CN115677846A - High affinity T cell receptors for the antigen SSX2 - Google Patents

High affinity T cell receptors for the antigen SSX2 Download PDF

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CN115677846A
CN115677846A CN202110849328.0A CN202110849328A CN115677846A CN 115677846 A CN115677846 A CN 115677846A CN 202110849328 A CN202110849328 A CN 202110849328A CN 115677846 A CN115677846 A CN 115677846A
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tcr
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黄金花
刘敏
张婷婷
翁志明
陈建君
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Xiangxue Life Science Technology Guangdong Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464484—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Abstract

The present invention provides a T Cell Receptor (TCR) having the property of binding to the AQIPEKIQK-HLA A1101 complex. The invention also provides multivalent TCR complexes, nucleic acid molecules encoding such TCRs, vectors comprising these nucleic acids, cells expressing such TCRs, and pharmaceutical compositions comprising the foregoing, which are useful for the diagnosis, treatment, and prevention of SSX2 positive diseases. The invention also provides methods of making such TCRs.

Description

High affinity T cell receptors for the antigen SSX2
Technical Field
The present invention relates to the field of biotechnology, and more specifically to a T Cell Receptor (TCR) capable of recognizing polypeptides derived from SSX2 proteins. The invention also relates to the preparation and use of said receptors.
Background
Only two types of molecules are able to recognize antigens in a specific manner. One of which is an immunoglobulin or antibody; the other is the T Cell Receptor (TCR), which is a cell membrane surface glycoprotein that exists as a heterodimer from the α chain/β chain or the γ chain/δ chain. The composition of the TCR repertoire of the immune system is generated by V (D) J recombination in the thymus, followed by positive and negative selection. In the peripheral environment, TCRs mediate the specific recognition of the major histocompatibility complex-peptide complex (pMHC) by T cells, and are therefore critical for the cellular immune function of the immune system.
TCRs are the only receptors for specific antigenic peptides presented on the Major Histocompatibility Complex (MHC), and such exogenous or endogenous peptides may be the only signs of cellular abnormalities. In the immune system, direct physical contact between T cells and Antigen Presenting Cells (APCs) is initiated by the binding of antigen-specific TCRs to pMHC complexes, and then other cell membrane surface molecules of both T cells and APCs interact, which causes a series of subsequent cell signaling and other physiological reactions, thereby allowing T cells of different antigen specificities to exert immune effects on their target cells.
The MHC class I and II molecular ligands corresponding to the TCR are also proteins of the immunoglobulin superfamily but are specific for presentation of antigens, with different individuals having different MHC, and thereby presenting different short peptides of a single protein antigen to the cell surface of the respective APC. Human MHC is often referred to as HLA gene or HLA complex.
SSX2 is a synovial sarcoma X breakpoint, also known as HOM-MEL-40.SSX2 is one of ten highly homologous nucleic acid proteins of the SSX family. SSX proteins are tumor testis antigens and are expressed only in tumor cells and in testicular germ cells that do not express MHC. SSX2 is expressed in a variety of human cancer cells, including, but not limited to, liver cancer, lung cancer, fibrosarcoma, breast cancer, colon cancer, prostate cancer. SSX2 is degraded to small polypeptides after intracellular production and is presented to the cell surface as a complex in association with MHC (major histocompatibility complex) molecules, AQIPEKIQK is a short peptide derived from the SSX2 antigen.
Thus, the AQIPEKIQK-HLA A1101 complex provides a marker for targeting of TCR to tumor cells. The TCR capable of combining the AQIPEKIQK-HLA A1101 complex has high application value in tumor treatment. For example, TCRs capable of targeting the tumor cell marker can be used to deliver cytotoxic or immunostimulatory agents to target cells, or to be transformed into T cells, enabling T cells expressing the TCR to destroy tumor cells for administration to patients in a therapeutic process known as adoptive immunotherapy. For the former purpose, the ideal TCR is of higher affinity, enabling the TCR to reside on the targeted cell for a long period of time. For the latter purpose, it is preferred to use a medium affinity TCR. Thus, those skilled in the art are working to develop TCRs that target tumor cell markers that can be used to meet different objectives.
Disclosure of Invention
It is an object of the present invention to provide a TCR with high affinity for the AQIPEKIQK-HLA A1101 complex.
It is a further object of the present invention to provide a method for preparing a TCR of the above type and uses thereof.
In a first aspect of the present invention there is provided a T Cell Receptor (TCR) comprising an alpha chain variable domain and a beta chain variable domain, which has the activity of binding to the AQIPEKIQK-HLA a1101 complex, and the amino acid sequence of the TCR alpha chain variable domain has at least 90% sequence homology with the amino acid sequence set forth in SEQ ID No. 1 and the amino acid sequence of the TCR beta chain variable domain has at least 90% sequence homology with the amino acid sequence set forth in SEQ ID No. 2.
In a preferred embodiment, the amino acid sequence of the TCR a chain variable domain and the amino acid sequence of the TCR β chain variable domain are not the amino acid sequence of the wild-type TCR a chain variable domain and the amino acid sequence of the wild-type TCR β chain variable domain at the same time.
In a further preferred embodiment, the amino acid sequence of the variable domain of the TCR alpha chain is not the amino acid sequence shown in SEQ ID NO. 1 and/or the amino acid sequence of the variable domain of the TCR beta chain is not the amino acid sequence shown in SEQ ID NO. 2.
In another preferred embodiment, the amino acid sequence of the α chain variable domain of the wild-type TCR is shown in SEQ ID NO 1; the amino acid sequence of the beta chain variable domain is shown as SEQ ID NO. 2.
In another preferred embodiment, the α chain variable domain of the TCR comprises an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the sequence set forth in SEQ ID No. 1. Preferably, the amino acid sequence of the variable domain of the TCR alpha chain has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO. 1.
In another preferred embodiment, the β chain variable domain of the TCR is an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology to the sequence set forth in SEQ ID No. 2. Preferably, the amino acid sequence of the variable domain of the TCR β chain has at least 95% sequence homology with the amino acid sequence set forth in SEQ ID NO. 2.
In another preferred embodiment, the amino acid sequence of the variable domain of the TCR α chain has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO. 1 and the amino acid sequence of the variable domain of the TCR β chain has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO. 2.
In another preferred embodiment, the TCR is mutated in CDR3 β of the variable domain of the TCR β chain, and the number of mutations is from 1 to 4, preferably 4.
In another preferred embodiment, the 3 CDRs of the TCR β chain variable domain: CDR1 beta is SGHVS; CDR2 beta is FQNEAQ; and CDR3 β is selected from ASSPTVGTSAYQF and ASARHAGTSAYNEQF.
In another preferred embodiment, the TCR α chain variable domain comprises 3 CDRs, CDR1 α is: SSYSPS, and CDR2 α is YTSAATLV, and CDR3 α: VVSSPGNTPLV.
In another preferred embodiment, the amino acid sequence of the variable domain of the TCR α chain is SEQ ID NO 1.
In another preferred embodiment, the reference sequence of the 3 CDR regions (complementarity determining regions) of the TCR α chain variable domain is as follows, CDR1 α: SSYSPS
CDR2α：YTSAATLV
CDR3 α: VVSSPGNTPLV, and contains at least one of the following mutations:
residues before mutation Post-mutation residues
Position 1Y of CDR2 α T or R or S or A
Position 2T of CDR2 α D or N or S or P or L or G
Position 3S of CDR2 α G or T or D or P or N or Y or H
4 th position A of CDR2 α Y or R or P or N or G
1 st position V of CDR3 alpha A
Position 2V of CDR3 alpha A or I
3 rd position S of CDR3 alpha D
Position 4P of CDR3 α I or L
7 th position T of CDR3 α G
Position 9L of CDR3 α Y
10 th position V of CDR3 alpha F
In another preferred embodiment, the reference sequence of the 3 CDR regions (complementarity determining regions) of the TCR β chain variable domain is as follows, CDR1 β: SGHVS
CDR2β：FQNEAQ
CDR3. Beta.: ASSPTVGTSAYNEQF, and CDR3 β contains at least one of the following mutations:
residues before mutation Post-mutation residues
3 rd position S of CDR3 beta A
Position 4P of CDR3 β R or E or Q or K or A or H
Position 5T of CDR3 beta H
V at position 6 of CDR3 β A or I
Preferably, the amino acid mutation in CDR3 β comprises:
residues before mutation Post-mutation residues
3 rd position S of CDR3 beta A
Position 5T of CDR3 beta H
In another preferred embodiment, the amino acid mutation sites of the variable domain of the TCR β chain are positions 3 and 5 of CDR3 β.
In another preferred embodiment, the TCR has at least 2-fold greater affinity for the AQIPEKIQK-HLA a1101 complex than a wild-type TCR.
In another preferred embodiment, the TCR is represented in SEQ ID NO:1 selected from one or more of Y50T/R/S/a, T51D/N/S/P/L/G, S52G/T/D/P/N/Y/H, a53Y/R/P/N/G, V92A, V93A/I, S94D, P95I/L, T98G, L100Y, V101F, wherein the numbering of the amino acid residues is as shown in SEQ ID No. 1.
In another preferred embodiment, the TCR is represented in SEQ ID NO:1 selected from one or more of S95A, P96R/E/Q/K/a/H, T97H, V98A/I, wherein the numbering of the amino acid residues is as shown in SEQ ID No. 2.
In another preferred embodiment, the TCR has CDRs selected from the group consisting of:
Figure BDA0003181782510000041
in another preferred embodiment, the TCR is soluble.
In another preferred embodiment, the TCR is an α β heterodimeric TCR comprising an α chain TRAC constant region sequence and a β chain TRBC1 or TRBC2 constant region sequence.
In another preferred embodiment, the TCR comprises (i) all or part of a TCR α chain, excluding its transmembrane domain, and (ii) all or part of a TCR β chain, excluding its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least part of the constant domain of the TCR chain.
In another preferred embodiment, the TCR comprises an artificial interchain disulfide bond between the α chain constant region and the β chain constant region.
In another preferred embodiment, the cysteine residues forming the artificial interchain disulfide bond between the constant regions of the TCR α and β chains are substituted at one or more groups of sites selected from:
thr48 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser57 of TRBC2 × 01 exon 1;
thr45 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser77 of TRBC2 × 01 exon 1;
tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC2 x 01;
thr45 of TRAC × 01 exon 1 and TRBC1 × 01 or Asp59 of TRBC2 × 01 exon 1;
ser15 of TRAC × 01 exon 1 and TRBC1 × 01 or TRBC2 × 01 exon 1 Glu15;
arg53 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser54 of TRBC2 × 01 exon 1;
pro89 of TRAC × 01 exon 1 and TRBC1 × 01 or Ala19 of TRBC2 × 01 exon 1;
and Tyr10 of exon 1 TRAC × 01 and TRBC1 × 01 or TRBC2 × 01 of Glu20 of exon 1.
In another preferred embodiment, the amino acid sequence of the α chain variable domain of the TCR is one of SEQ ID NOs 1, 5-17; and/or the amino acid sequence of the variable domain of the beta chain of the TCR is one of SEQ ID NO 2, 18-29.
In another preferred embodiment, the TCR is selected from the group consisting of:
Figure BDA0003181782510000051
in another preferred embodiment, the TCR is of human origin.
In another preferred embodiment, the TCR is a single chain TCR.
In another preferred embodiment, the TCR is a single chain TCR consisting of an alpha chain variable domain and a beta chain variable domain, the alpha and beta chain variable domains being linked by a flexible short peptide sequence (linker).
In another preferred embodiment, the TCR comprises an alpha chain constant region and a beta chain constant region, the alpha chain constant region being a murine constant region and/or the beta chain constant region being a murine constant region.
In another preferred embodiment, the C-or N-terminus of the α chain and/or β chain of the TCR is conjugated to a conjugate, preferably a detectable label, a therapeutic agent, a PK modifying moiety or a combination of any of these.
In another preferred embodiment, the therapeutic agent that binds to the TCR is an anti-CD 3 antibody linked to the C-or N-terminus of the α or β chain of the TCR.
In a second aspect of the invention, there is provided a multivalent TCR complex comprising at least two TCR molecules, at least one of which is a TCR according to the first aspect of the invention.
In a third aspect of the invention there is provided a nucleic acid molecule comprising a nucleic acid sequence encoding a TCR molecule according to the first aspect of the invention or a multivalent TCR complex according to the second aspect of the invention, or a complement thereof.
In a fourth aspect of the invention, there is provided a vector comprising the nucleic acid molecule of the third aspect of the invention.
In a fifth aspect of the invention, there is provided a host cell comprising a vector or chromosome of the fourth aspect of the invention and, integrated therein, an exogenous nucleic acid molecule of the third aspect of the invention.
In a sixth aspect of the invention there is provided an isolated cell expressing a TCR according to the first aspect of the invention, preferably the isolated cell is a T cell, NK cell or NKT cell, most preferably the isolated cell is a T cell.
In a seventh aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, or a cell according to the sixth aspect of the invention.
In an eighth aspect of the invention, there is provided a method of treating a disease, comprising administering to a subject in need thereof an amount of a TCR according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, or a cell according to the sixth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention, preferably wherein the disease is an SSX 2-positive tumour.
In a ninth aspect of the invention, there is provided the use of a TCR according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, or a cell according to the sixth aspect of the invention, in the manufacture of a medicament for the treatment of a tumour, preferably wherein the disease is a SSX2 positive tumour.
In a tenth aspect of the invention, there is provided a method of preparing a T cell receptor according to the first aspect of the invention, comprising the steps of:
(i) Culturing a host cell according to the fifth aspect of the invention, thereby expressing a T-cell receptor according to the first aspect of the invention;
(ii) Isolating or purifying said T cell receptor.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIGS. 1a and 1b show the amino acid sequences of the variable domains of wild-type TCR alpha chain, beta chain, respectively, capable of specifically binding to AQIPEKIQK-HLA A1101 complex.
FIGS. 2a and 2b show the amino acid sequences of soluble reference TCR α and β chains, respectively, of the invention.
Figures 3 (1) - (13) show the α chain variable domain amino acid sequences of the heterodimeric TCR with higher affinity for the AQIPEKIQK-HLA a1101 complex, respectively, with mutated residues underlined.
Figures 4 (1) - (12) show the β chain variable domain amino acid sequences of hetero-dimeric TCRs with higher affinity for the AQIPEKIQK-HLA a1101 complex, respectively, with mutated residues underlined.
Figure 5a and figure 5b show the extracellular amino acid sequences of wild type TCR alpha and beta chains, respectively, capable of specifically binding to the AQIPEKIQK-HLA a1101 complex.
FIGS. 6a and 6b show the amino acid sequences of wild type TCR alpha and beta chains, respectively, capable of specifically binding to AQIPEKIQK-HLA A1101 complex.
FIG. 7 is a graph of the binding curve of soluble reference TCR, i.e., wild-type TCR, to AQIPEKIQK-HLA A1101 complex.
Fig. 8a and 8b are experimental results of the activation function of effector cells transfected with the high affinity TCR of the present invention against short peptide-loaded T2 cells.
FIGS. 9a and 9b are experimental results of the activation function of effector cells transfected with the high affinity TCR of the invention against tumor cell lines.
FIGS. 10a and 10b are the results of LDH experiments on the killing function of effector cells transfected with the high affinity TCRs of the present invention against tumor cell lines.
FIG. 11 is a result of ELISpot assay for IFN-. Gamma.release from effector cells transfected with the high affinity TCR of the invention on human normal tissue cells.
Detailed Description
The present invention, through extensive and intensive studies, resulted in a T Cell Receptor (TCR) that recognizes the AQIPEKIQK short peptide (derived from SSX2 protein) presented as a peptide-HLA a1101 complex. The TCR has 3 CDR regions in its alpha chain variable domain:
CDR1α：SSYSPS
CDR2α：YTSAATLV
CDR3 α: a mutation in VVWSGNTPLV; and/or in the 3 CDR regions of its beta chain variable domain:
CDR1β：SGHVS
CDR2β：FQNEAQ
CDR3. Beta.: mutations occurred in ASSPTVGTSAYNEQF.
In another preferred embodiment, the affinity and/or binding half-life of the inventive TCR after mutation to the above AQIPEKIQK-HLA a1101 complex is at least 2-fold that of a wild-type TCR.
Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.
Term(s)
T Cell Receptor (TCR)
The TCR may be described using the international immunogenetics information system (IMGT). Native α β heterodimeric TCRs have an α chain and a β chain. In a broad sense, each chain comprises a variable region, a linker region and a constant region, and the beta chain also typically contains a short diversity region between the variable region and the linker region, but the diversity region is often considered part of the linker region. The TCR connecting region is defined by the unique TRAJ and TRBJ of IMGT, and the TCR constant region is defined by the TRAC and TRBC of IMGT.
Each variable region comprises 3 CDRs (complementarity determining regions) CDR1, CDR2 and CDR3, which are chimeric in the framework sequence. In the IMGT nomenclature, the different numbers of TRAV and TRBV refer to different types of V α and V β, respectively. In the IMGT system, the α chain constant domain has the following notation: TRAC 01, wherein "TR" represents a T cell receptor gene; "A" represents an alpha chain gene; c represents a constant region; ". 01" indicates allele 1. The beta-strand constant domain has the following symbols: TRBC1 x 01 or TRBC2 x 01, wherein "TR" denotes a T cell receptor gene; "B" represents a beta chain gene; c represents a constant region; "x01" indicates allele 1. The constant region of the alpha chain is uniquely defined, and in the form of the beta chain, there are two possible constant region genes, "C1" and "C2". The constant region gene sequences of the TCR alpha and beta chains can be obtained by those skilled in the art from published IMGT databases.
The α and β chains of a TCR are generally regarded as having two "domains" each, namely a variable domain and a constant domain. The variable domain is composed of linked variable regions and linked regions. Thus, in the description and claims of this application, the "TCR alpha chain variable domain" refers to the linked TRAV and TRAJ regions, and likewise the "TCR beta chain variable domain" refers to the linked TRBV and TRBD/TRBJ regions. The 3 CDRs of the TCR α chain variable domain are CDR1 α, CDR2 α and CDR3 α, respectively; the 3 CDRs of the variable domain of the TCR β chain are CDR1 β, CDR2 β and CDR3 β, respectively. The framework sequences of the TCR variable domains of the invention may be murine or human, preferably human. The constant domain of the TCR comprises an intracellular portion, a transmembrane region and an extracellular portion.
The alpha chain extracellular amino acid sequence and the beta chain extracellular amino acid sequence of the wild-type TCR are respectively SEQ ID NO:30 and SEQ ID NO:31 as shown in fig. 5a and 5 b. The TCR sequences used in the present invention are of human origin. The alpha chain amino acid sequence and the beta chain amino acid sequence of the wild-type TCR are respectively SEQ ID NO 32 and SEQ ID NO 33, as shown in FIG. 6a and FIG. 6 b. In the present invention, the terms "polypeptide of the invention", "TCR of the invention", "T cell receptor of the invention" are used interchangeably.
Natural interchain disulfide bond and artificial interchain disulfide bond
A set of disulfide bonds, referred to herein as "native interchain disulfide bonds," exist between the C α and C β chains of the membrane proximal region of native TCRs. In the present invention, the artificially introduced interchain covalent disulfide bond whose position is different from that of the natural interchain disulfide bond is referred to as an "artificial interchain disulfide bond".
For convenience of description, the positions of TRAC 01 and TRBC1 × 01 or TRBC2 × 01 amino acid sequences are numbered in the sequential order from the N-terminus to the C-terminus, for example, in TRBC1 × 01 or TRBC2 × 01, the 60 th amino acid in the sequential order from the N-terminus to the C-terminus is P (proline), and thus, in the present invention, it may be described as Pro60 of TRBC1 × 01 or TRBC2 × 01 exon 1, and also as the 60 th amino acid of TRBC1 × 01 or TRBC2 × 01 exon 1, and also as the Gln61 in the sequential order from the N-terminus to the C-terminus, and as the Gln61 of TRBC1 × 01 or TRBC2 × 01, and also as the Gln61 of TRBC1 × 01 or TRBC2 × 01 exon 1, and also as the TRBC1 × 01 or TRBC2 × 01, and so on. In the present invention, the position numbering of the amino acid sequences of the variable regions TRAV and TRBV follows the position numbering listed in IMGT. If an amino acid in TRAV, the position listed in IMGT is numbered 46, it is described herein as the 46 th amino acid of TRAV, and so on. In the present invention, the sequence position numbers of other amino acids are specifically described.
Tumor(s)
The term "tumor" is meant to include all types of cancer cell growth or carcinogenic processes, metastatic or malignantly transformed cells, tissues or organs, regardless of the type of pathology or the stage of infection. Examples of tumors include, without limitation: solid tumors, soft tissue tumors, and metastatic lesions. Examples of solid tumors include: malignancies of different organ systems, such as sarcomas, squamous carcinomas of the lung and cancers. For example: infected prostate, lung, breast, lymph, gastrointestinal (e.g., colon), and genitourinary tract (e.g., kidney, epithelial cells), pharynx. Squamous carcinoma of the lung includes malignant tumors, such as, for example, most cancers of the colon, rectum, renal cell, liver, lung, small cell, small intestine and esophagus. Metastatic lesions of the above-mentioned cancers can likewise be treated and prevented using the methods and compositions of the present invention.
Detailed Description
It is well known that the α chain variable domain and β chain variable domain of a TCR each contain 3 CDRs, similar to the complementarity determining regions of an antibody. CDR3 interacts with antigen short peptides and CDR1 and CDR2 interact with HLA. Thus, the CDRs of the TCR molecule determine their interaction with the antigen short peptide-HLA complex. The amino acid sequences of the alpha chain variable domain and the beta chain variable domain of the wild-type TCR capable of binding the antigen short peptide AQIPEKIQK and the HLA A1101 complex (i.e., AQIPEKIQK-HLA A1101 complex) are SEQ ID NO:1 and SEQ ID NO:2, the sequence is discovered by the inventor for the first time. It has the following CDR regions:
alpha chain variable domain CDR1 α: SSYSPS
CDR2α：YTSAATLV
CDR3α：VVSPGNTPLV
And the beta chain variable domain CDR1 β: SGHVS
CDR 2. Beta. Is: FQNEAQ and
CDR3β：ASSPTVGTSAYNEQF。
the invention obtains the TCR with improved affinity with AQIPEKIQK-HLA A1101 complex and improved cell function by carrying out mutation screening on the CDR region.
Further, the TCR of the invention is an α β heterodimeric TCR or a single chain TCR comprising an α chain variable domain and a β chain variable domain, the α chain variable domain of the TCR comprising at least 85% of the amino acid sequence set forth in SEQ ID No. 1; preferably, at least 90%; more preferably, at least 92%; more preferably, at least 94% (e.g., can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) of sequence homology; and/or the β chain variable domain of the TCR comprises at least 90%, preferably at least 92%, of the amino acid sequence set forth as SEQ ID No. 2; more preferably, at least 94% (e.g., may be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology) of sequence homology.
The wild type TCR β chain variable domain of SEQ ID NO:2, CDR1, CDR2 and CDR3 are located in SEQ ID NO:2 from bits 27-31, from bits 49-54, and from bits 93-107. Accordingly, the amino acid residue numbering adopts the numbering shown in SEQ ID NO. 1, 95S is the 3 rd position S of CDR3 beta, 96P is the 4 th position P of CDR3 beta, 97T is the 5 th position T of CDR3 beta, 98V is the 6 th position V of CDR3 beta.
Specifically, the specific form of the mutation in the variable domain of the beta strand includes one or more of S95A, P96R/E/Q/K/A/H, T97H, V98A/I.
The wild type TCR α chain variable domain of SEQ ID NO:1, i.e. CDR1, CDR2 and CDR3 are located in SEQ ID NO: bits 27-32, 50-57 and 92-101 of 1. Accordingly, the amino acid residue numbering is as shown in SEQ ID NO 1, wherein 50Y is the 1 st position Y of CDR2 alpha, 51T is the 2 nd position T of CDR2 alpha, 52S is the 3 rd position S of CDR2 alpha, 53A is the 4 th position A of CDR2 alpha, 92V is the 1 st position V of CDR3 alpha, 93V is the 2 nd position V of CDR3 alpha, 94S is the 3 rd position S of CDR3 alpha, 95P is the 4 th position P of CDR3 alpha, 98T is the 7 th position T of CDR3 alpha, 100L is the 9 th position L of CDR3 alpha, and 101V is the 10 th position V of CDR3 alpha.
Specifically, specific forms of the mutations described in the variable domains of the alpha chain include one or more of Y50T/R/S/A, T51D/N/S/P/L/G, S52G/T/D/P/N/Y/H, A53Y/R/P/N/G, V92A, V93A/I, S94D, P95I/L, T98G, L100Y, V101F.
It should be understood that the amino acid names herein are given by the international single english letter designation, and the three english letters abbreviation corresponding to the amino acid names are: ala (A), arg (R), asn (N), asp (D), cys (C), gln (Q), glu (E), gly (G), his (H), ile (I), leu (L), lys (K), met (M), phe (F), pro (P), ser (S), thr (T), trp (W), tyr (Y), val (V);
in the present invention, pro60 or 60P both represent proline at position 60. In addition, the present invention describes specific forms of the mutations in such a manner that "S95A" represents the substitution of S at position 95 with A, "V98A/I" represents the substitution of V at position 98 with A or with I, and so on.
According to the site-directed mutagenesis method well known to those skilled in the art, pro89 of exon 1 of α chain constant region TRAC × 01 of wild-type TCR is mutated to cysteine, ala19 of exon 1 of β chain constant region TRBC1 × 01 or TRBC2 × 01 is mutated to cysteine, to obtain a reference TCR whose amino acid sequences are SEQ ID No. 3 and SEQ ID NO:4, as shown in FIGS. 2a and 2b, the mutated cysteine residues are indicated in bold letters. The cysteine substitutions described above enable the formation of an artificial interchain disulfide bond between the constant regions of the α and β chains of the reference TCR to form a more stable soluble TCR, thereby enabling a more convenient assessment of the binding affinity and/or binding half-life between the TCR and the AQIPEKIQK-HLA a1101 complex. It will be appreciated that the CDR regions of the TCR variable region determine their affinity for the pMHC complex and therefore cysteine substitutions in the TCR constant region as described above do not have an effect on the binding affinity and/or binding half-life of the TCR. Thus, in the present invention, the binding affinity measured between the reference TCR and the AQIPEKIQK-HLA A1101 complex is considered to be the binding affinity between the wild-type TCR and the AQIPEKIQK-HLA A1101 complex. Similarly, if the binding affinity between the TCR of the invention and the AQIPEKIQK-HLA a1101 complex is measured to be at least 2 times greater than the binding affinity between the reference TCR and the AQIPEKIQK-HLA a1101 complex, i.e. equivalent to the binding affinity between the TCR of the invention and the AQIPEKIQK-HLA a1101 complex being at least 2 times greater than the binding affinity between the wild-type TCR and the AQIPEKIQK-HLA a1101 complex.
Binding affinity (equilibrium constant K to dissociation) can be determined by any suitable method D Inversely proportional) and binding half-life (denoted as T) 1/2 ) Such as by surface plasmon resonance. It will be appreciated that doubling the affinity of the TCR will result in K D And (4) halving. T is a unit of 1/2 Calculated as In2 divided by dissociation rate (K) off ). Thus, T 1/2 Doubling can result in K off The number is reduced by half. Preferably, the binding affinity or binding half-life of a given TCR is measured several times, e.g. 3 times or more, using the same assay protocol, and the results are averaged. In a preferred embodiment, the surface plasmon resonance (BIAcore) method of the examples herein is used to detect the affinity of soluble TCRs, provided that: the temperature is 25 ℃, and the PH value is 7.1-7.5. The method detects the dissociation equilibrium constant K of a reference TCR to AQIPEKIQK-HLA A1101 complex D 2.61E-03M, the dissociation equilibrium constant K of the wild-type TCR for the AQIPEKIQK-HLA A1101 complex is considered to be D Also 2.61E-03M. Affinity due to TCRDoubling will result in K D Halved, so if the dissociation equilibrium constant K of the high affinity TCR for the AQIPEKIQK-HLA A1101 complex is detected D At 2.61E-04M, this indicates that the affinity of the high affinity TCR for the AQIPEKIQK-HLA A1101 complex is 10 times greater than the affinity of the wild-type TCR for the AQIPEKIQK-HLA A1101 complex.
The mutation may be performed using any suitable method, including but not limited to those based on Polymerase Chain Reaction (PCR), cloning based on restriction enzymes, or Ligation Independent Cloning (LIC) methods. These methods are detailed in a number of standard molecular biology texts. For more details on Polymerase Chain Reaction (PCR) mutagenesis and Cloning by restriction enzymes, see Sambrook and Russell, (2001) Molecular Cloning-A Laboratory Manual (third edition) CSHL publisher. More information on the LIC method can be found (Rashtchian, (1995) Curr Opin Biotechnol6 (1): 30-6).
The method of producing the TCRs of the invention may be, but is not limited to, screening a diverse library of phage particles displaying such TCRs for a TCR with high affinity for the AQIPEKIQK-HLA-A1101 complex, as described in the literature (Li, et al (2005) Nature Biotech 23 (3): 349-354).
It will be appreciated that genes expressing the α and β chain variable domain amino acids of a wild type TCR, or genes expressing slightly modified α and β chain variable domain amino acids of a wild type TCR, may be used to make a template TCR. The alterations required to produce the high affinity TCRs of the invention are then introduced into the DNA encoding the variable domains of the template TCR.
The high affinity TCR of the invention comprises an alpha chain variable domain amino acid sequence of one of SEQ ID NO 1, 5-17; and/or the amino acid sequence of the variable domain of the beta chain of the TCR is one of SEQ ID NO 2, 18-29. The amino acid sequences of the α chain variable domain and β chain variable domain of the heterodimeric TCR molecules of the invention are preferably selected from table 1 below:
TABLE 1
Figure BDA0003181782510000121
For the purposes of the present invention, the inventive TCRs are moieties having at least one TCR α and/or TCR β chain variable domain. They typically comprise both a TCR α chain variable domain and a TCR β chain variable domain. They may be α β heterodimers or single chain forms or any other form that is stable. In adoptive immunotherapy, the full-length chain (comprising the cytoplasmic and transmembrane domains) of the α β heterodimeric TCR can be transfected. The TCRs of the invention are useful as targeting agents for delivering therapeutic agents to antigen presenting cells or in combination with other molecules to produce bifunctional polypeptides for targeting effector cells, where the TCRs are preferably in soluble form.
For stability, it is disclosed in the prior art that introduction of an artificial interchain disulfide bond between the α and β chain constant domains of the TCR enables soluble and stable TCR molecules to be obtained, as described in patent document PCT/CN 2015/093806. Thus, the inventive TCR may be one in which an artificial interchain disulfide bond is introduced between residues of the constant domains of its alpha and beta chains. Cysteine residues form an artificial interchain disulfide bond between the alpha and beta chain constant domains of the TCR. Cysteine residues may be substituted for other amino acid residues at appropriate positions in native TCRs to form artificial interchain disulfide bonds. For example, a disulfide bond is formed by substitution of Pro89 of TRAC × 01 exon 1 and substitution of Ala19 of TRBC1 × 01 or TRBC2 × 01 exon 1. Other sites for introducing cysteine residues to form disulfide bonds may also be: thr45 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser77 of TRBC2 × 01 exon 1; tyr10 of TRAC × 01 exon 1 and TRBC1 × 01 or TRBC2 × 01 exon 1 Ser17; thr45 of TRAC × 01 exon 1 and TRBC1 × 01 or Asp59 of TRBC2 × 01 exon 1; ser15 of TRAC × 01 exon 1 and TRBC1 × 01 or TRBC2 × 01 exon 1 Glu15; arg53 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser54 of TRBC2 × 01 exon 1; thr48 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser57 of TRBC2 × 01 exon 1; or Tyr10 of exon 1 of TRAC × 01 and TRBC1 × 01 or TRBC2 × 01, glu20 of exon 1. I.e., a cysteine residue, in place of any of the above-described alpha and beta chain constant domains. Deletion of the native interchain disulfide bonds can be achieved by truncating at most 15, or at most 10, or at most 8 or fewer amino acids at one or more of the C-termini of the TCR constant domains of the invention such that they do not include a cysteine residue, or by mutating a cysteine residue which forms a native interchain disulfide bond to another amino acid.
As described above, the TCRs of the invention may comprise an artificial interchain disulfide bond introduced between residues of the constant domains of their alpha and beta chains. It should be noted that the TCRs of the invention may each contain both TRAC constant domain sequences and TRBC1 or TRBC2 constant domain sequences, with or without the artificial disulfide bonds introduced as described above between the constant domains. The TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the TCR may be linked by the native interchain disulfide bonds present in the TCR.
In addition, for stability, PCT/CN2016/077680 also discloses that the introduction of an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of TCR can significantly improve TCR stability. Thus, the high affinity TCRs of the invention may also contain an artificial interchain disulfide bond between the α chain variable region and the β chain constant region. Specifically, the cysteine residues that form the artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR replace: amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01; amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01; amino acid 46 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01; or amino acid 47 of TRAV and amino acid 60 of exon 1 TRBC1 x 01 or TRBC2 x 01. Preferably, such a TCR may comprise (i) all or part of a TCR α chain, excluding its transmembrane domain, and (ii) all or part of a TCR β chain, excluding its transmembrane domain, wherein (i) and (ii) both comprise a variable domain and at least part of a constant domain of the TCR chain, the α chain forming a heterodimer with the β chain. More preferably, such a TCR may comprise the a chain variable domain and the β chain variable domain and all or part of the β chain constant domain, excluding the transmembrane domain, but which does not comprise the a chain constant domain, the a chain variable domain of the TCR forming a heterodimer with the β chain.
For stability, on the other hand, the inventive TCRs also include TCRs having mutations in their hydrophobic core region, preferably mutations that increase the stability of the inventive TCRs, as described in the patent publication WO 2014/206304. Such TCRs may be mutated at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or positions 3,5,7 from the last amino acid position of the short peptide of the alpha chain J gene (TRAJ), and/or positions 2,4,6 from the last amino acid position of the short peptide of the beta chain J gene (TRBJ), wherein the position numbering of the amino acid sequence is according to the position numbering listed in the International immunogenetic information System (IMGT). The above-mentioned international system of immunogenetics information is known to the skilled person and the position numbering of the amino acid residues of the different TCRs in IMGT can be derived from this database.
More specifically, the TCR with the mutated hydrophobic core region of the present invention may be a high stability single chain TCR comprising a flexible peptide chain connecting the variable domains of the α chain and β chain of the TCR. The CDR regions of the variable region of the TCR determine the affinity with the short peptide-HLA complex, and the mutation of the hydrophobic core can stabilize the TCR without affecting the affinity with the short peptide-HLA complex. It should be noted that the flexible peptide chain of the present invention can be any peptide chain suitable for linking the variable domains of TCR α and β chains.
The TCRs of the invention may also be provided in the form of multivalent complexes. Multivalent TCR complexes of the invention comprise polymers formed by association of two, three, four or more TCRs of the invention, such as might be formed by tetramer formation with the tetrameric domain of p53, or complexes formed by association of a plurality of TCRs of the invention with another molecule. The TCR complexes of the invention can be used to track or target cells presenting a particular antigen in vitro or in vivo, and can also be used to generate intermediates for other multivalent TCR complexes having such applications.
The TCRs of the invention may be used alone or in covalent or other association, preferably covalently, with a conjugate. The conjugates include a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the AQIPEKIQK-HLA-a1101 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.
Therapeutic agents that may be associated or conjugated with the TCRs of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, cancer metastasis reviews (Cancer metastasis) 24, 539); 2. biotoxics (Chaudhary et al, 1989, nature 339, 394, epel et al, 2002, cancer Immunology and Immunotherapy) 51, 565); 3. cytokines such as IL-2 etc (Gillies et al, 1992, journal of the national academy of sciences (PNAS) 89, 1428, card et al, 2004, cancer Immunology and Immunotherapy) 53, 345, hain et al, 2003, cancer Research (Cancer Research) 63, 3202); 4. antibody Fc fragment (Mosquera et al, 2005, journal Of Immunology 174, 4381); 5. antibody scFv fragments (Zhu et al, 1995, international Journal of Cancer 62, 319); 6. gold nanoparticles/nanorods (Lapotko et al, 2005, cancer communications (Cancer letters) 239, 36, huang et al, 2006, journal of the American Chemical Society 128, 2115); 7. viral particles (Peng et al, 2004, gene therapy) 11, 1234); 8. liposomes (Mamot et al, 2005, cancer research) 65, 11631); 9. nano magnetic particles; 10. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, and the like.
Antibodies or fragments thereof that bind to the TCRs of the invention include anti-T cell or NK-cell determining antibodies, such as anti-CD 3 or anti-CD 28 or anti-CD 16 antibodies, whose binding to the TCR directs effector cells to better target cells. A preferred embodiment is the binding of a TCR of the invention to an anti-CD 3 antibody or a functional fragment or variant of said anti-CD 3 antibody. Specifically, the fusion molecule of the TCR and the anti-CD 3 single-chain antibody comprises a TCR alpha chain variable domain amino acid sequence which is one of SEQ ID NO 1 and 5-17; and/or the amino acid sequence of the variable domain of the beta chain of the TCR is one of SEQ ID NO 2, 18-29.
The invention also relates to nucleic acid molecules encoding the inventive TCRs. The nucleic acid molecules of the invention may be in the form of DNA or in the form of RNA. The DNA may be the coding strand or the non-coding strand.
The full-length sequence of the nucleic acid molecule of the present invention or a fragment thereof can be obtained by, but not limited to, PCR amplification, recombination or artificial synthesis. At present, DNA sequences encoding the TCRs of the invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.
The invention also relates to vectors comprising the nucleic acid molecules of the invention, as well as to host cells genetically engineered with the vectors or coding sequences of the invention.
The invention also includes isolated cells, particularly T cells, expressing a TCR of the invention. There are many methods suitable for T cell transfection using DNA or RNA encoding the high affinity TCRs of the invention (e.g., robbins et al, (2008) j. Immunol.180: 6116-6131). T cells expressing the high affinity TCRs of the invention may be used for adoptive immunotherapy. One skilled in the art will be aware of many suitable methods for adoptive therapy (e.g., rosenberg et al, (2008) Nat Rev Cancer8 (4): 299-308).
The invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR of the invention, or a TCR complex of the invention, or a cell presenting a TCR of the invention.
The invention also provides a method of treating a disease comprising administering to a subject in need thereof an amount of a TCR of the invention, or a TCR complex of the invention, or a cell presenting a TCR of the invention, or a pharmaceutical composition of the invention.
In the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the structure and function of the protein. Thus, the TCR of the invention also includes TCRs in which up to 5, preferably up to 3, more preferably up to 2, most preferably 1 amino acid (especially outside the CDR regions) of the TCR of the invention has been replaced by amino acids of similar or analogous nature, and still retain its functionality.
The invention also includes TCRs that are slightly modified from the TCRs of the invention. Modified (generally without altering primary structure) forms include: chemically derivatized forms of the inventive TCR such as acetylation or carboxylation. Modifications also include glycosylation, such as those that result from glycosylation modifications made during synthesis and processing or during further processing steps of the inventive TCR. Such modification may be accomplished by exposing the TCR to an enzyme that effects glycosylation, such as mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are TCRs that have been modified to improve their resistance to proteolysis or to optimize solubility.
The TCRs of the invention, TCR complexes or TCR-transfected T cells of the invention can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier. The TCRs, multivalent TCR complexes or cells of the invention are typically provided as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier. The pharmaceutical composition may be in any suitable form (depending on the desired method of administration to the patient). It may be provided in unit dosage form, typically in a sealed container, and may be provided as part of a kit. Such kits (but not necessarily) include instructions for use. It may comprise a plurality of said unit dosage forms.
In addition, the TCRs of the invention may be used alone, or in combination or coupling with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).
The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. The term refers to such pharmaceutical carriers: they do not themselves induce the production of antibodies harmful to the individual receiving the composition and are not excessively toxic after administration. Such vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack pub. Co., n.j.1991). Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.
Pharmaceutically acceptable carriers in therapeutic compositions can comprise liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
Generally, the therapeutic compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for constitution with a solution or suspension, or liquid carrier, before injection, may also be prepared.
Once formulated, the compositions of the present invention may be administered by conventional routes including, but not limited to: intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably parenteral including subcutaneous, intramuscular, or intravenous. The subject to be prevented or treated may be an animal; especially a human.
When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition may be used depending on the use case. Preferably, injections, oral agents and the like are exemplified.
These pharmaceutical compositions may be formulated by mixing, dilution or dissolution according to a conventional method, and occasionally, suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizing agents are added, and the formulation process may be carried out in a conventional manner according to the dosage form.
The pharmaceutical compositions of the present invention may also be administered in the form of sustained release formulations. For example, the inventive TCR may be incorporated into a pellet or microcapsule carried by a slow release polymer, which pellet or microcapsule is then surgically implanted into the tissue to be treated. As examples of the sustained-release polymer, there may be exemplified ethylene-vinyl acetate copolymer, polyhydroxymethacrylate, polyacrylamide, polyvinylpyrrolidone, methyl cellulose, lactic acid polymer, lactic acid-glycolic acid copolymer and the like, and preferably biodegradable polymers such as lactic acid polymer and lactic acid-glycolic acid copolymer are exemplified.
When the pharmaceutical composition of the present invention is used for actual treatment, the TCR of the present invention or the TCR complex or the cells presenting the TCR of the present invention as an active ingredient can be reasonably determined according to the weight, age, sex, degree of symptoms of each patient to be treated, and the reasonable amount is finally decided by a physician.
The main advantages of the invention are:
(1) The inventive TCR is capable of specifically binding to the complex AQIPEKIQK-HLA a1101 and has an improved affinity compared to the wild type TCR.
(2) Effector cells transfected with the high affinity TCRs of the invention have a strong specific killing effect against target cells.
The following specific examples further illustrate the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example as described in Sambrook and Russell et al, molecular Cloning: A Laboratory Manual (third edition) (2001) CSHL Press, or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.
Materials and methods
The experimental materials used in the examples of the present invention are commercially available without specific description, for example, e.coli DH5 α is from Tiangen, e.coli BL21 (DE 3) is from Tiangen, e.coli Tuner (DE 3) is from Novagen, and plasmid pET28a is from Novagen; cell lines SK-MEL-28, SK-MEL-1, SNU423, HUCCT1, TT _ THYROID were obtained from Guangzhou Securio Biotech, inc., huh-1 was obtained from Nanjing Baibo Biotech, inc., and SK-MEL-5 and T2 were obtained from ATCC.
Example 1 binding characterization BIAcore analysis
BIAcore T200 real-time assay system was used to detect the binding activity of TCR molecules to the AQIPEKIQK-HLA-A1101 complex. Anti-streptavidin antibody (GenScript) was added to coupling buffer (10 mM sodium acetate buffer, pH 4.77), and then the antibody was passed through CM5 chip previously activated with EDC and NHS to immobilize the antibody on the chip surface, and finally the unreacted activated surface was blocked with ethanolamine hydrochloric acid solution to complete the coupling process at a coupling level of about 15,000 RU. The conditions are as follows: the temperature is 25 ℃, and the PH value is 7.1-7.5.
The low concentration of streptavidin was flowed over the surface of the antibody coated chip, then AQIPEKIQK-HLA-A1101 complex was flowed over the detection channel, the other channel served as a reference channel, and 0.05mM biotin was flowed over the chip at a flow rate of 10. Mu.L/min for 2min to block the remaining binding sites of streptavidin. The affinity was determined by single cycle kinetic assay by diluting TCR with HEPES-EP buffer (10 mM HEPES,150mM NaCl,3mM EDTA,0.005% P20, pH 7.4) to several different concentrations, sequentially flowing over the chip surface at a flow rate of 30. Mu.L/min for a binding time of 120s for each sample, and dissociating for 600s after the last sample. At the end of each assay run, the chip was regenerated with 10mM Gly-HCl pH 1.75. Kinetic parameters were calculated using BIAcore Evaluation software. The binding curves of the soluble reference TCR, i.e., the wild-type TCR, to the AQIPEKIQK-HLA a1101 complex are shown in figure 7.
The above AQIPEKIQK-HLA-A1101 complex is prepared as follows:
a. and (3) purification: collecting 100ml of E.coli bacterial liquid for inducing expression of heavy chain or light chain, centrifuging at 8000g at 4 ℃ for 10min, washing the bacterial cells once with 10ml of PBS, then resuspending the bacterial cells by vigorous shaking with 5ml of BugBuster Master Mix Extraction Reagents (Merck), carrying out rotary incubation at room temperature for 20min, centrifuging at 6000g at 4 ℃ for 15min, discarding supernatant, and collecting inclusion bodies.
Resuspending the inclusion body in 5ml of BugBuster Master Mix, and rotary incubating at room temperature for 5min; adding 30ml of 10-fold diluted BugBuster, uniformly mixing, and centrifuging at 4 ℃ at 6000g for 15min; discarding supernatant, adding 30ml of 10-fold diluted BugBuster to resuspend the inclusion bodies, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, repeating twice, adding 30ml of 20mM Tris-HCl with pH of 8.0 to resuspend the inclusion bodies, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, finally dissolving the inclusion bodies by using 20mM Tris-HCl 8M urea, detecting the purity of the inclusion bodies by SDS-PAGE, and detecting the concentration by using a BCA kit.
b. Renaturation: the synthesized short peptide AQIPEKIQK (Jiangsu Kingsry Biotech Co., ltd.) was dissolved in DMSO to a concentration of 20 mg/ml. Inclusion of light and heavy chains was solubilized using 8M urea, 20mM Tris pH 8.0, 10mM DTT, and further denatured by addition of 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA prior to renaturation. AQIPEKIQK peptide was added to a renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidative glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4 ℃) at 25mg/L (final concentration), followed by the addition of 20mg/L light chain and 90mg/L heavy chain in sequence (final concentration, heavy chain was added in three portions, 8 h/time), and the renaturation was performed at 4 ℃ for at least 3 days until completion, and SDS-PAGE checked for success or failure of the renaturation.
c. And (3) renaturation and purification: the renaturation buffer was replaced by dialysis against 10 volumes of 20mM Tris pH 8.0, at least twice to reduce the ionic strength of the solution sufficiently. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and then loaded on a HiTrap Q HP (GE general electric) anion exchange column (5 ml bed volume). Using an Akta purifier (GE general electric Co., ltd.), a linear gradient of 0-400mM NaCl prepared at 20mM Tris pH 8.0 was used to elute proteins, and pMHC was eluted at about 250mM NaCl, and the peak fractions were collected and the purity was checked by SDS-PAGE.
d. Biotinylation: the purified pMHC molecules were concentrated using Millipore ultrafiltration tubes while replacing the buffer with 20mM Tris pH 8.0, followed by addition of biotinylation reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50. Mu.M D-Biotin, 100. Mu.g/ml BirA enzyme (GST-BirA), incubation of the mixture overnight at room temperature, and SDS-PAGE to determine whether biotinylation was complete.
e. Purification of biotinylated complex: the biotinylated pMHC molecules were concentrated to 1ml using Millipore ultrafiltration tubes, the biotinylated pMHC was purified by gel filtration chromatography, and HiPrep was pre-equilibrated with filtered PBS using an Akta purifier (GE general electric Co., ltd.) TM A16/60S200 HR column (GE general electric) was loaded with 1ml of concentrated biotinylated pMHC and then eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules appeared as a unimodal elution at approximately 55 ml. The fractions containing the protein were pooled, concentrated using Millipore ultrafiltration tubes,the protein concentration was determined by BCA method (Thermo) and biotinylated pMHC molecules were stored in aliquots at-80 ℃ with the addition of the protease inhibitor cocktail (Roche).
Example 2 Generation of high affinity TCR
Phage display technology is one means of generating libraries of TCR high affinity variants to screen for high affinity variants. The TCR phage display and screening methods described by Li et al ((2005) Nature Biotech 23 (3): 349-354) were applied to wild-type TCR templates. The CDR regions of the template strand are mutated to create a library of high affinity TCRs and panning is performed using site-directed mutagenesis methods well known to those skilled in the art. Through multiple rounds of library construction, analysis and panning, the phage library has specific binding with corresponding antigen, and monoclonal is picked from the phage library and analyzed.
The affinity of the protein to AQIPEKIQK-HLA-A1101 complex is detected by BIAcore. The amino acid sequences of the alpha chain and beta chain variable domains of the wild-type TCR are shown in FIG. 1a (SEQ ID NO: 1) and FIG. 1b (SEQ ID NO: 2), respectively.
Extracellular sequence genes of TCR α and β chains to be expressed were synthesized and inserted into expression vector pET28a + (Novagene) by standard methods described in Molecular Cloning a Laboratory Manual (third edition, sambrook and Russell), with upstream and downstream Cloning sites being NcoI and NotI, respectively. Mutations in the CDR regions are introduced by overlap PCR (overlap PCR) which is well known to the person skilled in the art. The insert was confirmed by sequencing without error.
Example 3 expression, renaturation and purification of high affinity TCR
The expression vectors of TCR alpha and beta chains are respectively transformed into expression bacteria BL21 (DE 3) by a chemical transformation method, and the bacteria grow in LB culture solution and grow on OD 600 At 0.6 induction with final concentration of 0.5mM IPTG, inclusion bodies formed after expression of the α and β chains of the TCR were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution several times, and finally dissolved in 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA), 20mM Tris (pH 8.1).
The TCR α and β chains after lysis are separated by a 1:1 was rapidly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine,6.6mM ss-mericapoethylamine (4 ℃ C.) to a final concentration of 60mg/mL. After mixing, the solution was dialyzed against 10 volumes of deionized water (4 ℃ C.) and, after 12 hours, the deionized water was changed to a buffer (20mM Tris, pH 8.0) and dialysis was continued at 4 ℃ for 12 hours. The solution after completion of dialysis was filtered through a 0.45. Mu.M filter and then purified by an anion exchange column (HiTrap Q HP,5ml, GE Healthcare). The TCR eluted with peaks containing α and β dimers that were successfully renatured was confirmed by SDS-PAGE gel. The TCR was subsequently further purified by gel filtration chromatography (HiPrep 16/60, sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was greater than 90% as determined by SDS-PAGE and the concentration was determined by BCA method.
Example 4 BIAcore analysis results
The affinity of the α β heterodimeric TCR of the high affinity CDRs of the present invention to the AQIPEKIQK-HLA-a1101 complex was tested using the method described in example 1.
The amino acid sequences of the α chain and β chain variable domains of the novel TCR obtained by the invention are shown in FIGS. 3 (1) - (13) and FIGS. 4 (1) - (12), respectively. Expression vectors were constructed using the method described in example 2, and the high affinity mutated α β heterodimeric TCRs described above were expressed, renatured and purified using the method described in example 3, and then their affinity for AQIPEKIQK-HLA-a1101 complex was determined using BIAcore T200, as shown in table 2 below.
TABLE 2
Figure BDA0003181782510000191
Figure BDA0003181782510000201
As can be seen from table 2 above, the affinity of the heterodimeric TCR was at least 2-fold greater than the affinity of the wild-type TCR for the AQIPEKIQK-HLA-a1101 complex.
Example 5 expression, renaturation and purification of fusions of anti-CD 3 antibodies with high affinity α β hetero-dimeric TCRs
Fusion molecules were prepared by fusing an anti-CD 3 single chain antibody (scFv) to an α β heterodimeric TCR. The scFv of the anti-CD 3 is fused to the β chain of the TCR, which β chain may comprise the β chain variable domain of any of the above-described high affinity α β heterodimeric TCRs, and the TCR α chain of the fused molecule may comprise the α chain variable domain of any of the above-described high affinity α β heterodimeric TCRs.
Construction of fusion molecule expression vectors
1. Construction of alpha chain expression vector: the target gene carrying the alpha chain of the alpha beta heterodimeric TCR is subjected to double enzyme digestion by Nco I and Not I and is connected with a pET28a vector subjected to double enzyme digestion by Nco I and Not I. The ligation product was transformed into e.coli DH5 α, spread on LB plates containing kanamycin, cultured at 37 ℃ for overnight inversion, positive clones were selected for PCR screening, positive recombinants were sequenced, and after the correct sequence was determined, recombinant plasmids were extracted and transformed into e.coli Tuner (DE 3) for expression.
2. Construction of anti-CD 3 (scFv) -beta chain expression vector: primers were designed to link the anti-CD 3 scFv and the high affinity heterodimeric TCR β chain genes by overlap (overlap) PCR with the middle linker short peptide (linker) being ggggggs, and the gene fragments of the fusion proteins of the anti-CD 3 scFv and the high affinity heterodimeric TCR β chain were tagged with restriction endonuclease sites Nco i (CCATGG) and Not i (GCGGCCGC). The PCR amplification product was double-digested with Nco I and Not I, and ligated to pET28a vector double-digested with Nco I and Not I. The ligation product was transformed into e.coli DH5 α competent cells, coated with LB plates containing kanamycin, inverted cultured overnight at 37 ℃, positive clones were selected for PCR screening, positive recombinants were sequenced, and after the sequence was determined to be correct, recombinant plasmids were extracted and transformed into e.coli Tuner (DE 3) competent cells for expression.
Expression, renaturation and purification of fusion proteins
The expression plasmids were transformed into E.coli Tuner (DE 3) competent cells, respectively, and LB plates (kanamycin 50. Mu.g/mL) were plated and incubated at 37 ℃ overnight. The next day, the selected clones were inoculated into 10mL of LB liquid medium (kanamycin 50. Mu.g/mL) for 2-3 hours, inoculated into 1L of LB medium at a volume ratio of 1. After 4 hours of induction, the cells were harvested by centrifugation at 6000rpm for 10 min. The cells were washed once with PBS buffer and aliquoted, and 200mL of the cells from the bacterial culture were lysed with 5mL of BugBuster Master Mix (Merck) and the inclusion bodies were collected by centrifugation at 6000g for 15 min. 4 detergent washes were then performed to remove cell debris and membrane components. The inclusion bodies are then washed with a buffer such as PBS to remove detergents and salts. Finally, the inclusion bodies were dissolved in a buffer solution containing 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA), 2mM Tris, pH 8.1, and the inclusion body concentration was measured, and they were stored frozen at-80 ℃ after being dispensed.
The TCR α chain and the anti-CD 3 (scFv) - β chain after solubilization were separated by a 2:5 in 5M Urea (urea), 0.4M L-arginine (L-arginine), 2mM Tris pH 8.1,3.7mM cystamine,6.6mM β -mer peptide (4 ℃ C.), to final concentrations of 0.1mg/mL and 0.25mg/mL for α chain and anti-CD 3 (scFv) - β chain, respectively.
After mixing, the solution was dialyzed against 10 volumes of deionized water (4 ℃ C.) and, after 12 hours, the deionized water was changed to a buffer (10mM Tris, pH 8.0) and dialysis was continued at 4 ℃ for 12 hours. The solution after completion of dialysis was filtered through a 0.45. Mu.M filter and then purified by an anion exchange column (HiTrap Q HP 5ml, GE healthcare). The eluted peaks contain TCR alpha chain and anti-CD 3 (scFv) -beta chain dimers of which the renaturation was successful TCR alpha chain was confirmed by SDS-PAGE gel. The TCR fusion molecules were subsequently further purified by size exclusion chromatography (S-100/60, GE healthcare) and re-purified on an anion exchange column (HiTrap Q HP 5ml, GE healthcare). The purity of the purified TCR fusion molecule is greater than 90% as determined by SDS-PAGE and the concentration is determined by BCA.
Example 6 functional assay for activation of Effector cells transfected with high affinity TCRs of the invention against short peptide-loaded T2 cells
IFN-. Gamma.is a potent immunomodulatory factor produced by activated T lymphocytes, and thus the present example examined the IFN-. Gamma.counts by ELISPOT assays well known to those skilled in the art to verify the activation function and antigen specificity of cells transfected with the high affinity TCR of the invention. Transfection of the high affinity TCRs of the invention (TCR numbering and sequence numbers are given in Table 2) into the cells from the health lineageCD3 isolated from blood of volunteers + T cells as effector cells and CD3 transfected with other TCR (A6) or with wild type TCR (WT-TCR) from the same volunteer + T cells served as controls. The target cells used were T2-A11 (T2 cells transfected with HLA-A1101) loaded with the SSX2 antigen short peptide AQIPEKIQK, loaded with other antigen short peptides, or unloaded.
The experiment was carried out in two batches, the target cells used in the first batch being 1X 10 4 Single cell/well, effector cell 2X 10 3 Individual cells/well (calculated as transfection positivity); target cells for the second batch 2X 10 4 Single cell/well, effector cell 2X 10 3 Individual cells/well (calculated as positive transfection rate).
The following steps were carried out for both batches: firstly preparing an ELISPOT plate, adding target cells and effector cells into corresponding holes, then adding an SSX2 antigen short peptide AQIPEKIQK solution into an experimental group, adding other antigen short peptide solutions into a control group, and enabling the final concentration of the short peptide to be 10 -6 M, blank group add equal volume of medium and set two duplicate wells. Incubate overnight (37 ℃, 5%. On day 2 of the experiment, the plates were washed and subjected to secondary detection and color development, dried, and the spots formed on the membrane were counted using an immuno-spot plate READER (ELISPOT READER system; AID20 Co.).
The experimental results are shown in fig. 8a and fig. 8b, and aiming at the target cells loaded with the SSX2 antigen short peptide AQIPEKIQK, the effector cells transfected with the high affinity TCR of the invention have a more obvious activation effect than the effector cells transfected with the wild type TCR, while the effector cells transfected with other TCRs are inactive; meanwhile, effector cells transfected with the inventive TCR are substantially inactive against other antigen-loaded short peptide or unloaded target cells.
Example 7 Experimental of the activation function of Effector cells transfected with the high affinity TCR of the invention against cell lines
This example again demonstrates the activation function and specificity of effector cells transfected with the high affinity TCRs of the invention using tumor cell lines. Again, detection is by ELISPOT assays well known to those skilled in the art. The high affinity TCR (TCR number and sequence number thereof) of the inventionFrom table 2) transfected into CD3 isolated from blood of healthy volunteers + T cells as effector cells and CD3 transfected with other TCR (A6) or with wild type TCR (WT-TCR) from the same volunteer + T cells served as controls.
The positive tumor cell lines used were SK-MEL-28-SSX2 (SK-MEL-28 cell line transfected with SSX2 antigen), huh-1-A11 (Huh-1 cell line transfected with HLA-A1101), and the negative cell lines were SK-MEL-5, LCLs, SK-MEL-1, SNU423, HUCCT1.
The experiment was carried out in two batches, both of which were carried out with the following steps: first, an ELISPOT plate was prepared. ELISPOT plates were ethanol activated coated and incubated overnight at 4 ℃. Day 1 of the experiment, coating was removed, washed and blocked, incubated at room temperature for two hours, blocking solution removed, and the components of the experiment were added to ELISPOT plates: target cells are 2 x 10 4 2 x 10 effector cells per well 3 One/well (calculated as positive rate of transfection) and two duplicate wells were set. Incubation overnight (37 ℃,5% 2 ). On day 2 of the experiment, the plate was washed and subjected to secondary detection and color development, the plate was dried, and spots formed on the membrane were counted using an immuno spot plate READER (ELISPOT READER system; AID20 Co.).
The experimental results are shown in fig. 9a and 9b, and for the positive tumor cell line, the effector cells transfected with the high affinity TCR of the present invention still have a more significant activation effect than the effector cells transfected with the wild type TCR, while the effector cells transfected with other TCRs are inactive; at the same time, effector cells transfected with the high affinity TCRs of the invention are essentially inactive against SSX2 negative cell lines.
Example 8 killing function experiment of Effector cells transfected with high affinity TCR of the invention
This example demonstrates the killing function of cells transfected with the high affinity TCRs of the invention by measuring LDH release by nonradioactive cytotoxicity assays well known to those skilled in the art. The assay is a colorimetric substitution assay for the 51Cr release cytotoxicity assay that quantitatively measures LDH released after cell lysis. LDH released in the medium was detected using a 30-minute coupled enzymatic reaction in which LDH can convert a tetrazolium salt (INT) to red formazan (formazan). The amount of red product produced is proportional to the number of cells lysed. 490nm visible absorbance data can be collected using a standard 96-well plate reader. The formula is calculated as% cytotoxicity =100% × (experiment-effector cell spontaneous-target cell spontaneous)/(target cell maximal-target cell spontaneous).
LDH experiments of this example CD3 isolated from blood of healthy volunteers + T cells transfected with the high affinity TCR of the invention (TCR numbering and sequence number are shown in Table 2) as effector cells and CD3 transfected with other TCRs (A6) from the same volunteer + T cells served as controls. The positive tumor cell lines used were SK-MEL-28-SSX2, huh-1-A11, TT _ THYROID-A11 (TT _ THYROID cell line transfected with HLA-A1101), and the negative cell lines were SK-MEL-28, LCLs, SNU423, HUCCT1, TT _ THYROID.
The experiment was carried out in two batches, the target cells used in the first batch being 2X 10 4 Single cell/well, effector cell 2X 10 4 Individual cells/well (calculated as transfection positivity); 3X 10 target cells for the second batch 4 Single cell/well, effector cell 3X 10 4 Individual cells/well (calculated as positive transfection rate).
Both batches were subjected to the following experimental procedure: LDH plates are prepared first, target cells and effector cells are added to corresponding wells of the plates, and three multiple wells are set. Meanwhile, an effector cell spontaneous hole, a target cell maximum hole, a volume correction control hole and a culture medium background control hole are arranged. Incubation overnight (37 ℃,5% CO) 2 ). On day 2 of the experiment, color development was detected, and after termination of the reaction, the absorbance was recorded at 490nm using a microplate reader (Bioteck).
Experimental results as shown in fig. 10a and 10b, the effector cells transfected with the high affinity TCRs of the invention showed strong killing efficacy against positive tumor cell lines, whereas T cells transfected with other TCRs did not respond; meanwhile, the T cells transfected with the high affinity TCR of the present invention had essentially no killing of the negative tumor cell line, against the performance of SNU423 as background.
Example 9 demonstration of the specificity of effector cells transducing the high affinity TCRs of the invention on human Normal tissue cells
This example demonstrates the specificity of effector cells transduced with the high affinity TCRs of the invention using human normal tissue cells. Again by ELISPOT assays well known to those skilled in the art. The high affinity TCRs of the invention (TCR numbering and their sequence numbers are given in Table 2) were lentivirally transduced to CD3 isolated from blood of healthy volunteers + T cells as effector cells and CD3 transduced by other TCRs (A6) or by non-human (NC) transduction in the same volunteer + T cells served as controls.
The positive tumor cell line used in the experiment is Huh-1-A1101, the negative tumor cell line is SNU423, and the human normal tissue cells used in the experiment are: hCD34+ _ CB1-45A (human CD34+ progenitor cells), HAoEC-46A (human aortic endothelial cells), HAoEC-46B (human aortic endothelial cells), HAoAF-47 (human aortic adventitial fibroblasts), HAoAF-47-A1101, HCAEC-49A (human coronary endothelial cells), HCAEC-49B, HCF-51 (human cardiac fibroblasts), HCMEC-52A (human cardiac microvascular endothelial cells), HCMEC-52C, HUtMEC-55A (human uterine microvascular endothelial cells), HUtMEC-55A-A1101 (HUtMEC-55A transfected with HLA-A1101), HUtMEC-55B (human uterine microvascular endothelial cells) HUF-56 (human uterine fibroblast), HUtSMC-57 (human uterine smooth muscle cell), HUtSMC-57-A1101, HRCEpC-58 (human renal cortical epithelial cell), HRCEpC-58-A1101, HREpC-59 (human renal epithelial cell), HREpC-59-A1101, HCH-61A (human chondrocyte), HCH-61A-A1101, HCH-61B (human chondrocyte), HCH-61B-A1101 (HCH-61B transfected with HLA-A1101), HOB-63A (human osteoblast), HPF-64 (human pulmonary fibroblast), HPF-64-A1101, HBEpC-70A (human bronchial epithelial cell), HBEpC-70B (human bronchial epithelial cell), HBSMC-71 (human bronchial smooth muscle cells), HNEpC-73 (human nasal epithelial cells), HNEpC-73-A1101, HDMEC-74 (human dermal microvascular endothelial cells, adolescents), HDMEC-74-A1101, HDMEC-75 (human dermal microvascular endothelial cells, adult), HDMEC-75-A1101, NHDF-76B (human dermal fibroblasts, adult), HCM-Donor B (human cardiomyocytes), HUM183231 (human hepatocytes). The human normal tissue cells used were purchased from Promocell and Lonza.
First, an ELISPOT plate was prepared. ELISPOT flat plate ethanol activation bagIt was dried overnight at 4 ℃. On day 1 of the experiment, the coating was removed, washed and blocked, incubated for two hours at room temperature, the blocking solution removed, and the components of the assay added to an ELISPOT plate: target cells are 2 x 10 4 Cell/well, effector cell 2 x 10 3 One well (calculated as the positive rate of transduction) and two duplicate wells were set. Incubation overnight (37 ℃,5% CO) 2 ). On day 2 of the experiment, the plate was washed and subjected to secondary detection and color development, the plate was dried, and spots formed on the membrane were counted using an immuno spot plate READER (ELISPOT READER system; AID20 Co.).
The experimental results are shown in fig. 11, and the effector cells transduced the inventive high affinity TCR were inactive against human normal tissue cells, further verifying the high specificity of the inventive TCR.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.
SEQUENCE LISTING
<110> snow-fragrance Life sciences technology (Guangdong) Co., ltd
<120> high affinity T cell receptor for antigen SSX2
<130> 215729
<160> 33
<170> PatentIn version 3.3
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Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly
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Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly
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Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly
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Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly
1 5 10 15
Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser
20 25 30
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35 40 45
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50 55 60
Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser
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Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly
1 5 10 15
Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser
20 25 30
Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu
35 40 45
Lys Arg Ser Asp Pro Ala Thr Leu Val Lys Gly Ile Asn Gly Phe Glu
50 55 60
Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser
65 70 75 80
Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Val Val Ser Pro Gly
85 90 95
Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala
100 105 110
<210> 13
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> alpha chain variable domain amino acid sequence of TCR molecule
<400> 13
Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly
1 5 10 15
Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser
20 25 30
Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu
35 40 45
Lys Ser Pro Pro Asn Ala Thr Leu Val Lys Gly Ile Asn Gly Phe Glu
50 55 60
Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser
65 70 75 80
Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Val Val Ser Leu Gly
85 90 95
Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala
100 105 110
<210> 14
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> alpha chain variable domain amino acid sequence of TCR molecule
<400> 14
Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly
1 5 10 15
Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser
20 25 30
Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu
35 40 45
Lys Ala Leu Pro Asn Ala Thr Leu Val Lys Gly Ile Asn Gly Phe Glu
50 55 60
Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser
65 70 75 80
Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Val Val Ser Pro Gly
85 90 95
Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala
100 105 110
<210> 15
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> alpha chain variable domain amino acid sequence of TCR molecule
<400> 15
Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly
1 5 10 15
Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser
20 25 30
Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Gly Asn Gly Ala Thr Leu Val Lys Gly Ile Asn Gly Phe Glu
50 55 60
Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser
65 70 75 80
Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Val Val Ser Pro Gly
85 90 95
Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala
100 105 110
<210> 16
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> alpha chain variable domain amino acid sequence of TCR molecule
<400> 16
Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly
1 5 10 15
Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser
20 25 30
Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Ser Tyr Gly Ala Thr Leu Val Lys Gly Ile Asn Gly Phe Glu
50 55 60
Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser
65 70 75 80
Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Val Val Ser Pro Gly
85 90 95
Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala
100 105 110
<210> 17
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> alpha chain variable domain amino acid sequence of TCR molecule
<400> 17
Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly
1 5 10 15
Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser
20 25 30
Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Ser His Gly Ala Thr Leu Val Lys Gly Ile Asn Gly Phe Glu
50 55 60
Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser
65 70 75 80
Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Val Val Ser Pro Gly
85 90 95
Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala
100 105 110
<210> 18
<211> 117
<212> PRT
<213> Artificial Sequence
<220>
<223> beta chain variable domain amino acid sequence of TCR molecule
<400> 18
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ala Arg
85 90 95
His Ala Gly Thr Ser Ala Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr
100 105 110
Arg Leu Thr Val Leu
115
<210> 19
<211> 117
<212> PRT
<213> Artificial Sequence
<220>
<223> beta chain variable domain amino acid sequence of TCR molecule
<400> 19
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ala Arg
85 90 95
His Val Gly Thr Ser Ala Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr
100 105 110
Arg Leu Thr Val Leu
115
<210> 20
<211> 117
<212> PRT
<213> Artificial Sequence
<220>
<223> beta chain variable domain amino acid sequence of TCR molecule
<400> 20
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ala Glu
85 90 95
His Val Gly Thr Ser Ala Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr
100 105 110
Arg Leu Thr Val Leu
115
<210> 21
<211> 117
<212> PRT
<213> Artificial Sequence
<220>
<223> beta chain variable domain amino acid sequence of TCR molecule
<400> 21
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ala Gln
85 90 95
His Val Gly Thr Ser Ala Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr
100 105 110
Arg Leu Thr Val Leu
115
<210> 22
<211> 117
<212> PRT
<213> Artificial Sequence
<220>
<223> beta chain variable domain amino acid sequence of TCR molecule
<400> 22
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ala Lys
85 90 95
His Val Gly Thr Ser Ala Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr
100 105 110
Arg Leu Thr Val Leu
115
<210> 23
<211> 117
<212> PRT
<213> Artificial Sequence
<220>
<223> beta chain variable domain amino acid sequence of TCR molecule
<400> 23
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ala Ala
85 90 95
His Val Gly Thr Ser Ala Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr
100 105 110
Arg Leu Thr Val Leu
115
<210> 24
<211> 117
<212> PRT
<213> Artificial Sequence
<220>
<223> beta chain variable domain amino acid sequence of TCR molecule
<400> 24
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ser Arg
85 90 95
His Ala Gly Thr Ser Ala Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr
100 105 110
Arg Leu Thr Val Leu
115
<210> 25
<211> 117
<212> PRT
<213> Artificial Sequence
<220>
<223> beta chain variable domain amino acid sequence of TCR molecule
<400> 25
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ser Glu
85 90 95
His Ala Gly Thr Ser Ala Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr
100 105 110
Arg Leu Thr Val Leu
115
<210> 26
<211> 117
<212> PRT
<213> Artificial Sequence
<220>
<223> beta chain variable domain amino acid sequence of TCR molecule
<400> 26
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ala Arg
85 90 95
His Ile Gly Thr Ser Ala Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr
100 105 110
Arg Leu Thr Val Leu
115
<210> 27
<211> 117
<212> PRT
<213> Artificial Sequence
<220>
<223> beta chain variable domain amino acid sequence of TCR molecule
<400> 27
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ala Glu
85 90 95
His Ile Gly Thr Ser Ala Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr
100 105 110
Arg Leu Thr Val Leu
115
<210> 28
<211> 117
<212> PRT
<213> Artificial Sequence
<220>
<223> beta chain variable domain amino acid sequence of TCR molecule
<400> 28
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ala Gln
85 90 95
His Ile Gly Thr Ser Ala Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr
100 105 110
Arg Leu Thr Val Leu
115
<210> 29
<211> 117
<212> PRT
<213> Artificial Sequence
<220>
<223> beta chain variable domain amino acid sequence of TCR molecule
<400> 29
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ser His
85 90 95
Thr Val Gly Thr Ser Ala Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr
100 105 110
Arg Leu Thr Val Leu
115
<210> 30
<211> 206
<212> PRT
<213> Artificial Sequence
<220>
<223> "wild type TCR" alpha chain extracellular amino acid sequence
<400> 30
Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly
1 5 10 15
Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser
20 25 30
Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Thr Ser Ala Ala Thr Leu Val Lys Gly Ile Asn Gly Phe Glu
50 55 60
Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser
65 70 75 80
Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Val Val Ser Pro Gly
85 90 95
Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala
100 105 110
Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys
115 120 125
Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr
130 135 140
Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr
145 150 155 160
Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala
165 170 175
Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser
180 185 190
Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser
195 200 205
<210> 31
<211> 247
<212> PRT
<213> Artificial Sequence
<220>
<223> extracellular amino acid sequence of beta chain of "wild type TCR
<400> 31
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ser Pro
85 90 95
Thr Val Gly Thr Ser Ala Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr
100 105 110
Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val
115 120 125
Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala
130 135 140
Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu
145 150 155 160
Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp
165 170 175
Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys
180 185 190
Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg
195 200 205
Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp
210 215 220
Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala
225 230 235 240
Glu Ala Trp Gly Arg Ala Asp
245
<210> 32
<211> 253
<212> PRT
<213> Homo Sapiens
<400> 32
Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly
1 5 10 15
Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser
20 25 30
Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Thr Ser Ala Ala Thr Leu Val Lys Gly Ile Asn Gly Phe Glu
50 55 60
Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser
65 70 75 80
Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Val Val Ser Pro Gly
85 90 95
Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala
100 105 110
Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys
115 120 125
Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr
130 135 140
Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr
145 150 155 160
Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala
165 170 175
Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser
180 185 190
Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp
195 200 205
Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe
210 215 220
Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala
225 230 235 240
Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
245 250
<210> 33
<211> 296
<212> PRT
<213> Homo Sapiens
<400> 33
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ser Pro
85 90 95
Thr Val Gly Thr Ser Ala Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr
100 105 110
Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val
115 120 125
Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala
130 135 140
Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu
145 150 155 160
Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp
165 170 175
Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys
180 185 190
Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg
195 200 205
Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp
210 215 220
Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala
225 230 235 240
Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln
245 250 255
Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys
260 265 270
Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met
275 280 285
Val Lys Arg Lys Asp Ser Arg Gly
290 295

Claims (10)

1. A T Cell Receptor (TCR) comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, characterised in that it has activity to bind to the AQIPEKIQK-HLA a1101 complex and in that the amino acid sequence of the TCR alpha chain variable domain has at least 90% sequence homology with the amino acid sequence set out in SEQ ID No. 1 and the amino acid sequence of the TCR beta chain variable domain has at least 90% sequence homology with the amino acid sequence set out in SEQ ID No. 2.
2. A multivalent TCR complex comprising at least two TCR molecules, at least one of which is a TCR as claimed in claim 1.
3. A nucleic acid molecule comprising a nucleic acid sequence encoding a TCR as claimed in claim 1, or the complement thereof.
4. A vector comprising the nucleic acid molecule of claim 3.
5. A host cell comprising the vector of claim 4 or a nucleic acid molecule of claim 3 integrated into the chromosome.
6. An isolated cell expressing a TCR as claimed in claim 1.
7. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR as claimed in claim 1, or a TCR complex as claimed in claim 2, or a cell as claimed in claim 6.
8. A method of treating a disease comprising administering to a subject in need thereof a TCR as claimed in claim 1 or a TCR complex as claimed in claim 2 or a cell as claimed in claim 6 or a pharmaceutical composition as claimed in claim 7, preferably wherein the disease is an SSX2 positive tumour.
9. Use of a T cell receptor as claimed in claim 1, a TCR complex as claimed in claim 2 or a cell as claimed in claim 6 in the manufacture of a medicament for the treatment of a tumour, preferably wherein the tumour is an SSX2 positive tumour.
10. A method of preparing a T cell receptor according to claim 1, comprising the steps of:
(i) Culturing the host cell of claim 5 so as to express the T-cell receptor of claim 1;
(ii) Isolating or purifying said T cells.
CN202110849328.0A 2021-07-27 2021-07-27 High affinity T cell receptors for the antigen SSX2 Pending CN115677846A (en)

Priority Applications (2)

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CN202110849328.0A CN115677846A (en) 2021-07-27 2021-07-27 High affinity T cell receptors for the antigen SSX2
PCT/CN2022/107561 WO2023005859A1 (en) 2021-07-27 2022-07-25 High-affinity t-cell receptor for antigen ssx2

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110849328.0A CN115677846A (en) 2021-07-27 2021-07-27 High affinity T cell receptors for the antigen SSX2

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WO (1) WO2023005859A1 (en)

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1691964A (en) * 2002-09-06 2005-11-02 曼康公司 Epitope sequences
CN106336457B (en) * 2015-10-30 2018-03-06 广东香雪精准医疗技术有限公司 Identify the φt cell receptor of MAGE A3 antigen small peptides
CA3080546A1 (en) * 2017-10-03 2019-04-11 Juno Therapeutics, Inc. Hpv-specific binding molecules
CA3100775A1 (en) * 2018-05-18 2019-11-21 Children's National Medical Center Improved targeted t-cell therapy
CN112409474B (en) * 2019-08-23 2023-02-28 香雪生命科学技术（广东）有限公司 High-affinity TCR (T cell receptor) for identifying SSX2 antigen

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