino acid sequence of CDR1 a is DR[1 aX1 ][1 aX2][1 aX3][1 aX4] and the amino acid sequence of CDR2 a is I[2 aX1 ][2 aX2][2 aX3]GD, wherein [1 aX1 ] is V or L or M or H, and / or [1 aX2] is A or S or T or G, and / or [1 aX3] is Q or N or V or Y, and / or [1 aX4] is S or T or A or V, and / or [2 aX1 ] is Y or F, and / or [2 aX2] is S or N, and / or [2 aX3] is N or P.

[0040] In another preferred embodiment, the TCR has a CDR3a selected from the group consisting of: ATSDR[3aX1][3aX2]GAF[3aX3]E[3aX4][3aX5], wherein [3aX1] is Q or G, and / or [3aX2] is Y or F or Q or W, and / or [3aX3] is G or A, and / or [3aX4] is Q or Y, and / or [3aX5] is Y or H or F.

[0041] In another preferred embodiment, the TCR has a CDR3a selected from the group consisting of: ATSDR[3aX1][3aX2]GAF[3aX3]E[3aX4][3aX5], wherein [3aX1] is Q or G, and / or [3aX2] is Y or F or Q or W, and / or [3aX3] is G or A, and / or [3aX4] is Q or Y, and / or [3aX5] is Y or H or F.

[0042] In another preferred embodiment, the TCR has a CDR3a selected from the group consisting of: ATSDR[3aX1][3aX2]GAF[3aX3]E[3aX4][3aX5], wherein [3aX1] is Q or G, and / or [3aX2] is Y or F or Q or W, and / or [3aX3] is G or A, and / or [3aX4] is Q or Y, and / or [3aX5] is Y or H or F.

[0043] In another preferred embodiment, the TCR has a CDR3a selected from the group consisting of: ATSDR[3aX1][3aX2]GAF[3aX3]E[3aX4][3aX5], wherein [3aX1] is Q or G, and / or [3aX2] is Y or F or Q or W, and / or [3aX3] is G or A, and / or [3aX4] is Q or Y, and / or [3aX5] is Y or H or F.

[0044]

[0045] In another preferred embodiment, the TCR is soluble.

[0046] In another preferred embodiment, the TCR is an αβ heterodimeric TCR comprising an α chain TRAC constant region sequence and a β chain TRBC1 or TRBC2 constant region sequence.

[0047] In another preferred embodiment, the TCR comprises (i) all or a portion of a TCR α chain except for its transmembrane domain, and (ii) all or a portion of a TCR β chain except for its transmembrane domain, wherein both (i) and (ii) comprise a variable domain and at least a portion of a constant domain of a TCR chain.

[0048] In another preferred embodiment, the TCR has an artificial interchain disulfide bond between the constant regions of the α and β chains.

[0049] In another preferred embodiment, the cysteine residue that forms the artificial interchain disulfide bond between the constant regions of the α and β chains of the TCR is substituted for one or more of the following:

[0050] Thr48 of TRAC*01 exon 1 and Ser57 of TRBC1*01 or TRBC2*01 exon 1 ;

[0051] Thr45 of TRAC*01 exon 1 and Ser77 of TRBC1*01 or TRBC2*01 exon 1 ;

[0052] Tyr10 of TRAC*01 exon 1 and Ser17 of TRBC1*01 or TRBC2*01 exon 1 ;

[0053] Thr45 of TRAC*01 exon 1 and Asp59 of TRBC1*01 or TRBC2*01 exon 1 ;

[0054] Ser15 of TRAC*01 exon 1 and Glu15 of TRBC1*01 or TRBC2*01 exon 1 ;

[0055] Arg53 of TRAC*01 exon 1 and Ser54 of TRBC1*01 or TRBC2*01 exon 1 ;

[0056] Pro89 of TRAC*01 exon 1 and Ala19 of TRBC1*01 or TRBC2*01 exon 1 ;

[0057] Tyr10 of TRAC*01 exon 1 and Glu20 of TRBC1*01 or TRBC2*01 exon 1.

[0058] In another preferred embodiment, the alpha chain variable domain amino acid sequence of the TCR is one of SEQ ID NOs: 1, 13-21; and / or the beta chain variable domain amino acid sequence of the TCR is one of SEQ ID NOs: 2, 22-33.

[0059] In another preferred embodiment, the TCR is selected from the group consisting of:

[0060]

[0061]

[0062] In another preferred embodiment, the TCR is a single chain TCR.

[0063] In another preferred embodiment, the TCR is a single chain TCR consisting of an alpha chain variable domain and a beta chain variable domain connected by a flexible short peptide sequence (linker).

[0064] In another preferred embodiment, the C- or N-terminus of the alpha and / or beta chain of the TCR is conjugated to a conjugate, preferably the conjugate is a detectable label, a therapeutic agent, a PK modifying moiety or a combination of any of these.

[0065] In another preferred embodiment, the therapeutic agent conjugated to the TCR is an anti-CD3 antibody linked to the C- or N-terminus of the alpha or beta chain of the TCR.

[0066] In a second aspect of the application, there is provided a multivalent TCR complex comprising at least two TCR molecules, and wherein at least one of the TCR molecules is a TCR as defined in any of the preceding claims.

[0067] In a third aspect of the application, there is provided a nucleic acid molecule comprising a nucleic acid sequence encoding a TCR molecule as defined in the first aspect of the application or a multivalent TCR complex as defined in the second aspect of the application, or the complement thereof.

[0068] In a fourth aspect of the application, there is provided a vector comprising the nucleic acid molecule as defined in the third aspect of the application.

[0069] In a fifth aspect of the application, there is provided a host cell comprising the vector as defined in the fourth aspect of the application or having integrated into its chromosome the exogenous nucleic acid molecule as defined in the third aspect of the application.

[0070] In a sixth aspect of the application, there is provided an isolated cell expressing the TCR as defined in the first aspect of the application, preferably the isolated cell is a T cell, an NK cell and an NKT cell, most preferably the isolated cell is a T cell.

[0071] In a seventh aspect of the application, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR as defined in the first aspect of the application, or a TCR complex as defined in the second aspect of the application, or a cell as defined in the sixth aspect of the application.

[0072] In an eighth aspect of the application, there is provided a method of treating a disease, comprising administering to a subject in need thereof an effective amount of a TCR as defined in the first aspect of the application, or a TCR complex as defined in the second aspect of the application, or a cell as defined in the sixth aspect of the application, or a pharmaceutical composition as defined in the seventh aspect of the application, preferably the disease is an HPV16 E7 positive tumor, more preferably the tumor is cervical cancer.

[0073] In a ninth aspect, the present application provides the use of a TCR as defined in the first aspect of the application, or a TCR complex as defined in the second aspect of the application, or a cell as defined in the sixth aspect of the application, for the manufacture of a medicament for the treatment of a tumor, preferably the disease is a HPV16 E7 positive tumor, more preferably the tumor is cervical cancer.

[0074] In a tenth aspect, the present application provides a method for the manufacture of a T cell receptor as defined in the first aspect of the application, comprising the steps of:

[0075] (i) culturing a host cell as defined in the fifth aspect of the application, thereby expressing a T cell receptor as defined in the first aspect of the application;

[0076] (ii) isolating or purifying said T cell receptor.

[0077] It should be understood that, in the scope of the present application, the above technical features of the present application and the technical features specifically described in the following (e.g. in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0078] Figure 1a and Figure 1b show the amino acid sequences of the variable domains of the wild-type TCR alpha and beta chains, respectively, which are capable of specifically binding to the YMLDLQPET-HLA A0201 complex.

[0079] Figure 2a and Figure 2b are the amino acid sequences of the variable domains of the alpha and beta chains, respectively, of the single-chain template TCR constructed in the present application.

[0080] Figure 3a and Figure 3b are the DNA sequences of the variable domains of the alpha and beta chains, respectively, of the single-chain template TCR constructed in the present application.

[0081] Figure 4a and Figure 4b are the amino acid and DNA sequences, respectively, of the linker of the single-chain template TCR constructed in the present application.

[0082] Figure 5a , Figure 5b are the amino acid and DNA sequences, respectively, of the single-chain template TCR constructed in the present application.

[0083] Figure 6a and Figure 6b are the amino acid sequences of the soluble reference TCR alpha and beta chains, respectively, in the present application.

[0084] Figure 7(1)-(9) show the amino acid sequences of the variable domains of the alpha chain of the heterodimeric TCR with high affinity to the YMLDLQPET-HLA A0201 complex, respectively, with the mutated residues underlined.

[0085] Figure 8(1)-(12) show the amino acid sequences of the variable domains of the beta chain of the heterodimeric TCR with high affinity to the YMLDLQPET-HLA A0201 complex, respectively, with the mutated residues underlined.

[0086] Figure 9a and Figure 9b show the extracellular amino acid sequences of the wild-type TCR alpha and beta chains, respectively, which are capable of specifically binding to the YMLDLQPET-HLA A0201 complex.

[0087] Figure 10a and Figure 10b show the amino acid sequences of the wild-type TCR alpha and beta chains, respectively, which are capable of specifically binding to the YMLDLQPET-HLA A0201 complex.

[0088] Figure 11 is the binding curve of the soluble reference TCR, i.e. the wild-type TCR, to the YMLDLQPET-HLA A0201 complex.

[0089] Figure 12a and Figure 12b are the results of activation function experiments of effector cells transfected with the high affinity TCR of the application against T2 cells loaded with short peptides.

[0090] Figure 13a and Figure 13b are the results of activation function experiments of effector cells transfected with the high affinity TCR of the application against tumor cell lines.

[0091] Figure 14 are the results of killing function experiments of effector cells transfected with the high affinity TCR of the application against T2 cells loaded with short peptides in gradient.

[0092] Figure 15a , Figure 15b and Figure 15c are the results of killing function LDH experiments of effector cells transfected with the high affinity TCR of the application against tumor cell lines.

[0093] Figure 16a and Figure 16b are the results of killing function IncuCyte experiments of effector cells transfected with the high affinity TCR of the application against tumor cell lines. DETAILED DESCRIPTION

[0094] The present application, through extensive and in-depth research, obtains a high-affinity T cell receptor (TCR) for recognizing YMLDLQPET short peptide (derived from HPV16 E7 protein) presented in the form of peptide-HLA A0201 complex. The high-affinity TCR is mutated in the 3 CDR regions of the variable domain of its alpha chain:

[0095] CDR1α: DRVSQS

[0096] CDR2α: IYSNGD

[0097] CDR3α: AVNPRYGNKLV; and / or in the 3 CDR regions of the variable domain of its beta chain:

[0098] CDR1β: KGHDR

[0099] CDR2β: SFDVKD

[0100] CDR3β: ATSDRGQGAFGEQY; and, after mutation, the affinity and / or binding half-life of the TCR of the present application for the above YMLDLQPET-HLA A0201 complex is at least 2 times that of the wild-type TCR.

[0101] Before the present application is described, it is to be understood that this application is not limited to particular methods and experimental conditions described, as such methods and conditions can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present application will be limited only by the appended claims.

[0102] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0103] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are herein described.

[0104] The term

[0105] T cell receptor (TCR)

[0106] The TCRs can be described using the International Immunogenetics information system (IMGT). A native αβ heterodimeric TCR has an α chain and a β chain. Broadly, each chain comprises a variable region, a joining region, and a constant region, and the β chain usually also contains a short diversity region between the variable and joining regions, but this diversity region is often considered part of the joining region. The joining region of the TCR is determined by the unique IMGT TRAJ and TRBJ, and the constant region of the TCR is determined by IMGT TRAC and TRBC.

[0107] Each variable region comprises three CDRs (complementarity determining regions) embedded in a framework sequence, CDR1, CDR2, and CDR3. In the IMGT nomenclature, different numbers of TRAV and TRBV refer to different Va types and Vβ types, respectively. In the IMGT system, the α chain constant domain has the following notation: TRAC*01, where "TR" indicates T cell receptor gene; "A" indicates α chain gene; C indicates constant region; "*01" indicates allele 1. The β chain constant domain has the following notation: TRBC1*01 or TRBC2*01, where "TR" indicates T cell receptor gene; "B" indicates β chain gene; C indicates constant region; "*01" indicates allele 1. The constant region of the α chain is uniquely determined, and in the form of the β chain, there are two possible constant region genes "C1" and "C2". The constant region gene sequences of the TCR α and β chains can be obtained by the skilled person through the published IMGT database.

[0108] The α and β chains of the TCR are generally considered to each have two "domains", namely the variable domain and the constant domain. The variable domain is composed of the connected variable region and the joining region. Therefore, in the specification and claims of the present application, "TCR α chain variable domain" refers to the connected TRAV and TRAJ regions, and similarly, "TCR β chain variable domain" refers to the connected TRBV and TRBD / TRBJ regions. The three CDRs of the TCR α chain variable domain are CDR1α, CDR2α, and CDR3α, respectively; and the three CDRs of the TCR β chain variable domain are CDR1β, CDR2β, and CDR3β, respectively. The framework sequence of the TCR variable domain of the present application can be murine or human, and is preferably human. The constant domain of the TCR comprises an intracellular portion, a transmembrane region, and an extracellular portion.

[0109] In the present application, the amino acid sequences of the α and β chain variable domains of the wild-type TCR capable of binding to the YMLDLQPET-HLA A0201 complex are SEQ ID NO: 1 and SEQ ID NO: 2, respectively, as shown in Figure 1a and Figure 1bThe amino acid sequence of the alpha chain and the amino acid sequence of the beta chain of the soluble "reference TCR" described in the present application are SEQ ID NO: 11 and SEQ ID NO: 12, respectively, as shown in Figure 6a and Figure 6b The extracellular amino acid sequence of the alpha chain and the extracellular amino acid sequence of the beta chain of the "wild-type TCR" described in the present application are SEQ ID NO: 34 and SEQ ID NO: 35, respectively, as shown in Figure 9a and Figure 9b The TCR sequence used in the present application is of human origin. The amino acid sequence of the alpha chain and the amino acid sequence of the beta chain of the "wild-type TCR" described in the present application are SEQ ID NO: 36 and SEQ ID NO: 37, respectively, as shown in Figure 10a and 10b In the present application, the terms "polypeptide of the present application", "TCR of the present application", and "T cell receptor of the present application" are used interchangeably.

[0110] Natural interchain disulfide bond and artificial interchain disulfide bond

[0111] There is a group of disulfide bonds between the Cα and Cβ chains in the membrane proximal region of the native TCR, which is referred to as "natural interchain disulfide bond" in the present application. In the present application, the covalent disulfide bond artificially introduced and different from the position of the natural interchain disulfide bond is referred to as "artificial interchain disulfide bond".

[0112] For convenience of description, the position numbering of the amino acid sequence of TRAC*01 and TRBC1*01 or TRBC2*01 in the present application is in the order of N-terminal to C-terminal, for example, in TRBC1*01 or TRBC2*01, the 60th amino acid in the order of N-terminal to C-terminal is P (proline), which can be described as Pro60 of exon 1 of TRBC1*01 or TRBC2*01 in the present application, or it can be expressed as the 60th amino acid of exon 1 of TRBC1*01 or TRBC2*01, for another example, in TRBC1*01 or TRBC2*01, the 61st amino acid in the order of N-terminal to C-terminal is Q (glutamine), which can be described as Gln61 of exon 1 of TRBC1*01 or TRBC2*01 in the present application, or it can be expressed as the 61st amino acid of exon 1 of TRBC1*01 or TRBC2*01, and so on. In the present application, the position numbering of the amino acid sequence of the variable region TRAV and TRBV is in accordance with the position numbering listed in IMGT, for example, a certain amino acid in TRAV, the position numbering listed in IMGT is 46, which is described as the 46th amino acid of TRAV in the present application, and so on. In the present application, the sequence position numbering of other amino acids is as specified.

[0113] tumor

[0114] The term "tumor" refers to all types of cancerous cell growth or oncogenic processes, metastatic tissue or malignantly transformed cells, tissues or organs, regardless of histopathologic type or stage of invasiveness. Examples of tumors include, without limitation, solid tumors, soft tissue tumors, and metastatic lesions. Examples of solid tumors include malignant tumors of different organ systems, such as sarcomas, lung squamous carcinoma and carcinoma. For example: prostate, lung, breast, lymph, gastrointestinal (e.g., colon), and genitourinary tract (e.g., kidney), epithelial cells, pharynx. Lung squamous carcinoma includes malignant tumors such as, for example, colon cancer, rectal cancer, renal cell cancer, liver cancer, non-small cell lung cancer, small bowel cancer and esophageal cancer. Metastatic lesions of the above cancers can also be treated and prevented using the methods and compositions of the present application. DETAILED DESCRIPTION

[0116] It is known that the alpha chain variable domain and the beta chain variable domain of a TCR each contain 3 CDRs, analogous to the complementarity determining regions of an antibody. CDR3 interacts with the antigenic short peptide, and CDR1 and CDR2 interact with the HLA. Thus, the CDRs of a TCR molecule determine its interaction with the antigenic short peptide-HLA complex. The alpha chain variable domain amino acid sequence and the beta chain variable domain amino acid sequence of a wild-type TCR capable of binding to the antigenic short peptide YMLDLQPET in complex with HLA A0201 (i.e., YMLDLQPET-HLA A0201 complex) are SEQ ID NO: 1 and SEQ ID NO: 2, respectively, which sequences were first discovered by the present inventors. It has the following CDR regions:

[0117] CDR1a: DRVSQS

[0118] CDR2a: IYSNGD

[0119] CDR3a: AVNPRYGNKLV

[0120] CDR1b: KGHDR

[0121] CDR2b: SFDVKD and

[0122] CDR3b: ATSDRGQGAFGEQY.

[0123] The present application obtained, through mutation screening of the above CDR regions, a high affinity TCR with an affinity to the YMLDLQPET-HLA A0201 complex that is at least 2 times higher than the affinity of the wild-type TCR to the YMLDLQPET-HLA A0201 complex.

[0124] Further, the TCR of the present application is an αβ heterodimeric TCR, the α chain variable domain of the TCR comprises an amino acid sequence having at least 85%; preferably, at least 90%; more preferably, at least 92%; more preferably, at least 94% (e.g., can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) sequence homology to the amino acid sequence set forth in SEQ ID NO: 1; and / or the β chain variable domain of the TCR comprises an amino acid sequence having at least 90%, preferably, at least 92%; more preferably, at least 94% (e.g., can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology) sequence homology to the amino acid sequence set forth in SEQ ID NO: 2.

[0125] Further, the TCR of the present application is a single chain TCR, the α chain variable domain of the TCR comprises an amino acid sequence having at least 85%, preferably, at least 90%; more preferably, at least 92%; most preferably, at least 94% (e.g., can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) sequence homology to the amino acid sequence set forth in SEQ ID NO: 3; and / or the β chain variable domain of the TCR comprises an amino acid sequence having at least 85%, preferably, at least 90%; more preferably, at least 92%; most preferably, at least 94% (e.g., can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) sequence homology to the amino acid sequence set forth in SEQ ID NO: 4.

[0126] The 3 CDRs of the wild type TCR α chain variable domain of SEQ ID NO: 1, i.e., CDR1, CDR2 and CDR3, are located at positions 27-32, 50-55 and 90-100 of SEQ ID NO: 1, respectively. Accordingly, the amino acid residue numbering is taken from the numbering set forth in SEQ ID NO: 1, 29V is the 3rd V of CDR1α, 30S is the 4th S of CDR1α, 31Q is the 5th Q of CDR1α, 32S is the 6th S of CDR1α, 51Y is the 2nd Y of CDR2α, 52S is the 3rd S of CDR2α, 53N is the 4th N of CDR2α.

[0127] The present application provides a TCR having the property of binding to YMLDLQPET-HLA A0201 complex, and comprising an alpha chain variable domain and a beta chain variable domain, wherein the TCR is mutated in the alpha chain variable domain shown in SEQ ID NO: 1, and the mutated amino acid residue sites include one or more of 29V, 30S, 31Q, 32S, 51Y, 52S and 53N, wherein the amino acid residue numbering is shown in SEQ ID NO: 1.

[0128] Preferably, the mutated TCR alpha chain variable domain comprises one or more amino acid residues selected from the group consisting of 29L or 29M or 29H; 30A or 30T or 30G; 31N or 31V or 31Y; 32A or 32T or 32V; 51F; 52N; 53P, wherein the amino acid residue numbering is shown in SEQ ID NO: 1.

[0129] More specifically, the specific forms of the mutations in the alpha chain variable domain include one or more of V29L / M / H, S30A / T / G, Q31N / V / Y, S32A / T / V, Y51F, S52N and N53P.

[0130] In the present application, the 3 CDRs of the wild-type TCR beta chain variable domain SEQ ID NO: 2, i.e. CDR1, CDR2 and CDR3, are located at positions 27-31, 49-54 and 92-105 of SEQ ID NO: 2, respectively. Accordingly, 97G is the 6th G of CDR3β, 98Q is the 7th Q of CDR3β, 102G is the 11th G of CDR3β, 104Q is the 13th Q of CDR3β and 105Y is the 14th Y of CDR3β, with the amino acid residue numbering shown in SEQ ID NO: 2.

[0131] The present application provides a TCR having the property of binding to YMLDLQPET-HLA A0201 complex, and comprising a beta chain variable domain and a beta chain variable domain, wherein the TCR is mutated in the beta chain variable domain shown in SEQ ID NO: 2, and the mutated amino acid residue sites include one or more of 97G, 98Q, 102G, 104Q and 105Y, wherein the amino acid residue numbering is shown in SEQ ID NO: 2.

[0132] Preferably, the mutated TCR beta chain variable domain comprises one or more amino acid residues selected from the group consisting of 97Q; 98Y or 98W or 98F; 102A; 104Y; 105F or 105H, wherein the amino acid residue numbering is shown in SEQ ID NO: 2.

[0133] More specifically, the specific forms of the mutations in the beta chain variable domain include one or more of G97Q, Q98Y / W / F, G102A, Q104Y, Y105F / H.

[0134] It should be understood that the amino acid names herein are in the single letter code which is internationally accepted, and the corresponding amino acid names in the three letter code are: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gin (Q), Glu (E), Gly (G), His (H), He (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), Val (V);

[0135] In the present application, Pro60 or 60P both represent the 60th proline. In addition, the specific forms of the mutations in the present application are expressed as "G97Q" which means that the G at the 97th position is substituted by Q, and the like.

[0136] According to the method of site-directed mutation well known to those skilled in the art, Thr48 of the wild type TCR alpha chain constant region TRAC*01 exon 1 is mutated to cysteine, and Ser57 of the beta chain constant region TRBC1*01 or TRBC2*01 exon 1 is mutated to cysteine, i.e. to obtain the reference TCR, the amino acid sequences of which are SEQ ID NO: 11 and SEQ ID NO: 12, respectively, as shown in SEQ ID NO: 1 and SEQ ID NO: 2. Figure 6a and Figure 6bAs shown, the mutated cysteine residues are indicated by bold letters. The cysteine substitutions in the constant regions of the alpha and beta chains of the reference TCR allow for the formation of an artificial interchain disulfide bond, which results in a more stable soluble TCR, and thus allows for more convenient assessment of the binding affinity and / or binding half-life of the TCR for the YMLDLQPET-HLA A0201 complex. It will be appreciated that the CDR regions of the variable regions of the TCR determine the affinity of the TCR for the pMHC complex, and thus the cysteine substitutions in the constant regions of the TCR do not affect the binding affinity and / or binding half-life of the TCR. Therefore, in the present application, the measured binding affinity of the reference TCR for the YMLDLQPET-HLA A0201 complex is considered to be the binding affinity of the wild-type TCR for the YMLDLQPET-HLA A0201 complex. Similarly, if the measured binding affinity of the TCR of the application for the YMLDLQPET-HLA A0201 complex is at least 10 times the binding affinity of the reference TCR for the YMLDLQPET-HLA A0201 complex, this is considered to be equivalent to the binding affinity of the TCR of the application for the YMLDLQPET-HLA A0201 complex being at least 10 times the binding affinity of the wild-type TCR for the YMLDLQPET-HLA A0201 complex.

[0137] The binding affinity (and dissociation equilibrium constant K D ) and binding half-life (T 1 / 2 ) can be determined by any suitable method, such as by detection using surface plasmon resonance technology. It will be appreciated that doubling the affinity of the TCR will result in halving of K D . T 1 / 2 is calculated as In 2 divided by the off-rate (K off ). Therefore, doubling T 1 / 2 will result in halving of K off . Preferably, the binding affinity or binding half-life of a given TCR is determined using the same assay format on several occasions, such as 3 or more occasions, and the average value is taken. In a preferred embodiment, the affinity of the soluble TCR is determined using the surface plasmon resonance (BIAcore) method described in the Examples herein, at a temperature of 25°C and a pH of 7.1-7.5. This method determined the dissociation equilibrium constant K D of the reference TCR for the YMLDLQPET-HLA A0201 complex to be 9.37E-05 M, i.e. 93.7 μM, and this is considered to be the dissociation equilibrium constant K D of the wild-type TCR for the YMLDLQPET-HLA A0201 complex in the present application. Since doubling the affinity of the TCR will result in halving of K DThe value is halved, so if the dissociation equilibrium constant K of the high-affinity TCR on the YMLDLQPET-HLA A0201 complex is detected, it is because... D The value is 9.37E-06M, or 9.37 μM, indicating that the high-affinity TCR has 10 times the affinity for the YMLDLQPET-HLA A0201 complex compared to the wild-type TCR. Those skilled in the art are well aware of K... D The conversion relationship between units of value, i.e., 1M = 10 6 μM, 1μM = 1000nM, 1nM = 1000pM. In this invention, the affinity of the TCR to the YMLDLQPET-HLA A0201 complex is at least twice that of the wild-type TCR.

[0138] Mutation can be performed using any suitable method, including but not limited to those based on polymerase chain reaction (PCR), restriction enzyme cloning, or ligation-independent cloning (LIC). These methods are detailed in many standard molecular biology textbooks. More details on polymerase chain reaction (PCR) mutagenesis and restriction enzyme cloning can be found in Sambrook and Russell, (2001) Molecular Cloning - A Laboratory Manual (3rd ed.), CSHL. More information on the LIC method can be found in (Rashtchian, (1995) Curr Opin Biotechnol 6(1):30-6).

[0139] The method for generating the TCR of the present invention may be, but is not limited to, screening a TCR with high affinity for the YMLDLQPET-HLA-A0201 complex from a diverse library of phage particles exhibiting such TCRs, as described in the literature (Li, et al (2005) Nature Biotech 23(3):349-354).

[0140] It should be understood that genes expressing the α and β chain variable domain amino acids of wild-type TCR, or genes expressing the α and β chain variable domain amino acids of a slightly modified wild-type TCR, can be used to prepare template TCRs. The necessary modifications to generate the high-affinity TCR of this invention are then introduced into the DNA encoding the variable domain of this template TCR.

[0141] The high affinity TCR of the present application comprises an alpha chain variable domain amino acid sequence that is one of SEQ ID NOs: 1, 13-21; and / or a beta chain variable domain amino acid sequence of the TCR is one of SEQ ID NOs: 2, 22-33. In the present application, the amino acid sequences of the alpha chain variable domain and the beta chain variable domain forming the heterodimeric TCR molecule are preferably selected from Table 1 below:

[0142] Table 1

[0143]

[0144]

[0145] For the purposes of the present application, the TCRs are moieties having at least one TCR alpha and / or TCR beta chain variable domain. They usually comprise both a TCR alpha chain variable domain and a TCR beta chain variable domain. They can be in the form of an alpha beta heterodimer or in a single chain form or in any other form that is capable of stable existence. In adoptive immunotherapy, the full length chains of the alpha beta heterodimeric TCRs, including the cytoplasmic and transmembrane domains, can be transfected. The TCRs of the present application can be used as targeting agents for the delivery of therapeutic agents to antigen presenting cells or in combination with other molecules to make bifunctional polypeptides for the directional effect of effector cells, in which case the TCRs are preferably in soluble form.

[0146] For stability, it is disclosed in the prior art that introducing an artificial interchain disulfide bond between the constant domains of the alpha and beta chains of a TCR can obtain a soluble and stable TCR molecule, as described in patent document PCT / CN2015 / 093806. Therefore, the TCR of the present application can be a TCR in which an artificial interchain disulfide bond is introduced between the residues of the constant domains of its alpha and beta chains. The cysteine residues form an artificial interchain disulfide bond between the constant domains of the alpha and beta chains of the TCR. The cysteine residues can replace other amino acid residues at suitable sites in the native TCR to form an artificial interchain disulfide bond. For example, Thr48 of exon 1 of TRAC*01 and Ser57 of exon 1 of TRBC1*01 or TRBC2*01 are replaced to form a disulfide bond. Other sites where cysteine residues are introduced to form a disulfide bond can also be: Thr45 of exon 1 of TRAC*01 and Ser77 of exon 1 of TRBC1*01 or TRBC2*01; Tyr10 of exon 1 of TRAC*01 and Ser17 of exon 1 of TRBC1*01 or TRBC2*01; Thr45 of exon 1 of TRAC*01 and Asp59 of exon 1 of TRBC1*01 or TRBC2*01; Ser15 of exon 1 of TRAC*01 and Glu15 of exon 1 of TRBC1*01 or TRBC2*01; Arg53 of exon 1 of TRAC*01 and Ser54 of exon 1 of TRBC1*01 or TRBC2*01; Pro89 of exon 1 of TRAC*01 and Ala19 of exon 1 of TRBC1*01 or TRBC2*01; or Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01. That is, the cysteine residues replace any of the above-mentioned groups of sites in the constant domains of the alpha and beta chains. One or more C-terminal ends of the constant domains of the TCR of the present application can be truncated by at most 15, or at most 10, or at most 8 or fewer amino acids so that they do not include the cysteine residues to achieve the purpose of deleting the natural interchain disulfide bond, or the above-mentioned purpose can also be achieved by mutating the cysteine residues that form the natural interchain disulfide bond to another amino acid.

[0147] As described above, the TCR of the present application can comprise an artificial interchain disulfide bond introduced between the residues of the constant domains of its alpha and beta chains. It should be noted that the TCR of the present application can contain the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence, with or without the artificial disulfide bond introduced as described above between the constant domains. The TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the TCR can be connected by the natural interchain disulfide bond present in the TCR.

[0148] In addition, for stability, patent document PCT / CN2016 / 077680 also discloses that introducing an artificial interchain disulfide bond between the variable region of the alpha chain and the constant region of the beta chain of the TCR can significantly improve the stability of the TCR. Therefore, the high-affinity TCR of the present application can also contain an artificial interchain disulfide bond between the variable region of the alpha chain and the constant region of the beta chain. Specifically, the cysteine residues that form the artificial interchain disulfide bond between the variable region of the alpha chain and the constant region of the beta chain are substituted for: the 46th amino acid of TRAV and the 60th amino acid of TRBC1*01 or TRBC2*01 exon 1; the 47th amino acid of TRAV and the 61st amino acid of TRBC1*01 or TRBC2*01 exon 1; the 46th amino acid of TRAV and the 61st amino acid of TRBC1*01 or TRBC2*01 exon 1; or the 47th amino acid of TRAV and the 60th amino acid of TRBC1*01 or TRBC2*01 exon 1. Preferably, such a TCR can comprise (i) all or part of the TCR alpha chain except for its transmembrane domain, and (ii) all or part of the TCR beta chain except for its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least part of the constant domain of the TCR chain, and the alpha chain and the beta chain form a heterodimer. More preferably, such a TCR can comprise the alpha chain variable domain and the beta chain variable domain and all or part of the beta chain constant domain except for the transmembrane domain, but it does not contain the alpha chain constant domain, and the alpha chain variable domain of the TCR forms a heterodimer with the beta chain.

[0149] For stability, on the other hand, the TCR of the present application also includes TCRs that have mutations in their hydrophobic core regions, and the mutations in these hydrophobic core regions are preferably mutations that can improve the stability of the TCR of the present application, as described in patent document WO2014 / 206304. Such TCRs can have mutations in the following positions of the hydrophobic core of their variable domains: (alpha and / or beta chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and / or alpha chain J gene (TRAJ) short peptide amino acid positions -3, -5, -7 from the end, and / or beta chain J gene (TRBJ) short peptide amino acid positions -2, -4, -6 from the end, wherein the position numbering of the amino acid sequence is according to the position numbering listed in the International Immunogenetics Information System (IMGT). Those skilled in the art are aware of the above-mentioned International Immunogenetics Information System, and can obtain the position numbering of the amino acid residues of different TCRs in IMGT from this database.

[0150] More specifically, the TCR in the present application whose hydrophobic core region is mutated can be a high-stability single-chain TCR composed of a flexible peptide chain connecting the variable domains of the alpha and beta chains of the TCR. The CDR regions of the variable domains of the TCR determine the affinity between the TCR and the short peptide-HLA complex, and the mutation of the hydrophobic core can make the TCR more stable without affecting the affinity between the TCR and the short peptide-HLA complex. It should be noted that the flexible peptide chain in the present application can be any peptide chain suitable for connecting the variable domains of the alpha and beta chains of the TCR. The template chain for screening high-affinity TCR constructed in Example 1 of the present application is a high-stability single-chain TCR containing the above-mentioned mutation of the hydrophobic core. The use of a TCR with higher stability can more conveniently evaluate the affinity between the TCR and the YMLDLQPET-HLA-A0201 complex.

[0151] The CDR regions of the alpha and beta chain variable domains of the single-chain template TCR are exactly the same as those of the wild-type TCR. That is, the three CDRs of the alpha chain variable domain are CDR1a: DRVSQS; CDR2a: IYSNGD; and CDR3a: AVNPRYGNKLV, and the three CDRs of the beta chain variable domain are CDR1b: KGHDR; CDR2b: SFDVKD; and CDR3b: ATSDRGQGAFGEQY. The amino acid sequence (SEQ ID NO: 9) and the nucleotide sequence (SEQ ID NO: 10) of the single-chain template TCR are shown in Figure 5a and 5b respectively. The single-chain TCR composed of the alpha and beta chain variable domains with high affinity for the YMLDLQPET-HLA A0201 complex is screened in this way.

[0152] The alpha beta heterodimer with high affinity for the YMLDLQPET-HLA-A0201 complex in the present application is obtained by transferring the CDR regions of the alpha and beta chain variable domains of the screened high-affinity single-chain TCR to the corresponding positions of the wild-type TCR alpha chain variable domain (SEQ ID NO: 1) and the beta chain variable domain (SEQ ID NO: 2).

[0153] The TCR of the present application can also be provided in the form of a multivalent complex. The multivalent TCR complex of the present application comprises a multimer formed by the association of two, three, four or more TCRs of the present application, such as a tetramer using the tetramerization domain of p53, or a complex formed by the association of multiple TCRs of the present application with another molecule. The TCR complex of the present application can be used to track or target cells presenting a specific antigen in vitro or in vivo, and can also be used as an intermediate for producing other multivalent TCR complexes having such applications.

[0154] The TCRs of the application can be used alone or can be covalently or otherwise associated, preferably covalently, with a conjugate. The conjugate includes a detectable label (for diagnostic purposes, where the TCR is used to detect the presence of cells presenting the YMLDLQPET-HLA-A0201 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of these.

[0155] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agents, or enzymes capable of producing a detectable product.

[0156] Therapeutic agents that can be conjugated or coupled to the TCRs of the application include, but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539); 2. Biological toxins (Chaudhary et al., 1989, Nature 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy 51, 565); 3. Cytokines such as IL-2 and the like (Gillies et al., 1992, PNAS 89, 1428; Card et al., 2004, Cancer Immunology and Immunotherapy 53, 345; Halin et al., 2003, Cancer Research 63, 3202); 4. Antibody Fc fragments (Mosquera et al., 2005, The Journal Of Immunology 174, 4381); 5. Antibody scFv fragments (Zhu et al., 1995, International Journal of Cancer 62, 319); 6. Gold nanoparticles / nanorods (Lapotko et al., 2005, Cancer letters 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115); 7. Virus particles (Peng et al., 2004, Gene therapy 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research 65, 11631); 9. Nanomagnetic particles; 10. Prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. Chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticle, etc.

[0157] Antibodies or fragments thereof that bind to the TCR of the present application include anti-T cell or NK-cell determining antibodies, such as anti-CD3 or anti-CD28 or anti-CD16 antibodies, the binding of which to the TCR is capable of directing the effector cell to better target the target cell. One preferred embodiment is the TCR of the present application in combination with an anti-CD3 antibody or a functional fragment or variant of said anti-CD3 antibody. In particular, the fusion molecule of the TCR of the present application with an anti-CD3 single chain antibody comprises a variable domain of the alpha chain of the TCR having an amino acid sequence selected from one of SEQ ID NO: 1, 13-21; and / or a variable domain of the beta chain of the TCR having an amino acid sequence selected from one of SEQ ID NO: 2, 22-33.

[0158] The present application also relates to nucleic acid molecules encoding the TCR of the present application. The nucleic acid molecules of the present application can be in the form of DNA or RNA. The DNA can be the coding strand or the non-coding strand. For example, the nucleic acid sequence encoding the TCR of the present application can be identical to the nucleic acid sequence shown in the figures of the present application or a degenerate variant thereof. To illustrate the meaning of "degenerate variant", as used herein, a "degenerate variant" in the present application refers to a nucleic acid sequence that encodes a protein sequence having SEQ ID NO: 3, but differs from the sequence of SEQ ID NO: 5.

[0159] The nucleic acid molecules of the present application, either in their full-length sequence or fragments thereof, can be obtained by, but not limited to, PCR amplification, recombination or artificial synthesis. At present, it is possible to obtain the DNA sequence encoding the TCR of the present application (or fragments thereof, or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0160] The present application also relates to vectors comprising the nucleic acid molecules of the present application, and host cells genetically engineered with the vectors or coding sequences of the present application.

[0161] The present application also includes isolated cells, particularly T cells, expressing the TCR of the present application. There are many methods suitable for transfecting T cells with DNA or RNA encoding the high affinity TCR of the present application (e.g., Robbins et al., (2008) J. Immunol. 180:6116-6131). T cells expressing the high affinity TCR of the present application can be used for adoptive immunotherapy. Those skilled in the art are aware of many suitable methods for performing adoptive therapy (e.g., Rosenberg et al., (2008) Nat Rev Cancer 8(4): 299-308).

[0162] The present application also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR of the present application, or a TCR complex of the present application, or a cell presenting a TCR of the present application.

[0163] The present application also provides a method of treating a disease comprising administering to a subject in need thereof an effective amount of a TCR of the present application, or a TCR complex of the present application, or a cell presenting a TCR of the present application, or a pharmaceutical composition of the present application.

[0164] In the art, substitutions of amino acids with similar or analogous properties do not usually alter the function of a protein. Addition of one or several amino acids at the C-terminus and / or N-terminus also usually does not alter the structure and function of a protein. Thus, the TCR of the present application also includes a TCR of the present application in which up to 5, preferably up to 3, more preferably up to 2, most preferably 1 amino acid, especially amino acids outside the CDR regions, are replaced by amino acids of similar or analogous properties, and which still retains its functionality.

[0165] The present application also includes TCRs which are slightly modified versions of the TCRs of the present application. Modifications (which usually do not alter the primary structure) include chemically derivatized versions of the TCRs of the present application, such as acetylated or carboxylated versions. Modifications also include glycosylated versions, such as those produced by glycosylation modifications during synthesis and processing or further processing steps of the TCRs of the present application. Such modifications can be accomplished by exposing the TCRs to enzymes which glycosylate or deglycosylate, such as mammalian glycosylation enzymes. Modified versions also include sequences with phosphorylated amino acid residues, such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are TCRs which have been modified to improve their resistance to proteolysis or to optimize solubility.

[0166] The TCRs, TCR complexes or TCR-transfected T cells of the present application can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier. The TCRs, multivalent TCR complexes or cells of the present application are typically provided as part of a sterile pharmaceutical composition, which generally includes a pharmaceutically acceptable carrier. The pharmaceutical composition can be in any suitable form (depending on the desired method of administration to a patient). It can be provided in unit dosage form, typically in a sealed container, and can be provided as part of a kit. Such kits (but not necessarily) include instructions for use. It can include a plurality of said unit dosage forms.

[0167] In addition, the TCRs of the present application can be used alone or in combination or conjugation with other therapeutic agents (e.g. formulated in the same pharmaceutical composition).

[0168] The pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent. This term refers to carriers that are nontoxic to the cell or organism to be treated and do not interfere with the effectiveness of the therapeutic combination being administered. These carriers are well known to those skilled in the art. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Such carriers include, but are not limited to, saline, buffered fluid, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.

[0169] The pharmaceutically acceptable carrier in the therapeutic composition can contain liquids such as water, saline, glycerol, and ethanol. Additionally, auxiliary substances can be present in these carriers such as wetting or emulsifying agents, pH buffering substances, and the like.

[0170] Generally, the therapeutic composition can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles; or as lyophilized powders.

[0171] Once the composition of the present application is formulated, it can be administered by a variety of conventional routes including, but not limited to, intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably parenterally including subcutaneous, intramuscular, or intravenous. The subject to be prevented or treated can be an animal; particularly a human.

[0172] When the pharmaceutical composition of the present application is used for actual treatment, various pharmaceutical compositions in different dosage forms can be used depending on the use. Preferably, examples include injections, oral agents, and the like.

[0173] These pharmaceutical compositions can be prepared by mixing, diluting or dissolving according to a conventional method, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers, and co-solvents, and the preparation process can be performed in a conventional manner according to the dosage form.

[0174] The pharmaceutical composition of the present application can also be administered in the form of a sustained-release agent. For example, the TCR of the present application can be incorporated into a pellet or microcapsule using a sustained-release polymer as a carrier, and the pellet or microcapsule is then implanted into the tissue to be treated by surgery. As examples of the sustained-release polymer, mention can be made of ethylene-vinyl acetate copolymer, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, lactic acid-glycolic acid copolymer, and the like, and preferably mention can be made of biodegradable polymers such as lactic acid polymer and lactic acid-glycolic acid copolymer.

[0175] When the pharmaceutical composition of the present application is used for actual treatment, the TCR or TCR complex of the present application or the cell presenting the TCR of the present application as an active ingredient can be appropriately determined depending on the body weight, age, sex, and degree of symptoms of each patient to be treated, and the reasonable amount is finally determined by a physician.

[0176] The main advantages of the present application are:

[0177] (1) The high-affinity TCR of the present application has an affinity for the YMLDLQPET-HLA-A0201 complex and / or a binding half-life which is at least 2 times that of the wild-type TCR.

[0178] (2) The high-affinity TCR of the present application can specifically bind to the YMLDLQPET-HLA-A0201, and the cell transfected with the high-affinity TCR of the present application can be specifically activated.

[0179] (3) The effector cell transfected with the high-affinity TCR of the present application has a strong specific killing effect.

[0180] The following specific examples further illustrate the present application. It is to be understood that these examples are merely illustrative of the present application and do not in any way limit the scope of the present application. The experimental methods in the following examples, for which specific conditions are not mentioned, are generally performed according to the conventional conditions, for example, the conditions described in (Sambrook and Russell et al., Molecular Cloning - A Laboratory Manual (3rd ed.) (2001) CSHL Press), or the conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are by weight.

[0181] Materials and Methods

[0182] The experimental materials used in the embodiments of the present application are commercially available unless otherwise specified, wherein, the E. coli DH5a is purchased from Tiangen, the E. coli BL21(DE3) is purchased from Tiangen, the E. coli Tuner(DE3) is purchased from Novagen, and the plasmid pET28a is purchased from Novagen.

[0183] Production of a stable single-chain TCR template chain of the hydrophobic core mutation

[0184] According to the method of site-directed mutation, a stable single-chain TCR molecule is constructed by connecting the variable domains of TCR alpha and beta chains with a flexible short peptide (linker) according to the description in the patent document WO2014 / 206304, and the amino acid and DNA sequences thereof are SEQ ID NO: 9 and SEQ ID NO: 10, respectively, as shown in Figure 5a and Figure 5b The amino acid sequences of the variable domain of the alpha chain (SEQ ID NO: 3) and the variable domain of the beta chain (SEQ ID NO: 4) of the template chain are shown in Figure 2a and 2b The corresponding DNA sequences thereof are SEQ ID NO: 5 and SEQ ID NO: 6, respectively, as shown in Figure 3a and 3b The amino acid sequence and the DNA sequence of the flexible short peptide (linker) are SEQ ID NO: 7 and 8, respectively, as shown in Figure 4a and 4b .

[0185] The target gene carrying the template chain is double-digested with NcoI and NotI, and then connected with the pET28a vector double-digested with NcoI and NotI. The ligation product is transformed into E. coli DH5a, and then coated on a LB plate containing kanamycin and incubated at 37°C overnight. Positive clones are selected by PCR, and the positive recombinants are sequenced to determine the correct sequence. After the correct sequence is determined, the recombinant plasmid is extracted and transformed into E. coli BL21(DE3) for expression.

[0186] Expression, refolding and purification of the stable single-chain TCR constructed in Example 1

[0187] The BL21(DE3) colonies containing the recombinant plasmid pET28a-template chain prepared in Example 1 are inoculated into LB medium containing kanamycin, and incubated at 37°C until the OD 600The cells were harvested by centrifugation at 5000 rpm for 15 min and the cell pellet was lysed with Bugbuster Master Mix (Merck). The inclusion bodies were recovered by centrifugation at 6000 rpm for 15 min and washed with Bugbuster (Merck) to remove cell debris and membrane components. The inclusion bodies were recovered by centrifugation at 6000 rpm for 15 min. The inclusion bodies were dissolved in buffer (20 mM Tris-HCl pH 8.0, 8 M urea) and centrifuged at high speed to remove insoluble material. The supernatant was aliquoted and stored at -80°C.

[0188] To 5 mg of solubilized single-chain TCR inclusion body protein, 2.5 mL of buffer (6 M Gua-HCl, 50 mM Tris-HCl pH 8.1, 100 mM NaCl, 10 mM EDTA) was added, and DTT was added to a final concentration of 10 mM, and the mixture was treated at 37°C for 30 min. The treated single-chain TCR was added dropwise to 125 mL of refolding buffer (100 mM Tris-HCl pH 8.1, 0.4 M L-arginine, 5 M urea, 2 mM EDTA, 6.5 mM β-mercaptoethylamine, 1.87 mM Cystamine) using a syringe, and the mixture was stirred at 4°C for 10 min. The refolding solution was then placed in a cellulose membrane dialysis bag with a cut-off of 4 kDa, and the dialysis bag was placed in 1 L of pre-cooled water and stirred slowly at 4°C overnight. After 17 h, the dialysis solution was replaced with 1 L of pre-cooled buffer (20 mM Tris-HCl pH 8.0), and dialysis was continued at 4°C for 8 h. The dialysis solution was then replaced with fresh buffer and dialysis was continued overnight. After 17 h, the sample was filtered through a 0.45 μm filter membrane, degassed under vacuum, and passed through an anion exchange column (HiTrap Q HP, GE Healthcare) using a linear gradient of 0-1 M NaCl in 20 mM Tris-HCl pH 8.0 as the eluent. The eluted fractions were analyzed by SDS-PAGE, and the fraction containing the single-chain TCR was concentrated and further purified using a gel filtration column (Superdex 75 10 / 300, GE Healthcare). The target fraction was also analyzed by SDS-PAGE.

[0189] The eluted fractions for BIAcore analysis were further tested for purity using gel filtration. The conditions were: chromatographic column Agilent Bio SEC-3 (300 A, ), mobile phase 150 mM phosphate buffer, flow rate 0.5 mL / min, column temperature 25°C, and UV detection wavelength 214 nm.

[0190] Example 3 Binding characterization

[0191] BIAcore analysis

[0192] The binding activity of TCR molecules to YMLDLQPET-HLA-A0201 complex was detected using BIAcore T200 real-time analysis system. Antibodies against streptavidin (GenScript) were added into coupling buffer (10 mM sodium acetate buffer, pH 4.77), then the antibodies were flowed through a CM5 chip which was previously activated with EDC and NHS, so that the antibodies were immobilized on the chip surface, and finally the unreacted activated surface was blocked with ethanolamine hydrochloride solution, completing the coupling process, and the coupling level was about 15,000 RU. The conditions were: temperature 25°C, pH 7.1-7.5.

[0193] Low concentration of streptavidin was flowed through the antibody-coated chip surface, then YMLDLQPET-HLA-A0201 complex was flowed through the detection channel, and the other channel was used as a reference channel, and then 0.05 mM biotin was flowed through the chip at a flow rate of 10 μL / min for 2 min to block the remaining binding sites of streptavidin. The affinity was determined by single-cycle kinetic analysis method, and the TCR was diluted into several different concentrations with HEPES-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, pH 7.4), and then flowed through the chip surface at a flow rate of 30 μL / min, and the binding time of each injection was 120 s, and after the last injection, the dissociation time was 600 s. After each round of determination, the chip was regenerated with 10 mM Gly-HCl at pH 1.75. The kinetic parameters were calculated using BIAcore Evaluation software.

[0194] The preparation process of the above YMLDLQPET-HLA-A0201 complex is as follows:

[0195] a. Purification: 100 ml of E. coli broth induced to express heavy chain or light chain was collected, centrifuged at 8000 g at 4°C for 10 min, then the bacterial body was washed once with 10 ml of PBS, then the bacterial body was resuspended by vigorous shaking with 5 ml of BugBuster Master Mix Extraction Reagents (Merck), and incubated at room temperature for 20 min, then centrifuged at 6000 g at 4°C for 15 min, and the supernatant was discarded, and the inclusion body was collected.

[0196] Resuspend the inclusion bodies in 5 ml BugBuster Master Mix, rotate incubate at room temperature for 5 min; add 30 ml BugBuster diluted 10 times, mix well, centrifuge at 6000 g for 15 min at 4°C; discard the supernatant, resuspend the inclusion bodies with 30 ml BugBuster diluted 10 times, mix well, centrifuge at 6000 g for 15 min at 4°C, repeat twice, resuspend the inclusion bodies with 30 ml 20 mM Tris-HCl pH 8.0, mix well, centrifuge at 6000 g for 15 min at 4°C, finally dissolve the inclusion bodies with 20 mM Tris-HCl 8 M urea, detect the purity of the inclusion bodies by SDS-PAGE, and measure the concentration by BCA kit.

[0197] b. Refolding: Dissolve the synthetic short peptide YMLDLQPET (Beijing SAB Biosciences Co., Ltd.) in DMSO to a concentration of 20 mg / ml. Dissolve the inclusion bodies of the light chain and the heavy chain with 8 M urea, 20 mM Tris pH 8.0, 10 mM DTT, and add 3 M guanidine hydrochloride, 10 mM sodium acetate, 10 mM EDTA before refolding to further denature. Add YMLDLQPET peptide at 25 mg / L (final concentration) to the refolding buffer (0.4 M L-arginine, 100 mM Tris pH 8.3, 2 mM EDTA, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, 0.2 mM PMSF, cool to 4°C), then add 20 mg / L of the light chain and 90 mg / L of the heavy chain (final concentration, add the heavy chain in three times, 8 h / time) in turn, and perform refolding at 4°C for at least 3 days to completion, and detect whether refolding is successful by SDS-PAGE.

[0198] c. Post-refolding purification: Replace the refolding buffer with 10 volumes of 20 mM Tris pH 8.0 for dialysis, and replace the buffer at least twice to sufficiently reduce the ionic strength of the solution. After dialysis, filter the protein solution with a 0.45 μm cellulose acetate filter, and then load onto a HiTrap Q HP (GE General Electric Co.) anion exchange column (5 ml bed volume). Use an Akta purifier (GE General Electric Co.) to elute the protein with a 0-400 mM NaCl linear gradient prepared with 20 mM Tris pH 8.0, and the pMHC is eluted at about 250 mM NaCl, collect the peak fractions, and detect the purity by SDS-PAGE.

[0199] d. Biotinylation: Purified pMHC molecules were concentrated using Millipore ultrafiltration tubes while simultaneously replacing the buffer with 20 mM Tris pH 8.0, then biotinylation reagents were added 0.05 M Bicine pH 8.3, 10 mM ATP, 10 mM MgOAc, 50 μM D-Biotin, 100 μg / ml BirA enzyme (GST-BirA), and the mixture was incubated overnight at room temperature. Biotinylation was checked by SDS-PAGE.

[0200] e. Purification of biotinylated complex: Biotinylated pMHC molecules were concentrated to 1 ml using Millipore ultrafiltration tubes, and the biotinylated pMHC was purified using gel filtration chromatography on an Akta purifier (GE Healthcare) using a HiPrep 16 / 60 S200 HR column (GE Healthcare) pre-equilibrated with filtered PBS. The concentrated biotinylated pMHC molecules were loaded onto the column, and then eluted with PBS at a flow rate of 1 ml / min. The biotinylated pMHC molecules eluted as a single peak at approximately 55 ml. The fractions containing protein were pooled, concentrated using Millipore ultrafiltration tubes, and the protein concentration was determined by BCA assay (Thermo). The biotinylated pMHC molecules were aliquoted and stored at -80°C with protease inhibitor cocktail (Roche). TM 16 / 60 S200 HR column (GE Healthcare), and then eluted with PBS at a flow rate of 1 ml / min. The biotinylated pMHC molecules eluted as a single peak at approximately 55 ml. The fractions containing protein were pooled, concentrated using Millipore ultrafiltration tubes, and the protein concentration was determined by BCA assay (Thermo). The biotinylated pMHC molecules were aliquoted and stored at -80°C with protease inhibitor cocktail (Roche).

[0201] Example 4. Generation of high affinity TCRs

[0202] Bacteriophage display technology is a means to generate libraries of TCR high affinity variants for screening of high affinity variants. The TCR bacteriophage display and screening method described by Li et al. ((2005) Nature Biotech 23(3):349-354) was applied to the single chain TCR template of Example 1. Libraries of high affinity TCRs were created by mutating the CDR regions of the template chain and panned. After several rounds of panning, the phage libraries all had specific binding to the corresponding antigen, and single clones were picked and analyzed.

[0203] The CDR regions of the high affinity single chain TCRs screened were mutated into the corresponding positions of the variable domains of the αβ heterodimeric TCRs, and the affinities to the YMLDLQPET-HLA-A0201 complex were tested by BIAcore. The introduction of the CDR region high affinity mutations was performed by site-directed mutagenesis methods well known to those skilled in the art. The amino acid sequences of the α chain and β chain variable domains of the wild type TCRs are shown in SEQ ID NO: 1 and 2, respectively. Figure 1a (SEQ ID NO: 1) and 1b (SEQ ID NO: 2), respectively.

[0204] It should be noted that, in order to obtain a more stable soluble TCR and to more conveniently assess the binding affinity and / or binding half-life between the TCR and the YMLDLQPET-HLAA0201 complex, the αβ heterodimeric TCR can be a TCR in which a cysteine ​​residue is introduced into the constant region of both the α and β chains to form an artificial interchain disulfide bond. In this embodiment, the amino acid sequences of the TCR α and β chains after the introduction of the cysteine ​​residue are as follows: Figure 6a As shown in (SEQ ID NO:11) and 6b (SEQ ID NO:12), the introduced cysteine ​​residues are indicated by bold letters.

[0205] The extracellular sequences of the TCRα and β chains to be expressed were synthesized and inserted into the expression vector pET28a+ (Novagene) using standard methods described in *Molecular Cloning: A Laboratory Manual* (3rd edition, Sambrook and Russell). The upstream and downstream cloning sites were NcoI and NotI, respectively. Mutations in the CDR region were introduced using overlap PCR, a method well-known to those skilled in the art. The inserted fragments were confirmed to be correct by sequencing.

[0206] Example 5: Expression, refolding, and purification of high-affinity TCRs

[0207] The expression vectors for the TCRα and β chains were transformed into expression bacteria BL21(DE3) via chemical transformation. The bacteria were grown in LB medium and cultured at OD. 600 When the TCR expression rate was 0.6, inclusion bodies were induced with a final concentration of 0.5 mM IPTG. The inclusion bodies were extracted using BugBuster Mix (Novagene) and repeatedly washed with BugBuster solution. Finally, the inclusion bodies were dissolved in 6 M guanidine hydrochloride, 10 mM dithiothreitol (DTT), 10 mM EDTA, and 20 mM Tris (pH 8.1).

[0208] The solubilized TCR α and β chains were mixed at a 1 : 1 mass ratio in 5 M urea, 0.4 M arginine, 20 mM Tris (pH 8.1), 3.7 mM cystamine, 6.6 mM β-mercaptoethylamine (4°C) to a final concentration of 60 mg / mL. After mixing, the solution was dialyzed (4°C) against 10 volumes of deionized water for 12 hours, and then against buffer (20 mM Tris, pH 8.0) for another 12 hours at 4°C. The dialyzed solution was filtered through a 0.45 μM filter and purified by anion exchange column (HiTrap Q HP, 5 ml, GE Healthcare). The eluted peak containing the refolded α and β dimeric TCR was confirmed by SDS-PAGE. The TCR was further purified by gel filtration chromatography (HiPrep 16 / 60, Sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was greater than 90% as determined by SDS-PAGE, and the concentration was determined by BCA method.

[0209] Example 6 BIAcore analysis results

[0210] The affinity of the αβ heterodimeric TCR with the introduced high affinity CDR regions for the YMLDLQPET-HLA-A0201 complex was determined using the method described in Example 3.

[0211] The present application provides new TCR α chain and β chain variable domain amino acid sequences, as shown in Figures 7(1)-(9) and 8(1)-(12), respectively. Since the CDR regions of a TCR molecule determine its affinity for the corresponding pMHC complex, one skilled in the art would expect that the αβ heterodimeric TCR with the introduced high affinity mutation points would also have high affinity for the YMLDLQPET-HLA-A0201 complex. The expression vector was constructed using the method described in Example 4, and the above-mentioned αβ heterodimeric TCR with the introduced high affinity mutation was expressed, refolded and purified using the method described in Example 5, and then the affinity of the TCR for the YMLDLQPET-HLA-A0201 complex was determined using the BIAcore T200, as shown in Table 2 below.

[0212] Table 2

[0213]

[0214]

[0215] As shown in Table 2 above, the affinity of the heterodimeric TCR is at least 2 times that of the wild-type TCR for the YMLDLQPET-HLA-A0201 complex.

[0216] Expression, refolding and purification of the fusion of anti-CD3 antibody and high affinity αβ heterodimeric TCR

[0217] A fusion molecule was prepared by fusing a single chain antibody (scFv) of anti-CD3 with an αβ heterodimeric TCR. The scFv of anti-CD3 was fused with the β chain of the TCR, which can comprise the β chain variable domain of any of the above high affinity αβ heterodimeric TCRs, and the TCR α chain of the fusion molecule can comprise the α chain variable domain of any of the above high affinity αβ heterodimeric TCRs.

[0218] Construction of the fusion molecule expression vector

[0219] 1. Construction of the α chain expression vector: the target gene carrying the α chain of the αβ heterodimeric TCR was double digested with Nco I and Not I, and ligated with the pET28a vector double digested with Nco I and Not I. The ligation product was transformed into E. coli DH5α, spread on LB plate containing kanamycin, and incubated at 37°C overnight. Positive clones were picked and subjected to PCR screening. The positive recombinants were sequenced to confirm the correct sequence, and the recombinant plasmid was extracted and transformed into E. coli Tuner (DE3) for expression.

[0220] 2. Construction of the anti-CD3 (scFv)-β chain expression vector: by the method of overlap PCR, the primer was designed to link the anti-CD3 scFv and the high affinity heterodimeric TCR β chain gene, with the linker GGGGS (SEQ ID NO: 31) in the middle, and the gene fragment of the fusion protein of the scFv of anti-CD3 and the β chain of the high affinity heterodimeric TCR was made to carry the restriction enzyme sites Nco I (CCATGG (SEQ ID NO: 32)) and Not I (GCGGCCGC (SEQ ID NO: 33)). The PCR amplification product was double digested with Nco I and Not I, and ligated with the pET28a vector double digested with Nco I and Not I. The ligation product was transformed into E. coli DH5α competent cells, spread on LB plate containing kanamycin, and incubated at 37°C overnight. Positive clones were picked and subjected to PCR screening. The positive recombinants were sequenced to confirm the correct sequence, and the recombinant plasmid was extracted and transformed into E. coli Tuner (DE3) competent cells for expression.

[0221] Expression, refolding and purification of the fusion protein

[0222] The expression plasmids were transformed into E. coli Tuner (DE3) competent cells respectively, and spread on LB plates (kanamycin 50 μg / mL) and incubated at 37°C overnight. The next day, the clones were inoculated into 10 mL LB liquid medium (kanamycin 50 μg / mL) and incubated for 2-3 h, then inoculated into 1 L LB medium at a volume ratio of 1:100, and incubated until the OD600 was 0.5-0.8. The expression of the target protein was induced by adding IPTG at a final concentration of 1 mM. After 4 hours of induction, the cells were harvested by centrifugation at 6000 rpm for 10 min. The bacterial cells were washed once with PBS buffer, and the bacterial cells equivalent to 200 mL of bacterial culture were lysed with 5 mL BugBuster Master Mix (Merck). The inclusion bodies were collected by centrifugation at 6000 g for 15 min. Then, four detergent washes were performed to remove cell debris and membrane components. Then, the inclusion bodies were washed with a buffer such as PBS to remove the detergent and salt. Finally, the inclusion bodies were dissolved in a buffer solution containing 6 M guanidine hydrochloride, 10 mM dithiothreitol (DTT), 10 mM ethylenediaminetetraacetic acid (EDTA), 20 mM Tris, pH 8.1, and the concentration of the inclusion bodies was determined. The inclusion bodies were aliquoted and stored at -80°C.

[0223] The solubilized TCR a chain and anti-CD3 (scFv)-β chain were mixed at a mass ratio of 2:5 in 5 M urea, 0.4 M L-arginine, 20 mM Tris pH 8.1, 3.7 mM cystamine, 6.6 mM β-mercapoethylamine (4°C) at a final concentration of 0.1 mg / mL and 0.25 mg / mL for the a chain and anti-CD3 (scFv)-β chain, respectively.

[0224] After mixing, the solution was dialyzed in 10 volumes of deionized water (4°C) for 12 hours, and then the deionized water was replaced with buffer (10 mM Tris, pH 8.0) and dialyzed at 4°C for another 12 hours. After dialysis, the solution was filtered through a 0.45 μM filter, and then purified by anion exchange column (HiTrap Q HP 5 ml, GE healthcare). The elution peak contained the TCR a chain and anti-CD3 (scFv)-β chain dimer, which was confirmed by SDS-PAGE. The TCR fusion molecule was then further purified by size exclusion chromatography (S-100 16 / 60, GE healthcare) and anion exchange column (HiTrap Q HP 5 ml, GE healthcare). The purity of the purified TCR fusion molecule was greater than 90% as determined by SDS-PAGE, and the concentration was determined by BCA method.

[0225] Example 8 Activation function experiment of effector cells transfected with high affinity TCRs of the application on T2 cells loaded with short peptides

[0226] IFN-γ is a powerful immunomodulatory factor produced by activated T lymphocytes, and this example verifies the activation function and antigen specificity of cells transfected with high affinity TCRs of the application by detecting the number of IFN-γ through an ELISPOT experiment well known to those skilled in the art. CD3+ T cells isolated from the blood of a healthy volunteer were transfected with high affinity TCRs of the application as effector cells, and CD3+ T cells of the same volunteer transfected with another TCR (A6) were used as a control. The target cells used were T2 cells loaded with the HPV16 E7 antigen short peptide YMLDLQPET, T2 cells loaded with other short peptides, and empty T2 cells.

[0227] The following experiments were performed in two batches (I), (II) in succession:

[0228] (I) The high affinity TCRs can be found in Table 2, respectively,

[0229]

[0230] First, the ELISPOT plate was prepared. The ELISPOT plate was activated and coated with ethanol, and incubated overnight at 4°C. On the first day of the experiment, the coating solution was removed, washed and blocked, and incubated at room temperature for two hours. The blocking solution was removed, and each component of the experiment was added to the ELISPOT plate: target cells at 1*10 4 cells / well, effector cells at 1*10 3 cells / well (calculated as the positive rate of the antibody), and two duplicate wells were set up. The corresponding short peptide was added to make the final concentration of the short peptide in the ELISPOT plate 1*10 -6 M. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, wash the plate and perform secondary detection and color development. Dry the plate, and count the spots formed on the membrane using an immunospot plate reader (ELISPOT READER system; AID20 company).

[0231] (II) The high affinity TCRs can be found in Table 2, respectively,

[0232]

[0233] Each component added to the ELISPOT plate was: target cells at 1*10 4 cells / well, effector cells at 2*10 3 cells / well (calculated as the positive rate of the antibody), and two duplicate wells were set up. The remaining steps were the same as in Example (I).

[0234] The experimental results are shown in Figure 12a and Figure 12b As shown in the above tables, the effector cells transfected with the high-affinity TCRs of the present application have obvious activation effect on the target cells loaded with the HPV16 E7 antigen short peptide YMLDLQPET, while the effector cells transfected with other TCRs have no activation effect; at the same time, the effector cells transfected with the high-affinity TCRs of the present application have no activation effect on the target cells loaded with other short peptides or empty.

[0235] Example 9 Activation function experiment of effector cells transfected with high-affinity TCRs of the present application on tumor cell lines

[0236] This example uses tumor cell lines to verify again the activation function and specificity of effector cells transfected with high-affinity TCRs of the present application. The detection is also performed by the ELISPOT experiment well known to those skilled in the art. The CD3+T cells isolated from the blood of a healthy volunteer are transfected with the high-affinity TCRs of the present application as effector cells, and the CD3+T cells of the same volunteer transfected with other TCRs (A6) or wild-type TCRs (WT-TCR) are used as negative controls. The following experiments are performed in two batches (I) and (II) in sequence:

[0237] The high-affinity TCRs used in this batch can be known from Table 2, which are TCR1 (α chain variable domain SEQ ID NO: 1, β chain variable domain SEQ ID NO: 22), TCR2 (α chain variable domain SEQ ID NO: 1, β chain variable domain SEQ ID NO: 23) and TCR3 (α chain variable domain SEQ ID NO: 1, β chain variable domain SEQ ID NO: 24), respectively.

[0238] The HPV16 E7 positive tumor cell lines used in this batch are A375-E7 (HPV16 E7 overexpression) and HK-2, and the negative tumor cell lines are SK-MEL-28-E7 (HPV16 E7 overexpression), SK-MEL-28 and A375.

[0239] The high-affinity TCRs used in this batch can be known from Table 2, which are

[0240]

[0241] The HPV16 E7 positive tumor cell line used in this batch is A375-E7 (HPV16 E7 overexpression), and the negative tumor cell lines are A375 and SK-MEL-28.

[0242] Both batches were subjected to the following steps: First, ELISPOT plates were prepared. ELISPOT plates were ethanol-activated coated, 4°C overnight. On the first day of the experiment, the coating solution was removed, the plates were washed and blocked, incubated at room temperature for two hours, the blocking solution was removed, and the various components of the experiment were added to the ELISPOT plates: target cells were 2*10 4 cells / well, effector cells were 2*10 3 cells / well (calculated as the positive rate of the antibody), and two replicates were set up. Incubation was carried out overnight (37°C, 5% CO2). On the second day of the experiment, the plates were washed and subjected to secondary detection and color development, the plates were dried, and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID20 company).

[0243] The experimental results are shown in Figure 13a and Figure 13b , which show that, against HPV16 E7 positive tumor cell lines, effector cells transfected with the high-affinity TCR of the present application have a more obvious activation effect than effector cells transfected with wild type, while effector cells transfected with other TCRs have no activation state; at the same time, effector cells transfected with the high-affinity TCR of the present application have essentially no activation effect on HPV16 E7 negative tumor cell lines.

[0244] Example 10 Killing function experiment of effector cells transfected with the high-affinity TCR of the present application against T2 cells loaded with gradient short peptides

[0245] Lactate dehydrogenase (LDH) is abundant in the cytoplasm, and under normal circumstances cannot pass through the cell membrane, but when the cell is damaged or dies, it can be released outside the cell, at which time the activity of LDH in the cell culture medium is proportional to the number of cell deaths. In this embodiment, the release of LDH was determined by a non-radioactive cytotoxicity experiment well known to those skilled in the art, thereby verifying the killing function of cells transfected with the TCR of the present application. This experiment is a colorimetric alternative to the 51Cr release cytotoxicity test, which quantitatively measures the LDH released after cell lysis. A 30-minute coupled enzyme reaction is used to detect the LDH released in the culture medium, in which the enzyme can convert a tetrazolium salt (INT) to a red formazan. The amount of red product generated is proportional to the number of lysed cells. Standard 96-well plate readers can be used to collect 490 nm visible light absorbance data. The calculation formula is: % cytotoxicity = 100% x (experimental - effector cell spontaneous - target cell spontaneous) / (target cell maximum - target cell spontaneous).

[0246] This example transfects the high affinity TCR of the present application into CD3+T cells isolated from the blood of a healthy volunteer as effector cells, and uses CD3+T cells of the same volunteer that are either empty-transfected (NC) or transfected with other TCRs (A6) as negative controls. The high affinity TCRs and their numbers are known from Table 2, and are TCR1 (alpha chain variable domain SEQ ID NO: 1, beta chain variable domain SEQ ID NO: 22) and TCR2 (alpha chain variable domain SEQ ID NO: 1, beta chain variable domain SEQ ID NO: 23), respectively. The target cells are T2 cells loaded with the YMLDLQPET peptide, loaded with other short peptides, or empty-loaded.

[0247] First, prepare the LDH plate. Add 3 x 10 4 cells / well of target cells, 3 x 10 4 cells / well of effector cells (calculated according to the positive rate of antibodies) to the corresponding wells, then add HPV16 E7 antigen short peptide YMLDLQPET to the experimental group, and make the final concentration of the short peptide in the ELISPOT plate 1 x 10 -15 M to 1 x 10 -8 M, a total of 8 gradients; add other short peptides to the control group, and make the final concentration of the short peptide 1 x 10 -9 M to 1 x 10 -8 M, a total of 2 gradients, and set three replicate wells for each. At the same time, set effector cell spontaneous wells, target cell spontaneous wells, target cell maximum wells, volume correction control wells, and medium background control wells. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, detect color development, and after stopping the reaction, record the absorbance value at 490 nm using an enzyme label instrument (Bioteck).

[0248] The experimental results are shown in Figure 14 Table 3. For target cells loaded with HPV16 E7 antigen short peptide YMLDLQPET at different concentrations, effector cells transfected with the high affinity TCR of the present application showed strong killing effect, and reacted at a relatively low concentration of the above-mentioned specific short peptide, while effector cells transfected with other TCRs or empty-transfected effector cells had basically no killing effect. At the same time, effector cells transfected with the high affinity TCR of the present application had no killing effect on target cells loaded with other short peptides or empty-loaded target cells.

[0249] Example 11 Killing function experiment of effector cells transfected with the high affinity TCR of the present application against tumor cell lines

[0250] This example also demonstrates the killing function of the cells transfected with the TCR of the present application by measuring the release of LDH through non-radioactive cytotoxicity assays well known to those skilled in the art. The LDH assay of this example uses CD3+T cells isolated from the blood of a healthy volunteer transfected with the high affinity TCR of the present application as effector cells, and CD3+T cells of the same volunteer transfected with another TCR (A6) or wild type TCR (WT-TCR) as negative controls.

[0251] The following experiments were performed in three batches (I), (II), (III) in succession:

[0252] (I) The high affinity TCRs and their numbers are known from Table 2, which are TCR1 (alpha chain variable domain SEQ ID NO: 1, beta chain variable domain SEQ ID NO: 22), TCR2 (alpha chain variable domain SEQ ID NO: 1, beta chain variable domain SEQ ID NO: 23) and TCR3 (alpha chain variable domain SEQ ID NO: 1, beta chain variable domain SEQ ID NO: 24) respectively. The HPV16 E7 positive tumor cell lines used in this batch are CASKI and HK-2, and the negative tumor cell lines are SK-MEL-28, MEL526 and HCC827. The experimental steps are as follows: first prepare the LDH plate, and add the components of each group to the plate in the following order: 2x10 4 4 cells / well (calculated according to the positive rate of antibody) to the corresponding wells, and set up three replicate wells. At the same time, set up effector cell spontaneous wells, target cell spontaneous wells, target cell maximum wells, volume correction control wells and medium background control wells. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, detect color development, and after stopping the reaction, record the absorbance value at 490 nm using an enzyme marker (Bioteck).

[0253] (II) The high affinity TCRs and their numbers are known from Table 2, which are TCR5 (alpha chain variable domain SEQ ID NO: 14, beta chain variable domain SEQ ID NO: 2) and TCR9 (alpha chain variable domain SEQ ID NO: 15, beta chain variable domain SEQ ID NO: 24) respectively. The HPV16 E7 positive tumor cell line used in this batch is CASKI, and the negative tumor cell line is HCCC9810. The components added to the plate are as follows: 3x10 4 4 cells / well, effector cells 3x10

[0254] ​​(III) the high affinity TCRs and their numbering are TCR17 (alpha chain variable domain SEQ ID NO: 1, beta chain variable domain SEQ ID NO: 26), TCR19 (alpha chain variable domain SEQ ID NO: 1, beta chain variable domain SEQ ID NO: 28) and TCR22 (alpha chain variable domain SEQ ID NO: 1, beta chain variable domain SEQ ID NO: 31) respectively, as known from Table 2. The HPV16 E7 positive tumor cell line used in this batch was CASKI, and the negative tumor cell lines were A375 and SiHa. The components added to the plate were as follows: target cells 3 x 10 4 effector cells 3 x 10 4 cells / well (calculated as the percentage of antibody positivity), and the rest of the experimental procedure was the same as in Example (I).

[0255] The experimental results are shown in Figure 15a , Figure 15b and Figure 15c The effector cells transfected with the high affinity TCRs of the application still showed strong killing potency against the HPV16 E7 positive tumor cell line, and the killing function was significantly stronger than that of the T cells transfected with the wild type TCR, while the T cells transfected with other TCRs were basically not reactive; at the same time, the T cells transfected with the high affinity TCRs of the application had basically no killing effect on the negative tumor cell lines, further demonstrating the good specific killing function of the cells transfected with the high affinity TCRs of the application.

[0256] Example 12 Killing function verification of effector cells transfected with the high affinity TCR molecules of the application against tumor cell lines (IncuCyte experiment)

[0257] This example further verified the good specific killing effect of the effector cells transfected with the high affinity TCRs of the application on target cells and their sensitivity through the IncuCyte experiment, which is well known to those skilled in the art. IncuCyte is a functional analysis system that can automatically analyze images at different time points through real-time microscopic shooting in an incubator, and quantify real-time apoptosis numbers.

[0258] CD3+T cells isolated from the blood of a healthy volunteer were randomly selected as effector cells, and CD3+T cells transfected with another TCR (A6) or empty transfection (NC) of the same volunteer were used as controls. The TCRs and their numbers are known from Table 2, which are TCR1 (alpha chain variable domain SEQ ID NO: 1, beta chain variable domain SEQ ID NO: 22) and TCR2 (alpha chain variable domain SEQ ID NO: 1, beta chain variable domain SEQ ID NO: 23), respectively. Among the target cell lines, A375-E7 (HPV16 E7 overexpression) is a positive tumor cell line; A375 is a negative tumor cell line as a control.

[0259] On the first day of the experiment, the target cells were digested and centrifuged; resuspended with complete medium of RPMI1640+10%FBS without phenol red, and the target cells were evenly plated in a 96-well plate: 2*10 4 On the first day of the experiment, the target cells were digested and centrifuged; resuspended with complete medium of RPMI1640+10%FBS without phenol red, and the target cells were evenly plated in a 96-well plate: 2*10 4 On the first day of the experiment, the target cells were digested and centrifuged; resuspended with complete medium of RPMI1640+10%FBS without phenol red, and the target cells were evenly plated in a 96-well plate: 2*10

[0260] The experimental results are shown in Figure 16a and Figure 16b The cells transfected with the high-affinity TCRs of the present application can show strong and effective killing effect in a short period of time against HPV16 E7 positive tumor cell lines, while the effector cells transfected with other TCRs have no killing effect; at the same time, the cells transfected with the high-affinity TCRs of the present application have no killing effect on negative tumor cells.

[0261] All the documents mentioned in the present application are incorporated by reference in the present application, as if each document is individually incorporated by reference. In addition, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the above teachings of the present application, and these equivalent forms also fall within the scope defined by the claims attached hereto. SEQUENCE LISTING <110> Guangdong Xiangxue Precision Medical Technology Co., Ltd. <120> A high affinity T cell receptor recognizing an HPV antigen <130> P2020-1132 <160> 37 <170> SIPOSequenceListing 1.0 <210> 1 <211> 111 <212> PRT <213> Artificial Sequence (Artificial Sequence) <400> 1 Gln Lys Glu Val Glu Gin Asn Ser Gly Pro Leu Ser Val Pro Glu Gly 1 5 10 15 Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Val Ser Gin Ser 20 25 30 Phe Phe Trp Tyr Arg Gin Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met 35 40 45 Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gin 50 55 60 Leu Asn Lys Ala Ser Gin Tyr Val Ser Leu Leu Ile Arg Asp Ser Gin 65 70 75 80 Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Pro Arg Tyr Gly 85 90 95 Asn Lys Leu Val Phe Gly Ala Gly Thr Ile Leu Arg Val Lys Ser 100 105 110 <210> 2 <211> 115 <212> PRT <213> Artificial Sequence <400> 2 Asp Ala Asp Val Thr Gin Thr Pro Arg Asn Arg He Thr Lys Thr Gly 1 5 10 15 Lys Arg He Met Leu Glu Cys Ser Gin Thr Lys Gly His Asp Arg Met 20 25 30 Tyr Trp Tyr Arg Gin Asp Pro Gly Leu Gly Leu Arg Leu He Tyr Tyr 35 40 45 Ser Phe Asp Val Lys Asp He Asn Lys Gly Glu He Ser Asp Gly Tyr 50 55 60 Ser Val Ser Arg Gin Ala Gin Ala Lys Phe Ser Leu Ser Leu Glu Ser 65 70 75 80 Ala He Pro Asn Gin Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg 85 90 95 Gly Gin Gly Ala Phe Gly Glu Gin Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr 115 <210> 3 <211> 111 <212> PRT <213> Artificial Sequence <400> 3 Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly 1 5 10 15 Glu Asn Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Val Ser Gln Ser 20 25 30 Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met 35 40 45 Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln 50 55 60 Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Val Gln 65 70 75 80 Pro Ser Asp Ser Ala Thr Tyr Phe Cys Ala Val Asn Pro Arg Tyr Gly 85 90 95 Asn Lys Leu Val Phe Gly Ala Gly Thr Lys Leu Arg Val Lys Ser 100 105 110 <210> 4 <211> 115 <212> PRT <213> Artificial Sequence <400> 4 Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Leu Ser Val Lys Thr Gly 1 5 10 15 Lys Arg Val Thr Leu Glu Cys Ser Gln Thr Lys Gly His Asp Arg Met 20 25 30 Tyr Trp Tyr Arg Gin Asp Pro Gly Gin Gly Leu Arg Leu He Tyr Tyr 35 40 45 Ser Phe Asp Val Lys Asp He Asn Lys Gly Glu He Ser Asp Arg Tyr 50 55 60 Ser Val Ser Arg Gin Ala Gin Ala Lys Phe Ser Leu Ser He Gin Ser 65 70 75 80 Val Glu Pro Asn Asp Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg 85 90 95 Gly Gin Gly Ala Phe Gly Glu Gin Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr 115 <210> 5 <211> 333 <212> DNA <213> Artificial Sequence <400> 5 caaaaagaag ttgaacagaa tagtggcccg ctgagtgtgc cggaaggtga aaatgtgagt 60 attaattgta cctatagcga tcgcgttagt cagagctttt tctggtatcg tcagtatagc 120 ggtaaaagcc cggaactgat tatgagtatc tatagcaatg gcgataaaga agatggccgc 180 tttaccgcac agctgaataa ggcaagccag tatgtgagcc tgctgattcg cgatgtgcag 240 ccgagtgata gtgcaaccta tttttgtgca gtgaatccgc gttatggcaa taagctggtt 300 tttggtgccg gcaccaaact gcgcgttaaa agc 333 <210> 6 <211> 345 <212> DNA <213> Artificial Sequence <400> 6 gacgcagatg ttacccagac cccgcgtaat ctgagcgtga aaaccggcaa acgcgtgacc 60 ctggaatgca gtcagaccaa aggccatgat cgcatgtatt ggtatcgtca agatccgggt 120 cagggcctgc gtctgatcta ttatagcttt gatgttaaag acatcaacaa gggcgaaatt 180 agtgatcgtt atagcgttag tcgtcaggcc caggccaaat tttcactgag tattgaaagc 240 gttgaaccga atgataccgc cctgtatttt tgcgcaacca gcgatcgcgg ccagggtgcc 300 tttggcgaac agtattttgg cccgggtacc cgcctgaccg ttacc 345 <210> 7 <211> 24 <212> PRT <213> Artificial Sequence <400> 7 Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly 1 5 10 15 Gly Gly Ser Glu Gly Gly Thr Gly 20 <210> 8 <211> 72 <212> DNA <213> Artificial Sequence <400> 8 ggtggcggta gcgaaggtgg cggtagtgaa ggtggcggca gtgaaggtgg tggcagcgaa 60 ggtggtaccg gt 72 <210> 9 <211> 250 <212> PRT <213> Artificial Sequence <400> 9 Gln Lys Glu Val Glu Gin Asn Ser Gly Pro Leu Ser Val Pro Glu Gly 1 5 10 15 Glu Asn Val Ser lie Asn Cys Thr Tyr Ser Asp Arg Val Ser Gin Ser 20 25 30 Phe Phe Trp Tyr Arg Gin Tyr Ser Gly Lys Ser Pro Glu Leu lie Met 35 40 45 Ser lie Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gin 50 55 60 Leu Asn Lys Ala Ser Gin Tyr Val Ser Leu Leu lie Arg Asp Val Gin 65 70 75 80 Pro Ser Asp Ser Ala Thr Tyr Phe Cys Ala Val Asn Pro Arg Tyr Gly 85 90 95 Asn Lys Leu Val Phe Gly Ala Gly Thr Lys Leu Arg Val Lys Ser Gly 100 105 110 Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly 115 120 125 Gly Ser Glu Gly Gly Thr Gly Asp Ala Asp Val Thr Gln Thr Pro Arg 130 135 140 Asn Leu Ser Val Lys Thr Gly Lys Arg Val Thr Leu Glu Cys Ser Gln 145 150 155 160 Thr Lys Gly His Asp Arg Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln 165 170 175 Gly Leu Arg Leu Ile Tyr Tyr Ser Phe Asp Val Lys Asp Ile Asn Lys 180 185 190 Gly Glu Ile Ser Asp Arg Tyr Ser Val Ser Arg Gln Ala Gln Ala Lys 195 200 205 Phe Ser Leu Ser Ile Glu Ser Val Glu Pro Asn Asp Thr Ala Leu Tyr 210 215 220 Phe Cys Ala Thr Ser Asp Arg Gly Gln Gly Ala Phe Gly Glu Gln Tyr 225 230 235 240 Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 245 250 <210> 10 <211> 750 <212> DNA <213> Artificial Sequence <400> 10 caaaaagaag ttgaacagaa tagtggcccg ctgagtgtgc cggaaggtga aaatgtgagt 60 attaattgta cctatagcga tcgcgttagt cagagctttt tctggtatcg tcagtatagc 120 ggtaaaagcc cggaactgat tatgagtatc tatagcaatg gcgataaaga agatggccgc 180 tttaccgcac agctgaataa ggcaagccag tatgtgagcc tgctgattcg cgatgtgcag 240 ccgagtgata gtgcaaccta tttttgtgca gtgaatccgc gttatggcaa taagctggtt 300 tttggtgccg gcaccaaact gcgcgttaaa agcggtggcg gtagcgaagg tggcggtagt 360 gaaggtggcg gcagtgaagg tggtggcagc gaaggtggta ccggtgacgc agatgttacc 420 cagaccccgc gtaatctgag cgtgaaaacc ggcaaacgcg tgaccctgga atgcagtcag 480 accaaaggcc atgatcgcat gtattggtat cgtcaagatc cgggtcaggg cctgcgtctg 540 atctattata gctttgatgt taaagacatc aacaagggcg aaattagtga tcgttatagc 600 gttagtcgtc aggcccaggc caaattttca ctgagtattg aaagcgttga accgaatgat 660 accgccctgt atttttgcgc aaccagcgat cgcggccagg gtgcctttgg cgaacagtat 720 tttggcccgg gtacccgcct gaccgttacc 750 <210> 11 <211> 206 <212> PRT <213> Artificial Sequence <400> 11 Met Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu 1 5 10 15 Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Val Ser Gln 20 25 30 Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile 35 40 45 Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala 50 55 60<​​​​​​​​​​​​Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys 115 120 125 Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr 130 135 140 Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr 145 150 155 160 Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala 165 170 175 Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser 180 185 190 Ile Ile Pro Glu Asp Thr Phe Phe Cys Ser Pro Glu Ser Ser 195 200 205 <210> 12 <211> 246 <212> PRT <213> Artificial Sequence <400> 12 Met Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr Lys Thr 1 5 10 15 Gly Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His Asp Arg 0 25 30 Met Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr 35 40 45 Tyr Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser Asp Gly 50 55 60 Tyr Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser Leu Glu 65 70 75 80 Ser Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp 85 90 95 Arg Gly Gln Gly Ala Phe Gly Glu Gln Tyr Phe Gly Pro Gly Thr Arg 100 105 110 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 115 120 125 Val Phe Glu Pro Ser Glu Cys Glu Ile Ser His Thr Gln Lys Ala Thr 130 135 140 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 145 150 155 160 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 165 170 175 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu 180 185 190 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 195 200 205 His Phe Arg Cys Gin Val Gin Phe Tyr Gly Leu Ser Glu Asn Asp Glu 210 215 220 Trp Thr Gin Asp Arg Ala Lys Pro Val Thr Gin He Val Ser Ala Glu 225 230 235 240 Ala Trp Gly Arg Ala Asp 245 <210> 13 <211> 111 <212> PRT <213> Artificial Sequence <400> 13 Gln Lys Glu Val Glu Gin Asn Ser Gly Pro Leu Ser Val Pro Glu Gly 1 5 10 15 Ala He Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Leu Ser Gin Thr 20 25 30 Phe Phe Trp Tyr Arg Gin Tyr Ser Gly Lys Ser Pro Glu Leu He Met 35 40 45 Ser He Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gin 50 55 60 Leu Asn Lys Ala Ser Gin Tyr Val Ser Leu Leu He Arg Asp Ser Gin 65 70 75 80 Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Pro Arg Tyr Gly 85 90 95 Asn Lys Leu Val Phe Gly Ala Gly Thr Ile Leu Arg Val Lys Ser 100 105 110 <210> 14 <211> 111 <212> PRT <213> Artificial Sequence <400> 14 Gln Lys Glu Val Glu Gin Asn Ser Gly Pro Leu Ser Val Pro Glu Gly 1 5 10 15 Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Leu Ala Gin Ser 20 25 30 Phe Phe Trp Tyr Arg Gin Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met 35 40 45 Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gin 50 55 60 Leu Asn Lys Ala Ser Gin Tyr Val Ser Leu Leu Ile Arg Asp Ser Gin 65 70 75 80 Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Pro Arg Tyr Gly 85 90 95 Asn Lys Leu Val Phe Gly Ala Gly Thr Ile Leu Arg Val Lys Ser 100 105 110 <210> 15 <211> 111 <212> PRT <213> Artificial Sequence <400> 15 Gln Lys Glu Val Glu Gin Asn Ser Gly Pro Leu Ser Val Pro Glu Gly 1 5 10 15 Ala lie Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Leu Thr Tyr Val 20 25 30 Phe Phe Trp Tyr Arg Gin Tyr Ser Gly Lys Ser Pro Glu Leu lie Met 35 40 45 Ser lie Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gin 50 55 60 Leu Asn Lys Ala Ser Gin Tyr Val Ser Leu Leu lie Arg Asp Ser Gin 65 70 75 80 Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Pro Arg Tyr Gly 85 90 95 Asn Lys Leu Val Phe Gly Ala Gly Thr lie Leu Arg Val Lys Ser 100 105 110 <210> 16 <211> 111 <212> PRT <213> Artificial Sequence <400> 16 Gln Lys Glu Val Glu Gin Asn Ser Gly Pro Leu Ser Val Pro Glu Gly 1 5 10 15 Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Val Ser Gin Ser 20 25 30 Phe Phe Trp Tyr Arg Gin Tyr Ser Gly Lys Ser Pro Gin Leu Ile Met 35 40 45 Ser Ile Phe Asn Pro Gly Asp Lys Gin Asp Gly Gin Phe Thr Gin Gin 50 55 60 Leu Asn Lys Ala Ser Gin Tyr Val Ser Leu Leu Ile Arg Asp Ser Gin 65 70 75 80 Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Pro Gin Tyr Gly 85 90 95 Asn Lys Leu Val Phe Gly Ala Gly Thr Ile Leu Gin Val Lys Gin 100 105 110 <210> 17 <211> 111 <212> PRT <213> Artificial Sequence <400> 17 Gln Lys Gin Val Gin Gin Asn Ser Gin Pro Leu Ser Val Pro Gin Gin 1 5 10 15 Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Met Ala Asn Ser 20 25 30 Phe Phe Trp Tyr Arg Gin Tyr Ser Gly Lys Ser Pro Gin Leu Ile Met 35 40 45 Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln 50 55 60 Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln 65 70 75 80 Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Pro Arg Tyr Gly 85 90 95 Asn Lys Leu Val Phe Gly Ala Gly Thr Ile Leu Arg Val Lys Ser 100 105 110 <210> 18 <211> 111 <212> PRT <213> Artificial Sequence <400> 18 Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly 1 5 10 15 Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg His Ala Asn Thr 20 25 30 Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met 35 40 45 Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln 50 55 60 Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln 65 70 75 80 Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Pro Arg Tyr Gly 85 90 95 Asn Lys Leu Val Phe Gly Ala Gly Thr Ile Leu Arg Val Lys Ser 100 105 110 <210> 19 <211> 111 <212> PRT <213> Artificial Sequence <400> 19 Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly 1 5 10 15 Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Leu Ala Val Thr 20 25 30 Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met 35 40 45 Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln 50 55 60 Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln 65 70 75 80 Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Pro Arg Tyr Gly 85 90 95 Asn Lys Leu Val Phe Gly Ala Gly Thr Ile Leu Arg Val Lys Ser 100 105 110 <210> 20 <211> 111 <212> PRT <213> Artificial Sequence <400> 20 Gln Lys Glu Val Glu Gin Asn Ser Gly Pro Leu Ser Val Pro Glu Gly 1 5 10 15 Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Leu Ala Asn Val 20 25 30 Phe Phe Trp Tyr Arg Gin Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met 35 40 45 Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gin 50 55 60 Leu Asn Lys Ala Ser Gin Tyr Val Ser Leu Leu Ile Arg Asp Ser Gin 65 70 75 80 Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Pro Arg Tyr Gly 85 90 95 Asn Lys Leu Val Phe Gly Ala Gly Thr Ile Leu Arg Val Lys Ser 100 105 110 <210> 21 <211> 111 <212> PRT <213> Artificial Sequence <400> 21 Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly 1 5 10 15 Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Met Gly Asn Ala 20 25 30 Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met 35 40 45 Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln 50 55 60 Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln 65 70 75 80 Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Pro Arg Tyr Gly 85 90 95 Asn Lys Leu Val Phe Gly Ala Gly Thr Ile Leu Arg Val Lys Ser 100 105 110 <210> 22 <211> 115 <212> PRT <213> Artificial Sequence <400> 22 Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr Lys Thr Gly 1 5 10 15 Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His Asp Arg Met 20 25 30 Tyr Trp Tyr Arg Gin Asp Pro Gly Leu Gly Leu Arg Leu lie Tyr Tyr 35 40 45 Ser Phe Asp Val Lys Asp lie Asn Lys Gly Glu lie Ser Asp Gly Tyr 50 55 60 Ser Val Ser Arg Gin Ala Gin Ala Lys Phe Ser Leu Ser Leu Glu Ser 65 70 75 80 Ala lie Pro Asn Gin Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg 85 90 95 Gln Tyr Gly Ala Phe Gly Glu Gin Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr 115 <210> 23 <211> 115 <212> PRT <213> Artificial Sequence <400> 23 Asp Ala Asp Val Thr Gin Thr Pro Arg Asn Arg lie Thr Lys Thr Gly 1 5 10 15 Lys Arg lie Met Leu Glu Cys Ser Gin Thr Lys Gly His Asp Arg Met 20 25 30 Tyr Trp Tyr Arg Gin Asp Pro Gly Leu Gly Leu Arg Leu lie Tyr Tyr 35 40 45 Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser Asp Gly Tyr 50 55 60 Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser Leu Glu Ser 65 70 75 80 Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg 85 90 95 Gln Phe Gly Ala Phe Gly Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr 115 <210> 24 <211> 115 <212> PRT <213> Artificial Sequence <400> 24 Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr Lys Thr Gly 1 5 10 15 Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His Asp Arg Met 20 25 30 Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Tyr 35 40 45 Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser Asp Gly Tyr 50 55 60 Ser Val Ser Arg Gin Ala Gin Ala Lys Phe Ser Leu Ser Leu Glu Ser 65 70 75 80 Ala lie Pro Asn Gin Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg 85 90 95 Gln Gin Gly Ala Phe Gly Glu Gin Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr 115 <210> 25 <211> 115 <212> PRT <213> Artificial Sequence (Artificial Sequence) <400> 25 Asp Ala Asp Val Thr Gin Thr Pro Arg Asn Arg lie Thr Lys Thr Gly 1 5 10 15 Lys Arg lie Met Leu Glu Cys Ser Gin Thr Lys Gly His Asp Arg Met 20 25 30 Tyr Trp Tyr Arg Gin Asp Pro Gly Leu Gly Leu Arg Leu lie Tyr Tyr 35 40 45 Ser Phe Asp Val Lys Asp lie Asn Lys Gly Glu lie Ser Asp Gly Tyr 50 55 60 Ser Val Ser Arg Gin Ala Gin Ala Lys Phe Ser Leu Ser Leu Glu Ser 65 70 75 80 Ala lie Pro Asn Gin Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg 85 90 95 Gln Gin Gly Ala Phe Gly Glu Gin Phe Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr 115 <210> 26 <211> 115 <212> PRT <213> Artificial Sequence (Artificial Sequence) <400> 26 Asp Ala Asp Val Thr Gin Thr Pro Arg Asn Arg lie Thr Lys Thr Gly 1 5 10 15 Lys Arg lie Met Leu Glu Cys Ser Gin Thr Lys Gly His Asp Arg Met 20 25 30 Tyr Trp Tyr Arg Gin Asp Pro Gly Leu Gly Leu Arg Leu lie Tyr Tyr 35 40 45 Ser Phe Asp Val Lys Asp lie Asn Lys Gly Glu lie Ser Asp Gly Tyr 50 55 60 Ser Val Ser Arg Gin Ala Gin Ala Lys Phe Ser Leu Ser Leu Glu Ser 65 70 75 80 Ala lie Pro Asn Gin Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg 85 90 95 Gln Trp Gly Ala Phe Gly Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr 115 <210> 27 <211> 115 <212> PRT <213> Artificial Sequence (Artificial Sequence) <400> 27 Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr Lys Thr Gly 1 5 10 15 Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His Asp Arg Met 20 25 30 Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Tyr 35 40 45 Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser Asp Gly Tyr 50 55 60 Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser Leu Glu Ser 65 70 75 80 Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg 85 90 95 Gly Gln Gly Ala Phe Gly Glu Tyr His Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr 115 <210> 28 <211> 115 <212> PRT <213> Artificial Sequence <400> 28 Asp Ala Asp Val Thr Gin Thr Pro Arg Asn Arg He Thr Lys Thr Gly 1 5 10 15 Lys Arg He Met Leu Glu Cys Ser Gin Thr Lys Gly His Asp Arg Met 20 25 30 Tyr Trp Tyr Arg Gin Asp Pro Gly Leu Gly Leu Arg Leu He Tyr Tyr 35 40 45 Ser Phe Asp Val Lys Asp He Asn Lys Gly Glu He Ser Asp Gly Tyr 50 55 60 Ser Val Ser Arg Gin Ala Gin Ala Lys Phe Ser Leu Ser Leu Glu Ser 65 70 75 80 Ala He Pro Asn Gin Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg 85 90 95 Gly Gin Gly Ala Phe Gly Glu Tyr Phe Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr 115 <210> 29 <211> 115 <212> PRT <213> Artificial Sequence <400> 29 Asp Ala Asp Val Thr Gin Thr Pro Arg Asn Arg He Thr Lys Thr Gly 1 5 10 15 Lys Arg He Met Leu Glu Cys Ser Gin Thr Lys Gly His Asp Arg Met 20 25 30 Tyr Trp Tyr Arg Gin Asp Pro Gly Leu Gly Leu Arg Leu He Tyr Tyr 35 40 45 Ser Phe Asp Val Lys Asp He Asn Lys Gly Glu He Ser Asp Gly Tyr 50 55 60 Ser Val Ser Arg Gin Ala Gin Ala Lys Phe Ser Leu Ser Leu Glu Ser 65 70 75 80 Ala He Pro Asn Gin Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg 85 90 95 Gly Gin Gly Ala Phe Ala Glu Tyr Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr 115 <210> 30 <211> 115 <212> PRT <213> Artificial Sequence <400> 30 Asp Ala Asp Val Thr Gin Thr Pro Arg Asn Arg He Thr Lys Thr Gly 1 5 10 15 Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His Asp Arg Met 20 25 30 Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Tyr 35 40 45 Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser Asp Gly Tyr 50 55 60 Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser Leu Glu Ser 65 70 75 80 Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg 85 90 95 Gly Gln Gly Ala Phe Ala Glu Tyr His Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr 115 <210> 31 <211> 115 <212> PRT ​​​​​​​​​​​​Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Tyr 35 40 45 Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser Asp Gly Tyr 50 55 60 Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser Leu Glu Ser 65 70 75 80 Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg 85 90 95 Gly Gln Gly Ala Phe Ala Glu Tyr Phe Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr 115 <210> 32 <211> 115<00012​​​​​​​​​​​​​​​​​​Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser Asp Gly Tyr 50 55 60 Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser Leu Glu Ser 65 70 75 80 Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg 85 90 95 Gly Gln Gly Ala Phe Gly Glu Tyr Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr 115 <210> 33 <211> 115 <212> PRT <213> Artificial Sequence <400> 33 Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr Lys Thr Gly 1 5 10 15 Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His Asp Arg Met 20 25 30 Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Tyr 35 40 45 Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser Asp Gly Tyr 50 55 60 Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser Leu Glu Ser 65 70 75 80 Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg 85 90 95 Gly Gln Gly Ala Phe Gly Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr 115 <210> 34 <211> 205 <212> PRT <213> Artificial Sequence <400> 34 Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly 1 5 10 15 Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Val Ser Gln Ser 20 25 30 Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met 35 40 45 Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln 50 55 60 Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln 65 70 75 80 Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Pro Arg Tyr Gly 85 90 95 Asn Lys Leu Val Phe Gly Ala Gly Thr Ile Leu Arg Val Lys Ser Tyr 100 105 110 Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser 115 120 125 Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn 130 135 140 Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val 145 150 155 160<                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      &emsp​​​​​​​​​​​​​​​​​​​​​​​​1 5 10 15 Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His Asp Arg Met 20 25 30 Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Tyr 35 40 45 Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser Asp Gly Tyr 50 55 60 Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser Leu Glu Ser 65 70 75 80 Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg 85 90 95 Gly Gln Gly Ala Phe Gly Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val 115 120 125 Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu 130 135 140 Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp 145 150 155 160 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 165 170 175 Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser 180 185 190 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His 195 200 205 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp 210 215 220​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Leu Asn Lys Ala Ser Gin Tyr Val Ser Leu Leu lie Arg Asp Ser Gin 65 70 75 80 Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Pro Arg Tyr Gly 85 90 95 Asn Lys Leu Val Phe Gly Ala Gly Thr lie Leu Arg Val Lys Ser Tyr 100 105 110 lie Gin Asn Pro Asp Pro Ala Val Tyr Gin Leu Arg Asp Ser Lys Ser 115 120 125 Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gin Thr Asn 130 135 140 Val Ser Gin Ser Lys Asp Ser Asp Val Tyr lie Thr Asp Lys Thr Val 145 150 155 160 Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp 165 170 175 Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser lie 180 185 190 lie Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val 195 200 205 Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gin 210 215 220 Asn Leu Ser Val lie Gly Phe Arg lie Leu Leu Leu Lys Val Ala Gly 225 230 235 240 Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 245 250 <210> 37 <211> 294 <212> PRT <213> Artificial Sequence <400> 37 Asp Ala Asp Val Thr Gin Thr Pro Arg Asn Arg lie Thr Lys Thr Gly 1 5 10 15 Lys Arg lie Met Leu Glu Cys Ser Gin Thr Lys Gly His Asp Arg Met 20 25 30 Tyr Trp Tyr Arg Gin Asp Pro Gly Leu Gly Leu Arg Leu lie Tyr Tyr 35 40 45 Ser Phe Asp Val Lys Asp lie Asn Lys Gly Glu lie Ser Asp Gly Tyr 50 55 60 Ser Val Ser Arg Gin Ala Gin Ala Lys Phe Ser Leu Ser Leu Glu Ser 65 70 75 80 Ala lie Pro Asn Gin Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg 85 90 95 Gly Gin Gly Ala Phe Gly Glu Gin Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val 115 120 125 Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu 130 135 140 Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp 145 150 155 160 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 165 170 175 Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser 180 185 190 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His 195 200 205 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp 210 215 220 Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala 225 230 235 240 Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly 245 250 255 Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 260 265 270 Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys 275 280 285 Arg Lys Asp Ser Arg Gly 290

Claims

1. A T cell receptor (TCR) comprising: said TCR having CDRs selected from the group consisting of:

2. A TCR as claimed in claim 1, wherein said TCR comprising (i) a TCR alpha chain variable domain and all or part of a TCR alpha chain constant region except the transmembrane domain; and (ii) a TCR beta chain variable domain and all or part of a TCR beta chain constant region except the transmembrane domain.

3. A TCR as claimed in claim 1, wherein said TCR comprising an alpha chain constant region and a beta chain constant region, and said alpha chain constant region and said beta chain constant region comprise an artificial interchain disulfide bond.

4. A TCR as claimed in claim 3, wherein, The cysteine residue that forms the artificial interchain disulfide bond between the constant regions of the alpha and beta chains of said TCR is substituted for one or more sets of positions selected from the group consisting of: Thr48 of TRAC*01 exon 1 and Ser57 of TRBC1*01 or TRBC2*01 exon 1; Thr45 of TRAC*01 exon 1 and Ser77 of TRBC1*01 or TRBC2*01 exon 1; Tyr10 of TRAC*01 exon 1 and Ser17 of TRBC1*01 or TRBC2*01 exon 1; Thr45 of TRAC*01 exon 1 and Asp59 of TRBC1*01 or TRBC2*01 exon 1; Ser15 of TRAC*01 exon 1 and Glu15 of TRBC1*01 or TRBC2*01 exon 1; Arg53 of TRAC*01 exon 1 and Ser54 of TRBC1*01 or TRBC2*01 exon 1; Pro89 of TRAC*01 exon 1 and Ala19 of TRBC1*01 or TRBC2*01 exon 1; and Tyr10 of TRAC*01 exon 1 and Glu20 of TRBC1*01 or TRBC2*01 exon 1. said alpha chain variable domain amino acid sequence of said TCR is one of SEQ ID NOs: 1, 13-21; and / or said beta chain variable domain amino acid sequence of said TCR is one of SEQ ID NOs: 2, 22-33.

5. A TCR as claimed in claim 1, wherein the CDR3a sequence is SEQ ID NO:

1. said TCR is selected from the group consisting of:

6. A TCR as claimed in claim 1, wherein said TCR is a single chain TCR.

7. A TCR as claimed in claim 1, wherein said TCR is a single chain TCR consisting of an alpha chain variable domain and a beta chain variable domain connected by a flexible short peptide sequence.

8. A TCR as claimed in claim 7, wherein comprises at least two TCR molecules, and at least one of the TCR molecules is the TCR of claim 1.

9. A multivalent TCR complex, characterized in that, said nucleic acid molecule comprises a nucleic acid sequence encoding the TCR of any one of claims 1-8, or the complement thereof.

10. A nucleic acid molecule, characterized in that, said vector comprises the nucleic acid molecule of claim 10.

11. A vector, characterized in that, said host cell comprises the vector of claim 11 or the exogenous nucleic acid molecule of claim 10 integrated into the chromosome.

12. A host cell, characterized in that, said cell expresses the TCR of any one of claims 1-8.

13. An isolated cell, wherein, said isolated cell is a T cell.

14. The isolated cell of claim 13, wherein, said composition comprises a pharmaceutically acceptable carrier and the TCR of any one of claims 1-8, or the TCR complex of claim 9, or the cell of claim 13.

15. A pharmaceutical composition comprising, a medicament for use in the treatment of a tumor, said tumor being cervical cancer.

16. Use of the T cell receptor of any one of claims 1 to 8, the TCR complex of claim 9 or the cell of claim 13, characterized in that, comprising the steps of:

17. A method of making a T cell receptor of any one of claims 1-8, wherein, ​ (i) culturing the host cell of claim 12, thereby expressing the T cell receptor of any one of claims 1-8; (ii) isolating or purifying the T cell receptor.

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