CN116769017A

CN116769017A - MART-1 (27-35) epitope specific T cell receptor and application thereof

Info

Publication number: CN116769017A
Application number: CN202210234400.3A
Authority: CN
Inventors: 徐曲苗; 梁艳玲; 朱琳楠; 王飞; 董旋; 王凯; 顾颖; 侯勇
Original assignee: BGI Shenzhen Co Ltd
Current assignee: BGI Shenzhen Co Ltd
Priority date: 2022-03-10
Filing date: 2022-03-10
Publication date: 2023-09-19

Abstract

The invention belongs to the technical field of biology, and particularly relates to a T cell receptor capable of specifically recognizing MART-1 (27-35) epitope and related application thereof. By using the T cell receptor of the invention, cells carrying MART-1 (27-35) antigen peptide can be effectively killed, thereby realizing the treatment of melanoma.

Description

MART-1 (27-35) epitope specific T cell receptor and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a MART-1 (27-35) epitope specific T cell receptor and application thereof.

Background

Melanoma, also known as malignant melanoma, is a type of malignant tumor derived from melanocytes, and is commonly found in the skin, mucous membranes, the choroid of the eye, and other sites. Melanoma is the tumor species with the highest degree of malignancy of the skin, and is prone to distant metastasis. Among asians and colored ethnicities, melanoma of the primary skin accounts for 50% to 70%, and excessive ultraviolet irradiation in the european white race is one of clear causes. Ultraviolet light can cause skin burn and induce DNA mutation, thereby inducing melanoma. In addition, photosensitive skin, those with a high family history of moles or dysplasias are all high risk groups. The incidence of acromelamas in asia and africa is very small and the etiology is still unknown.

Tumor-associated antigens are normal, non-mutated self-proteins that are selectively expressed or overexpressed on cancer, and can be targeted epitopes for tumor immunotherapy. MART-1 (melanoma antigen recognized by T cells 1) antigenic peptides are known to be expressed on melanoma and healthy melanocytes in skin, eyes and ears, to be low-expressed on endometrium, ovary, lymph node and testis, and to be high-expressed in melanoma cells. MART-1 is highly expressed, for example, in uveal melanoma lesions (uveal melana lesions). MART-1 is therefore a very good epitope for immunotherapy of melanoma. Furthermore, melan-A can also be used for detection of vascular smooth muscle lipoma protein and mRNA levels. Melan-A/MART-1 26-35 (EAAGIGILTV) epitope of decapeptides and Melan-A/MART-1 27-35 (LAGIGILTV) anti-nonapeptideThe pro-epitope is the two major epitopes of MART-1. The nonapeptide has been shown to have strong immunodominance and has been shown to be specific for CD8 ⁺ Cytolytic T Lymphocytes (CTLs) most commonly recognize peptides. DMF4 and DMF 5T cell receptor (TCR) that have been identified so far recognize the same MART-1:27-35 epitopes, and has cross-reactivity. In clinical trials, the therapeutic approach of adoptive transfer of MART-1F5 TCR engineered peripheral blood mononuclear cells resulted in good results with tumor regression reaching 69%. Another clinical response showed that 6 out of 20 patients treated with DMF5 TCR (30%) experienced an objective anti-tumor response as defined by RECIST criteria. Therefore, the development of an adoptive therapy method for anti-MART-1 TCR genetically engineered lymphocytes has important significance and promotion effect on the immunotherapy of tumors, particularly melanoma.

Therefore, there is a need in the art to develop an anti-MART-1 TCR genetically engineered lymphocyte adoptive therapy for effectively treating melanoma.

Disclosure of Invention

As described above, the present invention aims to provide an adoptive therapeutic method for anti-MART-1 TCR genetically engineered lymphocytes to effectively treat melanoma.

Accordingly, in a first aspect, the present invention provides a T Cell Receptor (TCR) that specifically recognizes an epitope of MART-1 (27-35), comprising an alpha chain comprising three complementarity determining regions CDR1 alpha, CDR2 alpha and CDR3 alpha, and a beta chain comprising three complementarity determining regions CDR1 beta, CDR2 beta and CDR3 beta, and the amino acid sequences of CDR1 alpha, CDR2 alpha, CDR3 alpha, CDR1 beta, CDR2 beta and CDR3 beta being as shown in SEQ ID NO:1-6, SEQ ID NO:7-12, SEQ ID NO:13-18 or SEQ ID NO:19-24, respectively, or being a variant having at least one amino acid change as compared to said amino acid sequences and retaining the ability to specifically recognize an epitope of MART-1 (27-35).

In one embodiment, the alpha chain variable region comprises the amino acid sequence shown as SEQ ID NO. 25, SEQ ID NO. 33, SEQ ID NO. 41 or SEQ ID NO. 49 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 25, SEQ ID NO. 33, SEQ ID NO. 41 or SEQ ID NO. 49.

In one embodiment, the β chain variable region comprises the amino acid sequence shown as SEQ ID NO. 26, SEQ ID NO. 34, SEQ ID NO. 42 or SEQ ID NO. 50 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 26, SEQ ID NO. 34, SEQ ID NO. 42 or SEQ ID NO. 50.

In one embodiment, the alpha chain variable region and the beta chain variable region are each linked to a constant region. In a preferred embodiment, the amino acid sequence of the constant region linked to the alpha chain variable region is as shown in SEQ ID NO. 57. In yet another preferred embodiment, the amino acid sequence of the constant region linked to the β chain variable region is as shown in SEQ ID NO. 58.

In one embodiment, each of the α chain and the β chain is further fused to a signal peptide. In a preferred embodiment, the amino acid sequence of the signal peptide fused to the alpha chain is shown in SEQ ID NO 27, SEQ ID NO 35, SEQ ID NO 43 or SEQ ID NO 51. In yet another preferred embodiment, the amino acid sequence of the signal peptide fused to the β chain is as shown in SEQ ID NO. 28, SEQ ID NO. 36, SEQ ID NO. 44 or SEQ ID NO. 52.

In one embodiment, the alpha chain fused to the signal peptide and the beta chain fused to the signal peptide are linked together via a 2A peptide, such as a P2A peptide. In a preferred embodiment, the amino acid sequence of the T Cell Receptor (TCR) is as shown in SEQ ID NO. 29, SEQ ID NO. 37, SEQ ID NO. 45 or SEQ ID NO. 53.

In a second aspect, the invention provides a nucleic acid molecule encoding a T Cell Receptor (TCR) of the first aspect of the invention.

In one embodiment, the alpha chain variable region of the T Cell Receptor (TCR) is encoded by the nucleic acid sequence set forth in SEQ ID NO. 30, SEQ ID NO. 38, SEQ ID NO. 46 or SEQ ID NO. 54 or a degenerate sequence thereof.

In one embodiment, the beta chain variable region of the T Cell Receptor (TCR) is encoded by the nucleic acid sequence set forth in SEQ ID NO. 31, SEQ ID NO. 39, SEQ ID NO. 47 or SEQ ID NO. 55 or a degenerate sequence thereof.

In one embodiment, the T Cell Receptor (TCR) is encoded by the nucleic acid sequence set forth in SEQ ID NO. 32, SEQ ID NO. 40, SEQ ID NO. 48 or SEQ ID NO. 56 or a degenerate sequence thereof.

In a third aspect, the present invention provides a vector comprising a nucleic acid molecule of the second aspect of the invention.

In one embodiment, the vector is a viral vector such as a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral expression vector.

In a fourth aspect, the invention provides an effector cell comprising a T cell receptor of the first aspect of the invention, a nucleic acid molecule of the second aspect of the invention, or a vector of the third aspect of the invention.

In one embodiment, the cells are T cells, natural killer cells, human embryonic stem cells, lymphoid progenitor cells, and/or T cell precursors, such as cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells.

In a fifth aspect, the invention provides a method of killing a melanoma cell comprising contacting the T cell receptor of the first aspect of the invention or the effector cell of the fourth aspect of the invention with a melanoma cell.

In a preferred embodiment, the melanoma cells are MART-1 positive, HLA:A0201 typed melanoma cells.

In a sixth aspect, the invention provides a method of treating melanoma comprising administering to a patient suffering from melanoma a T cell receptor according to the first aspect of the invention or an effector cell according to the fourth aspect of the invention.

In a preferred embodiment, the melanoma is MART-1 positive, HLA:A0201 typed melanoma.

In a seventh aspect, the invention provides the use of a T cell receptor of the first aspect of the invention, a nucleic acid molecule of the second aspect of the invention, a vector of the third aspect of the invention, or an effector cell of the fourth aspect of the invention in the manufacture of a medicament for the treatment of melanoma.

In an eighth aspect, the invention provides a kit comprising a T cell receptor of the first aspect of the invention, a nucleic acid molecule of the second aspect of the invention, a vector of the third aspect of the invention, or an effector cell of the fourth aspect of the invention.

By using the T cell receptor of the invention, cells carrying MART-1 (27-35) antigen peptide can be effectively killed, thereby realizing the treatment of melanoma.

Drawings

In order to more clearly illustrate the examples of the invention or the technical solutions of the prior art, the drawings which are required to be used in the examples are briefly described below, it being evident that the drawings in the following description are only a part of examples of the invention and that other embodiments can be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the results of MART-1 specific T cell function detection by Elispot.

FIG. 2 shows the results of sorting specific T cells by MART-1 tetramer flow cytometry.

FIG. 3 shows the clonotype ratio and Top 10 TCR. Alpha. And. Beta. Chain pairing information for each clonotype in single cell V (D) J sequencing results.

FIG. 4 shows a TCR lentiviral shuttle plasmid vector construction map.

FIG. 5 shows the results of TCR-T build positive rate detection.

FIG. 6 shows the results of Elispot detection of MART-1 specific T cell function.

FIG. 7 shows the results of detection of TCR3 TCR-T cytokine secretion by flow cytometry analysis.

FIG. 8 shows the results of detection of TCR4-T cytokine secretion by flow cytometry analysis.

FIG. 9 shows the results of detection of TCR-T killing of target cells by flow cytometry analysis.

Detailed Description

The present invention will now be described more fully hereinafter. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments that can be obtained by a person skilled in the art based on the embodiments of the present invention are within the scope of the present invention.

Accordingly, in a first aspect, the present invention provides a T Cell Receptor (TCR) which specifically recognizes an epitope of MART-1 (27-35).

As is known in the art, a T Cell Receptor (TCR) is a specific receptor present on the surface of T cells and responsible for recognizing antigens presented by the Major Histocompatibility Complex (MHC). T Cell Receptors (TCRs) are heterodimers, consisting of two distinct subunits, of which 95% are composed of alpha and beta chains and 5% are composed of gamma and delta chains, and this ratio varies as a result of ontogeny or disease. For the α and β chains, each can be further divided into Variable region (V region), constant region (C region), transmembrane region and cytoplasmic region, wherein V region is a key site for specific recognition of antigen peptide/histocompatibility antigen complex (MHC). The V regions (vα and vβ) in turn each have three hypervariable regions CDR1, CDR2, CDR3, with the greatest variation in CDR3, which directly determines the antigen binding specificity of the TCR.

MART-1, chinese, is called T cell recognized melanoma antigen 1, a tumor associated antigen cloned from melanoma cell lines. It was found that amino acids 27-35 of MART-1 are epitopes on the tumor associated antigen and the amino acid sequence is LAGIGILTV.

The T Cell Receptor (TCR) of the invention is a T Cell Receptor (TCR) specifically recognizing an epitope of MART-1 (27-35), comprising an alpha chain comprising three complementarity determining regions CDR1 alpha, CDR2 alpha and CDR3 alpha, and a beta chain comprising three complementarity determining regions CDR1 beta, CDR2 beta and CDR3 beta, and having amino acid sequences of CDR1 alpha, CDR2 alpha, CDR3 alpha, CDR1 beta, CDR2 beta and CDR3 beta as shown in SEQ ID NO:1-6, SEQ ID NO:7-12, SEQ ID NO:13-18 or SEQ ID NO:19-24, respectively, or being a variant having at least one amino acid change as compared to said amino acid sequences and retaining the ability to specifically recognize an epitope of MART-1 (27-35).

The "at least one amino acid change" may be one, two or three amino acid changes, and the "amino acid change" may be a conservative substitution of an amino acid (conservative substitution). By "conservative substitution" is meant that one amino acid in a protein is replaced by another, chemically similar amino acid, e.g., a polar (nonpolar) amino acid replaces another polar (nonpolar) amino acid. Conservative substitutions generally do not alter or only slightly alter the nature of the protein. Common conservative substitutions include: the aromatic amino acids phenylalanine (Phe), tryptophan (Trp) and tyrosine (Tyr), the hydrophobic amino acids leucine (Leu), isoleucine (Ile) and valine (Val), the polar amino acids glutamine (Gln) and asparagine (Asn), the basic amino acids lysine (Lys), arginine (Arg) and histidine (His), the acidic amino acids aspartic acid (Asp) and glutamic acid (Glu), and the hydroxy amino acids serine (Ser) and threonine (Thr).

As described above, the α and β chains that make up the T Cell Receptor (TCR) each include a variable region (V region), where the V region is a critical site for specific recognition of the antigenic peptide/histocompatibility antigen complex (MHC). In the present invention, the alpha chain variable region comprising a T Cell Receptor (TCR) of the invention may comprise the amino acid sequence shown as SEQ ID NO. 25, SEQ ID NO. 33, SEQ ID NO. 41 or SEQ ID NO. 49 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 25, SEQ ID NO. 33, SEQ ID NO. 41 or SEQ ID NO. 49, and the beta chain variable region may comprise the amino acid sequence shown as SEQ ID NO. 26, SEQ ID NO. 34, SEQ ID NO. 42 or SEQ ID NO. 50 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 26, SEQ ID NO. 34, SEQ ID NO. 42 or SEQ ID NO. 50.

Herein, the expression "at least 80% sequence identity" means that the variant sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, even 100% sequence identity compared to the reference sequence.

Preferably, the alpha chain variable region comprising a T Cell Receptor (TCR) of the invention may comprise the amino acid sequence shown in SEQ ID NO. 25 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 25, and the beta chain variable region may comprise the amino acid sequence shown in SEQ ID NO. 26 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 26. This TCR receptor is also referred to herein as TCR1.

Preferably, the alpha chain variable region comprising a T Cell Receptor (TCR) of the invention may comprise the amino acid sequence shown in SEQ ID NO. 33 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 33 and the beta chain variable region may comprise the amino acid sequence shown in SEQ ID NO. 34 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 34. The TCR receptor is also referred to herein as TCR2.

Preferably, the alpha chain variable region comprising a T Cell Receptor (TCR) of the invention may comprise the amino acid sequence shown in SEQ ID NO. 41 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 41, and the beta chain variable region may comprise the amino acid sequence shown in SEQ ID NO. 42 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 42. This TCR receptor is also referred to herein as TCR3.

Preferably, the alpha chain variable region comprising a T Cell Receptor (TCR) of the invention may comprise the amino acid sequence shown in SEQ ID NO. 49 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 49 and the beta chain variable region may comprise the amino acid sequence shown in SEQ ID NO. 50 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 50. This TCR receptor is also referred to herein as TCR4.

As described above, T Cell Receptors (TCRs) contain constant regions in addition to variable regions. Thus, the α chain variable region and the β chain variable region can each be linked to a constant region to effectively increase the expression efficiency of the exogenous T cell receptor. The constant region may have the same species source as the variable region, or may have a different species source, but preferably has a different species source; for example, the constant region is derived from a mouse. Preferably, the amino acid sequence of the constant region linked to the alpha chain variable region is as shown in SEQ ID NO. 57. Preferably, the amino acid sequence of the constant region linked to the β chain variable region is as shown in SEQ ID NO. 58.

In addition, in some cases, the alpha and beta chains may each be further fused to a signal peptide for directing the transfer of the newly synthesized alpha and beta chains to the secretory pathway. The signal peptide is typically a peptide chain of 5-30 amino acids in length. As an example, the amino acid sequence of the signal peptide fused to the alpha chain of the T Cell Receptor (TCR) of the invention is shown in SEQ ID NO 27, SEQ ID NO 35, SEQ ID NO 43 or SEQ ID NO 51, and the amino acid sequence of the signal peptide fused to the beta chain of the T Cell Receptor (TCR) of the invention is shown in SEQ ID NO 28, SEQ ID NO 36, SEQ ID NO 44 or SEQ ID NO 52.

Preferably, the alpha chain variable region shown in SEQ ID NO. 25 is fused to the signal peptide shown in SEQ ID NO. 27 and the beta chain variable region shown in SEQ ID NO. 26 is fused to SEQ ID NO. 28.

Preferably, the alpha chain variable region shown in SEQ ID NO. 33 is fused to the signal peptide shown in SEQ ID NO. 35 and the beta chain variable region shown in SEQ ID NO. 34 is fused to the signal peptide shown in SEQ ID NO. 36.

Preferably, the alpha chain variable region shown in SEQ ID NO. 41 is fused to the signal peptide shown in SEQ ID NO. 43, and the beta chain variable region shown in SEQ ID NO. 42 is fused to the signal peptide shown in SEQ ID NO. 44.

Preferably, the alpha chain variable region shown in SEQ ID NO. 49 is fused to the signal peptide shown in SEQ ID NO. 51, and the beta chain variable region shown in SEQ ID NO. 50 is fused to the signal peptide shown in SEQ ID NO. 52.

Furthermore, the alpha and beta chains may be linked together by a 2A peptide, such as a P2A peptide, optionally by a 2A peptide, such as a P2A peptide.

The 2A peptide is a short peptide of viral origin, approximately 18-25 amino acids in length, and is commonly referred to as a "self-cleaving" peptide, which allows the production of multiple proteins from a single transcript. The 2A peptide does not completely "self-cleave" but rather works by allowing the ribosome to skip synthesis of glycine and proline peptide bonds at the C-terminus of the 2A element, ultimately resulting in separation of the 2A sequence end and downstream products. There are four commonly used 2A peptides, P2A, T2A, E a and F2A, respectively, derived from four different viruses. The P2A peptide is a typical self-cleaving polypeptide derived from porcine teschovirus (Porcine teschovirus, PTV, a picornavirus), the cleavage efficiency of the 2A peptide is high, the balance of the expression of the upstream and downstream genes is good, so that in the process of in vivo translation in eukaryotes, folded 2A causes steric hindrance to ribosomal peptide groups, and the nucleophilic attack of Pro-tRNA amino groups cannot be completed, thus 2A-ester bonds cannot be formed, and therefore cleavage can occur between the 19 th amino acid and the 20 th amino acid, an upstream protein fused with the tail of the 2A polypeptide is released, a fusion protein with 2A is formed, and at the same time, the ribose can continue to translate the downstream protein, and a complete downstream protein with one proline at the N end is formed.

As an example, the T Cell Receptor (TCR) of the invention may have an amino acid sequence as shown in SEQ ID NO. 29, SEQ ID NO. 37, SEQ ID NO. 45 or SEQ ID NO. 53, respectively.

The nucleic acid molecules may encode respective Complementarity Determining Regions (CDRs) of the α and β chain variable regions of a T Cell Receptor (TCR), e.g., CDR1 α, CDR2 α, CDR3 α, CDR1 β, CDR2 β and CDR3 β as shown in SEQ ID NO 1-6, SEQ ID NO 7-12, SEQ ID NO 13-18 or SEQ ID NO 19-24, respectively. By way of example, the nucleic acid molecule may comprise the nucleotide sequence shown in SEQ ID NOS: 65-70 or a degenerate sequence thereof for encoding CDR1 alpha, CDR2 alpha, CDR3 alpha, CDR1 beta, CDR2 beta and CDR3 beta in TCR 1. By way of example, the nucleic acid molecule may comprise the nucleotide sequence shown in SEQ ID NOS: 71-76 or degenerate sequences thereof for encoding CDR1 alpha, CDR2 alpha, CDR3 alpha, CDR1 beta, CDR2 beta and CDR3 beta in TCR 2. By way of example, the nucleic acid molecule may comprise the nucleotide sequence shown in SEQ ID NOS 77-82 or a degenerate sequence thereof for encoding CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β in TCR 3. By way of example, the nucleic acid molecule may comprise the nucleotide sequence shown in SEQ ID NOS: 83-88 or a degenerate sequence thereof for encoding CDR1 alpha, CDR2 alpha, CDR3 alpha, CDR1 beta, CDR2 beta and CDR3 beta in TCR 4.

The nucleic acid molecules may encode T Cell Receptor (TCR) alpha and beta chain variable regions. As an example, the nucleic acid molecule may comprise the nucleic acid sequence shown as SEQ ID NO. 30, SEQ ID NO. 38, SEQ ID NO. 46 or SEQ ID NO. 54 or a degenerate sequence thereof for encoding the alpha chain of the T Cell Receptor (TCR) of the invention. As an example, the nucleic acid molecule may comprise the nucleic acid sequence shown as SEQ ID NO. 31, SEQ ID NO. 39, SEQ ID NO. 47 or SEQ ID NO. 55 or a degenerate sequence thereof for encoding the beta chain of the T Cell Receptor (TCR) of the invention. It will be appreciated that in some cases, the nucleic acid molecule may comprise both of the above-described nucleic acid sequences encoding tcra and tcrp, respectively, or degenerate sequences thereof. As an example, the nucleic acid molecule may comprise the nucleic acid sequence shown as SEQ ID NO. 32, SEQ ID NO. 40, SEQ ID NO. 48 or SEQ ID NO. 56 or a degenerate sequence thereof for encoding a T Cell Receptor (TCR) of the invention.

The nucleic acid molecule may also encode a constant region linked to an alpha and/or beta chain variable region, e.g., a constant region linked to an alpha variable region as shown in SEQ ID NO:57, and/or a constant region linked to a beta variable region as shown in SEQ ID NO: 58.

The nucleic acid molecule may also encode a signal peptide fused to an alpha and/or beta chain, for example, a signal peptide for an alpha chain as shown in SEQ ID NO 27, SEQ ID NO 35, SEQ ID NO 43, or SEQ ID NO 51, and/or a signal peptide for a beta chain as shown in SEQ ID NO 28, SEQ ID NO 36, SEQ ID NO 44, or SEQ ID NO 52.

Vector (Vector) refers to a self-replicating DNA molecule capable of transferring a DNA fragment (gene of interest) into a recipient cell in a genetically engineered recombinant DNA technology, wherein a piece of DNA is excised without affecting its replication, and can be used to replace or insert exogenous (gene of interest) DNA to bring the DNA of interest into a host cell. Commonly used vectors are plasmids, phages, viruses and the like. In the present invention, the vector may express the T Cell Receptor (TCR) of the present invention, or an α or β chain thereof, or a fusion protein thereof fused to a signal peptide, or the like. The vector may be a viral vector, for example, a retrovirus vector, a lentivirus vector, an adenovirus vector, or an adeno-associated virus expression vector, but is not limited thereto. Any vector that can be used to express the protein of the invention can be used in the present invention.

In a fourth aspect, the invention provides an effector cell. Effector cells (effector cells) generally refer to immune cells involved in clearing foreign antigens and performing effector functions in immune responses, such as plasma cells, cytotoxic T cells, NK cells, APSC multipotent cells, mast cells, etc.

In the present invention, the effector cell may comprise the T cell receptor of the first aspect of the invention, the nucleic acid molecule of the second aspect of the invention, or the vector of the third aspect of the invention.

As an example, the cells may be T cells, natural killer cells, human embryonic stem cells, lymphoprogenitor cells, and/or T cell precursor cells, and the T cells may be cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells, but are not limited thereto.

In a fifth aspect, the invention provides a method of killing a melanoma cell comprising contacting the T cell receptor of the first aspect of the invention or the effector cell of the fifth aspect of the invention with a melanoma cell.

In this context, the term "killing melanoma cells" refers to the effect of the T cell receptor or effector cells of the invention on being able to partially or fully eliminate the melanoma cells after contact with the melanoma cells. In the present invention, the melanoma cells may include in-vivo and ex-vivo melanoma cells, and the method may be used for therapeutic purposes, but also for non-therapeutic purposes such as scientific research purposes, etc. Furthermore, preferably, the melanoma cells are MART-1 positive, HLA:A0201 typed melanoma cells.

In a sixth aspect, the application provides a method of treating melanoma comprising administering to a patient suffering from melanoma a T cell receptor according to the first aspect of the application or an effector cell according to the fourth aspect of the application. Preferably, the method of the application for treating melanoma is particularly suitable for treating MART-1 positive, HLA:A0201-type melanoma.

In a seventh aspect, the application provides the use of a T cell receptor of the first aspect of the application, a nucleic acid molecule of the second aspect of the application, a vector of the third aspect of the application, or an effector cell of the fourth aspect of the application in the manufacture of a medicament for the treatment of melanoma. Preferably, the melanoma is MART-1 positive, HLA:A0201 typed melanoma.

As demonstrated in the examples section of the present application, the T cell receptor of the present application or effector cells carrying the T cell receptor, upon contact with melanoma cells, can bind to and kill the melanoma cells while secreting cytokines such as α -tumor necrosis factor, γ -interferon, etc. Thus, the T cell receptor or the effector cell carrying the T cell receptor can be used for effectively treating melanoma or preparing medicines for treating melanoma.

Examples will be provided below to better understand the present invention. It should be understood that the invention is not limited to the following specific embodiments; modifications and improvements may be made to the invention without departing from the spirit and scope of the invention, and remain within the scope of the invention.

In addition, the experimental methods used in the following examples are all conventional methods unless otherwise specified; the test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all set up in triplicate and the results averaged.

Examples

Example 1 Induction and functional detection of MART-1 (27-35) specific T cells

Collecting 50-100ml HLA-A 0201 typed healthy human peripheral blood, centrifuging at 200g to obtain lower blood cells, adding a corresponding volume of Ficoll separating liquid after being resuspended by PBS, sucking out a mononuclear cell layer by a suction pipe after density gradient centrifugation, transferring the mononuclear cell layer into a centrifuge tube, centrifuging after being resuspended by sterile PBS, repeatedly washing for one time, and removing redundant Ficoll separating liquid and platelets to obtain peripheral blood mononuclear cells.

Separation of CD14 Using magnetic beads ⁺ Monocytes (dendritic cell (DC) precursor cells) and CD8 ⁺ T cells. CD8 ⁺ T cell cryopreservation, CD14 ⁺ Monocytes were induced for 3 days by Granulocyte-macrophage colony stimulating factor (Granulocyte-macrophage Colony Stimulating Factor, GM-CSF) in combination with Interleukin-4 (IL-4), induced to differentiate into immature DC cells, and then added with cytokines that promote maturation of the DC cells to differentiate the DC cells into mature DC cells, and then added with MART-1 (27-35) (LAGIGILTV) antigen polypeptide and the DC cells were co-cultured to prepare mature DC cells loaded with MART-1 (27-35) antigen.

Resuscitation CD8 1 day in advance ⁺ T cells, according to DC: CD8 ⁺ T cells at a density of 1:6-1:8 were seeded into cell culture plates, cultured in T009 medium containing IL-21 for 3 days, and then exchanged with medium containing IL-2, IL-7 and IL-15 to induce MART-1 specific CD8 ⁺ T cell expansion. In the culturing process, cell liquid is changed every 3 days, IL-2, IL-7 and IL-15 are properly supplemented, and cell state is observed, and MART-1 specific CD8 is obtained by enlarging culture ⁺ The T-cell population of the cell,and to perform an Elispot for functional detection. For details of Elispot, see example 6 below.

FIG. 1 shows the results of MART-1 specific T cell function detection by Elispot. As shown in FIG. 1, compared to the control group (control group MART-1 specific T cells reacted with T2-loaded unrelated polypeptides), the experimental group MART-1 antigen polypeptide specific T cells can efficiently recognize T2-loaded polypeptides (target cells) and secrete IFN-gamma, demonstrating that this population of cells is functional.

Example 2 MART-1 specific T cell sorting

Cells positive for function verified by Elispot were obtained and counted, and the number of cells taken out for flow sorting was 1×10 ⁶ Tube, add 1ml PBS to resuspend, centrifuge at 4℃500g, 5min, carefully discard supernatant, add 200. Mu.l PBS to resuspend; MART-1 tetramer (10. Mu.l/ml) was added to the tube for staining, mixed well and reacted at 4℃for 30min; after the reaction time, 1ml of PBS was added for resuspension, centrifuged at 500g for 5min at 4℃and the supernatant carefully discarded, 200. Mu.l of PBS was added for resuspension, and the mixture was placed on ice for flow detection; after flow loading, specific T cells against MART-1 (27-35) antigenic peptides were selected for subsequent single cell sequencing.

FIG. 2 shows the results of sorting specific T cells by MART-1 tetramer flow cytometry. As shown, T cells stimulated with MART-1 (27-35) antigen polypeptide antigen can detect 4.84% of positive tumor-specific T cells with tetramers, with the black boxes shown as sorted populations of cells of interest.

Example 3, 10X Genomics-based high throughput single cell TCR V (D) J full length sequencing droplet microfluidic technology based on the GemCode platform of 10X Genomics gel beads (beads), the positive single cell suspension obtained in example 2, a reverse transcription reaction system, and oil were passed together through an extremely fine channel to yield tiny droplets of water-in-oil. Separate oil droplets containing one Gel bead, one cell and a reverse transcription reaction system, namely GEMS (Gel Beads-In-Emulsions) can be isolated. The GEMS themselves are separate spaces, each containing a unique Barcoded bead. Each read carries a different tag sequence (Barcode) primer for labeling the transcriptome of each single cell, and the source cells of each read can be tracked after a later analysis. The hundreds of thousands of droplets generated can be collected in a single tube for reverse transcription reaction to generate a full-length cDNA product, enrichment of target fragments is carried out twice, TCR fragment amplification is carried out by using TCR specific primers (primers in a 10X Genomics high-throughput single-cell TCR V (D) J full-length sequencing kit), V (D) J fragments are enriched from the cDNA amplified product, and V (D) J library construction and sequencing are carried out.

FIG. 3 shows the paired TCR alpha and TCR beta sequence information for MART-1 specific T cells. Based on TCR cloning frequency, the inventors synthesized the 4 highest frequency paired TCR α and TCR β genes, i.e., cloototype 1-4, for subsequent functional validation.

EXAMPLE 4 construction of MART-1 specific TCR lentiviral shuttle plasmid vector

The problem of mismatch of the exogenous TCR transferred into T cells with the endogenous TCR can affect the expression of the exogenous TCR on T cells. Studies have reported that linking the variable region of the transferred TCR with the constant region of mice can effectively increase the expression efficiency of the exogenous TCR (Foley KC et al (2017) Mol Ther: oncolytics 5:105-115). Based on the 4 most frequent MART-1 specific TCRs obtained in sequencing, their αβ variable region sequences were fused to the mouse α constant region (SEQ ID NO: 57) and β2 constant region (SEQ ID NO: 58), respectively, to give TCR α and TCR β chains, and the TCR β and TCR α chains were linked with the P2A sequence (GSGATNFSLLKQAGDVEENPGP) of the internal self-cleaving porcine teschovirus, as shown in FIG. 4. The inventors placed tcrp before the P2A sequence because tcrp was longer than tcrp, placing the tcrp gene before the P2A sequence facilitated TCR expression and assembly. The inventors cloned the codon-optimized exogenous TCR gene fragments (SEQ ID NO: 32, SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO: 56) between BamHI and EcoRI cleavage sites of pRRLSIN.cPPT.PGK-GFP.WPRE lentiviral shuttle plasmid vector (purchased from Addgene) by means of restriction ligation (BamHI and EcoRI restriction enzyme cleavage) after gene synthesis, and replaced EGFP gene in the vector with TCR gene.

Example 5 preparation of MART-1 specific TCR transduced T cells (TCR-T)

TCR transduction of T cells is achieved by infecting human T cells with a lentiviral vector carrying the TCR gene of interest. Briefly, lentiviral shuttle plasmids carrying the TCR gene of interest and secondary lentiviral packaging plasmids (PsPAX 2, pmd2.G, available from Addgene) were transferred into 293T cells by Polyethylenimine (PEI) transfection, and replication-defective lentiviruses were released into the supernatant, which was collected 48-72 hours after transfection and concentrated by ultracentrifugation. The concentrated lentiviruses were subjected to titer assays and T cells of the 0201 type isolated from PBMC were infected at a MOI (Multiplicity of infection ). The transduced cells were expanded 24 hours after infection with media containing IL-2, IL-7, IL-15 cytokines. TCR expression positivity was detected 3-5 days post infection with anti-murine tcrp constant region antibodies and tetramer staining.

FIG. 5 shows the results of TCR-T build positive rate detection. The inventors named TCR1, TCR2, TCR3 and TCR4 in the order of higher cloning frequency, respectively, tested TCR-T construct positive rate using mouse TCR beta antibody after lentiviral infection, as shown in the results of FIG. 5, and subsequently tested the function of TCR-T constructed from these 4 TCR sequences.

Example 6 TCR transduced T cell function assay

To confirm that the TCR-T cells prepared in the present invention have antigen-specific activity, the present inventors conducted T cell function experiments under antigen stimulation.

1) Detection of TCR-T secretion of IFN-gamma cytokines by ELISPot

The enzyme-linked immunosorbent spot (ELISPot) assay is a detection method for quantitatively measuring the cytokine secretion frequency of single cells. The detection principle is that the cytokine produced by T lymphocyte after specific antigen stimulation is captured by the specific monoclonal antibody coated on the membrane. The captured cytokine was bound to a secondary antibody that labels alkaline phosphatase, developed via a 5-bromo-4-chloro-3-indole-phosphate/azocyclotetrazole (BCIP/NBT) substrate, and purple spots appearing at the bottom of the wells represent cytokine-producing cells.

The specific experimental procedure is as follows:

target cells were prepared on the first day: HLA-0201-typed T2 cells (purchased from ATCC) were counted, the required number of cells was removed, centrifuged at 400g for 5min at room temperature and resuspended in serum-free IMDM medium.

Target cells carry antigen polypeptides: the appropriate well plate and loading volume were determined based on the number of cells removed, and MART-1 (27-35) epitope polypeptide was formulated to 10. Mu.g/. Mu.l, added to the loading volume at 1000X, resuspended, and incubated at 37℃at 5% CO ₂ Incubator culture for 4 hours. Control groups loaded with no polypeptide but dimethyl sulfoxide (Dimethyl sulfoxide, DMSO) were also set.

Preparation of effector cells (TCR transduced T cells): effector cells were counted, desired cells were removed, centrifuged at 300g for 10min at room temperature, resuspended in 2% bovine serum T009 medium, and placed on ice.

Washing the plate: when the antigen load was left for 45min, the reaction well plate in the Human IFN-. Gamma.ELISPot.kit (Mabtech) was removed from the ultra clean bench, PBS was added, the wells were then left to stand for 30 seconds, the liquid was removed, after five repetitions of this action, 10% Fetal Bovine Serum (FBS) RPMI 1640 medium was added at 100. Mu.l/well, at 37℃with 5% CO ₂ Incubator incubate for 30min.

Sample adding: after the antigen loading time was over, T2 cells in the well plate were purged with 2% bovine serum T009 medium, centrifuged at 400g for 5min at room temperature, resuspended and counted with 2% bovine serum T009 medium, and brought to the corresponding concentration. After 50. Mu.l/well of effector cells were added to the reaction well plate, 50. Mu.l/well of target cell suspension, 20000 effector cells/well, 5000 target cells/well were added. Placing in 37 ℃ and 5% CO ₂ Incubator culture for 16-48h.

After the incubation time, the rest of the experimental procedure was performed according to the ELISpot kit instructions.

FIG. 6 shows the results of MART-1 specific T cell function detection by Elispot. As can be seen from the figure, TCR3 and TCR4 TCR-T cells effectively recognized T2 cells loaded with MART-1 (27-35) epitope polypeptide (experimental group in the figure) and secreted IFN- γ, demonstrating that TCR3 and TCR4, the 2 population of TCRT cells, are functional, compared to the control group (control group MART-1TCR-T cells reacted with DMSO-only T2 cells).

2) Detection of TCR-T cytokine secretion by flow assays

Intracellular flow assays are methods for detecting cytokine secretion from different cell subsets using anti-cytokine antibodies in combination with cell surface or intracellular specific subset markers.

The specific experimental procedure is as follows:

resting effector cells on the first day (TCR-T): the desired effector cells were removed, centrifuged, resuspended in cytokine-free medium, plated into corresponding well plates and allowed to rest for 24-48 hours.

The following day the target cells are loaded with antigen polypeptide: t2 cells were counted, the desired number of cells was removed, centrifuged at 400g for 5min and resuspended in 10% bovine serum IMDM medium. Determining proper pore plate and loading volume according to the number of the removed cells, preparing MART-1 epitope polypeptide into 10 mug/μl, adding into the loading volume according to 1000X, re-suspending, and mixing at 37deg.C with 5% CO ₂ Incubator culture overnight. Meanwhile, a control group loaded with no polypeptide and DMSO alone was set.

The third day effector cells were co-cultured with the target cells: after the antigen loading time was over, T2 cells in the well plate were purged with 2% bovine serum T009 medium, centrifuged at 400g for 5min at room temperature, resuspended in 2% bovine serum T009 medium, counted and adjusted to the corresponding concentration. Effector cells were counted, desired cells were removed, centrifuged at 300g for 10min at room temperature, resuspended in 2% bovine serum T009 medium, counted, and the concentration adjusted. After adding effector cells at 500. Mu.l/well to a 12-well plate, 500. Mu.l/well of target cell suspension was added, 7.5X10 s per well ⁵ Target cells, 3X 10 ⁶ The effector cells were placed in 5% CO at 37 ℃ ₂ Incubator incubates for 4-6h.

Flow-through dyeing: the co-incubated cells were collected from the well plate, centrifuged, resuspended in 200 μl PBS, surface stained with tetramer, fixed-plated using cell fixing/plating kit (BD), pipeted as necessary for staining, and stained with the corresponding cytokine antibody.

FIGS. 7 and 8 show the detection of TCR-T cytokine secretion by flow assays. As shown, TCR-T cells of TCR3 (FIG. 7) and TCR4 (FIG. 8) are able to efficiently recognize T2 cells loaded with MART-1 (27-35) epitope polypeptides and secrete IFN-gamma (interferon gamma) and TNF-alpha (tumor necrosis factor alpha) compared to the control group (control group MART-1TCR-T cells reacted with T2 cells plus DMSO only), demonstrating that the 2 population of TCR-T cells are functional. The results of the flow assay and cytokine secretion statistics are shown in fig. 7 and 8.

3) Detection of killing of target cells by TCR-T by flow analysis

The detection of cell killing effect by flow analysis is simpler, more convenient and faster than the traditional isotope labeling method. The principle is that the target cells are fluorescently marked by using hydroxy fluorescein diacetate succinimidyl ester (5, 6-carboxyfluorescein diacetate, succinimidyl ester, CFSE), and then the target cells are incubated with the effector cells, and then the activity rate of the target cells is detected, so that the killing function of the effector cells is reflected.

The specific experimental procedure is as follows:

target cells load antigen polypeptide on the first day: t2 cells were counted, the desired number of cells was removed, centrifuged at 400g for 5min and resuspended in 10% bovine serum IMDM medium. Determining appropriate well plate and loading volume according to the number of cells removed, preparing MART-1 epitope polypeptide into 10 μg/μl, adding 1000X into the loading volume, re-suspending, and mixing at 37deg.C with 5% CO ₂ Incubator culture overnight. Meanwhile, a control group loaded with no polypeptide and DMSO alone was set.

The following day effector cells were co-cultured with target cells: after the antigen loading time is over, CFSE fluorescent dye is added into target cells, the target cells are stained for 30 minutes, 10% bovine serum IMDM culture medium is added for cleaning, excessive dye is removed through centrifugation, then culture medium is added for resuspension cleaning, centrifugation is carried out at room temperature of 400g and 5min, and after centrifugation, 5% bovine serum AIM-V culture medium is used for resuspension, counting is carried out, and the concentration is adjusted to the corresponding concentration. Effector cells were counted, desired cells were removed, centrifuged at 300g for 10min at room temperature, resuspended in 2% bovine serum T009 medium, counted and concentration adjusted. After adding effector cells at 200. Mu.l/well to a 48-well plate, 200. Mu.l/well of target cell suspension was added, 5X 10 per well ⁴ The number of target cells to be treated is,1×10 ⁶ the effector cells were placed in 5% CO at 37 ℃ ₂ Incubator incubates for 4-6h.

Flow analysis: after incubation, the well plate was placed on ice, and Propidium Iodide (PI) dye was added before flow loading, and thoroughly dispersed and mixed. Flow-through assay fluorescence signals were collected in Fluorescein Isothiocyanate (FITC) and phycoerythrin-texas red (PE-texas red) channels.

Figure 9 shows the statistical results of detection of TCR-cell killing of target cells by flow-through analysis. As shown, these 4 MART-1TCR-T cells can efficiently recognize T2 cells loaded with MART-1 (27-35) epitope polypeptides and have specific killing effects compared to the control group (control group MART-1TCR-T cells reacted with T2 cells plus DMSO only).

The above is only a preferred embodiment of the present invention, but is not limited thereto. Any modification, equivalent replacement, improvement, etc. of the present invention within the spirit and principle of the present invention should be included in the protection scope of the present invention.

The sequence is briefly described:

sequence list:

SED ID NO 1: TCR1 CDR1 alpha amino acid sequence: NSASQS

SED ID NO 2: TCR1 CDR2 alpha amino acid sequence: vYSSGN

SED ID NO 3: TCR1 CDR3 alpha amino acid sequence: VADSGGGADGLT

SED ID NO 4: TCR1 CDR1 β amino acid sequence: SGHDN

SED ID NO 5: TCR1 CDR2 β amino acid sequence: fVKESK

SED ID NO 6: TCR1 CDR3 β amino acid sequence: ASSHQGLGTEAF

SED ID NO 7: TCR2 CDR1 alpha amino acid sequence: SIFNT

SED ID NO 8: TCR2 CDR2 alpha amino acid sequence: LYKAGEL

SED ID NO 9: TCR2 CDR3 alpha amino acid sequence: AGKNTDKLI

SED ID NO 10: TCR2 CDR1 β amino acid sequence: MDHEN

SED ID NO 11: TCR2 CDR2 β amino acid sequence: SYDVKM

SED ID NO 12: TCR2 CDR3 β amino acid sequence: ASSFPLLGGYT

SED ID NO 13: TCR3 CDR1 alpha amino acid sequence: DRVSQS (digital video signal system)

SED ID NO 14: TCR3 CDR2 alpha amino acid sequence: IYSNGD

SEQ ID NO. 15: TCR3 CDR3 a amino acid sequence: AVSGGGYSTLT

SED ID NO 16: TCR3 CDR1 β amino acid sequence: LGHDT

SED ID NO 17: TCR3 CDR2 β amino acid sequence: YNNKEL

SED ID NO 18: TCR3 CDR3 β amino acid sequence: ASSWGTDTQY

SED ID NO 19: TCR4 CDR1 alpha amino acid sequence: NSAFQY

SED ID NO 20: TCR4 CDR2 alpha amino acid sequence: tysgn

SED ID NO. 21: TCR4 CDR3 alpha amino acid sequence: AMSFMSSGSARQLT

SED ID NO. 22: TCR4 CDR1 β amino acid sequence: MDHEN

SED ID NO 23: TCR4 CDR2 β amino acid sequence: SYDVKM

SED ID NO 24: TCR4 CDR3 β amino acid sequence: ASTFTSSYNSPLH

SEQ ID NO. 25: TCR1 alpha variable region amino acid sequence

RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQ LNRASQYISLLIRDSKLSDSATYLCVADSGGGADGLTFGKGTHLIIQP

SEQ ID NO. 26: TCR 1. Beta. Variable region amino acid sequence

EAGVTQFPSHSVIEKGQTVTLRCDPISGHDNLYWYRRVMGKEIKFLLHFVKESKQDESGMPNN RFLAERTGGTYSTLKVQPAELEDSGVYFCASSHQGLGTEAFFGQGTRLTVV

SEQ ID NO. 27: signal peptide sequences for TCR1 alpha variable region amino acid sequences

MMISLRVLLVILWLQLSWVWSQ

SEQ ID NO. 28: signal peptide sequences for the amino acid sequence of the TCR1 beta variable region

MVSRLLSLVSLCLLGAKHI

SEQ ID NO. 29: amino acid sequence of TCR1 expression element

MVSRLLSLVSLCLLGAKHIEAGVTQFPSHSVIEKGQTVTLRCDPISGHDNLYWYRRVMGKEIKF LLHFVKESKQDESGMPNNRFLAERTGGTYSTLKVQPAELEDSGVYFCASSHQGLGTEAFFGQG TRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVS TDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAE AWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNSRAKRSGSGA TNFSLLKQAGDVEENPGPMMISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNC TYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYL CVADSGGGADGLTFGKGTHLIIQPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMES GTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDM NLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS

SEQ ID NO. 30: TCR1 alpha variable region nucleic acid sequences

AGGAAGGAGGTGGAGCAGGACCCCGGACCTTTCAATGTGCCAGAGGGCGCCACCGTGGCC TTTAACTGCACATACTCCAATTCTGCCAGCCAGTCCTTCTTTTGGTATCGGCAGGATTGTAG AAAGGAGCCCAAGCTGCTGATGAGCGTGTACTCCTCTGGAAACGAGGACGGCCGGTTCAC CGCACAGCTGAATAGAGCCTCCCAGTACATCTCTCTGCTGATCAGGGACAGCAAGCTGTCT GATAGCGCCACATATCTGTGCGTGGCAGATTCCGGAGGAGGAGCAGACGGCCTGACCTTTG GCAAGGGCACACACCTGATCATCCAGCCC

SEQ ID NO. 31: TCR 1. Beta. Variable region nucleic acid sequences

GAGGCAGGAGTGACCCAGTTCCCATCCCACTCTGTGATCGAGAAGGGCCAGACCGTGACA CTGAGATGTGATCCCATCTCCGGCCACGACAACCTGTACTGGTATCGGAGAGTGATGGGCA AGGAGATCAAGTTCCTGCTGCACTTTGTGAAGGAGTCTAAGCAGGATGAGAGCGGCATGCC TAACAATAGGTTCCTGGCAGAGAGGACCGGAGGAACATACTCTACCCTGAAGGTGCAGCC AGCAGAGCTGGAGGACAGCGGCGTGTATTTTTGCGCAAGCTCCCACCAGGGACTGGGAAC CGAGGCCTTCTTTGGCCAGGGCACAAGGCTGACCGTGGTG

SEQ ID NO. 32: construction of TCR1 into nucleic acid sequences contained in lentiviral vectors

ATGGTGAGCCGGCTGCTGAGCCTGGTGTCCCTGTGCCTGCTGGGAGCAAAGCACATCGAGG CAGGAGTGACCCAGTTCCCATCCCACTCTGTGATCGAGAAGGGCCAGACCGTGACACTGAG ATGTGATCCCATCTCCGGCCACGACAACCTGTACTGGTATCGGAGAGTGATGGGCAAGGAG ATCAAGTTCCTGCTGCACTTTGTGAAGGAGTCTAAGCAGGATGAGAGCGGCATGCCTAACA ATAGGTTCCTGGCAGAGAGGACCGGAGGAACATACTCTACCCTGAAGGTGCAGCCAGCAG AGCTGGAGGACAGCGGCGTGTATTTTTGCGCAAGCTCCCACCAGGGACTGGGAACCGAGG CCTTCTTTGGCCAGGGCACAAGGCTGACCGTGGTGGAGGATCTGCGCAACGTGACACCCCC TAAGGTGTCCCTGTTCGAGCCATCTAAGGCCGAGATCGCCAATAAGCAGAAGGCCACCCTG GTGTGCCTGGCAAGGGGCTTCTTTCCCGATCACGTGGAGCTGAGCTGGTGGGTGAACGGCA AGGAGGTGCACTCTGGCGTGAGCACCGACCCTCAGGCCTACAAGGAGTCCAATTACTCTTA TTGCCTGTCTAGCAGGCTGCGCGTGTCCGCCACATTTTGGCACAACCCCAGGAATCACTTCC GCTGTCAGGTGCAGTTTCACGGCCTGAGCGAGGAGGATAAGTGGCCTGAGGGCTCCCCAA AGCCAGTGACCCAGAATATCTCTGCCGAGGCATGGGGAAGAGCAGACTGCGGAATCACAA GCGCCTCCTATCACCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAA GGCCACACTGTATGCCGTGCTGGTGTCCGGCCTGGTGCTGATGGCCATGGTGAAGAAGAAG AACTCTAGGGCAAAGAGATCTGGCAGCGGAGCAACCAATTTCAGCCTGCTGAAGCAGGCA GGCGACGTGGAGGAGAACCCTGGACCAATGATGATCTCCCTGAGGGTGCTGCTGGTCATCC TGTGGCTGCAGCTGTCTTGGGTGTGGAGCCAGAGGAAGGAGGTGGAGCAGGACCCCGGAC CTTTCAATGTGCCAGAGGGCGCCACCGTGGCCTTTAACTGCACATACTCCAATTCTGCCAGC CAGTCCTTCTTTTGGTATCGGCAGGATTGTAGAAAGGAGCCCAAGCTGCTGATGAGCGTGT ACTCCTCTGGAAACGAGGACGGCCGGTTCACCGCACAGCTGAATAGAGCCTCCCAGTACAT CTCTCTGCTGATCAGGGACAGCAAGCTGTCTGATAGCGCCACATATCTGTGCGTGGCAGAT TCCGGAGGAGGAGCAGACGGCCTGACCTTTGGCAAGGGCACACACCTGATCATCCAGCCC GACATCCAGAACCCAGAGCCCGCCGTGTATCAGCTGAAGGACCCTCGCAGCCAGGATAGC ACCCTGTGCCTGTTCACAGACTTTGATAGCCAGATCAATGTGCCTAAGACCATGGAGTCCG GCACATTCATCACCGACAAGACAGTGCTGGATATGAAGGCCATGGACTCCAAGTCTAACGG CGCCATCGCCTGGAGCAATCAGACCTCCTTCACATGCCAGGATATCTTTAAGGAGACCAAC GCCACATACCCTAGCTCCGACGTGCCATGTGATGCCACCCTGACAGAGAAGTCCTTCGAGA CCGACATGAACCTGAATTTTCAGAACCTGTCTGTGATGGGCCTGAGAATCCTGCTGCTGAA GGTGGCCGGCTTTAATCTGCTGATGACACTGAGACTGTGGTCTAGCTGA

SEQ ID NO. 33: TCR2 alpha variable region amino acid sequence

NQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGIT RKDSFLNISASIPSDVGIYFCAGKNTDKLIFGTGTRLQVFP

SEQ ID NO. 34: TCR2 beta variable region amino acid sequences

DVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPE GYSVSREKKERFSLILESASTNQTSMYLCASSFPLLGGYTFGSGTRLTVV

SEQ ID NO. 35: signal peptide sequences for TCR2 alpha variable region amino acid sequences

MLLEHLLIILWMQLTWVSGQQL

SEQ ID NO. 36: signal peptide sequences for TCR2 beta variable region amino acid sequences

MGIRLLCRVAFCFLAVGLV

SEQ ID NO. 37: amino acid sequence of TCR2 expression element

MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLG LRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSFPLLGGYTFGSGT RLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVST DPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEA WGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNSRAKRSGSGAT NFSLLKQAGDVEENPGPMLLEHLLIILWMQLTWVSGQQLNQSPQSMFIQEGEDVSMNCTSSSIF NTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGKN TDKLIFGTGTRLQVFPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTV LDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLS VMGLRILLLKVAGFNLLMTLRLWSS

SEQ ID NO. 38: TCR2 alpha variable region nucleic acid sequences

AATCAGTCTCCCCAGAGCATGTTCATCCAGGAGGGCGAGGACGTGAGCATGAACTGTACCA GCTCCTCTATCTTTAATACATGGCTGTGGTACAAGCAGGACCCCGGAGAGGGACCCGTGCT GCTGATCGCCCTGTATAAGGCAGGAGAGCTGACAAGCAACGGCCGGCTGACCGCACAGTT CGGAATCACAAGAAAGGATTCCTTTCTGAATATCAGCGCCTCCATCCCCTCTGACGTGGGC ATCTACTTCTGCGCCGGCAAGAACACCGATAAGCTGATCTTCGGCACCGGCACACGGCTGC AGGTGTTTCCC

SEQ ID NO. 39: TCR 2. Beta. Variable region nucleic acid sequences

GACGTGAAGGTGACCCAGAGCTCCCGGTACCTGGTGAAGAGAACAGGCGAGAAGGTGTTC CTGGAGTGCGTGCAGGACATGGATCACGAGAACATGTTTTGGTATAGGCAGGACCCCGGA CTGGGACTGAGACTGATCTACTTCTCCTATGATGTGAAGATGAAGGAGAAGGGCGACATCC CAGAGGGCTACTCCGTGTCTAGGGAGAAGAAGGAGCGGTTCAGCCTGATCCTGGAGAGCG CCTCCACCAATCAGACAAGCATGTACCTGTGCGCCTCTAGCTTCCCACTGCTGGGCGGCTAT ACCTTTGGCAGCGGCACCAGGCTGACAGTGGTG

SEQ ID NO. 40: TCR2 construction into a lentiviral vector comprising a nucleic acid sequence:

ATGGGCATCCGGCTGCTGTGCAGAGTGGCCTTCTGTTTTCTGGCCGTGGGCCTGGTGGACGT GAAGGTGACCCAGAGCTCCCGGTACCTGGTGAAGAGAACAGGCGAGAAGGTGTTCCTGGA GTGCGTGCAGGACATGGATCACGAGAACATGTTTTGGTATAGGCAGGACCCCGGACTGGG ACTGAGACTGATCTACTTCTCCTATGATGTGAAGATGAAGGAGAAGGGCGACATCCCAGAG GGCTACTCCGTGTCTAGGGAGAAGAAGGAGCGGTTCAGCCTGATCCTGGAGAGCGCCTCCA CCAATCAGACAAGCATGTACCTGTGCGCCTCTAGCTTCCCACTGCTGGGCGGCTATACCTTT GGCAGCGGCACCAGGCTGACAGTGGTGGAGGACCTGCGCAACGTGACACCCCCTAAGGTG TCTCTGTTCGAGCCCAGCAAGGCCGAGATCGCCAATAAGCAGAAGGCCACCCTGGTGTGCC TGGCAAGGGGCTTCTTTCCTGACCACGTGGAGCTGTCCTGGTGGGTGAACGGCAAGGAGGT GCACTCCGGCGTGTCTACCGATCCACAGGCCTACAAGGAGAGCAATTACTCCTATTGCCTG TCCTCTCGGCTGAGAGTGAGCGCCACATTTTGGCACAACCCAAGGAATCACTTCCGCTGTC AGGTGCAGTTTCACGGCCTGTCCGAGGAGGACAAGTGGCCAGAGGGCTCTCCAAAGCCAG TGACCCAGAACATCAGCGCCGAGGCATGGGGAAGGGCAGATTGCGGCATCACATCTGCCA GCTATCACCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCAC ACTGTATGCCGTGCTGGTGTCCGGCCTGGTGCTGATGGCCATGGTGAAGAAGAAGAACTCT AGGGCAAAGCGGAGCGGCTCTGGAGCAACCAATTTCAGCCTGCTGAAGCAGGCAGGCGAC GTGGAGGAGAACCCTGGACCAATGCTGCTGGAGCACCTGCTGATCATCCTGTGGATGCAGC TGACCTGGGTGTCCGGCCAGCAGCTGAATCAGTCTCCCCAGAGCATGTTCATCCAGGAGGG CGAGGACGTGAGCATGAACTGTACCAGCTCCTCTATCTTTAATACATGGCTGTGGTACAAG CAGGACCCCGGAGAGGGACCCGTGCTGCTGATCGCCCTGTATAAGGCAGGAGAGCTGACA AGCAACGGCCGGCTGACCGCACAGTTCGGAATCACAAGAAAGGATTCCTTTCTGAATATCA GCGCCTCCATCCCCTCTGACGTGGGCATCTACTTCTGCGCCGGCAAGAACACCGATAAGCT GATCTTCGGCACCGGCACACGGCTGCAGGTGTTTCCCGATATCCAGAATCCCGAGCCTGCC GTGTATCAGCTGAAGGACCCTAGATCCCAGGATAGCACCCTGTGCCTGTTCACCGACTTTG ATAGCCAGATCAACGTGCCTAAGACCATGGAGTCCGGCACCTTTATCACAGACAAGACCGT GCTGGATATGAAGGCCATGGACTCTAAGAGCAACGGCGCCATCGCCTGGAGCAATCAGAC ATCCTTCACCTGCCAGGACATCTTTAAGGAGACAAATGCCACCTACCCTAGCTCCGACGTG CCATGTGATGCCACCCTGACAGAGAAGTCCTTCGAGACCGATATGAACCTGAATTTTCAGA ACCTGTCTGTGATGGGCCTGAGAATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGAT GACACTGCGCCTGTGGTCTAGCTGA

SEQ ID NO. 41: amino acid sequence of TCR3 alpha variable region

QKEVEQNSGPLSVPEGAIASLNCTYSDRVSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQ LNKASQYVSLLIRDSQPSDSATYLCAVSGGGYSTLTFGKGTMLLVSP

SEQ ID NO. 42: TCR3 beta variable region amino acid sequences

DTAVSQTPKYLVTQMGNDKSIKCEQNLGHDTMYWYKQDSKKFLKIMFSYNNKELIINETVPNR FSPKSPDKAHLNLHINSLELGDSAVYFCASSWGTDTQYFGPGTRLTVL

SEQ ID NO. 43: signal peptide sequences for the amino acid sequence of the TCR3 alpha variable region

MMKSLRVLLVILWLQLSWVWSQ

SEQ ID NO. 44: signal peptide sequences for TCR3 beta variable region amino acid sequences

MGCRLLCCVVFCLLQAGPL

SEQ ID NO. 45: amino acid sequence of TCR3 expression element

MGCRLLCCVVFCLLQAGPLDTAVSQTPKYLVTQMGNDKSIKCEQNLGHDTMYWYKQDSKKF LKIMFSYNNKELIINETVPNRFSPKSPDKAHLNLHINSLELGDSAVYFCASSWGTDTQYFGPGTR LTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTD PQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAW GRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNSRAKRSGSGATNF SLLKQAGDVEENPGPMMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYS DRVSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCA VSGGGYSTLTFGKGTMLLVSPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFI TDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLN FQNLSVMGLRILLLKVAGFNLLMTLRLWSS

SEQ ID NO. 46: TCR3 alpha variable region nucleic acid sequences

ATGATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCTGGGTTTGGAG CCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGC CTCTCTCAACTGCACTTACAGTGACCGAGTTTCCCAGTCCTTCTTCTGGTACAGACAATATT CTGGGAAAAGCCCTGAGTTGATAATGTCCATATACTCCAATGGTGACAAAGAAGATGGAA GGTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCA GCCCAGTGATTCAGCCACCTACCTCTGTGCCGTGAGCGGTGGAGGATACAGCACCCTCACC TTTGGGAAGGGGACTATGCTTCTAGTCTCTCCA

SEQ ID NO. 47: TCR 3. Beta. Variable region nucleic acid sequences

ATGGGCTGCAGGCTCCTCTGCTGTGTGGTCTTCTGCCTCCTCCAAGCAGGTCCCTTGGACAC AGCTGTTTCCCAGACTCCAAAATACCTGGTCACACAGATGGGAAACGACAAGTCCATTAAA TGTGAACAAAATCTGGGCCATGATACTATGTATTGGTATAAACAGGACTCTAAGAAATTTC TGAAGATAATGTTTAGCTACAATAATAAGGAGCTCATTATAAATGAAACAGTTCCAAATCG CTTCTCACCTAAATCTCCAGACAAAGCTCACTTAAATCTTCACATCAATTCCCTGGAGCTTG GTGACTCTGCTGTGTATTTCTGTGCCAGCAGTTGGGGCACAGACACGCAGTATTTTGGCCCA GGCACCCGGCTGACAGTGCTC

SEQ ID NO. 48: construction of TCR3 into nucleic acid sequences contained in lentiviral vectors

ATGGGCTGCAGGCTGCTGTGCTGCGTGGTGTTCTGCCTGCTGCAGGCAGGACCACTGGATA CAGCCGTGAGCCAGACCCCCAAGTACCTGGTGACACAGATGGGCAACGACAAGTCCATCA AGTGCGAGCAGAATCTGGGCCACGATACCATGTACTGGTATAAGCAGGACTCCAAGAAGT TCCTGAAGATCATGTTCTCTTACAACAATAAGGAGCTGATCATCAACGAGACCGTGCCTAA TCGCTTCAGCCCAAAGTCCCCCGATAAGGCCCACCTGAACCTGCACATCAATAGCCTGGAG CTGGGCGATTCCGCCGTGTACTTCTGCGCCAGCTCCTGGGGCACCGACACACAGTATTTTG GACCAGGAACCAGGCTGACAGTGCTGGAGGACCTGCGCAACGTGACACCCCCTAAGGTGT CTCTGTTTGAGCCAAGCAAGGCCGAGATCGCCAATAAGCAGAAGGCCACCCTGGTGTGCCT GGCAAGGGGCTTCTTTCCCGATCACGTGGAGCTGAGCTGGTGGGTGAACGGCAAGGAGGT GCACTCTGGCGTGAGCACAGACCCTCAGGCCTACAAGGAGTCCAATTACTCTTATTGCCTG TCTAGCCGGCTGAGAGTGTCCGCCACCTTCTGGCACAACCCCCGGAATCACTTCAGATGTC AGGTGCAGTTTCACGGCCTGAGCGAGGAGGATAAGTGGCCTGAGGGCTCCCCAAAGCCCG TGACACAGAACATCTCTGCCGAGGCATGGGGAAGGGCAGACTGCGGAATCACCTCTGCCA GCTATCACCAGGGCGTGCTGAGCGCCACAATCCTGTACGAGATCCTGCTGGGCAAGGCCAC CCTGTATGCCGTGCTGGTGTCTGGCCTGGTGCTGATGGCCATGGTGAAGAAGAAGAACAGC AGGGCAAAGCGGAGCGGCTCTGGAGCCACCAATTTTTCCCTGCTGAAGCAGGCCGGCGAT GTGGAGGAGAATCCTGGCCCAATGATGAAGAGCCTGAGAGTGCTGCTGGTCATCCTGTGGC TGCAGCTGAGCTGGGTGTGGTCCCAGCAGAAGGAGGTGGAGCAGAACTCTGGACCTCTGA GCGTGCCAGAGGGAGCAATCGCCTCTCTGAATTGTACATACAGCGACCGGGTGAGCCAGTC CTTCTTTTGGTACAGACAGTATAGCGGCAAGTCCCCAGAGCTGATCATGTCTATCTACAGC AACGGCGACAAGGAGGATGGCCGGTTCACCGCCCAGCTGAATAAGGCCTCCCAGTACGTG AGCCTGCTGATCAGAGACAGCCAGCCCTCTGATAGCGCCACATACCTGTGCGCCGTGTCCG GAGGAGGATATTCTACCCTGACATTTGGCAAGGGCACCATGCTGCTGGTGTCCCCTGACAT CCAGAACCCCGAGCCTGCCGTGTACCAGCTGAAGGACCCAAGATCCCAGGATAGCACCCT GTGCCTGTTCACCGACTTTGATAGCCAGATCAATGTGCCCAAGACCATGGAGTCCGGCACC TTCATCACAGACAAGACCGTGCTGGATATGAAGGCCATGGACTCCAAGTCTAACGGCGCCA TCGCCTGGAGCAATCAGACATCCTTCACCTGCCAGGATATCTTTAAGGAGACAAACGCCAC CTATCCCTCCTCTGACGTGCCTTGTGATGCCACCCTGACAGAGAAGTCTTTCGAGACAGAC ATGAACCTGAATTTTCAGAACCTGTCCGTGATGGGCCTGAGAATCCTGCTGCTGAAGGTGG CCGGCTTTAATCTGCTGATGACCCTGCGCCTGTGGAGCTCCTGA

SEQ ID NO. 49: TCR4 alpha variable region amino acid sequence

QKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKEDGRFTA QVDKSSKYISLFIRDSQPSDSATYLCAMSFMSSGSARQLTFGSGTQLTVLP

SEQ ID NO. 50: TCR 4. Beta. Variable region amino acid sequence

DVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPE GYSVSREKKERFSLILESASTNQTSMYLCASTFTSSYNSPLHFGNGTRLTVT

SEQ ID NO. 51: signal peptide sequences for TCR4 alpha variable region amino acid sequences

MMKSLRVLLVILWLQLSWVWSQ

SEQ ID NO. 52: signal peptide sequences for TCR4 beta variable region amino acid sequences

MGIRLLCRVAFCFLAVGLV

SEQ ID NO. 53:amino acid sequence of TCR4 expression element

MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLG LRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASTFTSSYNSPLHFGN GTRLTVTEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGV STDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAE AWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNSRAKRSGSGA TNFSLLKQAGDVEENPGPMMKSLRVLLVILWLQLSWVWSQQKEVEQDPGPLSVPEGAIVSLNC TYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSAT YLCAMSFMSSGSARQLTFGSGTQLTVLPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPK TMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSF ETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS

SEQ ID NO. 54: TCR4 alpha variable region nucleic acid sequences

ATGATGAAATCCTTGAGAGTTTTACTGGTGATCCTGTGGCTTCAGTTAAGCTGGGTTTGGAG CCAACAGAAGGAGGTGGAGCAGGATCCTGGACCACTCAGTGTTCCAGAGGGAGCCATTGT TTCTCTCAACTGCACTTACAGCAACAGTGCTTTTCAATACTTCATGTGGTACAGACAGTATT CCAGAAAAGGCCCTGAGTTGCTGATGTACACATACTCCAGTGGTAACAAAGAAGATGGAA GGTTTACAGCACAGGTCGATAAATCCAGCAAGTATATCTCCTTGTTCATCAGAGACTCACA GCCCAGTGATTCAGCCACCTACCTCTGTGCAATGAGCTTCATGTCTTCTGGTTCTGCAAGGC AACTGACCTTTGGATCTGGGACACAATTGACTGTTTTACCT

SEQ ID NO. 55: TCR 4. Beta. Variable region nucleic acid sequences

ATGGGAATCAGGCTCCTCTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCTCGTAGATGT GAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGGAGAGAAAGTTTTTCTGGA ATGTGTCCAGGATATGGACCATGAAAATATGTTCTGGTATCGACAAGACCCAGGTCTGGGG CTACGGCTGATCTATTTCTCATATGATGTTAAAATGAAAGAAAAAGGAGATATTCCTGAGG GGTACAGTGTCTCTAGAGAGAAGAAGGAGCGCTTCTCCCTGATTCTGGAGTCCGCCAGCAC CAACCAGACATCTATGTACCTCTGTGCCAGCACCTTTACCAGCTCCTATAATTCACCCCTCC ACTTTGGGAACGGGACCAGGCTCACTGTGACA

SEQ ID NO. 56: construction of TCR4 into nucleic acid sequences contained in lentiviral vectors

ATGGGCATCCGGCTGCTGTGCAGAGTGGCCTTCTGTTTTCTGGCCGTGGGCCTGGTGGATGT GAAGGTGACACAGAGCTCCAGGTACCTGGTGAAGCGCACCGGCGAGAAGGTGTTCCTGGA GTGCGTGCAGGACATGGATCACGAGAACATGTTTTGGTATAGGCAGGACCCTGGACTGGG ACTGAGGCTGATCTACTTCAGCTATGATGTGAAGATGAAGGAGAAGGGCGACATCCCAGA GGGCTACTCCGTGTCTCGGGAGAAGAAGGAGAGATTTTCCCTGATCCTGGAGAGCGCCTCC ACCAATCAGACAAGCATGTACCTGTGCGCCTCCACCTTCACATCTAGCTATAACAGCCCTCT GCACTTTGGCAATGGCACACGGCTGACCGTGACAGAGGATCTGAGAAACGTGACCCCCCCT AAGGTGTCCCTGTTCGAGCCCTCTAAGGCCGAGATCGCCAATAAGCAGAAGGCCACCCTGG TGTGCCTGGCAAGGGGCTTCTTTCCTGATCACGTGGAGCTGTCCTGGTGGGTGAACGGCAA GGAGGTGCACAGCGGCGTGTCCACAGACCCACAGGCCTACAAGGAGTCTAATTACAGCTA TTGCCTGTCCTCTCGGCTGAGAGTGAGCGCCACCTTTTGGCACAACCCAAGGAATCACTTCC GCTGTCAGGTGCAGTTTCACGGCCTGTCTGAGGAGGATAAGTGGCCAGAGGGCAGCCCAA AGCCAGTGACACAGAACATCTCCGCCGAGGCATGGGGAAGGGCAGACTGCGGCATCACCT CTGCCAGCTATCACCAGGGCGTGCTGTCCGCCACAATCCTGTACGAGATCCTGCTGGGCAA GGCCACCCTGTATGCCGTGCTGGTGTCCGGCCTGGTGCTGATGGCCATGGTGAAGAAGAAG AACTCTAGGGCAAAGCGGAGCGGCTCTGGAGCAACCAATTTCAGCCTGCTGAAGCAGGCA GGCGATGTGGAGGAGAACCCTGGACCAATGATGAAGTCCCTGAGAGTGCTGCTGGTCATCC TGTGGCTGCAGCTGAGCTGGGTGTGGTCCCAGCAGAAGGAGGTGGAGCAGGACCCCGGAC CTCTGAGCGTGCCAGAGGGAGCAATCGTGTCCCTGAACTGTACCTACAGCAATTCCGCCTT CCAGTACTTCATGTGGTACCGGCAGTATAGCAGAAAGGGCCCTGAGCTGCTGATGTACACA TATAGCTCCGGCAATAAGGAGGATGGCCGGTTCACCGCCCAGGTGGACAAGTCTAGCAAG TACATCTCTCTGTTTATCAGAGACAGCCAGCCATCTGATAGCGCCACATATCTGTGCGCCAT GTCCTTCATGTCCTCTGGCTCTGCCAGGCAGCTGACCTTTGGCAGCGGAACCCAGCTGACA GTGCTGCCAGACATCCAGAACCCAGAGCCCGCCGTGTACCAGCTGAAGGACCCTCGCTCCC AGGATAGCACCCTGTGCCTGTTCACCGACTTTGATTCTCAGATCAATGTGCCCAAGACCAT GGAGAGCGGCACCTTCATCACAGACAAGACCGTGCTGGATATGAAGGCCATGGACTCCAA GTCTAACGGCGCCATCGCCTGGTCTAATCAGACAAGCTTCACCTGCCAGGATATCTTTAAG GAGACAAACGCCACCTACCCTAGCTCCGACGTGCCATGTGATGCCACCCTGACAGAGAAGT CCTTCGAGACAGACATGAACCTGAATTTTCAGAACCTGTCTGTGATGGGCCTGAGAATCCT GCTGCTGAAGGTGGCCGGCTTTAATCTGCTGATGACCCTGCGCCTGTGGTCTAGCTGA

SEQ ID NO. 57: constant region amino acid sequence linked to TCR1 alpha variable region

DIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIA WSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNL LMTLRLWSS

SEQ ID NO. 58: constant region amino acid sequence linked to TCR 1. Beta. Variable region

EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAY KESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRAD CGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS

SEQ ID NO 59: CDR1 a nucleotide sequence of TCR 1: AATTCTGCCAGCCAGTCC

SEQ ID NO. 60: CDR2 a nucleotide sequence of TCR 1: GTGTACTCCTCTGGAAAC

SEQ ID NO. 61: CDR3 a nucleotide sequence of TCR 1:

GTGGCAGATTCCGGAGGAGGAGCAGACGGCCTGACC

SEQ ID NO. 62: CDR1 β nucleotide sequence of TCR 1: TCCGGCCACGACAAC

SEQ ID NO. 63: CDR2 β nucleotide sequence of TCR 1: TTTGTGAAGGAGTCTAAG

SEQ ID NO. 64: CDR3 β nucleotide sequence of TCR 1:

GCAAGCTCCCACCAGGGACTGGGAACCGAGGCCTTC

SEQ ID NO. 65: CDR1 a nucleotide sequence of TCR 2: TCTATCTTTAATACA

SEQ ID NO. 66: CDR2 a nucleotide sequence of TCR 2: CTGTATAAGGCAGGAGAGCTG

SEQ ID NO. 67: CDR3 a nucleotide sequence of TCR 2: GCCGGCAAGAACACCGATAAGCTGATC

SEQ ID NO. 68: CDR1 β nucleotide sequence of TCR 2: ATGGATCACGAGAAC

SEQ ID NO. 69: CDR2 β nucleotide sequence of TCR 2: TCCTATGATGTGAAGATG

SEQ ID NO. 70: CDR3 β nucleotide sequence of TCR 2: GCCTCTAGCTTCCCACTGCTGGGCGGCTATACC

SEQ ID NO. 71: CDR1 a nucleotide sequence of TCR 3: GACCGGGTGAGCCAGTCC

SEQ ID NO. 72: CDR2 a nucleotide sequence of TCR 3: ATCTACAGCAACGGCGAC

SEQ ID NO. 73: CDR3 a nucleotide sequence of TCR 3: GCCGTGTCCGGAGGAGGATATTCTACCCTGACA

SEQ ID NO. 74: CDR1 β nucleotide sequence of TCR 3: CTGGGCCACGATACC

SEQ ID NO. 75: CDR2 β nucleotide sequence of TCR 3: TACAACAATAAGGAGCTG

SEQ ID NO. 76: CDR3 β nucleotide sequence of TCR 3: GCCAGCTCCTGGGGCACCGACACACAGTAT

SEQ ID NO. 77: CDR1 a nucleotide sequence of TCR 4: AATTCCGCCTTCCAGTAC

SEQ ID NO. 78: CDR2 a nucleotide sequence of TCR 4: ACATATAGCTCCGGCAAT

SEQ ID NO. 79: CDR3 a nucleotide sequence of TCR 4:

GCCATGTCCTTCATGTCCTCTGGCTCTGCCAGGCAGCTGACC

SEQ ID NO. 80: CDR1 β nucleotide sequence of TCR 4: ATGGATCACGAGAAC

SEQ ID NO. 81: CDR2 β nucleotide sequence of TCR 4: AGCTATGATGTGAAGATG

SEQ ID NO. 82: CDR3 β nucleotide sequence of TCR 4:

GCCTCCACCTTCACATCTAGCTATAACAGCCCTCTGCAC。

sequence listing

<110> Shenzhen Hua institute of great life science

<120> MART-1 (27-35) epitope specific T cell receptor and uses thereof

<141> 2022-03-01

<160> 82

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 1

Asn Ser Ala Ser Gln Ser

1 5

<210> 2

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 2

Val Tyr Ser Ser Gly Asn

1 5

<210> 3

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 3

Val Ala Asp Ser Gly Gly Gly Ala Asp Gly Leu Thr

1 5 10

<210> 4

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 4

Ser Gly His Asp Asn

1 5

<210> 5

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 5

Phe Val Lys Glu Ser Lys

1 5

<210> 6

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 6

Ala Ser Ser His Gln Gly Leu Gly Thr Glu Ala Phe

1 5 10

<210> 7

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 7

Ser Ile Phe Asn Thr

1 5

<210> 8

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 8

Leu Tyr Lys Ala Gly Glu Leu

1 5

<210> 9

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 9

Ala Gly Lys Asn Thr Asp Lys Leu Ile

1 5

<210> 10

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 10

Met Asp His Glu Asn

1 5

<210> 11

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 11

Ser Tyr Asp Val Lys Met

1 5

<210> 12

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 12

Ala Ser Ser Phe Pro Leu Leu Gly Gly Tyr Thr

1 5 10

<210> 13

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 13

Asp Arg Val Ser Gln Ser

1 5

<210> 14

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 14

Ile Tyr Ser Asn Gly Asp

1 5

<210> 15

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 15

Ala Val Ser Gly Gly Gly Tyr Ser Thr Leu Thr

1 5 10

<210> 16

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 16

Leu Gly His Asp Thr

1 5

<210> 17

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 17

Tyr Asn Asn Lys Glu Leu

1 5

<210> 18

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 18

Ala Ser Ser Trp Gly Thr Asp Thr Gln Tyr

1 5 10

<210> 19

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 19

Asn Ser Ala Phe Gln Tyr

1 5

<210> 20

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 20

Thr Tyr Ser Ser Gly Asn

1 5

<210> 21

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 21

Ala Met Ser Phe Met Ser Ser Gly Ser Ala Arg Gln Leu Thr

1 5 10

<210> 22

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 22

Met Asp His Glu Asn

1 5

<210> 23

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 23

Ser Tyr Asp Val Lys Met

1 5

<210> 24

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 24

Ala Ser Thr Phe Thr Ser Ser Tyr Asn Ser Pro Leu His

1 5 10

<210> 25

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 25

Arg Lys Glu Val Glu Gln Asp Pro Gly Pro Phe Asn Val Pro Glu Gly

1 5 10 15

Ala Thr Val Ala Phe Asn Cys Thr Tyr Ser Asn Ser Ala Ser Gln Ser

20 25 30

Phe Phe Trp Tyr Arg Gln Asp Cys Arg Lys Glu Pro Lys Leu Leu Met

35 40 45

Ser Val Tyr Ser Ser Gly Asn Glu Asp Gly Arg Phe Thr Ala Gln Leu

50 55 60

Asn Arg Ala Ser Gln Tyr Ile Ser Leu Leu Ile Arg Asp Ser Lys Leu

65 70 75 80

Ser Asp Ser Ala Thr Tyr Leu Cys Val Ala Asp Ser Gly Gly Gly Ala

85 90 95

Asp Gly Leu Thr Phe Gly Lys Gly Thr His Leu Ile Ile Gln Pro

100 105 110

<210> 26

<211> 114

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 26

Glu Ala Gly Val Thr Gln Phe Pro Ser His Ser Val Ile Glu Lys Gly

1 5 10 15

Gln Thr Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Asp Asn Leu

20 25 30

Tyr Trp Tyr Arg Arg Val Met Gly Lys Glu Ile Lys Phe Leu Leu His

35 40 45

Phe Val Lys Glu Ser Lys Gln Asp Glu Ser Gly Met Pro Asn Asn Arg

50 55 60

Phe Leu Ala Glu Arg Thr Gly Gly Thr Tyr Ser Thr Leu Lys Val Gln

65 70 75 80

Pro Ala Glu Leu Glu Asp Ser Gly Val Tyr Phe Cys Ala Ser Ser His

85 90 95

Gln Gly Leu Gly Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr

100 105 110

Val Val

<210> 27

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 27

Met Met Ile Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu

1 5 10 15

Ser Trp Val Trp Ser Gln

20

<210> 28

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 28

Met Val Ser Arg Leu Leu Ser Leu Val Ser Leu Cys Leu Leu Gly Ala

1 5 10 15

Lys His Ile

<210> 29

<211> 603

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 29

Met Val Ser Arg Leu Leu Ser Leu Val Ser Leu Cys Leu Leu Gly Ala

1 5 10 15

Lys His Ile Glu Ala Gly Val Thr Gln Phe Pro Ser His Ser Val Ile

20 25 30

Glu Lys Gly Gln Thr Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His

35 40 45

Asp Asn Leu Tyr Trp Tyr Arg Arg Val Met Gly Lys Glu Ile Lys Phe

50 55 60

Leu Leu His Phe Val Lys Glu Ser Lys Gln Asp Glu Ser Gly Met Pro

65 70 75 80

Asn Asn Arg Phe Leu Ala Glu Arg Thr Gly Gly Thr Tyr Ser Thr Leu

85 90 95

Lys Val Gln Pro Ala Glu Leu Glu Asp Ser Gly Val Tyr Phe Cys Ala

100 105 110

Ser Ser His Gln Gly Leu Gly Thr Glu Ala Phe Phe Gly Gln Gly Thr

115 120 125

Arg Leu Thr Val Val Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val

130 135 140

Ser Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala

145 150 155 160

Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu

165 170 175

Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp

180 185 190

Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg

195 200 205

Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg

210 215 220

Cys Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu

225 230 235 240

Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly

245 250 255

Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu

260 265 270

Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr

275 280 285

Ala Val Leu Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys Lys

290 295 300

Asn Ser Arg Ala Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu

305 310 315 320

Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Met Ile

325 330 335

Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser Trp Val

340 345 350

Trp Ser Gln Arg Lys Glu Val Glu Gln Asp Pro Gly Pro Phe Asn Val

355 360 365

Pro Glu Gly Ala Thr Val Ala Phe Asn Cys Thr Tyr Ser Asn Ser Ala

370 375 380

Ser Gln Ser Phe Phe Trp Tyr Arg Gln Asp Cys Arg Lys Glu Pro Lys

385 390 395 400

Leu Leu Met Ser Val Tyr Ser Ser Gly Asn Glu Asp Gly Arg Phe Thr

405 410 415

Ala Gln Leu Asn Arg Ala Ser Gln Tyr Ile Ser Leu Leu Ile Arg Asp

420 425 430

Ser Lys Leu Ser Asp Ser Ala Thr Tyr Leu Cys Val Ala Asp Ser Gly

435 440 445

Gly Gly Ala Asp Gly Leu Thr Phe Gly Lys Gly Thr His Leu Ile Ile

450 455 460

Gln Pro Asp Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp

465 470 475 480

Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser

485 490 495

Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp

500 505 510

Lys Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala

515 520 525

Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys

530 535 540

Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr

545 550 555 560

Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn

565 570 575

Leu Ser Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe

580 585 590

Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

595 600

<210> 30

<211> 333

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 30

aggaaggagg tggagcagga ccccggacct ttcaatgtgc cagagggcgc caccgtggcc 60

tttaactgca catactccaa ttctgccagc cagtccttct tttggtatcg gcaggattgt 120

agaaaggagc ccaagctgct gatgagcgtg tactcctctg gaaacgagga cggccggttc 180

accgcacagc tgaatagagc ctcccagtac atctctctgc tgatcaggga cagcaagctg 240

tctgatagcg ccacatatct gtgcgtggca gattccggag gaggagcaga cggcctgacc 300

tttggcaagg gcacacacct gatcatccag ccc 333

<210> 31

<211> 342

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 31

gaggcaggag tgacccagtt cccatcccac tctgtgatcg agaagggcca gaccgtgaca 60

ctgagatgtg atcccatctc cggccacgac aacctgtact ggtatcggag agtgatgggc 120

aaggagatca agttcctgct gcactttgtg aaggagtcta agcaggatga gagcggcatg 180

cctaacaata ggttcctggc agagaggacc ggaggaacat actctaccct gaaggtgcag 240

ccagcagagc tggaggacag cggcgtgtat ttttgcgcaa gctcccacca gggactggga 300

accgaggcct tctttggcca gggcacaagg ctgaccgtgg tg 342

<210> 32

<211> 1812

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 32

atggtgagcc ggctgctgag cctggtgtcc ctgtgcctgc tgggagcaaa gcacatcgag 60

gcaggagtga cccagttccc atcccactct gtgatcgaga agggccagac cgtgacactg 120

agatgtgatc ccatctccgg ccacgacaac ctgtactggt atcggagagt gatgggcaag 180

gagatcaagt tcctgctgca ctttgtgaag gagtctaagc aggatgagag cggcatgcct 240

aacaataggt tcctggcaga gaggaccgga ggaacatact ctaccctgaa ggtgcagcca 300

gcagagctgg aggacagcgg cgtgtatttt tgcgcaagct cccaccaggg actgggaacc 360

gaggccttct ttggccaggg cacaaggctg accgtggtgg aggatctgcg caacgtgaca 420

ccccctaagg tgtccctgtt cgagccatct aaggccgaga tcgccaataa gcagaaggcc 480

accctggtgt gcctggcaag gggcttcttt cccgatcacg tggagctgag ctggtgggtg 540

aacggcaagg aggtgcactc tggcgtgagc accgaccctc aggcctacaa ggagtccaat 600

tactcttatt gcctgtctag caggctgcgc gtgtccgcca cattttggca caaccccagg 660

aatcacttcc gctgtcaggt gcagtttcac ggcctgagcg aggaggataa gtggcctgag 720

ggctccccaa agccagtgac ccagaatatc tctgccgagg catggggaag agcagactgc 780

ggaatcacaa gcgcctccta tcaccagggc gtgctgagcg ccaccatcct gtacgagatc 840

ctgctgggca aggccacact gtatgccgtg ctggtgtccg gcctggtgct gatggccatg 900

gtgaagaaga agaactctag ggcaaagaga tctggcagcg gagcaaccaa tttcagcctg 960

ctgaagcagg caggcgacgt ggaggagaac cctggaccaa tgatgatctc cctgagggtg 1020

ctgctggtca tcctgtggct gcagctgtct tgggtgtgga gccagaggaa ggaggtggag 1080

caggaccccg gacctttcaa tgtgccagag ggcgccaccg tggcctttaa ctgcacatac 1140

tccaattctg ccagccagtc cttcttttgg tatcggcagg attgtagaaa ggagcccaag 1200

ctgctgatga gcgtgtactc ctctggaaac gaggacggcc ggttcaccgc acagctgaat 1260

agagcctccc agtacatctc tctgctgatc agggacagca agctgtctga tagcgccaca 1320

tatctgtgcg tggcagattc cggaggagga gcagacggcc tgacctttgg caagggcaca 1380

cacctgatca tccagcccga catccagaac ccagagcccg ccgtgtatca gctgaaggac 1440

cctcgcagcc aggatagcac cctgtgcctg ttcacagact ttgatagcca gatcaatgtg 1500

cctaagacca tggagtccgg cacattcatc accgacaaga cagtgctgga tatgaaggcc 1560

atggactcca agtctaacgg cgccatcgcc tggagcaatc agacctcctt cacatgccag 1620

gatatcttta aggagaccaa cgccacatac cctagctccg acgtgccatg tgatgccacc 1680

ctgacagaga agtccttcga gaccgacatg aacctgaatt ttcagaacct gtctgtgatg 1740

ggcctgagaa tcctgctgct gaaggtggcc ggctttaatc tgctgatgac actgagactg 1800

tggtctagct ga 1812

<210> 33

<211> 105

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 33

Asn Gln Ser Pro Gln Ser Met Phe Ile Gln Glu Gly Glu Asp Val Ser

1 5 10 15

Met Asn Cys Thr Ser Ser Ser Ile Phe Asn Thr Trp Leu Trp Tyr Lys

20 25 30

Gln Asp Pro Gly Glu Gly Pro Val Leu Leu Ile Ala Leu Tyr Lys Ala

35 40 45

Gly Glu Leu Thr Ser Asn Gly Arg Leu Thr Ala Gln Phe Gly Ile Thr

50 55 60

Arg Lys Asp Ser Phe Leu Asn Ile Ser Ala Ser Ile Pro Ser Asp Val

65 70 75 80

Gly Ile Tyr Phe Cys Ala Gly Lys Asn Thr Asp Lys Leu Ile Phe Gly

85 90 95

Thr Gly Thr Arg Leu Gln Val Phe Pro

100 105

<210> 34

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 34

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His Glu Asn Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Phe Pro

85 90 95

Leu Leu Gly Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr Val Val

100 105 110

<210> 35

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 35

Met Leu Leu Glu His Leu Leu Ile Ile Leu Trp Met Gln Leu Thr Trp

1 5 10 15

Val Ser Gly Gln Gln Leu

20

<210> 36

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 36

Met Gly Ile Arg Leu Leu Cys Arg Val Ala Phe Cys Phe Leu Ala Val

1 5 10 15

Gly Leu Val

<210> 37

<211> 595

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 37

Met Gly Ile Arg Leu Leu Cys Arg Val Ala Phe Cys Phe Leu Ala Val

1 5 10 15

Gly Leu Val Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys

20 25 30

Arg Thr Gly Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His

35 40 45

Glu Asn Met Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu

50 55 60

Ile Tyr Phe Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro

65 70 75 80

Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile

85 90 95

Leu Glu Ser Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser

100 105 110

Ser Phe Pro Leu Leu Gly Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu

115 120 125

Thr Val Val Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu

130 135 140

Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu

145 150 155 160

Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp

165 170 175

Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln

180 185 190

Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg

195 200 205

Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln

210 215 220

Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser

225 230 235 240

Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala

245 250 255

Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu Ser Ala

260 265 270

Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val

275 280 285

Leu Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys Lys Asn Ser

290 295 300

Arg Ala Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys

305 310 315 320

Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Leu Leu Glu His

325 330 335

Leu Leu Ile Ile Leu Trp Met Gln Leu Thr Trp Val Ser Gly Gln Gln

340 345 350

Leu Asn Gln Ser Pro Gln Ser Met Phe Ile Gln Glu Gly Glu Asp Val

355 360 365

Ser Met Asn Cys Thr Ser Ser Ser Ile Phe Asn Thr Trp Leu Trp Tyr

370 375 380

Lys Gln Asp Pro Gly Glu Gly Pro Val Leu Leu Ile Ala Leu Tyr Lys

385 390 395 400

Ala Gly Glu Leu Thr Ser Asn Gly Arg Leu Thr Ala Gln Phe Gly Ile

405 410 415

Thr Arg Lys Asp Ser Phe Leu Asn Ile Ser Ala Ser Ile Pro Ser Asp

420 425 430

Val Gly Ile Tyr Phe Cys Ala Gly Lys Asn Thr Asp Lys Leu Ile Phe

435 440 445

Gly Thr Gly Thr Arg Leu Gln Val Phe Pro Asp Ile Gln Asn Pro Glu

450 455 460

Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu

465 470 475 480

Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn Val Pro Lys Thr Met

485 490 495

Glu Ser Gly Thr Phe Ile Thr Asp Lys Thr Val Leu Asp Met Lys Ala

500 505 510

Met Asp Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser

515 520 525

Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser

530 535 540

Ser Asp Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr

545 550 555 560

Asp Met Asn Leu Asn Phe Gln Asn Leu Ser Val Met Gly Leu Arg Ile

565 570 575

Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu

580 585 590

Trp Ser Ser

595

<210> 38

<211> 315

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 38

aatcagtctc cccagagcat gttcatccag gagggcgagg acgtgagcat gaactgtacc 60

agctcctcta tctttaatac atggctgtgg tacaagcagg accccggaga gggacccgtg 120

ctgctgatcg ccctgtataa ggcaggagag ctgacaagca acggccggct gaccgcacag 180

ttcggaatca caagaaagga ttcctttctg aatatcagcg cctccatccc ctctgacgtg 240

ggcatctact tctgcgccgg caagaacacc gataagctga tcttcggcac cggcacacgg 300

ctgcaggtgt ttccc 315

<210> 39

<211> 336

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 39

gacgtgaagg tgacccagag ctcccggtac ctggtgaaga gaacaggcga gaaggtgttc 60

ctggagtgcg tgcaggacat ggatcacgag aacatgtttt ggtataggca ggaccccgga 120

ctgggactga gactgatcta cttctcctat gatgtgaaga tgaaggagaa gggcgacatc 180

ccagagggct actccgtgtc tagggagaag aaggagcggt tcagcctgat cctggagagc 240

gcctccacca atcagacaag catgtacctg tgcgcctcta gcttcccact gctgggcggc 300

tatacctttg gcagcggcac caggctgaca gtggtg 336

<210> 40

<211> 1788

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 40

atgggcatcc ggctgctgtg cagagtggcc ttctgttttc tggccgtggg cctggtggac 60

gtgaaggtga cccagagctc ccggtacctg gtgaagagaa caggcgagaa ggtgttcctg 120

gagtgcgtgc aggacatgga tcacgagaac atgttttggt ataggcagga ccccggactg 180

ggactgagac tgatctactt ctcctatgat gtgaagatga aggagaaggg cgacatccca 240

gagggctact ccgtgtctag ggagaagaag gagcggttca gcctgatcct ggagagcgcc 300

tccaccaatc agacaagcat gtacctgtgc gcctctagct tcccactgct gggcggctat 360

acctttggca gcggcaccag gctgacagtg gtggaggacc tgcgcaacgt gacaccccct 420

aaggtgtctc tgttcgagcc cagcaaggcc gagatcgcca ataagcagaa ggccaccctg 480

gtgtgcctgg caaggggctt ctttcctgac cacgtggagc tgtcctggtg ggtgaacggc 540

aaggaggtgc actccggcgt gtctaccgat ccacaggcct acaaggagag caattactcc 600

tattgcctgt cctctcggct gagagtgagc gccacatttt ggcacaaccc aaggaatcac 660

ttccgctgtc aggtgcagtt tcacggcctg tccgaggagg acaagtggcc agagggctct 720

ccaaagccag tgacccagaa catcagcgcc gaggcatggg gaagggcaga ttgcggcatc 780

acatctgcca gctatcacca gggcgtgctg agcgccacca tcctgtacga gatcctgctg 840

ggcaaggcca cactgtatgc cgtgctggtg tccggcctgg tgctgatggc catggtgaag 900

aagaagaact ctagggcaaa gcggagcggc tctggagcaa ccaatttcag cctgctgaag 960

caggcaggcg acgtggagga gaaccctgga ccaatgctgc tggagcacct gctgatcatc 1020

ctgtggatgc agctgacctg ggtgtccggc cagcagctga atcagtctcc ccagagcatg 1080

ttcatccagg agggcgagga cgtgagcatg aactgtacca gctcctctat ctttaataca 1140

tggctgtggt acaagcagga ccccggagag ggacccgtgc tgctgatcgc cctgtataag 1200

gcaggagagc tgacaagcaa cggccggctg accgcacagt tcggaatcac aagaaaggat 1260

tcctttctga atatcagcgc ctccatcccc tctgacgtgg gcatctactt ctgcgccggc 1320

aagaacaccg ataagctgat cttcggcacc ggcacacggc tgcaggtgtt tcccgatatc 1380

cagaatcccg agcctgccgt gtatcagctg aaggacccta gatcccagga tagcaccctg 1440

tgcctgttca ccgactttga tagccagatc aacgtgccta agaccatgga gtccggcacc 1500

tttatcacag acaagaccgt gctggatatg aaggccatgg actctaagag caacggcgcc 1560

atcgcctgga gcaatcagac atccttcacc tgccaggaca tctttaagga gacaaatgcc 1620

acctacccta gctccgacgt gccatgtgat gccaccctga cagagaagtc cttcgagacc 1680

gatatgaacc tgaattttca gaacctgtct gtgatgggcc tgagaatcct gctgctgaag 1740

gtggccggct tcaatctgct gatgacactg cgcctgtggt ctagctga 1788

<210> 41

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 41

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 Ser Gly Gly Gly Tyr

85 90 95

Ser Thr Leu Thr Phe Gly Lys Gly Thr Met Leu Leu Val Ser Pro

100 105 110

<210> 42

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 42

Asp Thr Ala Val Ser Gln Thr Pro Lys Tyr Leu Val Thr Gln Met Gly

1 5 10 15

Asn Asp Lys Ser Ile Lys Cys Glu Gln Asn Leu Gly His Asp Thr Met

20 25 30

Tyr Trp Tyr Lys Gln Asp Ser Lys Lys Phe Leu Lys Ile Met Phe Ser

35 40 45

Tyr Asn Asn Lys Glu Leu Ile Ile Asn Glu Thr Val Pro Asn Arg Phe

50 55 60

Ser Pro Lys Ser Pro Asp Lys Ala His Leu Asn Leu His Ile Asn Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Trp Gly

85 90 95

Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu

100 105 110

<210> 43

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 43

Met Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu

1 5 10 15

Ser Trp Val Trp Ser Gln

20

<210> 44

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 44

Met Gly Cys Arg Leu Leu Cys Cys Val Val Phe Cys Leu Leu Gln Ala

1 5 10 15

Gly Pro Leu

<210> 45

<211> 600

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 45

Met Gly Cys Arg Leu Leu Cys Cys Val Val Phe Cys Leu Leu Gln Ala

1 5 10 15

Gly Pro Leu Asp Thr Ala Val Ser Gln Thr Pro Lys Tyr Leu Val Thr

20 25 30

Gln Met Gly Asn Asp Lys Ser Ile Lys Cys Glu Gln Asn Leu Gly His

35 40 45

Asp Thr Met Tyr Trp Tyr Lys Gln Asp Ser Lys Lys Phe Leu Lys Ile

50 55 60

Met Phe Ser Tyr Asn Asn Lys Glu Leu Ile Ile Asn Glu Thr Val Pro

65 70 75 80

Asn Arg Phe Ser Pro Lys Ser Pro Asp Lys Ala His Leu Asn Leu His

85 90 95

Ile Asn Ser Leu Glu Leu Gly Asp Ser Ala Val Tyr Phe Cys Ala Ser

100 105 110

Ser Trp Gly Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr

115 120 125

Val Leu Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe

130 135 140

Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val

145 150 155 160

Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp

165 170 175

Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Ala

180 185 190

Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val

195 200 205

Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val

210 215 220

Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro

225 230 235 240

Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp

245 250 255

Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu Ser Ala Thr

260 265 270

Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu

275 280 285

Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys Lys Asn Ser Arg

290 295 300

Ala Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln

305 310 315 320

Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Met Lys Ser Leu Arg

325 330 335

Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser Trp Val Trp Ser Gln

340 345 350

Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly

355 360 365

Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Val Ser Gln Ser

370 375 380

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

385 390 395 400

Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

405 410 415

Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln

420 425 430

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Ser Gly Gly Gly Tyr

435 440 445

Ser Thr Leu Thr Phe Gly Lys Gly Thr Met Leu Leu Val Ser Pro Asp

450 455 460

Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser

465 470 475 480

Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn

485 490 495

Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Thr Val

500 505 510

Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala Trp

515 520 525

Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr Asn

530 535 540

Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr Glu

545 550 555 560

Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Ser Val

565 570 575

Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu

580 585 590

Met Thr Leu Arg Leu Trp Ser Ser

595 600

<210> 46

<211> 399

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 46

atgatgaaat ccttgagagt tttactagtg atcctgtggc ttcagttgag ctgggtttgg 60

agccaacaga aggaggtgga gcagaattct ggacccctca gtgttccaga gggagccatt 120

gcctctctca actgcactta cagtgaccga gtttcccagt ccttcttctg gtacagacaa 180

tattctggga aaagccctga gttgataatg tccatatact ccaatggtga caaagaagat 240

ggaaggttta cagcacagct caataaagcc agccagtatg tttctctgct catcagagac 300

tcccagccca gtgattcagc cacctacctc tgtgccgtga gcggtggagg atacagcacc 360

ctcacctttg ggaaggggac tatgcttcta gtctctcca 399

<210> 47

<211> 390

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 47

atgggctgca ggctcctctg ctgtgtggtc ttctgcctcc tccaagcagg tcccttggac 60

acagctgttt cccagactcc aaaatacctg gtcacacaga tgggaaacga caagtccatt 120

aaatgtgaac aaaatctggg ccatgatact atgtattggt ataaacagga ctctaagaaa 180

tttctgaaga taatgtttag ctacaataat aaggagctca ttataaatga aacagttcca 240

aatcgcttct cacctaaatc tccagacaaa gctcacttaa atcttcacat caattccctg 300

gagcttggtg actctgctgt gtatttctgt gccagcagtt ggggcacaga cacgcagtat 360

tttggcccag gcacccggct gacagtgctc 390

<210> 48

<211> 1803

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 48

atgggctgca ggctgctgtg ctgcgtggtg ttctgcctgc tgcaggcagg accactggat 60

acagccgtga gccagacccc caagtacctg gtgacacaga tgggcaacga caagtccatc 120

aagtgcgagc agaatctggg ccacgatacc atgtactggt ataagcagga ctccaagaag 180

ttcctgaaga tcatgttctc ttacaacaat aaggagctga tcatcaacga gaccgtgcct 240

aatcgcttca gcccaaagtc ccccgataag gcccacctga acctgcacat caatagcctg 300

gagctgggcg attccgccgt gtacttctgc gccagctcct ggggcaccga cacacagtat 360

tttggaccag gaaccaggct gacagtgctg gaggacctgc gcaacgtgac accccctaag 420

gtgtctctgt ttgagccaag caaggccgag atcgccaata agcagaaggc caccctggtg 480

tgcctggcaa ggggcttctt tcccgatcac gtggagctga gctggtgggt gaacggcaag 540

gaggtgcact ctggcgtgag cacagaccct caggcctaca aggagtccaa ttactcttat 600

tgcctgtcta gccggctgag agtgtccgcc accttctggc acaacccccg gaatcacttc 660

agatgtcagg tgcagtttca cggcctgagc gaggaggata agtggcctga gggctcccca 720

aagcccgtga cacagaacat ctctgccgag gcatggggaa gggcagactg cggaatcacc 780

tctgccagct atcaccaggg cgtgctgagc gccacaatcc tgtacgagat cctgctgggc 840

aaggccaccc tgtatgccgt gctggtgtct ggcctggtgc tgatggccat ggtgaagaag 900

aagaacagca gggcaaagcg gagcggctct ggagccacca atttttccct gctgaagcag 960

gccggcgatg tggaggagaa tcctggccca atgatgaaga gcctgagagt gctgctggtc 1020

atcctgtggc tgcagctgag ctgggtgtgg tcccagcaga aggaggtgga gcagaactct 1080

ggacctctga gcgtgccaga gggagcaatc gcctctctga attgtacata cagcgaccgg 1140

gtgagccagt ccttcttttg gtacagacag tatagcggca agtccccaga gctgatcatg 1200

tctatctaca gcaacggcga caaggaggat ggccggttca ccgcccagct gaataaggcc 1260

tcccagtacg tgagcctgct gatcagagac agccagccct ctgatagcgc cacatacctg 1320

tgcgccgtgt ccggaggagg atattctacc ctgacatttg gcaagggcac catgctgctg 1380

gtgtcccctg acatccagaa ccccgagcct gccgtgtacc agctgaagga cccaagatcc 1440

caggatagca ccctgtgcct gttcaccgac tttgatagcc agatcaatgt gcccaagacc 1500

atggagtccg gcaccttcat cacagacaag accgtgctgg atatgaaggc catggactcc 1560

aagtctaacg gcgccatcgc ctggagcaat cagacatcct tcacctgcca ggatatcttt 1620

aaggagacaa acgccaccta tccctcctct gacgtgcctt gtgatgccac cctgacagag 1680

aagtctttcg agacagacat gaacctgaat tttcagaacc tgtccgtgat gggcctgaga 1740

atcctgctgc tgaaggtggc cggctttaat ctgctgatga ccctgcgcct gtggagctcc 1800

tga 1803

<210> 49

<211> 114

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 49

Gln Lys Glu Val Glu Gln Asp Pro Gly Pro Leu Ser Val Pro Glu Gly

1 5 10 15

Ala Ile Val Ser Leu Asn Cys Thr Tyr Ser Asn Ser Ala Phe Gln Tyr

20 25 30

Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys Gly Pro Glu Leu Leu Met

35 40 45

Tyr Thr Tyr Ser Ser Gly Asn Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

Val Asp Lys Ser Ser Lys Tyr Ile Ser Leu Phe Ile Arg Asp Ser Gln

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Met Ser Phe Met Ser Ser

85 90 95

Gly Ser Ala Arg Gln Leu Thr Phe Gly Ser Gly Thr Gln Leu Thr Val

100 105 110

Leu Pro

<210> 50

<211> 114

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 50

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His Glu Asn Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Thr Phe Thr

85 90 95

Ser Ser Tyr Asn Ser Pro Leu His Phe Gly Asn Gly Thr Arg Leu Thr

100 105 110

Val Thr

<210> 51

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 51

Met Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu

1 5 10 15

Ser Trp Val Trp Ser Gln

20

<210> 52

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 52

Met Gly Ile Arg Leu Leu Cys Arg Val Ala Phe Cys Phe Leu Ala Val

1 5 10 15

Gly Leu Val

<210> 53

<211> 606

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 53

Met Gly Ile Arg Leu Leu Cys Arg Val Ala Phe Cys Phe Leu Ala Val

1 5 10 15

Gly Leu Val Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys

20 25 30

Arg Thr Gly Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His

35 40 45

Glu Asn Met Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu

50 55 60

Ile Tyr Phe Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro

65 70 75 80

Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile

85 90 95

Leu Glu Ser Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser

100 105 110

Thr Phe Thr Ser Ser Tyr Asn Ser Pro Leu His Phe Gly Asn Gly Thr

115 120 125

Arg Leu Thr Val Thr Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val

130 135 140

Ser Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala

145 150 155 160

Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu

165 170 175

Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp

180 185 190

Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg

195 200 205

Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg

210 215 220

Cys Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu

225 230 235 240

Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly

245 250 255

Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu

260 265 270

Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr

275 280 285

Ala Val Leu Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys Lys

290 295 300

Asn Ser Arg Ala Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu

305 310 315 320

Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Met Lys

325 330 335

Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser Trp Val

340 345 350

Trp Ser Gln Gln Lys Glu Val Glu Gln Asp Pro Gly Pro Leu Ser Val

355 360 365

Pro Glu Gly Ala Ile Val Ser Leu Asn Cys Thr Tyr Ser Asn Ser Ala

370 375 380

Phe Gln Tyr Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys Gly Pro Glu

385 390 395 400

Leu Leu Met Tyr Thr Tyr Ser Ser Gly Asn Lys Glu Asp Gly Arg Phe

405 410 415

Thr Ala Gln Val Asp Lys Ser Ser Lys Tyr Ile Ser Leu Phe Ile Arg

420 425 430

Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Met Ser Phe

435 440 445

Met Ser Ser Gly Ser Ala Arg Gln Leu Thr Phe Gly Ser Gly Thr Gln

450 455 460

Leu Thr Val Leu Pro Asp Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln

465 470 475 480

Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp

485 490 495

Phe Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe

500 505 510

Ile Thr Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser

515 520 525

Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp

530 535 540

Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys

545 550 555 560

Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn

565 570 575

Phe Gln Asn Leu Ser Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val

580 585 590

Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

595 600 605

<210> 54

<211> 408

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 54

atgatgaaat ccttgagagt tttactggtg atcctgtggc ttcagttaag ctgggtttgg 60

agccaacaga aggaggtgga gcaggatcct ggaccactca gtgttccaga gggagccatt 120

gtttctctca actgcactta cagcaacagt gcttttcaat acttcatgtg gtacagacag 180

tattccagaa aaggccctga gttgctgatg tacacatact ccagtggtaa caaagaagat 240

ggaaggttta cagcacaggt cgataaatcc agcaagtata tctccttgtt catcagagac 300

tcacagccca gtgattcagc cacctacctc tgtgcaatga gcttcatgtc ttctggttct 360

gcaaggcaac tgacctttgg atctgggaca caattgactg ttttacct 408

<210> 55

<211> 399

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 55

atgggaatca ggctcctctg tcgtgtggcc ttttgtttcc tggctgtagg cctcgtagat 60

gtgaaagtaa cccagagctc gagatatcta gtcaaaagga cgggagagaa agtttttctg 120

gaatgtgtcc aggatatgga ccatgaaaat atgttctggt atcgacaaga cccaggtctg 180

gggctacggc tgatctattt ctcatatgat gttaaaatga aagaaaaagg agatattcct 240

gaggggtaca gtgtctctag agagaagaag gagcgcttct ccctgattct ggagtccgcc 300

agcaccaacc agacatctat gtacctctgt gccagcacct ttaccagctc ctataattca 360

cccctccact ttgggaacgg gaccaggctc actgtgaca 399

<210> 56

<211> 1821

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 56

atgggcatcc ggctgctgtg cagagtggcc ttctgttttc tggccgtggg cctggtggat 60

gtgaaggtga cacagagctc caggtacctg gtgaagcgca ccggcgagaa ggtgttcctg 120

gagtgcgtgc aggacatgga tcacgagaac atgttttggt ataggcagga ccctggactg 180

ggactgaggc tgatctactt cagctatgat gtgaagatga aggagaaggg cgacatccca 240

gagggctact ccgtgtctcg ggagaagaag gagagatttt ccctgatcct ggagagcgcc 300

tccaccaatc agacaagcat gtacctgtgc gcctccacct tcacatctag ctataacagc 360

cctctgcact ttggcaatgg cacacggctg accgtgacag aggatctgag aaacgtgacc 420

ccccctaagg tgtccctgtt cgagccctct aaggccgaga tcgccaataa gcagaaggcc 480

accctggtgt gcctggcaag gggcttcttt cctgatcacg tggagctgtc ctggtgggtg 540

aacggcaagg aggtgcacag cggcgtgtcc acagacccac aggcctacaa ggagtctaat 600

tacagctatt gcctgtcctc tcggctgaga gtgagcgcca ccttttggca caacccaagg 660

aatcacttcc gctgtcaggt gcagtttcac ggcctgtctg aggaggataa gtggccagag 720

ggcagcccaa agccagtgac acagaacatc tccgccgagg catggggaag ggcagactgc 780

ggcatcacct ctgccagcta tcaccagggc gtgctgtccg ccacaatcct gtacgagatc 840

ctgctgggca aggccaccct gtatgccgtg ctggtgtccg gcctggtgct gatggccatg 900

gtgaagaaga agaactctag ggcaaagcgg agcggctctg gagcaaccaa tttcagcctg 960

ctgaagcagg caggcgatgt ggaggagaac cctggaccaa tgatgaagtc cctgagagtg 1020

ctgctggtca tcctgtggct gcagctgagc tgggtgtggt cccagcagaa ggaggtggag 1080

caggaccccg gacctctgag cgtgccagag ggagcaatcg tgtccctgaa ctgtacctac 1140

agcaattccg ccttccagta cttcatgtgg taccggcagt atagcagaaa gggccctgag 1200

ctgctgatgt acacatatag ctccggcaat aaggaggatg gccggttcac cgcccaggtg 1260

gacaagtcta gcaagtacat ctctctgttt atcagagaca gccagccatc tgatagcgcc 1320

acatatctgt gcgccatgtc cttcatgtcc tctggctctg ccaggcagct gacctttggc 1380

agcggaaccc agctgacagt gctgccagac atccagaacc cagagcccgc cgtgtaccag 1440

ctgaaggacc ctcgctccca ggatagcacc ctgtgcctgt tcaccgactt tgattctcag 1500

atcaatgtgc ccaagaccat ggagagcggc accttcatca cagacaagac cgtgctggat 1560

atgaaggcca tggactccaa gtctaacggc gccatcgcct ggtctaatca gacaagcttc 1620

acctgccagg atatctttaa ggagacaaac gccacctacc ctagctccga cgtgccatgt 1680

gatgccaccc tgacagagaa gtccttcgag acagacatga acctgaattt tcagaacctg 1740

tctgtgatgg gcctgagaat cctgctgctg aaggtggccg gctttaatct gctgatgacc 1800

ctgcgcctgt ggtctagctg a 1821

<210> 57

<211> 137

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 57

Asp Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg

1 5 10 15

Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile

20 25 30

Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Thr

35 40 45

Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala

50 55 60

Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr

65 70 75 80

Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr

85 90 95

Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Ser

100 105 110

Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu

115 120 125

Leu Met Thr Leu Arg Leu Trp Ser Ser

130 135

<210> 58

<211> 173

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<400> 58

Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro

1 5 10 15

Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val Cys Leu

20 25 30

Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

35 40 45

Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Ala Tyr Lys

50 55 60

Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala

65 70 75 80

Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe

85 90 95

His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro Lys Pro

100 105 110

Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly

115 120 125

Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu Ser Ala Thr Ile Leu

130 135 140

Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser

145 150 155 160

Gly Leu Val Leu Met Ala Met Val Lys Lys Lys Asn Ser

165 170

<210> 59

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 59

aattctgcca gccagtcc 18

<210> 60

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 60

gtgtactcct ctggaaac 18

<210> 61

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 61

gtggcagatt ccggaggagg agcagacggc ctgacc 36

<210> 62

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 62

tccggccacg acaac 15

<210> 63

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 63

tttgtgaagg agtctaag 18

<210> 64

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 64

gcaagctccc accagggact gggaaccgag gccttc 36

<210> 65

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 65

tctatcttta ataca 15

<210> 66

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 66

ctgtataagg caggagagct g 21

<210> 67

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 67

gccggcaaga acaccgataa gctgatc 27

<210> 68

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 68

atggatcacg agaac 15

<210> 69

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 69

tcctatgatg tgaagatg 18

<210> 70

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 70

gcctctagct tcccactgct gggcggctat acc 33

<210> 71

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 71

gaccgggtga gccagtcc 18

<210> 72

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 72

atctacagca acggcgac 18

<210> 73

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 73

gccgtgtccg gaggaggata ttctaccctg aca 33

<210> 74

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 74

ctgggccacg atacc 15

<210> 75

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 75

tacaacaata aggagctg 18

<210> 76

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 76

gccagctcct ggggcaccga cacacagtat 30

<210> 77

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 77

aattccgcct tccagtac 18

<210> 78

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 78

acatatagct ccggcaat 18

<210> 79

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 79

gccatgtcct tcatgtcctc tggctctgcc aggcagctga cc 42

<210> 80

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 80

atggatcacg agaac 15

<210> 81

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 81

agctatgatg tgaagatg 18

<210> 82

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<400> 82

gcctccacct tcacatctag ctataacagc cctctgcac 39

Claims

1. A T Cell Receptor (TCR) that specifically recognizes a MART-1 (27-35) epitope, comprising an alpha chain and a beta chain, wherein the alpha chain comprises three complementarity determining regions CDR1 alpha, CDR2 alpha and CDR3 alpha, the beta chain comprises three complementarity determining regions CDR1 beta, CDR2 beta and CDR3 beta, and the amino acid sequences of CDR1 alpha, CDR2 alpha, CDR3 alpha, CDR1 beta, CDR2 beta and CDR3 beta are shown as SEQ ID NOs 1-6, 7-12, 13-18 or 19-24, respectively, or are variants having at least one amino acid change compared to the amino acid sequences and retaining the ability to specifically recognize a MART-1 (27-35) epitope.

2. The T Cell Receptor (TCR) according to claim 1, wherein the alpha chain variable region comprises the amino acid sequence shown in SEQ ID No. 25, SEQ ID No. 33, SEQ ID No. 41 or SEQ ID No. 49 or an amino acid sequence having at least 80% sequence identity to SEQ ID No. 25, SEQ ID No. 33, SEQ ID No. 41 or SEQ ID No. 49.

3. The T Cell Receptor (TCR) according to claim 1 or 2, wherein the β chain variable region comprises the amino acid sequence shown in SEQ ID No. 26, SEQ ID No. 34, SEQ ID No. 42 or SEQ ID No. 50 or an amino acid sequence having at least 80% sequence identity to SEQ ID No. 26, SEQ ID No. 34, SEQ ID No. 42 or SEQ ID No. 50.

4. A T Cell Receptor (TCR) according to any one of claims 1-3, wherein the alpha chain variable region and the beta chain variable region are each linked to a constant region; preferably, the amino acid sequence of the constant region linked to the alpha chain variable region is as shown in SEQ ID NO. 57; preferably, the amino acid sequence of the constant region linked to the β chain variable region is as shown in SEQ ID NO. 58.

5. The T Cell Receptor (TCR) of any one of claims 1-4, wherein the alpha chain and the beta chain are each further fused to a signal peptide; preferably, the amino acid sequence of the signal peptide fused with the alpha chain is shown as SEQ ID NO. 27, SEQ ID NO. 35, SEQ ID NO. 43 or SEQ ID NO. 51; preferably, the amino acid sequence of the signal peptide fused to the beta chain is shown as SEQ ID NO. 28, SEQ ID NO. 36, SEQ ID NO. 44 or SEQ ID NO. 52.

6. The T cell receptor of claim 5, wherein the alpha chain fused to the signal peptide and the beta chain fused to the signal peptide are linked together via a 2A peptide, such as a P2A peptide; preferably, the amino acid sequence of the T Cell Receptor (TCR) is shown as SEQ ID NO. 29, SEQ ID NO. 37, SEQ ID NO. 45 or SEQ ID NO. 53.

7. A nucleic acid molecule encoding the T Cell Receptor (TCR) of any one of claims 1-6.

8. The nucleic acid molecule of claim 7, wherein the alpha chain variable region of the T Cell Receptor (TCR) is encoded by the nucleic acid sequence set forth in SEQ ID No. 30, SEQ ID No. 38, SEQ ID No. 46 or SEQ ID No. 54, or degenerate sequences thereof.

9. The nucleic acid molecule of claim 7 or 8, wherein the β chain variable region of the T Cell Receptor (TCR) is encoded by the nucleic acid sequence set forth in SEQ ID No. 31, SEQ ID No. 39, SEQ ID No. 47 or SEQ ID No. 55, or degenerate sequences thereof.

10. The nucleic acid molecule of any one of claims 7-9, wherein the T Cell Receptor (TCR) is encoded by the nucleic acid sequence set forth in SEQ ID No. 32, SEQ ID No. 40, SEQ ID No. 48 or SEQ ID No. 56, or degenerate sequences thereof.

11. A vector comprising the nucleic acid molecule of any one of claims 7-10; preferably, the vector is a viral vector such as a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral expression vector.

12. An effector cell comprising the T cell receptor of any one of claims 1-6, the nucleic acid molecule of any one of claims 7-10, or the vector of claim 11.

13. The effector cell of claim 12, wherein the cell is a T cell, a natural killer cell, a human embryonic stem cell, a lymphoid progenitor cell, and/or a T cell precursor cell, such as a cytotoxic T cell, a helper T cell, a natural killer T cell, and a suppressor T cell.

14. A method of killing a melanoma cell comprising contacting the T cell receptor of any one of claims 1-6 or the effector cell of claim 12 or 13 with a melanoma cell; preferably, the melanoma cells are MART-1 positive, HLA:A0201 typed melanoma cells.

15. A method of treating melanoma comprising administering the T cell receptor of any one of claims 1-6 or the effector cell of claim 12 or 13 to a patient with melanoma; preferably, the melanoma is MART-1 positive, HLA:A0201 typed melanoma.

16. Use of the T cell receptor of any one of claims 1-6, the nucleic acid molecule of any one of claims 7-10, the vector of claim 11, or the effector cell of claim 12 or 13 in the manufacture of a medicament for the treatment of melanoma; preferably, the melanoma is MART-1 positive, HLA:A0201 typed melanoma.

17. A kit comprising the T cell receptor of any one of claims 1-6, the nucleic acid molecule of any one of claims 7-10, the vector of claim 11, or the effector cell of claim 12 or 13.