WO2023040946A1 - Tcr à haute affinité reconnaissant ssx2 - Google Patents

Tcr à haute affinité reconnaissant ssx2 Download PDF

Info

Publication number
WO2023040946A1
WO2023040946A1 PCT/CN2022/118984 CN2022118984W WO2023040946A1 WO 2023040946 A1 WO2023040946 A1 WO 2023040946A1 CN 2022118984 W CN2022118984 W CN 2022118984W WO 2023040946 A1 WO2023040946 A1 WO 2023040946A1
Authority
WO
WIPO (PCT)
Prior art keywords
tcr
seq
chain
variable domain
amino acid
Prior art date
Application number
PCT/CN2022/118984
Other languages
English (en)
Chinese (zh)
Inventor
陈少沛
李小林
刘敏
郑文静
唐先青
Original Assignee
香雪生命科学技术（广东）有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 香雪生命科学技术（广东）有限公司 filed Critical 香雪生命科学技术（广东）有限公司
Publication of WO2023040946A1 publication Critical patent/WO2023040946A1/fr

Links

Images

Classifications

    • 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
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present application belongs to the field of biotechnology, and more specifically relates to a T cell receptor (T cell receptor, TCR) capable of recognizing polypeptides derived from SSX2 protein.
  • T cell receptor T cell receptor, TCR
  • the present application also relates to the preparation and use of said receptors.
  • TCR T cell receptor
  • TCR is the sole receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC), and such exogenous or endogenous peptides may be the only sign of abnormalities in cells.
  • MHC major histocompatibility complex
  • the combination of antigen-specific TCR and pMHC complexes triggers direct physical contact between T cells and antigen-presenting cells (APCs), and then other cell membrane surface molecules of T cells and APCs interact. It causes a series of subsequent cell signal transmission and other physiological responses, so that T cells with different antigen specificities exert immune effects on their target cells.
  • APCs antigen-presenting cells
  • MHC class I and class II molecules corresponding to TCR are also proteins of the immunoglobulin superfamily, but they are specific for the presentation of antigens. Different individuals have different MHCs, so they can present different short sequences of a protein antigen. Peptides to the surface of the respective APC cells. Human MHC is often referred to as HLA genes or HLA complexes.
  • SSX2 is the synovial sarcoma X breakpoint, also known as HOM-MEL-40.
  • SSX2 is one of ten highly homologous nucleic acid proteins of the SSX family.
  • the SSX protein is a tumor-testis antigen expressed only in tumor cells and testicular germ cells without MHC expression.
  • 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. After SSX2 is produced in cells, it is degraded into small molecular polypeptides, and combines with MHC (major histocompatibility complex) molecules to form a complex, which is presented to the cell surface.
  • AQIPEKIQK (SEQ ID NO:47) is derived from SSX2 antigen of short peptides.
  • the AQIPEKIQK(SEQ ID NO:47)-HLA A1101 complex provides a TCR-targetable marker for tumor cells.
  • the TCR that can bind to the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex has high application value for the treatment of tumors.
  • a TCR capable of targeting this tumor cell marker can be used to deliver a cytotoxic or immunostimulatory agent to the target cell, or be transformed into a T cell, enabling the T cell expressing the TCR to destroy the tumor cell, so that in what is known as Administered to patients during the course of treatment with adoptive immunotherapy.
  • the ideal TCR is one with high affinity, allowing the TCR to reside on the targeted cell for a long period of time.
  • the use of intermediate affinity TCRs is preferred. Therefore, those skilled in the art are devoting themselves to developing TCRs targeting tumor cell markers that can be used for different purposes.
  • the application provides a TCR with high affinity for the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex.
  • the present application further provides a preparation method of the above-mentioned TCR and an application of the above-mentioned TCR.
  • the first aspect of the present application provides a T cell receptor (TCR) comprising an ⁇ -chain variable domain and a ⁇ -chain variable domain, which has the ability to bind to the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex activity, and the amino acid sequence of the TCR ⁇ chain variable domain has at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO: 1; and the amino acid sequence of the TCR ⁇ chain variable domain and SEQ ID NO: The amino acid sequences shown in 2 have at least 90% sequence homology.
  • TCR T cell receptor
  • the amino acid sequence of the variable domain of the TCR ⁇ chain and the amino acid sequence of the variable domain of the TCR ⁇ chain are different from the amino acid sequence of the variable domain of the wild-type TCR ⁇ chain and the amino acid sequence of the variable domain of the wild-type TCR ⁇ chain sequence.
  • the amino acid sequence of the TCR ⁇ chain variable domain is not the amino acid sequence shown in SEQ ID NO:1, or the amino acid sequence of the TCR ⁇ chain variable domain is not the amino acid sequence shown in SEQ ID NO:2 sequence.
  • the ⁇ -chain variable domain of the TCR comprises at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences with 98% or 99% sequence homology. Or relative to the sequence shown in SEQ ID NO: 1, there are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acid residue insertions, deletions, substitutions or combinations thereof.
  • the ⁇ chain variable domain of the TCR is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences with 98%, 99% or 100% sequence homology. Or relative to the sequence shown in SEQ ID NO: 1, there are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acid residue insertions, deletions, substitutions or combinations thereof.
  • the amino acid sequence of the variable domain of the TCR ⁇ 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 ⁇ chain is identical to that of SEQ ID NO: 1.
  • the amino acid sequence shown in ID NO:2 has at least 95% sequence homology.
  • the TCR is mutated in CDR3 ⁇ of the variable domain of the TCR ⁇ chain, and the number of mutations is 1-4, preferably 3 or 4.
  • the three CDRs of the TCR ⁇ chain variable domain are: CDR1 ⁇ : SGHVS (SEQ ID NO: 48); CDR2 ⁇ : FQNEAQ (SEQ ID NO: 49); and CDR3 ⁇ : ASSLRAGGNTIY (SEQ ID NO: :50);
  • the amino acid sequence of the TCR ⁇ chain variable domain is SEQ ID NO: 2.
  • CDR1 ⁇ in the variable domain of the TCR ⁇ chain is SSYSPS (SEQ ID NO:51)
  • CDR2 ⁇ is YTSAATLV (SEQ ID NO:52)
  • CDR3 ⁇ is selected from ALTLGNTPLV (SEQ ID NO:53), GMTLGNTPLV (SEQ ID NO:54) and AITLGNTPLV (SEQ ID NO:55).
  • amino acid mutation sites of the TCR ⁇ chain variable domain are positions 1, 2, 3 and 4 of CDR3 ⁇ .
  • amino acid mutation sites of the TCR ⁇ chain variable domain are the 2nd, 3rd and 4th positions of CDR3 ⁇ .
  • the reference sequences of the three CDR regions (complementarity determining regions) of the variable domain of the TCR ⁇ chain are as follows,
  • CDR1 ⁇ SSYSPS (SEQ ID NO:51);
  • CDR3 ⁇ GGSIGNTPLV (SEQ ID NO:56), and contains at least one of the following mutations:
  • amino acid mutation in CDR3 ⁇ comprises:
  • amino acid mutation in CDR3 ⁇ comprises:
  • amino acid mutation in the CDR3 ⁇ comprises:
  • amino acid mutation in CDR3 ⁇ comprises:
  • the reference sequences of the three CDR regions (complementarity determining regions) of the TCR ⁇ chain variable domain are as follows,
  • CDR1 ⁇ SGHVS (SEQ ID NO: 48);
  • CDR3 ⁇ ASSLRAGGNTIY (SEQ ID NO:50), and CDR3 ⁇ contains at least one of the following mutations:
  • the affinity between the TCR and the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex is at least 5 times that of the wild-type TCR.
  • the TCR is mutated in the ⁇ chain variable domain shown in SEQ ID NO: 1, and the mutation is selected from I95L/P/V/N, S94T/H, G93L/M/I One or several groups in /S, G92A, S28V/T/I, S30T/D, S32W/Y, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO:1.
  • the TCR is mutated in the ⁇ chain variable domain shown in SEQ ID NO: 2, and the mutation is selected from S95Q, L96A/E/P, R97Y/F/H/W, A98P , G100S, N101S/T, T102D, I103F, Y104T/H one or more groups, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO:2.
  • the TCR has a CDR selected from the group consisting of:
  • the TCR is soluble.
  • the TCR is an ⁇ heterodimeric TCR, comprising a TRAC constant region sequence of the ⁇ chain and a TRBC1 or TRBC2 constant region sequence of the ⁇ chain.
  • the TCR comprises (i) the TCR ⁇ chain variable domain and all or part of the TCR ⁇ chain constant region except its transmembrane domain; and (ii) the TCR ⁇ chain variable domain and its transmembrane domain All or part of the constant region of the TCR ⁇ chain outside the domain.
  • an artificial interchain disulfide bond is contained between the constant region of the ⁇ chain and the constant region of the ⁇ chain of the TCR.
  • cysteine residues forming an artificial interchain disulfide bond between the constant regions of the TCR ⁇ and ⁇ chains are substituted for one or more groups of sites selected from the following:
  • the amino acid sequence of the ⁇ -chain variable domain of the TCR is one of SEQ ID NO: 1, 13-30; and/or the amino acid sequence of the ⁇ -chain variable domain of the TCR is SEQ ID NO: 2.
  • SEQ ID NO: 1 One of 31-42.
  • the TCR is selected from the following group:
  • the TCR is of human origin.
  • the TCR is isolated and purified.
  • the TCR is a single-chain TCR.
  • the TCR is a single-chain TCR composed of an ⁇ -chain variable domain and a ⁇ -chain variable domain, and the ⁇ -chain variable domain and the ⁇ -chain variable domain are composed of a flexible short peptide sequence (linker )connect.
  • the TCR comprises an ⁇ -chain constant region and a ⁇ -chain constant region
  • the ⁇ -chain constant region is a mouse constant region and/or the ⁇ -chain constant region is a mouse constant region.
  • the TCR comprises an ⁇ -chain constant region and a ⁇ -chain constant region, the ⁇ -chain constant region is a murine constant region and/or the ⁇ -chain constant region is a murine constant region.
  • a conjugate is bound to the C- or N-terminus of the ⁇ chain and/or ⁇ chain of the TCR, preferably, the conjugate is a detectable marker or a therapeutic agent.
  • the therapeutic agent that binds to the TCR is an anti-CD3 antibody linked to the C- or N-terminus of the ⁇ or ⁇ chain of the TCR.
  • the second aspect of the present application provides a multivalent TCR complex, which comprises at least two TCR molecules, and at least one of the TCR molecules is the TCR described in the first aspect of the present application.
  • the third aspect of the present application provides a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule described in the first aspect of the present application or the multivalent TCR complex described in the second aspect of the present application or its complement sequence.
  • the fourth aspect of the present application provides a vector containing the nucleic acid molecule described in the third aspect of the present application.
  • the fifth aspect of the present application provides a host cell containing the vector described in the fourth aspect of the present application or the exogenous nucleic acid molecule described in the third aspect of the present application integrated into the chromosome.
  • the sixth aspect of the present application provides an isolated cell expressing the TCR described in the first aspect of the application, preferably, the isolated cell is a T cell, NK cell or NKT cell, more preferably , the isolated cells are T cells.
  • the seventh aspect of the present application provides a pharmaceutical composition, which contains a pharmaceutically acceptable carrier and the TCR described in the first aspect of the present application, or the TCR complex described in the second aspect of the present application, Or the cell described in the sixth aspect of the present application.
  • the eighth aspect of the present application provides a method for treating diseases, comprising administering an appropriate amount of the TCR described in the first aspect of the present application, or the TCR complex described in the second aspect of the present application, or the For the cell described in the sixth aspect of the application, or the pharmaceutical composition described in the seventh aspect of the application, preferably, the disease is a SSX2-positive tumor.
  • the ninth aspect of the present application provides the use of the TCR described in the first aspect of the present application, or the TCR complex described in the second aspect of the present application, or the use of the cells described in the sixth aspect of the present application, for preparing and treating tumors
  • the disease is a SSX2 positive tumor.
  • the tenth aspect of the present application provides a method for preparing the T cell receptor described in the first aspect of the present application, comprising the steps of:
  • Figure 1a and Figure 1b respectively show the amino acid sequences of the variable domains of the wild-type TCR ⁇ chain and ⁇ chain that can specifically bind to the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex.
  • Figure 2a and Figure 2b respectively show the amino acid sequence of the ⁇ -chain variable domain and the amino acid sequence of the ⁇ -chain variable domain of the single-chain template TCR constructed in the present application.
  • Figure 3a and Figure 3b are the DNA sequences of the ⁇ -chain variable domain and the ⁇ -chain variable domain of the single-chain template TCR constructed in the present application, respectively.
  • Figure 4a and Figure 4b are the amino acid sequence and DNA sequence of the linker of the single-chain template TCR constructed in the present application, respectively.
  • Figure 5a and Figure 5b are the amino acid sequence and DNA sequence of the single-stranded template TCR constructed in the present application, respectively.
  • Figure 6a and Figure 6b are the amino acid sequences of the soluble reference TCR ⁇ chain and ⁇ chain in this application, respectively.
  • Figure 7 (1)-(18) shows the ⁇ -chain variable domain amino acid sequence of the heterogeneous dimerization TCR with high affinity to AQIPEKIQK (SEQ ID NO:47)-HLA A1101 complex respectively, the residue of mutation is added underlined.
  • Figure 8 (1)-(12) shows the amino acid sequence of the ⁇ chain variable domain of the heterogeneous dimerization TCR with high affinity to AQIPEKIQK (SEQ ID NO:47)-HLA A1101 complex respectively, the residue of mutation is added underlined.
  • Figure 9a and Figure 9b respectively show the extracellular amino acid sequences of the wild-type TCR ⁇ chain and ⁇ chain that can specifically bind to the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex.
  • Figure 10a and Figure 10b respectively show the amino acid sequences of the wild-type TCR ⁇ chain and ⁇ chain that can specifically bind to the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex.
  • Figure 11 is the binding curve of the soluble reference TCR, that is, the wild-type TCR and the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex.
  • Figure 12 is the binding curve of soluble single-chain TCR and AQIPEKIQK (SEQ ID NO:47)-HLA A1101 complex.
  • Fig. 13 is the experimental result of the activation function of the effector cells transfected with the high-affinity TCR of the present application for T2 cells loaded with short peptides.
  • Fig. 14a, Fig. 14b and Fig. 14c are the experimental results of the activation function of the effector cells transfected with the high-affinity TCR of the present application for tumor cell lines.
  • FIGS 15a and 15b show the killing function LDH experiment results of the effector cells transfected with the high-affinity TCR of the present application for tumor cell lines.
  • TCR high-affinity T cell receptor
  • SEQ ID NO: 47 short peptide (derived from SSX2 protein), and the AQIPEKIQK (SEQ ID NO: 47) 47) Short peptides are presented as peptide-HLA A1101 complexes.
  • the high-affinity TCR has three CDR regions in its ⁇ -chain variable domain:
  • CDR1 ⁇ SSYSPS (SEQ ID NO:51);
  • CDR3 ⁇ Mutation in GGSIGNTPLV (SEQ ID NO:56);
  • CDR1 ⁇ SGHVS (SEQ ID NO: 48);
  • CDR3 ⁇ Mutation occurs in ASSLRAGGNTIY (SEQ ID NO:50); and, after the mutation, the affinity and/or binding half-life of the TCR of the present application to the above-mentioned AQIPEKIQK (SEQ ID NO:47)-HLA A1101 complex is at least 5 times that of the wild-type TCR times.
  • T cell receptor T cell receptor, TCR
  • TCRs can be described using the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • Natural ⁇ heterodimeric TCRs have an ⁇ chain and a ⁇ chain.
  • each chain comprises a variable region, a connecting region, and a constant region
  • the beta strands usually also contain a short variable region between the variable and connecting regions, but this variable region is often considered part of the connecting region.
  • the junction region of the TCR is determined by the unique TRAJ and TRBJ of IMGT, and the constant region of the TCR is determined by the TRAC and TRBC of IMGT.
  • Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, chimeric in the framework sequence.
  • TRAV and TRBV refer to different V ⁇ types and V ⁇ types, respectively.
  • the ⁇ -chain constant domain has the following symbols: TRAC*01, where "TR” indicates the T cell receptor gene; "A” indicates the ⁇ -chain gene; C indicates the constant region; "*01” indicates allele Gene 1.
  • the beta chain constant domain has the following symbols: TRBC1*01 or TRBC2*01, where "TR” indicates the T cell receptor gene; "B” indicates the beta chain gene; C indicates the constant region; "*01” indicates the 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". Those skilled in the art can obtain the constant region gene sequences of TCR ⁇ and ⁇ chains through the published IMGT database.
  • TCR ⁇ chain variable domain refers to the connected TRAV and TRAJ regions
  • TCR ⁇ chain variable domain refers to the connected TRBV and TRBD/TRBJ regions.
  • the three CDRs of the variable domain of the TCR ⁇ chain are CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ ; the three CDRs of the variable domain of the TCR ⁇ chain are CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ .
  • the framework sequence of the TCR variable domain of the present application can be of murine or human origin, preferably of human origin.
  • the constant domain of a TCR comprises an intracellular portion, a transmembrane region and an extracellular portion.
  • amino acid sequences of the variable domains of the ⁇ and ⁇ chains of the wild-type TCR capable of binding to the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex are SEQ ID NO: 1 and SEQ ID NO: 2, respectively, as shown in Figure 1a and shown in Figure 1b.
  • the ⁇ -chain amino acid sequence and ⁇ -chain amino acid sequence of the soluble "reference TCR" described in this application are SEQ ID NO: 11 and SEQ ID NO: 12, respectively, as shown in Figure 6a and Figure 6b.
  • the ⁇ -chain extracellular amino acid sequence and ⁇ -chain extracellular amino acid sequence of the "wild-type TCR” described in this application are SEQ ID NO: 43 and SEQ ID NO: 44, respectively, as shown in Figure 9a and Figure 9b.
  • the TCR sequences used in this application are of human origin.
  • the ⁇ -chain amino acid sequence and ⁇ -chain amino acid sequence of the "wild-type TCR” described in this application are SEQ ID NO: 45 and SEQ ID NO: 46, respectively, as shown in Figures 10a and 10b.
  • the terms "polypeptide of the present application", “TCR of the present application”, “T cell receptor of the present application” are used interchangeably.
  • the position numbers of the amino acid sequences of TRAC*01 and TRBC1*01 or TRBC2*01 in this application are numbered in sequence from the N-terminal to the C-terminal, such as in TRBC1*01 or TRBC2*01, according to the sequence from N
  • the 60th amino acid in the sequence from terminal to C-terminal is P (proline)
  • it can be described as Pro60 of exon 1 of TRBC1*01 or TRBC2*01 in this application and it can also be expressed as TRBC1* 01 or the 60th amino acid of exon 1 of TRBC2*01, and for example in TRBC1*01 or TRBC2*01
  • the 61st amino acid is Q (glutamine) in the order from N-terminal to C-terminal, then this
  • it can be described as Gln61 of exon 1 of TRBC1*01 or TRBC2*01, or it can be expressed as amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01,
  • the position numbers of the amino acid sequences of the variable regions TRAV and TRBV are according to the position numbers listed in IMGT. For example, if a certain amino acid in TRAV is numbered 46 in IMGT, it will be described as the 46th amino acid in TRAV in this application, and so on. In this application, if there are special instructions for the sequence position numbers of other amino acids, the special instructions shall be followed.
  • tumors include, but are not limited to: solid tumors, soft tissue tumors, and metastatic lesions.
  • solid tumors include: malignant tumors of different organ systems, such as sarcomas, squamous cell carcinomas of the lung, and carcinomas. Examples: Infected prostate, lung, breast, lymph, gastrointestinal (eg colon), and genitourinary tract (eg kidney, epithelium), pharynx.
  • Squamous cell carcinoma of the lung includes malignancies such as most colon, rectal, renal cell, liver, non-small cell carcinomas of the lung, small intestine, and esophagus. Metastatic lesions of the above cancers can also be treated and prevented by the methods and compositions of the present application.
  • ⁇ -chain variable domain and ⁇ -chain variable domain of TCR each contain 3 CDRs, which are similar to the complementarity determining regions of antibodies.
  • CDR3 interacts with antigenic short peptides
  • CDR1 and CDR2 interact with HLA. Therefore, the CDR of the TCR molecule determines its interaction with the antigen short peptide-HLA complex.
  • variable domain amino acid sequence and the ⁇ chain of the wild-type TCR that can bind to the antigenic short peptide AQIPEKIQK (SEQ ID NO: 47) and the HLA A1101 complex (that is, the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex)
  • the amino acid sequences of the variable domains are respectively SEQ ID NO: 1 and SEQ ID NO: 2, which were discovered by the inventors for the first time. It has the following CDR regions:
  • CDR1 ⁇ SSYSPS (SEQ ID NO:51);
  • CDR3 ⁇ GGSIGNTPLV (SEQ ID NO: 56);
  • CDR1 ⁇ SGHVS (SEQ ID NO: 48);
  • CDR3 ⁇ ASSLRAGGNTIY (SEQ ID NO: 50).
  • the present application has obtained the affinity for the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex by performing mutation screening on the above CDR region, which is at least 5 times higher affinity TCRs.
  • the TCR described in the present application is an ⁇ heterodimeric TCR
  • the ⁇ chain variable domain of the TCR comprises at least 85% of the amino acid sequence shown in SEQ ID NO:1; preferably, at least 90%; more preferably Preferably, at least 92%; most preferably, at least 94% (e.g., may be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% %, 99% sequence homology) amino acid sequence of sequence homology); and/or the ⁇ chain variable domain of the TCR comprises at least 90% of the amino acid sequence shown in SEQ ID NO:2, preferably , at least 92%; more preferably, at least 94% (e.g., may be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity Homology) amino acid sequence of sequence homology.
  • the TCR described in the present application is a single-chain TCR
  • the ⁇ -chain variable domain of the TCR comprises at least 85%, preferably at least 90%, of the amino acid sequence shown in SEQ ID NO:3; more preferably, at least 92%; most preferably, at least 94% (e.g., may be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% % sequence homology) amino acid sequence of sequence homology); and/or the beta chain variable domain of the TCR comprises at least 85%, preferably at least 90% of the amino acid sequence shown in SEQ ID NO:4 %; more preferably, at least 92%; most preferably, at least 94%; (e.g., may be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence of sequence homology).
  • the three CDRs of the wild-type TCR ⁇ chain variable domain SEQ ID NO: 1, namely CDR1, CDR2 and CDR3 are respectively located at positions 27-32, 50-57 and 92-101 of SEQ ID NO: 1 . Accordingly, the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 1, 95I is the fourth position I of CDR3 ⁇ , 94S is the third position S of CDR3 ⁇ , 93G is the second position G of CDR3 ⁇ , and 92G is the The first G of CDR3 ⁇ , 28S is the second S of CDR1 ⁇ , 30S is the fourth S of CDR1 ⁇ , and 32S is the sixth S of CDR1 ⁇ .
  • variable domain of the ⁇ chain examples include I95L/P/V/N, S94T/H, G93L/M/I/S, G92A, S28V/T/I, S30T/D, S32W/ One or more groups in Y.
  • the 3 CDRs of the wild-type TCR ⁇ chain variable domain SEQ ID NO: 2, namely CDR1, CDR2 and CDR3 are respectively located at positions 27-31, 49-54 and 93-104 of SEQ ID NO: 2 .
  • the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 1, 95S is the third S of CDR3 ⁇ , 96L is the fourth L of CDR3 ⁇ , 97R is the fifth R of CDR3 ⁇ , and 98A is The 6th A of CDR3 ⁇ , 100G is the 8th G of CDR3 ⁇ , 101N is the 9th N of CDR3 ⁇ , 102T is the 10th T of CDR3 ⁇ , 103I is the 11th I of CDR3 ⁇ , 104Y is It is the 12th Y of CDR3 ⁇ .
  • the specific form of the mutation in the variable domain of the ⁇ chain includes one of S95Q, L96A/E/P, R97Y/F/H/W, A98P, G100S, N101S/T, T102D, I103F, and Y104T/H group or groups.
  • amino acids in this article are identified by international single English letters, and the corresponding three-letter abbreviations of 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);
  • Pro60 or 60P both represent the 60th proline.
  • expression of the specific form of the mutation described in this application is such as "I95L/P/V/N" means that the I at the 95th position is replaced by L or by P or by V or by N, and so on .
  • the Thr48 of the exon 1 of the TRAC*01 exon of the wild-type TCR ⁇ chain constant region was mutated to cysteine, and the ⁇ chain constant region TRBC1*01 or TRBC2*01 exon 1 was mutated.
  • the Ser57 of Ser57 is mutated to cysteine to obtain a reference TCR, the amino acid sequences of which are SEQ ID NO: 11 and SEQ ID NO: 12, respectively, as shown in Figure 6a and Figure 6b, the mutated cysteine residue Indicated in bold letters.
  • the above-mentioned cysteine substitutions enable the formation of artificial interchain disulfide bonds between the constant regions of the ⁇ and ⁇ chains of the reference TCR to form a more stable soluble TCR, thereby enabling more convenient evaluation of TCR and AQIPEKIQK (SEQ ID NO :47)-Binding affinity and/or binding half-life between HLA A1101 complexes. It should be understood that the CDR region of the TCR variable region determines its affinity with the pMHC complex, therefore, the above-mentioned cysteine substitution in the TCR constant region will not affect the binding affinity and/or binding half-life of the TCR.
  • the measured binding affinity between the reference TCR and the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex is considered to be the binding affinity between the wild-type TCR and the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex. Binding affinity between complexes.
  • the binding affinity between the TCR of the present application and the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex is determined to be the binding affinity between the reference TCR and the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex.
  • the affinity is at least 5 times that of the wild-type TCR and the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex. At least 5 times the binding affinity between
  • Binding affinity (inversely proportional to the dissociation equilibrium constant KD ) and binding half-life (expressed as T 1/2 ) can be determined by any suitable method, such as by surface plasmon resonance techniques. It will be appreciated that doubling the affinity of a TCR will result in a halving of the KD . T 1/2 was calculated as In2 divided by the off-rate (K off ). Therefore, doubling T 1/2 causes K off to be halved.
  • the same assay protocol is used to detect the binding affinity or binding half-life of a given TCR several times, eg 3 times or more, and the results are averaged.
  • the surface plasmon resonance (BIAcore) method in the examples herein is used to detect the affinity of soluble TCR, and the conditions are: temperature 25° C., pH value 7.1-7.5.
  • This method detects that the dissociation equilibrium constant K D of the reference TCR to the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex is 9.07E-04M, that is, 907 ⁇ M.
  • the wild-type TCR has a negative effect on the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex.
  • the dissociation equilibrium constant K D of NO:47)-HLA A1101 complex was also 907 ⁇ M.
  • the affinity of the TCR to the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex is at least 5 times that of the wild-type TCR.
  • Mutations may be performed using any suitable method, including but not limited to those based on polymerase chain reaction (PCR), restriction enzyme based cloning or ligation independent cloning (LIC) methods. These methods are detailed in many standard molecular biology textbooks. More details on polymerase chain reaction (PCR) mutagenesis and restriction enzyme based cloning can be found in Sambrook and Russell, (2001) Molecular Cloning - A Laboratory Manual (Third Edition) CSHL publishing house. More information on the LIC method can be found in (Rashtchian, (1995) Curr Opin Biotechnol 6(1):30-6).
  • PCR polymerase chain reaction
  • LIC ligation independent cloning
  • the method for producing the TCR of the present application may be, but not limited to, screening a TCR with high affinity for the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex from a diversity library of phage particles displaying such TCR, such as Described in the literature (Li, et al (2005) Nature Biotech 23(3):349-354).
  • genes expressing the variable domain amino acids of the alpha and beta chains of wild-type TCRs or genes expressing the amino acids of the variable domains of the alpha and beta chains of wild-type TCRs with slight modifications can be used to make template TCRs.
  • the changes required to generate the high affinity TCRs of the present application are then introduced into the DNA encoding the variable domains of this template TCR.
  • the high-affinity TCR of the present application comprises the amino acid sequence of the ⁇ -chain variable domain as one of SEQ ID NO:1, 13-30; and/or the amino acid sequence of the ⁇ -chain variable domain of the TCR as SEQ ID NO:2, One of 31-42.
  • the amino acid sequences of the ⁇ -chain variable domain and the ⁇ -chain variable domain of the heterodimeric TCR molecule of the present application are preferably selected from the following table 1:
  • a TCR of the present application is a portion having at least one variable domain of a TCR alpha and/or TCR beta chain. They usually contain both TCR ⁇ chain variable domains and TCR ⁇ chain variable domains. They may be ⁇ heterodimers or single-chain forms or any other forms that can exist stably.
  • the full-length chain of the ⁇ heterodimeric TCR (including the cytoplasmic and transmembrane domains) can be transfected.
  • the TCR of the present application can be used as a targeting agent to deliver therapeutic agents to antigen-presenting cells or be combined with other molecules to prepare bifunctional polypeptides to target effector cells. At this time, TCR is preferably in a soluble form.
  • the TCR of the present application may be a TCR that introduces an artificial interchain disulfide bond between residues of its alpha and beta chain constant domains.
  • Cysteine residues form artificial interchain disulfide bonds between the alpha and beta chain constant domains of the TCR. Cysteine residues can be substituted for other amino acid residues at appropriate sites in native TCRs to form artificial interchain disulfide bonds.
  • substitution of Thr48 of exon 1 of TRAC*01 and substitution of Ser57 of exon 1 of TRBC1*01 or TRBC2*01 to form disulfide bonds can also be: Thr45 of TRAC*01 exon 1 and Ser77 of TRBC1*01 or TRBC2*01 exon 1; TRAC*01 exon Tyr10 of 1 and Ser17 of exon 1 of TRBC1*01 or TRBC2*01; Thr45 of exon 1 of TRAC*01 and Asp59 of exon 1 of TRBC1*01 or TRBC2*01; of exon 1 of TRAC*01 Ser15 and Glu15 of exon 1 of TRBC1*01 or TRBC2*01; Arg53 of exon 1 of TRAC*01 and Ser54 of exon 1 of TRBC1*01 or TRBC2*01; Pro89 of exon 1 of TRAC*01 and Ala19 of exon 1 of TRBC1*01 or TRBC2*01;
  • cysteine residues replace any one group of positions in the above-mentioned ⁇ and ⁇ chain constant domains.
  • a maximum of 15, or a maximum of 10, or a maximum of 8 or fewer amino acids may be truncated at one or more C-terminal ends of the TCR constant domains of the present application, so that it does not include a cysteine residue to achieve the deletion of the native
  • the purpose of interchain disulfide bonds can also be achieved by mutating the cysteine residue that forms the natural interchain disulfide bond to another amino acid.
  • the TCRs of the present application may contain artificial interchain disulfide bonds introduced between residues in the constant domains of their alpha and beta chains. It should be noted that the TCR of the present application can contain both the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence, with or without the introduced artificial disulfide bond between the constant domains as described above.
  • the TRAC constant domain sequence of the TCR and the TRBC1 or TRBC2 constant domain sequence may be linked by native interchain disulfide bonds present in the TCR.
  • the patent document PCT/CN2016/077680 also discloses that the introduction of an artificial interchain disulfide bond between the ⁇ -chain variable region and the ⁇ -chain constant region of TCR can significantly improve the stability of TCR. Therefore, the high-affinity TCR of the present application may also contain an artificial interchain disulfide bond between the variable region of the ⁇ chain and the constant region of the ⁇ chain.
  • cysteine residue that forms an artificial interchain disulfide bond between the ⁇ -chain variable region and the ⁇ -chain constant region of the TCR is substituted for: amino acid 46 of TRAV and TRBC1*01 or TRBC2* Amino acid 60 of exon 1 of 01; amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01; amino acid 46 of TRAV and exon of TRBC1*01 or TRBC2*01 Amino acid 61 of exon 1; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01.
  • such a TCR may comprise (i) all or part of a TCR alpha chain except for its transmembrane domain, and (ii) all or part of a TCR beta chain except for its transmembrane domain, wherein (i) and (ii ) all contain the variable domain of the TCR chain and at least a part of the constant domain, and the ⁇ chain and the ⁇ chain form a heterodimer.
  • such a TCR may comprise an ⁇ -chain variable domain and a ⁇ -chain variable domain and all or part of a ⁇ -chain constant domain except the transmembrane domain, but it does not contain an ⁇ -chain constant domain, and the ⁇ -chain of said TCR Chain variable domains form heterodimers with beta chains.
  • the TCRs of the present application also include TCRs that undergo mutations in their hydrophobic core regions, and these mutations in the hydrophobic core regions are preferably mutations that can improve the stability of the TCRs of the present application, as disclosed in Publication No. described in the patent literature of WO2014/206304.
  • TCRs may have mutations at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable domain amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or Or the penultimate 3rd, 5th, and 7th amino acid positions of the ⁇ -chain J gene (TRAJ) short peptide, and/or the penultimate 2nd, 4th, and 6th amino acid positions of the ⁇ -chain J gene (TRBJ) short peptide amino acid position, where the position number of the amino acid sequence By position number as listed in the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • the TCR with mutations in the hydrophobic core region of the present application may be a highly stable single-chain TCR composed of a flexible peptide chain connecting the variable domains of the ⁇ chain and the ⁇ chain of the TCR.
  • the CDR region of the TCR variable region determines its affinity with the short peptide-HLA complex.
  • the mutation of the hydrophobic core can make the TCR more stable, but it will not affect its affinity with the short peptide-HLA complex.
  • the flexible peptide chain in this application can be any peptide chain suitable for linking the variable domains of TCR ⁇ and ⁇ chains.
  • the template strand used for screening high-affinity TCR constructed in Example 1 of the present application is the above-mentioned highly stable single-chain TCR containing a hydrophobic core mutation. Using a TCR with higher stability can more conveniently evaluate the affinity between the TCR and the AQIPEKIQK(SEQ IDNO:47)-HLA A1101 complex.
  • the CDR regions of the ⁇ -chain variable domain and the ⁇ -chain variable domain of the single-chain template TCR are completely identical to the CDR regions of the wild-type TCR. That is, the three CDRs of the ⁇ -chain variable domain are CDR1 ⁇ : SSYSPS (SEQ ID NO:51); CDR2 ⁇ : YTSAATLV (SEQ ID NO:52); CDR3 ⁇ : GGSIGNTPLV (SEQ ID NO:56) and the ⁇ -chain variable domain
  • the three CDRs are CDR1 ⁇ : SGHVS (SEQ ID NO: 48); CDR2 ⁇ : FQNEAQ (SEQ ID NO: 49); CDR3 ⁇ : ASSLRAGGNTIY (SEQ ID NO: 50).
  • the amino acid sequence (SEQ ID NO: 9) and nucleotide sequence (SEQ ID NO: 10) of the single-stranded template TCR are shown in Figures 5a and 5b, respectively.
  • a single-chain TCR composed of an ⁇ -chain variable domain and a ⁇ -chain variable domain with high affinity to the AQIPEKIQK (SEQ ID NO:47)-HLA A1101 complex was screened out.
  • the ⁇ heterodimer with high affinity to the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex of the present application is obtained by combining the ⁇ and ⁇ chains of the screened high-affinity single-chain TCR The CDR region of the variable domain is transferred to the corresponding positions of the wild-type TCR ⁇ chain variable domain (SEQ ID NO:1) and the ⁇ chain variable domain (SEQ ID NO:2).
  • the TCRs of the present application may also be provided in the form of multivalent complexes.
  • the multivalent TCR complex of the present application comprises two, three, four or more multimers formed by combining the TCRs of the present application, such as the tetramerization domain of p53 can be used to produce tetramers, or multimers A complex formed by the combination of a TCR of the present application and another molecule.
  • the TCR complexes of the present application can be used to track or target cells presenting specific antigens in vitro or in vivo, and can also be used to generate intermediates for other multivalent TCR complexes with such applications.
  • the TCR of the present application can be used alone, and can also be combined with a conjugate in a covalent or other manner, preferably in a covalent manner.
  • the conjugates include detectable markers (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the AQIPEKIQK (SEQ ID NO:47)-HLA A1101 complex), therapeutic agents, PK (protein kinase) A modifying moiety or any combination of the above is combined or coupled.
  • Detectable labels for diagnostic purposes include, but are not limited to: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or products capable of producing detectable enzymes.
  • Therapeutic agents that can be combined or coupled with the TCR of the present application include but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews (Cancer metastasis reviews) 24, 539); 2. Biological toxicity (Chaudhary et al., 1989 , Nature (Nature) 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy (Cancer Immunology and Immunotherapy) 51, 565); 3. Cytokines such as IL-2 etc.
  • Gold nanoparticles /Nanorods (Lapotko et al., 2005, Cancer letters (Cancer letters) 239, 36; Huang et al., 2006, Journal of the American Chemical Society (Journal of the American Chemical Society) 128, 2115); 7. Virus particles (Peng et al., 2004 , Gene therapy (Gene therapy) 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research (Cancer research) 65, 11631); 9. Nanomagnetic particles; 10. Prodrug activating enzymes (for example, DT - diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)); 11. Chemotherapeutic agents (eg, cisplatin) or nanoparticles in any form, etc.
  • DTD DT - diaphorase
  • BPHL biphenylhydrolase-like protein
  • Antibodies or fragments thereof that bind to the TCR of the present application include anti-T cells or NK-cell determination antibodies, such as anti-CD3 or anti-CD28 or anti-CD16 antibodies, and the combination of the above-mentioned antibodies or fragments thereof and TCR can carry out effects on effector cells. Orientation to better target target cells.
  • a preferred embodiment is that the TCR of the present application is combined with an anti-CD3 antibody or a functional fragment or variant of the anti-CD3 antibody.
  • the fusion molecule of the TCR of the present application and the anti-CD3 single-chain antibody comprises an amino acid sequence selected from the variable domain of the TCR ⁇ chain as one of SEQ ID NO: 1, 13-30; and/or the variable ⁇ chain of the TCR
  • the domain amino acid sequence is one of SEQ ID NO:2, 31-42.
  • the present application also relates to nucleic acid molecules encoding the TCRs of the present application.
  • the nucleic acid molecules of the present application may be in the form of DNA or RNA.
  • DNA can be either the coding strand or the non-coding strand.
  • the nucleic acid sequence encoding the TCR of the present application may be the same as or a degenerate variant of the nucleic acid sequence shown in the drawings of the present application.
  • degenerate variant refers to a protein sequence that encodes a protein having SEQ ID NO:3, but has a sequence that is identical to the sequence of SEQ ID NO:5 different nucleic acid sequences.
  • the full-length sequence of the nucleic acid molecule of the present application or its fragments can usually be obtained by, but not limited to, PCR amplification, recombination or artificial synthesis.
  • the DNA sequence encoding the TCR of the present application (or its fragments, or its derivatives) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • the present application also relates to vectors comprising the nucleic acid molecules of the present application, and host cells produced by genetic engineering using the vectors or coding sequences of the present application.
  • the present application also includes isolated cells expressing the TCRs of the present application, especially T cells.
  • T cells There are many methods suitable for T cell transfection with DNA or RNA encoding the high-affinity TCR of the present application (eg, Robbins et al., (2008) J. Immunol. 180:6116-6131).
  • T cells expressing the high-affinity TCR of this application can be used for adoptive immunotherapy.
  • Many suitable methods of performing adoptive therapy are known to those skilled in the art (eg, Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).
  • the present application also provides a pharmaceutical composition, which contains a pharmaceutically acceptable carrier and the TCR of the present application, or the TCR complex of the present application, or a cell presenting the TCR of the present application.
  • the present application also provides a method for treating diseases, comprising administering an appropriate amount of the TCR of the present application, or the TCR complex of the present application, or the cells presenting the TCR of the present application, or the pharmaceutical composition of the present application to the subject in need of treatment.
  • the TCR of the present application also includes at most 5, preferably at most 3, more preferably at most 2, and most preferably 1 amino acid (especially the amino acid located outside the CDR region) of the TCR of the present application, which are similar in nature or similar amino acid replacement, and still be able to maintain its functional TCR.
  • the present application also includes the slightly modified TCR of the present application.
  • Modified (generally without altering the primary structure) forms include: chemically derivatized forms of the TCRs of the present application such as acetylation or carboxylation.
  • Modifications also include glycosylation, such as those TCRs produced by glycosylation modifications during the synthesis and processing of the TCRs of the present application or during further processing steps. This modification can be accomplished by exposing the TCR to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme.
  • Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are TCRs that have been modified to increase their resistance to proteolysis or to optimize solubility.
  • the TCR of the present application, the TCR complex or the T cells transfected by the TCR of the present application can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the TCRs, multivalent TCR complexes or cells of the present application are typically provided as part of a sterile pharmaceutical composition, which 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 can be presented in unit dosage form, usually in a hermetically sealed container, which can be provided as part of a kit. Such kits, but not necessarily, include instructions for use. It may comprise a plurality of such unit dosage forms.
  • the TCR of the present application can be used alone, or combined or coupled with other therapeutic agents (such as formulated in the same pharmaceutical composition).
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent.
  • pharmaceutical carriers which do not, by themselves, induce the production of antibodies deleterious to the individual receiving the composition and which are not unduly toxic upon administration. These 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 carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • Pharmaceutically acceptable carriers in therapeutic compositions can contain liquids, such as water, saline, glycerol and ethanol.
  • liquids such as water, saline, glycerol and ethanol.
  • auxiliary substances in these carriers such as wetting agents or emulsifying agents, pH buffering substances and the like.
  • therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution, or suspension, in liquid carriers prior to injection can also be prepared.
  • compositions of the present application may be administered by conventional routes including, but not limited to: intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical, preferably parenteral Including subcutaneous, intramuscular or intravenous.
  • the subject to be prevented or treated can be an animal; especially a human.
  • compositions of the present application When the pharmaceutical composition of the present application is used for actual treatment, various dosage forms of the pharmaceutical composition can be used according to the usage conditions. Preferably, injections, oral preparations and the like can be exemplified.
  • compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents, etc. (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a conventional manner depending on the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents, etc. (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a conventional manner depending on the dosage form.
  • the pharmaceutical composition of the present application can also be administered in the form of sustained release formulations.
  • the TCR of the present application can be incorporated into pills or microcapsules with a slow-release polymer as a carrier, and then the pills or microcapsules are surgically implanted into the tissue to be treated.
  • sustained-release polymers include ethylene-vinyl acetate copolymers, polyhydroxymethacrylate (polyhydrometaacrylate), polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, Lactic acid-glycolic acid copolymers and the like are preferably exemplified by biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.
  • the TCR or TCR complex of the present application or the cells presenting the TCR of the present application as the active ingredient can be selected according to the body weight, age, sex, and degree of symptoms of each patient to be treated. However, it should be reasonably determined, and finally the doctor will determine the reasonable dosage.
  • the affinity and/or binding half-life of the high-affinity TCR of the present application to the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex is at least 5 times that of the wild-type TCR.
  • the high-affinity TCR of the present application can specifically bind to the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101, and the cells transfected with the high-affinity TCR of the present application can be specifically activated.
  • E.coli DH5 ⁇ is purchased from Tiangen
  • E.coli BL21 (DE3) is purchased from Tiangen
  • E.coli Tuner (DE3) is purchased from From Novagen
  • plasmid pET28a was purchased from Novagen.
  • This application uses the method of site-directed mutagenesis, according to the patent document WO2014/206304, to construct a stable single-chain TCR molecule composed of a flexible short peptide (linker) connecting the variable domains of TCR ⁇ and ⁇ chains, its amino acid and DNA
  • the sequences are SEQ ID NO:9 and SEQ ID NO:10, respectively, as shown in Figure 5a and Figure 5b.
  • the amino acid sequences of the ⁇ -chain variable domain (SEQ ID NO:3) and the ⁇ -chain variable domain (SEQ ID NO:4) of the template chain are shown in Figures 2a and 2b; the corresponding DNA sequences are SEQ ID NO : 5 and SEQ ID NO: 6, as shown in Figures 3a and 3b; the amino acid sequence and DNA sequence of the flexible short peptide (linker) are respectively SEQ ID NO: 7 and SEQ ID NO: 8, as shown in Figures 4a and 4b .
  • the target gene carrying the template strand was digested with NcoI and NotI, and then ligated with the pET28a vector that had been digested with NcoI and NotI.
  • the ligation product was transformed into E.coli DH5 ⁇ , coated with kanamycin-containing LB plates, cultured upside down at 37°C overnight, and positive clones were picked for PCR screening, and the positive recombinants were sequenced, and the recombinant plasmids were extracted and transformed after confirming the sequence was correct to E.coli BL21(DE3) for expression.
  • Example 2 Expression, renaturation and purification of the stable single-chain TCR constructed in Example 1
  • the eluted fractions for BIAcore analysis were further tested for purity by gel filtration.
  • the conditions are: chromatographic column Agilent Bio SEC-3 (300A, 7.8 ⁇ 300mm), the mobile phase is 150mM phosphate buffer, the flow rate is 0.5mL/min, the column temperature is 25°C, and the ultraviolet detection wavelength is 214nm.
  • BIAcore T200 real-time analysis system was used to detect the binding activity of TCR molecule and AQIPEKIQK(SEQ ID NO:47)-HLA A1101 complex.
  • Add the anti-streptavidin antibody (GenScript) to the coupling buffer (10mM sodium acetate buffer, pH 4.77), and then flow the antibody through the CM5 chip activated with EDC and NHS in advance to immobilize the antibody on the chip surface, and finally block the unreacted activated surface with ethanolamine hydrochloric acid solution to complete the coupling process, and the coupling level is about 15000RU.
  • the conditions are: temperature 25°C, pH value 7.1-7.5.
  • a low concentration of streptavidin was flowed over the surface of the antibody-coated chip, and then the AQIPEKIQK(SEQ ID NO:47)-HLA A1101 complex was flowed through the detection channel, and the other channel was used as a reference channel, and then 0.05mM Biotin flowed through the chip at a flow rate of 10 ⁇ L/min for 2 min to block the remaining binding sites of streptavidin.
  • the binding time of each injection is 120s, and the dissociation time is 600s after the last injection.
  • 20mg/L light chain and 90mg/L heavy chain in sequence final concentration, heavy chain is
  • the protein-containing fractions were pooled, concentrated with Millipore ultrafiltration tubes, and the protein concentration was determined by the BCA method (Thermo).
  • the biotinylated pMHC molecules were subpackaged and stored at -80°C by adding protease inhibitor cocktail (Roche).
  • Phage display technology is a means to generate a library of TCR high-affinity variants to screen for high-affinity variants.
  • the TCR phage display and screening method described by Li et al. ((2005) Nature Biotech 23(3):349-354) was applied to the single-chain TCR template in Example 1.
  • a library of high-affinity TCRs is constructed and screened by mutating the CDR regions of the template strand. After several rounds of screening, the phage library has specific binding to the corresponding antigen, and single clones are picked and analyzed.
  • the CDR region mutation of the screened high-affinity single-chain TCR was introduced into the corresponding site of the variable domain of the ⁇ heterodimerized TCR, and its association with AQIPEKIQK(SEQ ID NO:47)-HLA A1101 was detected by BIAcore complex affinity.
  • the introduction of the above-mentioned high-affinity mutation point in the CDR region adopts the method of site-directed mutation well known to those skilled in the art.
  • the amino acid sequences of the ⁇ -chain and ⁇ -chain variable domains of the above wild-type TCR are shown in Figure 1a (SEQ ID NO: 1) and 1b (SEQ ID NO: 2), respectively.
  • the ⁇ heterodimeric TCR can be A cysteine residue was introduced into the constant regions of the ⁇ and ⁇ chains to form a TCR with artificial interchain disulfide bonds.
  • the amino acid sequences of the TCR ⁇ and ⁇ chains after the cysteine residues were introduced As shown in Figures 6a (SEQ ID NO: 11) and 6b (SEQ ID NO: 12), the introduced cysteine residues are indicated in bold letters.
  • the extracellular sequence genes of the TCR ⁇ and ⁇ chains to be expressed were synthesized and inserted into the expression vectors, respectively, by standard methods described in Molecular Cloning a Laboratory Manual (Third Edition, Sambrook and Russell) For pET28a+ (Novagene), the upstream and downstream cloning sites are NcoI and NotI, respectively. Mutations in the CDR regions were introduced by overlapping PCR (overlap PCR) well known to those skilled in the art. The insert was confirmed by sequencing.
  • the solution was filtered through a 0.45 ⁇ M filter membrane, and then purified by an anion exchange column (HiTrap Q HP, 5 mL, GE Healthcare).
  • the elution peaks containing TCRs of successfully refolded ⁇ and ⁇ dimers were confirmed by SDS-PAGE.
  • TCR was then further purified by gel filtration chromatography (HiPrep 16/60, SephacrylS-100HR, GE Healthcare). The purity of the purified TCR was determined by SDS-PAGE to be greater than 90%, and the concentration was determined by the BCA method.
  • Embodiment 6 BIAcore analysis result
  • Example 3 The method described in Example 3 was used to detect the affinity between the ⁇ heterodimeric TCR introduced into the high-affinity CDR region and the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex.
  • the present application obtains the amino acid sequence of the variable domain of the TCR ⁇ chain and the amino acid sequence of the variable domain of the TCR ⁇ chain, as shown in Figures 7 (1)-(18) and Figures 8 (1)-(12), respectively. Since the CDR region of the TCR molecule determines its affinity with the corresponding pMHC complex, those skilled in the art can expect that the ⁇ heterodimeric TCR introduced into the high-affinity mutation point also has the ability to AQIPEKIQK (SEQ ID NO: 47)-HLA High affinity for the A1101 complex.
  • Example 4 Using the method described in Example 4 to construct an expression vector, using the method described in Example 5 to express, anneal and purify the above-mentioned ⁇ heterodimeric TCR with a high-affinity mutation, and then use BIAcore T200 to determine its relationship with AQIPEKIQK( The affinity of SEQ ID NO:47)-HLA A1101 complex is shown in Table 2 below.
  • the affinity of the heterodimeric TCR is at least 5 times that of the wild-type TCR to the AQIPEKIQK (SEQ ID NO: 47)-HLA A1101 complex.
  • Example 7 Expression, refolding and purification of the fusion of anti-CD3 antibody and high-affinity ⁇ heterodimeric TCR
  • a fusion molecule was prepared by fusing an anti-CD3 single-chain antibody (scFv) to an ⁇ heterodimeric TCR.
  • the scFv of anti-CD3 is fused with the ⁇ chain of TCR.
  • the TCR ⁇ chain can contain any of the ⁇ chain variable domains of the above-mentioned high-affinity ⁇ heterodimeric TCR, and the TCR ⁇ chain of the fusion molecule can contain any of the above-mentioned high-affinity The ⁇ -chain variable domain of a sex ⁇ heterodimeric TCR.
  • the target gene carrying the ⁇ chain of ⁇ heterodimeric TCR was digested with NcoI and NotI, and then ligated with the pET28a vector that had been digested with NcoI and NotI.
  • the ligation product was transformed into E.coli DH5 ⁇ , spread on LB plates containing kanamycin, cultured upside down at 37°C overnight, picked positive clones for PCR screening, sequenced positive recombinants, and extracted recombinant plasmids after confirming the correct sequence Transformed into E.coli Tuner (DE3) for expression.
  • anti-CD3 (scFv)-beta chain expression vector by overlapping (overlap) PCR, design primers to connect anti-CD3 scFv and high-affinity heterogeneous dimeric TCR beta chain gene, the middle connection
  • the short peptide (linker) is GGGGS (SEQ ID NO: 84)
  • the gene fragment of the fusion protein of anti-CD3 scFv and high-affinity heterodimeric TCR ⁇ chain is carried with a restriction endonuclease site Nco I (CCATGG) and Not I (GCGGCCGC).
  • the PCR amplified product was digested with Nco I and Not I, and then ligated with the pET28a vector that had been digested with Nco I and Not I.
  • the ligation product was transformed into E.coli DH5 ⁇ competent cells, coated with LB plates containing kanamycin, cultured upside down at 37°C overnight, picked positive clones for PCR screening, sequenced the positive recombinants, and extracted after confirming the correct sequence
  • the recombinant plasmid was transformed into E.coli Tuner (DE3) competent cells for expression.
  • the expression plasmids were respectively transformed into E.coli Tuner (DE3) competent cells, spread on LB plates (kanamycin 50 ⁇ g/mL) and cultured at 37°C overnight. The next day, pick clones and inoculate them into 10 mL LB liquid medium (kanamycin 50 ⁇ g/mL) for 2-3 hours, then inoculate them into 1L LB medium at a volume ratio of 1:100, and continue to cultivate until the OD600 is 0.5-0.8, adding The final concentration was 1mM IPTG to induce the expression of the target protein. After 4 hours of induction, cells were harvested by centrifugation at 6000 rpm for 10 min. The cells were washed once with PBS buffer, and the cells were subpackaged.
  • E.coli Tuner E.coli Tuner
  • DTT dithiothreitol
  • EDTA ethylenediaminetetraacetic acid
  • the solution was filtered through a 0.45 ⁇ M filter membrane, and then purified by an anion exchange column (HiTrap Q HP 5 mL, GE healthcare).
  • the elution peak contained the successfully refolded TCR ⁇ chain and anti-CD3(scFv)- ⁇ chain dimer TCR confirmed by SDS-PAGE gel.
  • the TCR fusion molecule was then further purified by size exclusion chromatography (S-100 16/60, GE healthcare) and again by an anion exchange column (HiTrap Q HP 5 mL, GE healthcare).
  • the purity of the purified TCR fusion molecule is determined by SDS-PAGE to be greater than 90%, and the concentration is determined by the BCA method.
  • Example 8 Activation function experiment of effector cells transfected with high-affinity TCR of this application for T2 target cells loaded with short peptides
  • IFN- ⁇ is a powerful immunoregulatory factor produced by activated T lymphocytes. Therefore, in this example, the number of IFN- ⁇ spots is detected by the ELISPOT experiment well known to those skilled in the art to verify the presence of cells transfected with the high-affinity TCR of this application. Activation function and antigen specificity. TCR was randomly selected for the experiment, and the specific TCR sequence used can be learned from Table 2. TCR2, TCR4, TCR9, TCR21, TCR23, and TCR26 were transfected into CD3 + T cells isolated from the blood of healthy volunteers as effector cells.
  • CD3 + T cells transfected with other TCR A6 and CD3 + T cells transfected with wild-type TCR (WT-TCR) were used as controls in the same volunteer.
  • the target cells used were T2-A11 loaded with SSX2 antigen short peptide AQIPEKIQK (SEQ ID NO:47), T2-A11 loaded with other antigen short peptides, or empty T2-A11 (referring to transfected HLA-A1101 T2 cells, the same below).
  • ELISPOT plate First prepare the ELISPOT plate, first add 1 ⁇ 104 cells/well of target cells and 2 ⁇ 103 cells/well of effector cells (calculated according to the positive rate of transfection) into the corresponding wells, and then add SSX2 antigen short peptide to the experimental group AQIPEKIQK (SEQ ID NO:47), and its final concentration in the ELISPOT well plate was 1 ⁇ 10 -6 M; in the control group, other antigen short peptides were added, and its final concentration was 1 ⁇ 10 -6 M, And set up two multiple holes. Incubate overnight (37°C, 5% CO 2 ). On the second day of the experiment, the plate was washed for secondary detection and color development, the plate was dried, and the spots formed on the membrane were counted by an immunospot plate reader (ELISPOT READER system; AID20 company).
  • ELISPOT READER system AID20 company
  • the effector cells transfected with the high-affinity TCR of the present application have a higher effect than the effector cells transfected with the wild-type TCR. Obvious activation effect, while the effector cells transfected with other TCRs or empty transfection are inactive; at the same time, the effector cells transfected with the TCR of this application are inactive for target cells loaded with other antigen short peptides or empty.
  • Example 9 For the cell line, the activation function experiment of the effector cells transfected with the high-affinity TCR of the application
  • tumor cell lines were used to verify again the activation function and specificity of the effector cells transfected with the high-affinity TCR of the present application.
  • TCR molecules are randomly selected and detected by ELISPOT experiments well known to those skilled in the art.
  • the high-affinity TCR of this application was transfected into CD3 + T cells isolated from the blood of healthy volunteers as effector cells, and the same volunteer was transfected with other TCR (A6) or wild-type TCR (WT-TCR ) CD3 + T cells as a control.
  • the tumor cell lines used in the examples are SK-MEL-28, TT_THYROID, HUCC-T1, SNU423, SW620, Huh-1, LCLs-150909A, LCLs-151008A, respectively.
  • SK-MEL-28, TT_THYROID, HUCC-T1, and SNU423 were purchased from Guangzhou Saiku Biotechnology Co., Ltd.
  • Huh-1 was purchased from Nanjing Kebai Biotechnology Co., Ltd.
  • SW620 was purchased from ATCC.
  • the following three batches (I), (II), (III) were successively tested:
  • the high-affinity TCRs can be learned from Table 2, which are TCR2, TCR4, TCR5, and TCR23 respectively.
  • the SSX2 positive tumor cell line used in this batch is SK-MEL-28-SSX2 (SSX2 overexpression), and the negative tumor cell line is SK-MEL-28, HUCC-T1, SNU423, LCLs-150909A, LCLs-151008A,, TT_THYROID.
  • the high-affinity TCRs can be learned from Table 2, which are TCR1, TCR2, TCR20, TCR23, and TCR25, respectively.
  • the SSX2 positive tumor cell lines used in this batch were SK-MEL-28-SSX2 (SSX2 overexpression), TT_THYROID-A11, TT_THYROID, and the negative tumor cell lines were HUCC-T1, SNU423, SW620-A11, SW620.
  • the high-affinity TCR can be learned from Table 2, which is TCR3.
  • the SSX2-positive tumor cell lines used in this batch were SK-MEL-28-SSX2 (SSX2 overexpression), HUCC-T1-SSX2 (SSX2 overexpression), Huh-1-A11, and the negative tumor cell lines were HUCC-T1, SNU423.
  • ELISPOT plates were activated with ethanol and coated at 4°C overnight. On the first day of the experiment, remove the coating solution, wash and block, incubate at room temperature for two hours, remove the blocking solution, and add each component of the test to the ELISPOT plate: 2* 10 cells/well for target cells, 2* for effector cells 10 3 cells/well (calculated according to the positive rate of transfection), and set up two duplicate holes. Incubate overnight (37°C, 5% CO 2 ). On the second day of the experiment, the plate was washed for secondary detection and color development, the plate was dried, and the spots formed on the membrane were counted by an immunospot plate reader (ELISPOT READER system; AID20 company).
  • ELISPOT READER system AID20 company
  • Lactate dehydrogenase is abundant in the cytoplasm and cannot pass through the cell membrane under normal circumstances. When the cells are damaged or die, they can be released outside the cells. At this time, the activity of LDH in the cell culture medium is directly proportional to the number of dead cells.
  • the TCR molecules of the present application were randomly selected, and the release of LDH was measured through a non-radioactive cytotoxicity experiment well known to those skilled in the art, so as to verify the killing function of the cells transfected with the TCR of the present application.
  • CD3 + T cells isolated from the blood of healthy volunteers were used to transfect the high-affinity TCR of this application as effector cells, and CD3 + T cells transfected with other TCR (A6) from the same volunteer were used as negative control.
  • the tumor cell lines used in the examples are SK-MEL-28, TT_THYROID, HUCC-T1, Huh-1, and SNU423, among which, SK-MEL-28, TT_THYROID, HUCC-T1, and SNU423 were all purchased from Guangzhou Saiku Biotechnology Co., Ltd. Co., Ltd., Huh-1 was purchased from Nanjing Kebai Biotechnology Co., Ltd.
  • the following two batches (I), (II) were successively tested:
  • the high-affinity TCRs can be learned from Table 2, which are TCR1, TCR2, TCR20, TCR23, and TCR25, respectively.
  • the SSX2-positive tumor cell lines used in this batch were SK-MEL-28-SSX2 (SSX2 overexpression), TT_THYROID-A11, and the negative tumor cell lines were HUCC-T1, TT_THYROID.
  • the high-affinity TCR can be learned from Table 2, which is TCR3.
  • the SSX2-positive tumor cell lines used in this batch were SK-MEL-28-SSX2 (SSX2 overexpression), HUCC-T1-SSX2 (SSX2 overexpression), Huh-1-A11, and the negative tumor cell lines were HUCC-T1, SNU423.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Wood Science & Technology (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Oncology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un TCR à haute affinité reconnaissant SSX2, qui a la propriété de se lier à un complexe AQIPEKIQK (SEQ ID NO : 47)-HLA A1101. L'invention concerne également un complexe TCR multivalent, une molécule d'acide nucléique codant pour le TCR, un vecteur contenant l'acide nucléique, une cellule exprimant le TCR, et une composition pharmaceutique comprenant les matériaux décrits. Ils sont utilisés pour le diagnostic, le traitement et la prévention de maladies SSX2-positives. L'Invention concerne également un procédé de fabrication du TCR.
PCT/CN2022/118984 2021-09-17 2022-09-15 Tcr à haute affinité reconnaissant ssx2 WO2023040946A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111094910.7A CN115819555A (zh) 2021-09-17 2021-09-17 一种识别ssx2的高亲和力tcr
CN202111094910.7 2021-09-17

Publications (1)

Publication Number Publication Date
WO2023040946A1 true WO2023040946A1 (fr) 2023-03-23

Family

ID=85515322

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/118984 WO2023040946A1 (fr) 2021-09-17 2022-09-15 Tcr à haute affinité reconnaissant ssx2

Country Status (2)

Country Link
CN (1) CN115819555A (fr)
WO (1) WO2023040946A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030228634A1 (en) * 2001-11-07 2003-12-11 Simard John J.L. Expression vectors encoding epitopes of target-associated antigens and methods for their design
CN103124740A (zh) * 2010-09-21 2013-05-29 美国卫生和人力服务部 抗-ssx-2 t细胞受体和相关材料及使用方法
CN111954679A (zh) * 2017-10-03 2020-11-17 朱诺治疗学股份有限公司 Hpv特异性结合分子
CN112512538A (zh) * 2018-05-18 2021-03-16 国家儿童医疗中心 改进的靶向t细胞疗法
WO2021129559A1 (fr) * 2019-12-24 2021-07-01 南京北恒生物科技有限公司 Protéine de fusion de récepteur de lymphocytes t et son utilisation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030228634A1 (en) * 2001-11-07 2003-12-11 Simard John J.L. Expression vectors encoding epitopes of target-associated antigens and methods for their design
CN103124740A (zh) * 2010-09-21 2013-05-29 美国卫生和人力服务部 抗-ssx-2 t细胞受体和相关材料及使用方法
CN111954679A (zh) * 2017-10-03 2020-11-17 朱诺治疗学股份有限公司 Hpv特异性结合分子
CN112512538A (zh) * 2018-05-18 2021-03-16 国家儿童医疗中心 改进的靶向t细胞疗法
WO2021129559A1 (fr) * 2019-12-24 2021-07-01 南京北恒生物科技有限公司 Protéine de fusion de récepteur de lymphocytes t et son utilisation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DANIEL ABATE-DAGA, DANIEL E. SPEISER, NACHIMUTHU CHINNASAMY, ZHILI ZHENG, HUI XU, STEVEN A. FELDMAN, STEVEN A. ROSENBERG, RICHARD : "Development of a T Cell Receptor Targeting an HLA-A*0201 Restricted Epitope from the Cancer-Testis Antigen SSX2 for Adoptive Immunotherapy of Cancer", PLOS ONE, vol. 9, no. 3, pages 1 - 12, XP055224166, DOI: 10.1371/journal.pone.0093321 *
LI PENG, HUILING ZHENG: "Expression of Cancer Testis Antigen SSX2 and SCP-1 in Hepatocellular Carcinoma", NING XIA YI XUE ZA ZHI : SHUANG YUE KAN = NINGXIA MEDICAL JOURNAL, NING XIA YI XUE ZA ZHI BIAN JI WEI YUAN HUI BIAN JI, CN, vol. 36, no. 9, 28 February 2014 (2014-02-28), CN , pages 790 - 792, XP093047792, ISSN: 1001-5949, DOI: 10.13621/j.1001-5949.2014.09.0790 *

Also Published As

Publication number Publication date
CN115819555A (zh) 2023-03-21

Similar Documents

Publication Publication Date Title
WO2019109821A1 (fr) Récepteur de lymphocytes t à haute affinité dirigé contre prame
CN112759641B (zh) 一种识别Kras G12V的高亲和力TCR
CN110198953B (zh) 针对ny-eso的高亲和力tcr
WO2021185368A1 (fr) Tcr à haute affinité pour reconnaître un antigène afp
WO2021043284A1 (fr) Récepteur de lymphocytes t à haute affinité qui reconnaît ssx2
WO2021036924A1 (fr) Tcr à haute affinité pour reconnaître un antigène ssx2
WO2021032020A1 (fr) Récepteur de lymphocytes t à haute affinité pour la reconnaissance d'afp
TW202144403A (zh) 一種辨識ｈｐｖ１６的高親和力ｔｃｒ
WO2020182082A1 (fr) Tcr à haute affinité pour reconnaître un antigène afp
CN110938136B (zh) 一种识别afp抗原的高亲和力t细胞受体
WO2020098717A1 (fr) Tcr à haute affinité pour la reconnaissance d'afp
WO2019158084A1 (fr) Récepteur de lymphocytes t du hbs à haute affinité
WO2022022696A1 (fr) Tcr à haute affinité pour reconnaître l'afp
WO2021228255A1 (fr) Récepteur de lymphocytes t à haute affinité pouvant reconnaître un antigène afp
WO2023040946A1 (fr) Tcr à haute affinité reconnaissant ssx2
WO2023005859A1 (fr) Récepteur de lymphocytes t à haute affinité pour l'antigène ssx2
WO2022262842A1 (fr) Récepteur de lymphocytes t à haute affinité pour antigène afp
WO2023179768A1 (fr) Tcr à haute affinité pour identifier un antigène mage-a4, séquence et utilisation de celui-ci
WO2023221959A1 (fr) Récepteurs de lymphocytes t à haute affinité pour la reconnaissance de mage et leur utilisation
WO2022206860A1 (fr) Récepteur des lymphocytes t pour afp
WO2017041704A1 (fr) Récepteur de lymphocyte t ayant une affinité élevée de ciblage de polypeptide à chaîne courte d'antigène rhamm
WO2022166905A1 (fr) Tcr à haute affinité contre le hpv
CN117659163A (zh) 一种识别afp的高亲和力t细胞受体及其应用
CN116135879A (zh) 针对afp的高亲和力tcr

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22869314

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE