[go: up one dir, main page]

WO2021204287A1 - Tcr à haute affinité pour reconnaître hpv16 - Google Patents

Tcr à haute affinité pour reconnaître hpv16 Download PDF

Info

Publication number
WO2021204287A1
WO2021204287A1 PCT/CN2021/086455 CN2021086455W WO2021204287A1 WO 2021204287 A1 WO2021204287 A1 WO 2021204287A1 CN 2021086455 W CN2021086455 W CN 2021086455W WO 2021204287 A1 WO2021204287 A1 WO 2021204287A1
Authority
WO
WIPO (PCT)
Prior art keywords
tcr
variable domain
chain
chain variable
cdr3α
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/CN2021/086455
Other languages
English (en)
Chinese (zh)
Inventor
李懿
郭珊珊
战凯
黄娇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Xlifesc Ltd
Original Assignee
Xlifesc Ltd
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 Xlifesc Ltd filed Critical Xlifesc Ltd
Publication of WO2021204287A1 publication Critical patent/WO2021204287A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • 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
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • 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
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/892—Reproductive system [uterus, ovaries, cervix, testes]

Definitions

  • the present invention relates to the field of biotechnology, and more specifically to a T cell receptor (TCR) capable of recognizing a polypeptide derived from HPV16 E7 protein.
  • TCR T cell receptor
  • the invention also relates to the preparation and use of the receptor.
  • TCR T cell receptor
  • TCR is the only receptor for specific antigen peptides presented on the main histocompatibility complex (MHC). This exogenous or endogenous peptide may be the only sign of abnormal cells.
  • MHC main histocompatibility complex
  • APC antigen presenting cells
  • the MHC class I and class II molecular ligands corresponding to TCR are also proteins of the immunoglobulin superfamily but have specificity for the presentation of antigens. Different individuals have different MHCs, which can present different shortcomings in a protein antigen. Peptides to the surface of the respective APC cells. Human MHC is usually called HLA gene or HLA complex.
  • HPV16 E7 gene is one of the genes in the early region of the human papillomavirus (HPV) genome.
  • the E7 protein encoded by it is a small acidic protein containing about 100 amino acids.
  • High-risk HPV16 E7 protein is an important cause of cervical cancer, head and neck cancer, and anal cancer.
  • the newly synthesized antigen is processed into antigen peptides in the cytoplasm, and combined with MHC (major histocompatibility complex) molecules to form a complex, which is presented to the cell surface.
  • YMLDLQPET is a short peptide derived from HPV16 E7 antigen and is a target for the treatment of HPV16 E7 related diseases.
  • the YMLDLQPET-HLA A0201 complex provides a marker for TCR to target tumor cells.
  • the TCR that can be combined with YMLDLQPET-HLA A0201 complex has high application value for tumor treatment.
  • TCR that can target the tumor cell marker can be used to deliver cytotoxic or immunostimulant to target cells, or be transformed into T cells, so that T cells expressing the TCR can destroy tumor cells, so that they can be called Adoptive immunotherapy is given to patients during the course of treatment.
  • the ideal TCR has a higher affinity, so that the TCR can reside on the targeted cells for a long time.
  • it is preferable to use a medium-affinity TCR it is preferable to use a medium-affinity TCR. Therefore, those skilled in the art devote themselves to developing TCRs that can be used for different purposes to target tumor cell markers.
  • the purpose of the present invention is to provide a TCR with higher affinity to the YMLDLQPET-HLA A0201 complex.
  • Another object of the present invention is to provide a method for preparing the above-mentioned type of TCR and use of the above-mentioned type of TCR.
  • the first aspect of the present invention provides a T cell receptor (TCR), which has the activity of binding to the YMLDLQPET-HLA A0201 complex.
  • TCR T cell receptor
  • the T cell receptor comprises a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain, wherein it has the activity of binding YMLDLQPET-HLA A0201 complex, and the ⁇ chain of the TCR can be
  • the variable domain comprises an amino acid sequence having at least 90% sequence homology with the sequence shown in SEQ ID NO: 1
  • the TCR ⁇ chain variable domain comprises at least 90% sequence homology with the sequence shown in SEQ ID NO: 2 Sexual amino acid sequence.
  • the affinity of the TCR and the YMLDLQPET-HLA A0201 complex is at least twice that of the wild-type TCR.
  • the ⁇ chain variable domain of the TCR contains at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, and the sequence shown in SEQ ID NO:1. Amino acid sequence with 98% or 99% sequence homology.
  • the ⁇ chain variable domain of the TCR is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, and the sequence shown in SEQ ID NO: 2 Amino acid sequence with 98%, 99% or 100% sequence homology.
  • the alpha chain variable domain of the TCR comprises an amino acid sequence having at least 96% sequence homology with the sequence shown in SEQ ID NO:1.
  • the ⁇ -chain variable domain of the TCR comprises an amino acid sequence having at least 97% sequence homology with the sequence shown in SEQ ID NO: 2.
  • the reference sequence of the three CDR regions of the TCR ⁇ chain variable domain is as follows:
  • CDR3 ⁇ ALYNQGGKLI, and CDR3 ⁇ contains at least one of the following mutations:
  • variable domain of the TCR ⁇ chain in the variable domain of the TCR ⁇ chain, the number of amino acid mutations in CDR3 ⁇ is 2 or 3 or 4.
  • the reference sequence of the three CDR regions of the TCR ⁇ chain variable domain is as follows:
  • CDR3 ⁇ ALYNQGGKLI, and CDR3 ⁇ contains at least one of the following mutations:
  • the reference sequence of the three CDR regions of the TCR ⁇ chain variable domain is as follows:
  • CDR3 ⁇ ALYNQGGKLI, and CDR3 ⁇ contains at least one of the following mutations:
  • the ⁇ -chain variable domain of the TCR includes an amino acid sequence having at least 97% sequence homology with the sequence shown in SEQ ID NO: 2.
  • the reference sequences of the three CDR regions of the TCR ⁇ chain variable domain are as follows:
  • CDR3 ⁇ ALYNQGGKLI, and CDR3 ⁇ contains at least one of the following mutations:
  • the reference sequences of the three CDR regions of the TCR ⁇ chain variable domain are as follows:
  • CDR3 ⁇ ALYNQGGKLI, and CDR3 ⁇ contains at least one of the following mutations:
  • amino acid sequence of the variable domain of the TCR ⁇ chain is SEQ ID NO: 2.
  • the reference sequence of the three CDR regions of the TCR ⁇ chain variable domain is as follows:
  • CDR3 ⁇ ASSLLAGSYEQY, and CDR3 ⁇ contains at least one of the following mutations:
  • the TCR ⁇ chain variable domain comprises 3 CDR regions, and the amino acid sequences of the 3 CDR regions of the TCR ⁇ chain variable domain are as follows:
  • amino acid sequence of the variable domain of the TCR ⁇ chain is SEQ ID NO: 2.
  • the TCR ⁇ chain variable domain comprises three CDR regions, wherein CDR1 ⁇ is ATGYPS, and CDR2 ⁇ is ATKADDK.
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , wherein the amino acid sequence of CDR1 ⁇ is ATGYPS, the amino acid sequence of CDR2 ⁇ is ATKADDK, and the amino acid sequence of CDR3 ⁇ is: ALY[3 ⁇ X1][3 ⁇ X2] [3 ⁇ X3][3 ⁇ X4][3 ⁇ X5]LI, where [3 ⁇ X1] is N or L or A or V, [3 ⁇ X2] is Q or F or Y or W, [3 ⁇ X3] is G or N or S or D or A or V or T or H or M, [3 ⁇ X4] is G or A or N or S or F, and [3 ⁇ X5] is K or V or R or L or I or M or Q or S or N.
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , wherein the amino acid sequence of CDR1 ⁇ is ATGYPS, the amino acid sequence of CDR2 ⁇ is ATKADDK, and the amino acid sequence of CDR3 ⁇ is: ALY[3 ⁇ X1][3 ⁇ X2] [3 ⁇ X3][3 ⁇ X4][3 ⁇ X5]LI, where [3 ⁇ X1] is N or L or A or V.
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , wherein the amino acid sequence of CDR1 ⁇ is ATGYPS, the amino acid sequence of CDR2 ⁇ is ATKADDK, and the amino acid sequence of CDR3 ⁇ is: ALY[3 ⁇ X1][3 ⁇ X2] [3 ⁇ X3][3 ⁇ X4][3 ⁇ X5]LI, where [3 ⁇ X2] is Q or F or Y or W.
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , wherein the amino acid sequence of CDR1 ⁇ is ATGYPS, the amino acid sequence of CDR2 ⁇ is ATKADDK, and the amino acid sequence of CDR3 ⁇ is: ALY[3 ⁇ X1][3 ⁇ X2] [3 ⁇ X3][3 ⁇ X4][3 ⁇ X5]LI, where [3 ⁇ X3] is G or N or S or D or A or V or T or H or M.
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , wherein the amino acid sequence of CDR1 ⁇ is ATGYPS, the amino acid sequence of CDR2 ⁇ is ATKADDK, and the amino acid sequence of CDR3 ⁇ is: ALY[3 ⁇ X1][3 ⁇ X2] [3 ⁇ X3][3 ⁇ X4][3 ⁇ X5]LI, where [3 ⁇ X4] is G or A or N or S or F.
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , wherein the amino acid sequence of CDR1 ⁇ is ATGYPS, the amino acid sequence of CDR2 ⁇ is ATKADDK, and the amino acid sequence of CDR3 ⁇ is: ALY[3 ⁇ X1][3 ⁇ X2] [3 ⁇ X3][3 ⁇ X4][3 ⁇ X5]LI, where [3 ⁇ X5] is K or V or R or L or I or M or Q or S or N.
  • amino acid sequence of the CDR3 ⁇ is selected from: ALYLFNGKLI, ALYNYNAVLI, ALYNFSNRLI and ALYNFNSLLI.
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , wherein the amino acid sequence of CDR1 ⁇ is SGHTA, the amino acid sequence of CDR2 ⁇ is FQGTGA, and the amino acid sequence of CDR3 ⁇ is: ASSLLAGSY[3 ⁇ X1][3 ⁇ X2] [3 ⁇ X3], where [3 ⁇ X1] is E or Y or M, [3 ⁇ X2] is Q or V or L, and [3 ⁇ X3] is Y or E or S.
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , wherein the amino acid sequence of CDR1 ⁇ is SGHTA, the amino acid sequence of CDR2 ⁇ is FQGTGA, and the amino acid sequence of CDR3 ⁇ is: ASSLLAGSY[3 ⁇ X1][3 ⁇ X2] [3 ⁇ X3], where [3 ⁇ X1] is E or Y or M.
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , wherein the amino acid sequence of CDR1 ⁇ is SGHTA, the amino acid sequence of CDR2 ⁇ is FQGTGA, and the amino acid sequence of CDR3 ⁇ is: ASSLLAGSY[3 ⁇ X1][3 ⁇ X2] [3 ⁇ X3], where [3 ⁇ X2] is Q or V or L.
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , wherein the amino acid sequence of CDR1 ⁇ is SGHTA, the amino acid sequence of CDR2 ⁇ is FQGTGA, and the amino acid sequence of CDR3 ⁇ is: ASSLLAGSY[3 ⁇ X1][3 ⁇ X2] [3 ⁇ X3], where [3 ⁇ X3] is Y or E or S.
  • the TCR has a mutation in the ⁇ chain variable domain shown in SEQ ID NO:1, and the mutation is selected from N94L/A/V, Q95F/Y/W, G96N/S/D One or several groups in /A/V/T/H/M, G97A/N/S/F, K98V/R/L/I/M/Q/S/N, wherein the amino acid residue numbering adopts SEQ ID NO:1 The number shown.
  • the TCR has a mutation in the ⁇ chain variable domain shown in SEQ ID NO: 2, and the mutation is selected from one or more of E102Y/M, Q103V/L, and Y104S/E. Group, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 2.
  • the TCR has a CDR selected from the following group:
  • the TCR is soluble.
  • the TCR is an ⁇ heterodimeric TCR, which comprises an ⁇ chain TRAC constant region sequence and a ⁇ chain TRBC1 or TRBC2 constant region sequence.
  • the TCR comprises (i) all or part of the TCR ⁇ chain excluding its transmembrane domain, and (ii) all or part of the TCR ⁇ chain excluding its transmembrane domain, wherein (i) And (ii) both comprise the variable domain and at least a part of the constant domain of the TCR chain.
  • the ⁇ -chain constant region and the ⁇ -chain constant region of the TCR contain artificial interchain disulfide bonds.
  • cysteine residues forming artificial interchain disulfide bonds between the constant regions of the TCR ⁇ and ⁇ chains are substituted for one or more sets of sites selected from the following:
  • amino acid sequence of the alpha chain variable domain of the TCR is selected from: SEQ ID NO: 13-46; and/or the amino acid sequence of the beta chain variable domain of the TCR is SEQ ID NO: 2.
  • the TCR is selected from the following group:
  • 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.
  • a conjugate is bound to the C- or N-terminus of the ⁇ chain and/or ⁇ chain of the TCR.
  • the conjugate is a detectable label, a therapeutic agent, or a PK modification. Part or any combination of these substances.
  • 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 invention provides a multivalent TCR complex comprising at least two TCR molecules, and at least one of the TCR molecules is the TCR described in the first aspect of the present invention.
  • the third aspect of the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule according to the first aspect of the present invention or the multivalent TCR complex according to the second aspect of the present invention or its complement sequence.
  • the fourth aspect of the present invention provides a vector containing the nucleic acid molecule described in the third aspect of the present invention.
  • the fifth aspect of the present invention provides a host cell containing the vector of the fourth aspect of the present invention or the nucleic acid molecule of the third aspect of the present invention integrated into the chromosome.
  • the sixth aspect of the present invention provides an isolated cell expressing the TCR described in the first aspect of the present invention.
  • the seventh aspect of the present invention provides a pharmaceutical composition containing a pharmaceutically acceptable carrier and the TCR according to the first aspect of the present invention, or the TCR complex according to the second aspect of the present invention, Or the cell described in the sixth aspect of the present invention.
  • the eighth aspect of the present invention provides a method for treating diseases, comprising administering an appropriate amount of the TCR according to the first aspect of the present invention, or the TCR complex according to the second aspect of the present invention, or the present invention to a subject in need of treatment.
  • the disease is an HPV-positive tumor, and more preferably, the tumor is cervical cancer.
  • the ninth aspect of the present invention provides the use of the TCR according to the first aspect of the present invention, or the TCR complex according to the second aspect of the present invention, or the use of the cell according to the sixth aspect of the present invention, for preparing and treating tumors
  • the disease is an HPV-positive tumor, and more preferably, the tumor is cervical cancer.
  • the tenth aspect of the present invention provides a method for preparing the T cell receptor according to the first aspect of the present invention, including the steps:
  • Figure 1a and Figure 1b respectively show the amino acid sequences of wild-type TCR ⁇ and ⁇ chain variable domains that can specifically bind to the YMLDLQPET-HLA A0201 complex.
  • Figure 2a and Figure 2b are the amino acid sequence of the alpha variable domain and the amino acid sequence of the beta chain variable domain of the single-chain template TCR constructed in the present invention, respectively.
  • Figures 3a and 3b are respectively the DNA sequence of the ⁇ variable domain and the DNA sequence of the ⁇ chain variable domain of the single-stranded template TCR constructed in the present invention.
  • Figure 4a and Figure 4b are respectively the amino acid sequence and nucleotide sequence of the linker of the single-stranded template TCR constructed in the present invention.
  • Figure 5a and Figure 5b respectively show the amino acid sequence and DNA sequence of the single-stranded template TCR constructed in the present invention.
  • Figure 6a and Figure 6b respectively show the amino acid sequences of the soluble reference TCR alpha and beta chains of the present invention.
  • Figures 7(1)-(34) respectively show the amino acid sequence of the ⁇ chain variable domain of a heterodimeric TCR with high affinity for the YMLDLQPET-HLA A0201 complex, and the mutated residues are underlined.
  • Figures 8a-c respectively show the amino acid sequence of the ⁇ -chain variable domain of a heterodimeric TCR with high affinity for the YMLDLQPET-HLA A0201 complex, and the mutated residues are underlined.
  • Figures 9a and 9b respectively show the extracellular amino acid sequences of wild-type TCR ⁇ and ⁇ chains that can specifically bind to the YMLDLQPET-HLA A0201 complex.
  • Figures 10a and 10b respectively show the amino acid sequences of wild-type TCR ⁇ and ⁇ chains that can specifically bind to the YMLDLQPET-HLA A0201 complex.
  • Figure 11 is the binding curve of the soluble reference TCR, that is, wild-type TCR and YMLDLQPET-HLA A0201 complex.
  • Figures 12a and 12b are the results of the activation function experiment of the effector cells transfected with the high-affinity TCR of the present invention against T2 cells loaded with short peptides.
  • Figure 13 shows the results of the activation function experiment of the effector cells transfected with the high-affinity TCR of the present invention against tumor cell lines.
  • Figure 14 shows the results of the killing function experiment of the effector cells transfected with the high-affinity TCR of the present invention against T2 cells loaded with short peptides.
  • FIG. 15 shows the results of the killing function experiment (LDH experiment) of effector cells transfected with the high-affinity TCR of the present invention against tumor cell lines.
  • Figures 16a and 16b are the results of the killing function experiment (incuCyte experiment) of the effector cells transfected with the high-affinity TCR of the present invention against tumor cell lines.
  • the present invention has obtained a high-affinity T cell receptor (TCR) that recognizes the YMLDLQPET short peptide (derived from HPV16 E7 protein) in the form of a peptide-HLA A0201 complex Be presented.
  • TCR high-affinity T cell receptor
  • the high-affinity TCR is in the 3 CDR regions of its ⁇ chain variable domain:
  • CDR3 ⁇ A mutation in ALYNQGGKLI; and/or in the 3 CDR regions of the ⁇ chain variable domain:
  • CDR3 ⁇ Mutation occurs in ASSLLAGSYEQY; and after the mutation, the affinity and/or binding half-life of the TCR of the present invention for the YMLDLQPET-HLA A0201 complex is at least twice that of the wild-type TCR.
  • TCR T cell receptor
  • the International Immunogenetics Information System can be used to describe TCR.
  • the natural ⁇ heterodimeric TCR has an ⁇ chain and a ⁇ chain. Broadly speaking, each chain includes a variable region, a connecting region, and a constant region.
  • the beta chain usually also contains a short variable region between the variable region and the connecting region, but the variable region is often regarded as a part of the connecting region.
  • the unique TRAJ and TRBJ of IMGT are used to determine the connection region of TCR, and the TRAC and TRBC of IMGT are used to determine the constant region of TCR.
  • Each variable region contains 3 CDRs (complementarity determining regions), CDR1, CDR2, and CDR3, chimeric in the framework sequence.
  • the different numbers of TRAV and TRBV refer to different types of V ⁇ and V ⁇ , respectively.
  • the alpha chain constant domain has the following symbols: TRAC*01, where "TR” represents the T cell receptor gene; "A” represents the alpha chain gene; C represents the constant region; "*01” represents alleles Gene 1.
  • TRBC1*01 or TRBC2*01 where “TR” represents the T cell receptor gene; “B” represents the ⁇ -chain gene; C represents the constant region; “*01” represents the allele 1.
  • the constant region of the ⁇ chain is uniquely determined.
  • the alpha and beta chains of TCR are generally regarded as having two “domains” each, namely the variable domain and the constant domain.
  • the variable domain is composed of connected variable regions and linking regions. Therefore, in the specification and claims of this application, “TCR ⁇ chain variable domain” refers to the connected TRAV and TRAJ regions, and similarly, “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 ⁇ , respectively.
  • the framework sequence of the TCR variable domain of the present invention may be of murine or human origin, and is preferably of human origin.
  • the constant domain of TCR contains an intracellular part, a transmembrane region and an extracellular part.
  • the amino acid sequences of the alpha and beta chain variable domains of the wild-type TCR capable of binding to the YMLDLQPET-HLA A0201 complex are SEQ ID NO: 1 and SEQ ID NO: 2, respectively, as shown in Fig. 1a and Fig. 1b.
  • the alpha chain amino acid sequence and the beta chain amino acid sequence of the soluble "reference TCR" in the present invention are SEQ ID NO: 11 and SEQ ID NO: 12, respectively, as shown in Figure 6a and Figure 6b.
  • the extracellular amino acid sequence of the alpha chain and the extracellular amino acid sequence of the beta chain of the "wild-type TCR” in the present invention are respectively SEQ ID NO: 50 and SEQ ID NO: 51, as shown in Figure 9a and Figure 9b.
  • the TCR sequence used in the present invention is of human origin.
  • the alpha chain amino acid sequence and the beta chain amino acid sequence of the "wild-type TCR" in the present invention are SEQ ID NO: 52 and SEQ ID NO: 53, as shown in Figures 10a and 10b.
  • the terms "polypeptide of the present invention", “TCR of the present invention", and “T cell receptor of the present invention” are used interchangeably.
  • the position numbers of the amino acid sequence of TRAC*01 and TRBC1*01 or TRBC2*01 in the present invention are numbered in sequence from the N-terminus to the C-terminus.
  • TRBC1*01 or TRBC2*01 press from N
  • the 60th amino acid in the sequence from end to C end is P (proline)
  • Pro60 of TRBC1*01 or TRBC2*01 exon 1 in the present invention or it can be expressed as TRBC1* 01 or TRBC2*01 exon 1 of the 60th amino acid
  • the 61st amino acid is Q (glutamine) in sequence from N-terminal to C-terminal, then this
  • it can be described as Gln61 of TRBC1*01 or TRBC2*01 exon 1, or it can be expressed as the 61st amino acid of TRBC1*01 or TRBC2*01 exon 1, and so on.
  • the position numbers of the amino acid sequences of TRAV and TRBV in the variable regions follow the position numbers listed in IMGT.
  • the position number listed in IMGT is 46, it is described as the 46th amino acid of TRAV in the present invention, and the rest can be deduced by analogy.
  • the sequence position numbers of other amino acids have special instructions, follow the special instructions.
  • tumor is meant to include all types of cancer cell growth or carcinogenic processes, metastatic tissues or malignant transformed cells, tissues or organs, regardless of the pathological type or the stage of infection.
  • tumors include, without limitation, solid tumors, soft tissue tumors, and metastatic lesions.
  • solid tumors include: malignant tumors of different organ systems, such as sarcoma, lung squamous cell carcinoma, and cancer.
  • sarcoma for example: infected prostate, lung, breast, lymph, gastrointestinal (for example: colon), and genitourinary tract (for example: kidney, epithelial cells), pharynx.
  • Lung squamous cell carcinoma includes malignant tumors, for example, most colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer, and esophageal cancer.
  • malignant tumors for example, most colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer, and esophageal cancer.
  • the above-mentioned metastatic lesions of cancer can also be treated and prevented by the method and composition of the present invention.
  • the ⁇ 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 short antigen peptides
  • CDR1 and CDR2 interact with HLA. Therefore, the CDR of the TCR molecule determines its interaction with the antigen short peptide-HLA complex.
  • amino acid sequence of the alpha chain variable domain and the amino acid sequence of the beta chain variable domain of the wild-type TCR that can bind the antigen short peptide YMLDLQPET and HLA A0201 complex are SEQ ID NO:1 and SEQ, respectively ID NO: 2, this sequence is the first discovery by the inventor. It has the following CDR regions:
  • the present invention obtains a high-affinity TCR whose affinity with the YMLDLQPET-HLA A0201 complex is at least 2 times the affinity of the wild-type TCR with the YMLDLQPET-HLA A0201 complex through mutation screening of the above CDR regions.
  • the present invention provides a T cell receptor (TCR), which has the activity of binding YMLDLQPET-HLA A0201 complex.
  • the T cell receptor comprises a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain.
  • the TCR ⁇ chain variable domain comprises 3 CDR regions.
  • the reference sequences of the 3 CDR regions of the TCR ⁇ chain variable domain are as follows:
  • CDR3 ⁇ ALYNQGGKLI, and CDR3 ⁇ contains at least one of the following mutations:
  • the number of amino acid mutations in the CDR region of the TCR ⁇ chain may be 2, 3 or 4.
  • the TCR of the present invention is an ⁇ heterodimeric TCR
  • the ⁇ chain variable domain of the TCR contains at least 85% of the amino acid sequence shown in SEQ ID NO:1; preferably, at least 90%; more preferably Preferably, at least 92%; more preferably, at least 94% (eg, it can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% %, 99% sequence homology); and/or the ⁇ -chain variable domain of the TCR contains at least 90% of the amino acid sequence shown in SEQ ID NO: 2, preferably , At least 92%; more preferably, at least 94% (eg, it can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the same sequence Source) the amino acid sequence of sequence homology.
  • the TCR of the present invention is a single-chain TCR
  • the ⁇ -chain variable domain of the TCR contains at least 85%, preferably at least 90%, and more preferably at least the amino acid sequence shown in SEQ ID NO: 3; 92%; most preferably, at least 94% (eg, it can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% % Sequence homology); and/or the ⁇ -chain variable domain of the TCR contains at least 85% of the amino acid sequence shown in SEQ ID NO: 4, preferably at least 90%.
  • % more preferably, at least 92%; most preferably, at least 94%; (e.g., it can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% The sequence homology) of the sequence homology of the amino acid sequence.
  • the three CDRs of the wild-type TCR ⁇ chain variable domain SEQ ID NO:1, namely CDR1, CDR2, and CDR3 are located at positions 27-32, 50-56, and 91-100 of SEQ ID NO:1, respectively .
  • the amino acid residue numbering adopts the numbering shown in SEQ ID NO:1, 94N is the 4th N of CDR3 ⁇ , 95Q is the 5th Q of CDR3 ⁇ , and 96G is the 6th G and 97G of CDR3 ⁇ . It is the 7th G of CDR3 ⁇ , and 98K is the 8th K of CDR3 ⁇ .
  • the present invention provides a TCR with the characteristics of binding YMLDLQPET-HLA A0201 complex, and includes an ⁇ chain variable domain and a ⁇ chain variable domain, and is characterized in that the TCR is in the ⁇ chain variable shown in SEQ ID NO:1 A mutation occurs in the domain, and the mutated amino acid residue positions include one or more of 94N, 95Q, 96G, 97G, and 98K, wherein the numbering of the amino acid residues adopts the numbering shown in SEQ ID NO:1.
  • the TCR ⁇ chain variable domain after mutation includes one or more amino acid residues selected from the following group: 94L or 94A or 94V; 95F or 95Y or 95W; 96N or 96S or 96D or 96A or 96V or 96T Or 96H or 96M; 97A or 97N or 97S or 97F; 98V or 98R or 98L or 98I or 98M or 98Q or 98S or 98N, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO:1.
  • variable domain of the ⁇ chain include N94L/A/V, Q95F/Y/W, G96N/S/D/A/V/T/H/M, G97A/N/S One or several groups among /F, K98V/R/L/I/M/Q/S/N.
  • the three CDRs of the wild-type TCR ⁇ chain variable domain SEQ ID NO: 2, namely CDR1, CDR2, and CDR3 are located at positions 27-31, 49-54, and 93-104 of SEQ ID NO: 2, respectively . Accordingly, the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 2, 102E is the 10th position E of CDR3 ⁇ , 103Q is the 11th position Q of CDR3 ⁇ , and 104Y is the 12th position Y of CDR3 ⁇ .
  • the present invention provides a TCR that has the property of binding YMLDLQPET-HLA A0201 complex, and includes a ⁇ -chain variable domain and a ⁇ -chain variable domain, and is characterized in that the TCR is in the ⁇ -chain variable shown in SEQ ID NO: 2 A mutation occurs in the domain, and the mutated amino acid residue positions include one or more of 102E, 103Q and 104Y, wherein the numbering of the amino acid residues adopts the numbering shown in SEQ ID NO: 2.
  • the mutated TCR ⁇ chain variable domain includes one or more amino acid residues selected from the following group: 102Y or 102M, 103V or 103L, 104S or 104E, wherein the amino acid residue numbering adopts SEQ ID NO: The number shown in 2.
  • the specific form of the mutation in the ⁇ chain variable domain includes one or several groups of E102Y/M, Q103V/L, and Y104S/E.
  • the Thr48 of the wild-type TCR ⁇ chain constant region TRAC*01 exon 1 was mutated to cysteine, and the ⁇ chain constant region TRBC1*01 or TRBC2*01 exon 1
  • the Ser57 of Ser57 is mutated to cysteine to obtain the reference TCR. Its amino acid sequence is SEQ ID NO: 11 and SEQ ID NO: 12, respectively, as shown in Figure 6a and Figure 6b, the cysteine residue after mutation Expressed in bold letters.
  • the above cysteine substitution can form artificial interchain disulfide bonds between the constant regions of the ⁇ and ⁇ chains of the reference TCR to form a more stable soluble TCR, which makes it easier to evaluate the complex of TCR and YMLDLQPET-HLA A0201
  • the binding affinity and/or binding half-life between substances 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 YMLDLQPET-HLA A0201 complex is considered to be the binding affinity between the wild-type TCR and the YMLDLQPET-HLA A0201 complex.
  • the binding affinity between the TCR of the present invention and the YMLDLQPET-HLA A0201 complex is at least 10 times that of the reference TCR and the YMLDLQPET-HLA A0201 complex, it is equivalent to the TCR of the present invention and YMLDLQPET.
  • the binding affinity between -HLA A0201 complex is at least 10 times that between wild-type TCR and YMLDLQPET-HLA A0201 complex.
  • the binding affinity (inversely proportional to the dissociation equilibrium constant K D ) and the binding half-life (expressed as T 1/2 ) can be measured by any suitable method, such as detection by surface plasmon resonance technology. It should be understood that doubling the affinity of TCR will cause K D to be halved. T 1/2 is calculated as In2 divided by the dissociation rate (K off ). Therefore, doubling T 1/2 will cause K off to be halved.
  • the same test protocol is used to detect the binding affinity or binding half-life of a given TCR several times, for example, 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: a temperature of 25° C. and a pH of 7.1-7.5.
  • This method detects that the dissociation equilibrium constant K D of the reference TCR to the YMLDLQPET-HLA A0201 complex is 6.31E-05M, which is 63.1 ⁇ M, and the present invention considers the dissociation equilibrium of the wild-type TCR to the YMLDLQPET-HLA A0201 complex
  • the constant K D is also 63.1 ⁇ M.
  • the affinity to YMLDLQPET-HLA A0201 complex is 10 times that of wild-type TCR to YMLDLQPET-HLA A0201 complex.
  • the affinity of the TCR and YMLDLQPET-HLA A0201 complex is at least 2 times that of wild-type TCR; preferably, at least 5 times; more preferably, at least 10 times.
  • Any suitable method can be used for mutation, including but not limited to those based on polymerase chain reaction (PCR), cloning based on restriction enzymes, or ligation-independent cloning (LIC) methods.
  • PCR polymerase chain reaction
  • LIC ligation-independent cloning
  • the method for producing the TCR of the present invention can be, but is not limited to, screening a TCR with high affinity for the YMLDLQPET-HLA-A0201 complex from a diverse library of phage particles displaying such TCR, as shown in the literature (Li, et al. (2005) Nature Biotech 23(3):349-354).
  • genes expressing wild-type TCR alpha and beta chain variable domain amino acids or genes expressing slightly modified wild-type TCR alpha and beta chain variable domain amino acids can be used to prepare template TCRs.
  • the DNA encoding the variable domain of the template TCR is then introduced into the changes required to produce the high-affinity TCR of the present invention.
  • the high-affinity TCR of the present invention comprises one of the alpha chain variable domain amino acid sequence SEQ ID NO: 13-46 and/or the beta chain variable domain amino acid sequence SEQ ID NO: 2.
  • the amino acid sequences of the ⁇ -chain variable domain and ⁇ -chain variable domain forming the heterodimeric TCR molecule are preferably from Table 1 below:
  • the TCR of the present invention is a part having at least one TCR ⁇ and/or TCR ⁇ chain variable domain. They usually contain both the TCR ⁇ chain variable domain and the TCR ⁇ chain variable domain. They can be ⁇ heterodimers or single-stranded forms or any other forms that can exist stably. In adoptive immunotherapy, the full-length chain of ⁇ heterodimeric TCR (including cytoplasm and transmembrane domain) can be transfected.
  • the TCR of the present invention can be used as a targeting agent for delivering therapeutic agents to antigen-presenting cells or combined with other molecules to prepare bifunctional polypeptides to target effector cells. In this case, the TCR is preferably in a soluble form.
  • the prior art discloses that the introduction of artificial interchain disulfide bonds between the ⁇ and ⁇ chain constant domains of TCR can obtain soluble and stable TCR molecules, as described in patent document PCT/CN2015/093806 Narrated. Therefore, the TCR of the present invention may be a TCR in which an artificial interchain disulfide bond is introduced between the residues of the constant domain of its ⁇ and ⁇ chains. 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 positions in the natural TCR to form artificial interchain disulfide bonds.
  • Thr48 in TRAC*01 exon 1 and replacing Ser57 in TRBC1*01 or TRBC2*01 exon 1 to form a disulfide bond can also be: Thr45 of TRAC*01 exon 1 and TRBC1*01 or Ser77 of TRBC2*01 exon 1; TRAC*01 exon Tyr10 of 1 and Ser17 of TRBC1*01 or TRBC2*01 exon 1; Thr45 of TRAC*01 exon 1 and Asp59 of TRBC1*01 or TRBC2*01 exon 1; TRAC*01 exon 1 Ser15 and Glu15 of TRBC1*01 or TRBC2*01 exon 1; Arg53 of TRAC*01 exon 1 and Ser54 of TRBC1*01 or TRBC2*01 exon 1; Pro89 and Pro89 of TRAC*01 exon 1 Ala19 of TRBC1*01 or TRBC2*01 exon 1; or Tyr10 of TRAC*01 ex
  • cysteine residues replace any set of positions in the constant domains of the ⁇ and ⁇ chains.
  • One or more C-terminals of the TCR constant domain of the present invention can be truncated up to 15, or up to 10, or up to 8 or less amino acids so that it does not include cysteine residues to achieve the deletion of natural
  • the purpose of the interchain disulfide bond can also be achieved by mutating the cysteine residue that forms the natural interchain disulfide bond to another amino acid.
  • the TCR of the present invention may contain artificial interchain disulfide bonds introduced between the residues of the constant domains of its ⁇ and ⁇ chains. It should be noted that, with or without the introduced artificial disulfide bonds between the constant domains, the TCR of the present invention can contain the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence.
  • the TRAC constant domain sequence of TCR and the TRBC1 or TRBC2 constant domain sequence can be linked by natural interchain disulfide bonds present in the TCR.
  • patent document PCT/CN2016/077680 also discloses that the introduction of artificial interchain disulfide bonds between the ⁇ chain variable region and the ⁇ chain constant region of the TCR can significantly improve the stability of the TCR. Therefore, the high-affinity TCR of the present invention may also contain artificial interchain disulfide bonds between the ⁇ chain variable region and the ⁇ chain constant region.
  • cysteine residue that forms an artificial interchain disulfide bond between the ⁇ chain variable region and the ⁇ chain constant region of the TCR is substituted: the 46th amino acid of TRAV and TRBC1*01 or TRBC2* The 60th amino acid of 01 exon 1; the 47th amino acid of TRAV and the 61st amino acid of TRBC1*01 or TRBC2*01 exon 1; the 46th amino acid of TRAV and the TRBC1*01 or TRBC2*01 exon The 61st amino acid of sub 1; or the 47th amino acid of TRAV and the 60th amino acid of TRBC1*01 or TRBC2*01 exon 1.
  • such a TCR may comprise (i) all or part of the TCR ⁇ chain excluding its transmembrane domain, and (ii) all or part of the TCR ⁇ chain excluding its transmembrane domain, wherein (i) and (ii) ) Contains the variable domain and at least a part of the constant domain of the TCR chain, and the ⁇ chain and the ⁇ chain form a heterodimer. More preferably, such a TCR may include an ⁇ chain variable domain and a ⁇ chain variable domain and all or part of the ⁇ chain constant domain except the transmembrane domain, but it does not include the ⁇ chain constant domain. The chain variable domain and the ⁇ chain form a heterodimer.
  • the TCR of the present invention also includes TCRs with mutations in the hydrophobic core region.
  • These mutations in the hydrophobic core region are preferably mutations that can improve the stability of the TCR of the present invention, as described in Publication No. It is described in the patent document of WO2014/206304.
  • Such a TCR can be mutated at the following variable domain hydrophobic core positions: ( ⁇ and/or ⁇ chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/ Or alpha chain J gene (TRAJ) short peptide amino acid positions from the bottom 3, 5, and 7; According to the position number listed in the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • the TCR with a mutation in the hydrophobic core region of the present invention may be a highly stable single-chain TCR composed of a flexible peptide chain connecting the variable domains of the ⁇ and ⁇ chains of the TCR.
  • the CDR region of the TCR variable region determines its affinity with the short peptide-HLA complex. Mutations in 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 the present invention can be any peptide chain suitable for connecting the variable domains of the TCR ⁇ and ⁇ chains.
  • the template chain constructed in Example 1 of the present invention for screening high-affinity TCR is the above-mentioned high-stability single-chain TCR containing a hydrophobic core mutation. Using TCR with higher stability can more conveniently evaluate the affinity between TCR and YMLDLQPET-HLA-A0201 complex.
  • the CDR regions of the ⁇ -chain variable domain and ⁇ -chain variable domain of the single-chain template TCR are exactly the same as the CDR regions of the wild-type TCR. That is, the three CDRs of the ⁇ chain variable domain are CDR1 ⁇ : ATGYPS; CDR2 ⁇ : ATKADDK; CDR3 ⁇ : ALYNQGGKLI and the three CDRs of the ⁇ chain variable domain are CDR1 ⁇ : SGHTA; CDR2 ⁇ : FQGTGA; CDR3 ⁇ : ASSLLAGSYEQY.
  • 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. Based on this, a single-chain TCR composed of ⁇ -chain variable domain and ⁇ -chain variable domain with high affinity to YMLDLQPET-HLA A0201 complex was screened out.
  • the ⁇ heterodimer with high affinity to the YMLDLQPET-HLA-A0201 complex of the present invention is obtained by transferring the CDR regions of the ⁇ and ⁇ chain variable domains of the selected high-affinity single-chain TCR To the corresponding positions of the wild-type TCR ⁇ chain variable domain (SEQ ID NO: 1) and ⁇ chain variable domain (SEQ ID NO: 2).
  • the TCR of the present invention can also be provided in the form of a multivalent complex.
  • the multivalent TCR complex of the present invention comprises a polymer formed by combining two, three, four or more TCRs of the present invention.
  • the tetramerization domain of p53 can be used to generate a tetramer, or more A complex formed by combining the TCR of the present invention with another molecule.
  • the TCR complex of the present invention can be used to track or target cells presenting a specific antigen in vitro or in vivo, and can also be used to produce intermediates of other multivalent TCR complexes with such applications.
  • the TCR of the present invention can be used alone or combined with the conjugate in a covalent or other manner, preferably in a covalent manner.
  • the conjugate includes a detectable marker (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the YMLDLQPET-HLA-A0201 complex), a therapeutic agent, a PK (protein kinase) modified portion, or any of the above Combination of substances combined or coupled.
  • Detectable markers used for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computed tomography technology) contrast agents, or capable of producing detectable products Of enzymes.
  • Therapeutic agents that can be combined or coupled with the TCR of the present invention include but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539); 2. Biotoxicity (Chaudhary et al., 1989) , 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/Nano Stick (Lapotko et al., 2005, Cancer letters 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115); 7. Virus particles (Peng et al., 2004, Gene Treatment (Genetherapy) 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research (Cancer research) 65, 11631); 9. Nano magnetic particles; 10. Prodrug activating enzymes (for example, DT-cardiac Diazyme (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. Chemotherapeutics (for example, cisplatin) or any form of nanoparticles, etc.
  • DTD DT-cardiac Diazyme
  • BPHL biphenyl hydrolase-like protein
  • the antibodies or fragments thereof that bind to the TCR of the present invention include anti-T cell or NK-cell determining antibodies, such as anti-CD3 or anti-CD28 or anti-CD16 antibodies.
  • the combination of the above-mentioned antibodies or fragments with TCR can affect effector cells. Orientation to better target target cells.
  • a preferred embodiment is that the TCR of the present invention is combined with an anti-CD3 antibody or a functional fragment or variant of the anti-CD3 antibody.
  • the fusion molecule of the TCR and the anti-CD3 single chain antibody of the present invention includes one selected from the amino acid sequence of the variable domain of TCR ⁇ chain SEQ ID NO: 13-46, and the amino acid sequence of the variable domain of TCR ⁇ chain SEQ ID NO: 2.
  • the invention also relates to nucleic acid molecules encoding the TCR of the invention.
  • the nucleic acid molecule of the present invention may be in the form of DNA or RNA.
  • DNA can be a coding strand or a non-coding strand.
  • the nucleic acid sequence encoding the TCR of the present invention may be the same as the nucleic acid sequence shown in the drawings of the present invention or a degenerate variant.
  • degenerate variant refers to a protein sequence that encodes SEQ ID NO: 3, but has the same sequence as SEQ ID NO: 5 Different nucleic acid sequences.
  • the full-length sequence of the nucleic acid molecule of the present invention or its fragments can usually be obtained by but not limited to PCR amplification method, recombination method or artificial synthesis method.
  • the DNA sequence encoding the TCR (or a fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • the present invention also relates to a vector containing the nucleic acid molecule of the present invention, and a host cell produced by genetic engineering using the vector or coding sequence of the present invention.
  • the present invention also includes isolated cells expressing the TCR of the present invention, 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 invention (eg, Robbins et al., (2008) J. Immunol. 180: 6116-6131).
  • T cells expressing the high-affinity TCR of the present invention can be used for adoptive immunotherapy.
  • Those skilled in the art can know many suitable methods for adoptive therapy (eg, Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).
  • the present invention also provides a pharmaceutical composition containing a pharmaceutically acceptable carrier and the TCR of the present invention, or the TCR complex of the present invention, or a cell presenting the TCR of the present invention.
  • the present invention also provides a method for treating diseases, comprising administering an appropriate amount of the TCR of the present invention, or the TCR complex of the present invention, or cells presenting the TCR of the present invention, or the pharmaceutical composition of the present invention to a subject in need of treatment.
  • amino acids in this article are identified by internationally accepted 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 proline at position 60.
  • the specific form of the mutation in the present invention is expressed as "N93D” represents that the N at position 93 is replaced by D.
  • N93D/E represents that the N at position 93 is replaced by D or replaced by E. The rest can be deduced by analogy.
  • the TCR of the present invention also includes at most 5 of the TCR of the present invention, preferably at most 3, more preferably at most 2, and most preferably 1 amino acid (especially the amino acid located outside the CDR region), which are similar in nature. Or similar amino acids are replaced, and still maintain its functional TCR.
  • the present invention also includes a TCR slightly modified from the TCR of the present invention.
  • Modified (usually without changing the primary structure) forms include: chemically derived forms of the TCR of the present invention such as acetylation or carboxylation.
  • Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of the TCR of the present invention or in further processing steps. This modification can be accomplished by exposing the TCR to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase).
  • Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes TCR that has been modified to improve its resistance to proteolysis or optimize its solubility.
  • the TCR, TCR complex of the present invention or T cells transfected with the TCR of the present invention can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the TCR, multivalent TCR complex or cell of the present invention is usually provided as part of a sterile pharmaceutical composition, which usually includes a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be in any suitable form (depending on the desired method of administration to the patient). It can be provided in a unit dosage form, usually in a sealed container, and can be provided as part of a kit. Such kits (but not required) include instructions for use. It may include a plurality of such unit dosage forms.
  • the TCR of the present invention can be used alone, or can be combined or coupled with other therapeutic agents (for example, formulated in the same pharmaceutical composition).
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent.
  • pharmaceutical carriers that do not themselves induce the production of antibodies that are harmful to the individual receiving the composition, and do not have excessive toxicity after administration. These vectors are well known to those of ordinary skill in the art. A full 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.
  • the pharmaceutically acceptable carrier in the therapeutic composition may contain liquids such as water, saline, glycerol and ethanol.
  • these carriers may also contain auxiliary substances, such as wetting or emulsifying agents, and pH buffering substances.
  • the therapeutic composition can be made into an injectable, such as a liquid solution or suspension; it can also be made into a solid form suitable for being formulated into a solution or suspension in a liquid carrier before injection.
  • an injectable such as a liquid solution or suspension
  • it can also be made into a solid form suitable for being formulated into a solution or suspension in a liquid carrier before injection.
  • composition of the present invention can be administered by conventional routes, including (but not limited to): intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably gastrointestinal External includes subcutaneous, intramuscular or intravenous.
  • the object to be prevented or treated can be an animal; especially a human.
  • composition of the present invention When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition can be used according to the use situation. 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 (Isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the formulation process can be carried out in a customary manner according to the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic (Isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the formulation process can be carried out in a customary manner according to the dosage form.
  • the pharmaceutical composition of the present invention can also be administered in the form of a sustained-release formulation.
  • the TCR of the present invention can be incorporated into a pill or microcapsule with a sustained-release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated.
  • sustained-release polymers ethylene-vinyl acetate copolymers, 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 invention as the active ingredient or the cells presenting the TCR of the present invention can be based on the weight, age, sex, and degree of symptoms of each patient to be treated. The reasonable dosage is determined by the doctor in the end.
  • the affinity and/or binding half-life of the high-affinity TCR of the present invention for the YMLDLQPET-HLA-A0201 complex is at least 2 times, preferably at least 5 times, and more preferably at least 10 times that of wild-type TCR.
  • the high-affinity TCR of the present invention can specifically bind to the YMLDLQPET-HLA A0201, and the cells transfected with the high-affinity TCR of the present invention can be specifically activated and proliferate.
  • the effector cells transfected with the high-affinity TCR of the present invention have a strong killing effect on target cells.
  • E. coli DH5 ⁇ was purchased from Tiangen
  • E. coli BL21 (DE3) was purchased from Tiangen
  • E. coli Tuner (DE3) was purchased.
  • plasmid pET28a was purchased from Novagen.
  • Example 1 Stability of hydrophobic core mutations. Generation of single-stranded TCR template strands
  • the present invention uses the method of site-directed mutagenesis, according to the patent document WO2014/206304, to construct a stable single-stranded TCR molecule composed of a flexible short peptide (linker) connecting TCR ⁇ and ⁇ chain variable domains, its amino acids 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 ⁇ variable domain (SEQ ID NO: 3) and ⁇ variable domain (SEQ ID NO: 4) of the template chain are shown in Figures 2a and 2b; the corresponding DNA sequences are respectively SEQ ID NO: 5 And 6, as shown in Figures 3a and 3b; the amino acid sequence and DNA sequence of the flexible short peptide (linker) are SEQ ID NO: 7 and 8, respectively, as shown in Figures 4a and 4b.
  • the target gene carrying the template chain was digested with Nco I and Not I, and then ligated with the pET28a vector that was digested with Nco I and Not I.
  • the ligation product was transformed into E.coli DH5 ⁇ , spread on an LB plate containing kanamycin, incubated overnight at 37°C, and selected positive clones for PCR screening, and sequenced the positive recombinants to confirm the correct sequence and extract the recombinant plasmid for transformation To E.coli BL21(DE3), for expression.
  • Example 2 Expression, renaturation and purification of the stable single-chain TCR constructed in Example 1
  • the inclusion bodies were dissolved in a buffer (20mM Tris-HCl pH 8.0, 8M urea), centrifuged at a high speed to remove insoluble materials, the supernatant was quantified by BCA method, then aliquoted, and stored at -80°C for later use.
  • a syringe to drop the single-stranded TCR treated above into 125mL of refolding buffer (100mM Tris-HCl pH 8.1, 0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mM ⁇ -mercapthoethylamine, 1.87mM Cystamine), Stir at 4°C for 10 minutes, then put the refolding solution into a cellulose membrane dialysis bag with a cutoff of 4kDa, place the dialysis bag in 1L of pre-cooled water, and slowly stir overnight at 4°C.
  • refolding buffer 100mM Tris-HCl pH 8.1, 0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mM ⁇ -mercapthoethylamine, 1.87mM Cystamine
  • the collected elution fractions were analyzed by SDS-PAGE, and the fractions containing single-stranded TCR were concentrated and further purified with a gel filtration column (Superdex 75 10/300, GE Healthcare), and the target fractions were also analyzed by SDS-PAGE.
  • the eluted fractions used for BIAcore analysis were further tested for purity by gel filtration.
  • the conditions are: 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 UV detection wavelength is 214nm.
  • the BIAcore T200 real-time analysis system was used to detect the binding activity of TCR molecules and YMLDLQPET-HLA-A0201 complex.
  • the conditions are: the temperature is 25°C, and the pH is 7.1-7.5.
  • the binding time of each injection is 120s, and let it dissociate for 600s after the last injection.
  • the chip was regenerated with 10mM Gly-HCl pH 1.75. Use BIAcore Evaluation software to calculate kinetic parameters.
  • YMLDLQPET peptide at 25mg/L (final concentration) into the refolding buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4°C), then add 20mg/L light chain and 90mg/L heavy chain (final concentration, heavy chain is added three times, 8h/time), and renaturate at 4°C for at least 3 days Upon completion, SDS-PAGE will test whether the renaturation is successful.
  • Biotinylation Concentrate the purified pMHC molecules with a Millipore ultrafiltration tube, and replace the buffer with 20mM Tris pH 8.0, and then add the biotinylation reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50 ⁇ M D- Biotin, 100 ⁇ g/ml BirA enzyme (GST-BirA), incubate the mixture overnight at room temperature, SDS-PAGE to detect whether the biotinylation is complete.
  • the protein-containing fractions were combined, concentrated with a Millipore ultrafiltration tube, the protein concentration was determined by the BCA method (Thermo), and the protease inhibitor cocktail (Roche) was added to store the biotinylated pMHC molecules in aliquots at -80°C.
  • Phage display technology is a means to generate a library of TCR high-affinity variants to screen 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-stranded TCR template in Example 1.
  • a high-affinity TCR library was established and panning was performed. After several rounds of panning, the phage library has specific binding to the corresponding antigen, and a single clone is selected from it and analyzed.
  • the screened high-affinity single-chain TCR mutations in the CDR region were introduced into the corresponding positions of the variable domains of the ⁇ heterodimeric TCR, and the affinity with the YMLDLQPET-HLA-A0201 complex was detected by BIAcore.
  • the introduction of the above-mentioned high-affinity mutation points in the CDR region adopts a site-directed mutation method well known to those skilled in the art.
  • the amino acid sequences of the alpha chain and beta chain variable domains of the wild-type TCR are shown in Figure 1a (SEQ ID NO: 1) and 1b (SEQ ID NO: 2), respectively.
  • the ⁇ heterodimeric TCR can be constant in the ⁇ and ⁇ chains.
  • a cysteine residue is introduced into the regions to form the TCR of the artificial inter-chain disulfide bond.
  • the amino acid sequences of the TCR ⁇ and ⁇ chains after the introduction of cysteine residues are shown in Figure 6a (SEQ ID NO : 11) and shown in 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 vector by the standard method described in the "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 region are introduced by overlapping PCR (overlap PCR) well known to those skilled in the art. The inserted fragment was confirmed by sequencing.
  • TCR ⁇ and ⁇ chains were respectively transformed into expressing bacteria BL21(DE3) by chemical transformation method.
  • the ⁇ and ⁇ chains of TCR were expressed
  • the inclusion bodies formed later were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution.
  • the inclusion bodies were finally dissolved in 6M guanidine hydrochloride, 10mM dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA) ), 20mM Tris (pH 8.1).
  • the dissolved TCR ⁇ and ⁇ chains are quickly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine, 6.6mM ⁇ -mercapoethylamine (4°C) at a mass ratio of 1:1, and the final concentration is 60mg/mL.
  • 5M urea 20mM Tris (pH 8.1)
  • 20mM Tris 20mM Tris (pH 8.1)
  • cystamine 3.7mM cystamine
  • 6.6mM ⁇ -mercapoethylamine 4°C
  • the solution is filtered through a 0.45 ⁇ M filter membrane, and then purified by an anion exchange column (HiTrap Q HP, 5ml, GE Healthcare).
  • the eluted peak contains the successfully renatured ⁇ and ⁇ dimer TCR confirmed by SDS-PAGE gel.
  • TCR is then further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100 HR, 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.
  • Example 3 The method described in Example 3 was used to detect the affinity of the ⁇ heterodimeric TCR introduced into the high-affinity CDR region and the YMLDLQPET-HLA-A0201 complex.
  • the present invention obtains high-affinity TCR alpha and beta chain variable domain amino acid sequences, as shown in Figure 7 (1)- Figure 7 (34) and Figure 8a-c, 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 with high-affinity mutation points will also have high affinity for the YMLDLQPET-HLA-A0201 complex. .
  • Example 4 Use the method described in Example 4 to construct an expression vector, use the method described in Example 5 to express, renature and purify the above-mentioned ⁇ heterodimeric TCR with high affinity mutations, and then use BIAcore T200 to determine its relationship with YMLDLQPET-
  • the affinity of the HLA-A0201 complex is shown in Table 2 below.
  • the affinity of the heterodimeric TCR is at least twice the affinity of the wild-type TCR for the YMLDLQPET-HLA-A0201 complex.
  • Example 7 Expression, renaturation and purification of fusion of anti-CD3 antibody and high-affinity ⁇ heterodimeric TCR
  • the anti-CD3 single-chain antibody (scFv) is fused with ⁇ heterodimeric TCR to prepare a fusion molecule.
  • the anti-CD3 scFv is fused with the ⁇ chain of the TCR.
  • the TCR ⁇ chain may include any of the above-mentioned high-affinity ⁇ heterodimeric TCR ⁇ -chain variable domains
  • the TCR ⁇ chain of the fusion molecule may include any of the above-mentioned high-affinity The alpha chain variable domain of a sexual alpha beta heterodimeric TCR.
  • the construction of the ⁇ chain expression vector The target gene carrying the ⁇ chain of ⁇ heterodimeric TCR was digested with Nco I and Not I, and then linked to the pET28a vector that was digested with Nco I and Not I.
  • the ligation product was transformed into E.coli DH5 ⁇ , spread on an LB plate containing kanamycin, and incubated overnight at 37°C. Positive clones were selected for PCR screening, the positive recombinants were sequenced, and the recombinant plasmids were extracted after the sequence was confirmed. Transform into E.coli Tuner (DE3) for expression.
  • anti-CD3(scFv)- ⁇ chain expression vector by overlapping PCR method, design primers to connect anti-CD3scFv and high-affinity heterodimeric TCR ⁇ chain gene, and the middle connection is short
  • the peptide (linker) is GGGGS (SEQ ID NO: 31)
  • the gene fragment of the fusion protein of the anti-CD3 scFv and the high-affinity heterodimeric TCR ⁇ chain has the restriction endonuclease site Nco I ( CCATGG (SEQ ID NO: 32)) and Not I (GCGGCCGC (SEQ ID NO: 33)).
  • the PCR amplified product was digested with Nco I and Not I, and ligated with the pET28a vector digested with Nco I and Not I.
  • the ligation product was transformed into E.coli DH5 ⁇ competent cells, spread on LB plates containing kanamycin, incubated overnight at 37°C, picked positive clones for PCR screening, sequenced the positive recombinants, and extracted after confirming the correct sequence
  • the recombinant plasmid is 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 incubated overnight at 37°C. On the next day, pick the clones and inoculate them into 10mL LB liquid medium (kanamycin 50 ⁇ g/mL) for 2-3 hours, inoculate them into 1L LB medium at a volume ratio of 1:100, continue to cultivate until the OD600 is 0.5-0.8, add The final concentration is 1mM IPTG induces the expression of the target protein. After 4 hours of induction, the cells were harvested by centrifugation at 6000 rpm for 10 min. Wash the cells once with PBS buffer and separate the cells.
  • the dissolved TCR ⁇ chain and anti-CD3 (scFv)- ⁇ chain are quickly mixed with 5M urea (urea), 0.4M L-arginine (L-arginine), 20mM Tris pH 8.1, 3.7 at a mass ratio of 2:5 mM cystamine, 6.6mM ⁇ -mercapoethylamine (4°C), the final concentration of ⁇ chain and anti-CD3 (scFv)- ⁇ chain are 0.1 mg/mL and 0.25 mg/mL, respectively.
  • the TCR fusion molecule is then further purified by size exclusion chromatography (S-100 16/60, GE healthcare), and again purified by anion exchange column (HiTrap Q HP 5ml, GE healthcare).
  • the purity of the purified TCR fusion molecule was determined by SDS-PAGE to be greater than 90%, and the concentration was determined by the BCA method.
  • Example 8 For T2 cells loaded with short peptides, the activating function experiment of effector cells transfected with the high-affinity TCR of the present invention
  • This example verifies that the effector cells transfected with the high-affinity TCR of the present invention have a good specific activation effect on target cells.
  • the function and specificity of the high-affinity TCR of the present invention in cells are tested by the ELISPOT experiment well-known to those skilled in the art. Randomly select CD3+ T cells isolated from the blood of healthy volunteers by transfecting the high-affinity TCR of the present invention as effector cells, and use the same volunteer to transfect wild-type TCR (WT-TCR) and other TCRs (A6)
  • WT-TCR wild-type TCR
  • A6 wild-type TCR
  • the CD3+ T cells were used as a control group.
  • the target cells used were T2 cells loaded with HPV16 E7 antigen short peptide YMLDLQPET, and T2 cells loaded with other short peptides and empty T2 cells were used as controls.
  • TCR1 ⁇ chain variable domain SEQ ID NO: 13, ⁇ chain variable domain SEQ ID NO: 2)
  • TCR9 ⁇ chain variable domain SEQ ID NO: 21, ⁇ chain variable domain SEQ ID NO: 2)
  • TCR3 ⁇ chain variable domain SEQ ID NO: 15, ⁇ chain variable domain SEQ ID NO: 2)
  • TCR10 ⁇ chain variable domain SEQ ID NO: 22, ⁇ chain variable domain SEQ ID NO: 2)
  • TCR11 ⁇ chain variable domain SEQ ID NO: 23, ⁇ chain variable domain SEQ ID NO: 2)
  • TCR12 ⁇ chain variable domain SEQ ID NO: 24, ⁇ chain variable domain SEQ ID NO: 2)
  • TCR14 ⁇ chain variable domain SEQ ID NO: 26, ⁇ chain variable domain SEQ ID NO: 2)
  • TCR4 ⁇ chain variable domain SEQ ID NO: 16, ⁇ chain variable domain SEQ ID NO: 2)
  • TCR15 ⁇ chain variable domain SEQ ID NO: 27, ⁇ chain variable domain SEQ ID NO: 2)
  • TCR23 ⁇ chain variable domain SEQ ID
  • TCR6 ⁇ chain variable domain SEQ ID NO: 18, ⁇ chain variable domain SEQ ID NO: 2
  • TCR16 ⁇ chain variable domain SEQ ID NO: 28, ⁇ chain variable domain SEQ ID NO: 2
  • TCR22 ⁇ chain variable domain SEQ ID NO: 31, ⁇ chain variable domain SEQ ID NO: 2)
  • TCR7 ⁇ chain variable domain SEQ ID NO: 19, ⁇ chain variable domain SEQ ID NO: 2
  • TCR5 ⁇ chain variable domain SEQ ID NO: 17, ⁇ chain variable domain SEQ ID NO: 2).
  • ELISPOT plate was activated and coated with ethanol at 4°C overnight.
  • Add the corresponding short peptides so that the final concentration of the short peptides in the ELISPOT plate is 1 ⁇ 10 -6 M.
  • Incubate overnight 37°C, 5% CO 2 ).
  • the plate was washed and subjected to secondary detection and color development, the plate was dried, and then the spots formed on the membrane were counted with an immunospot plate reader (ELISPOT READER system; AID20 company).
  • T cells transfected with the high-affinity TCR of the present invention have a more obvious activation response than those transfected with wild-type T cells.
  • the T cells transfected with other TCRs have no activated state; at the same time, the T cells transfected with the high-affinity TCR of the present invention are not activated by T2 cells loaded with other short peptides or empty.
  • This example also verifies that the effector cells transfected with the high-affinity TCR of the present invention have a good specific activation effect on target cells.
  • the function and specificity of the high-affinity TCR of the present invention in cells are tested by the ELISPOT experiment well-known to those skilled in the art.
  • the CD3+T cells isolated from the blood of healthy volunteers with the high-affinity TCR of the present invention are randomly selected as effector cells, and the CD3+T cells transfected with other TCR (A6) from the same volunteer are used as the control group.
  • the high-affinity TCR and its number are known from Table 2.
  • TCR2 ⁇ chain variable domain SEQ ID NO: 14, ⁇ chain variable domain SEQ ID NO: 2
  • TCR6 ⁇ chain variable domain SEQ ID NO: 18, ⁇ chain variable domain SEQ ID NO: 2
  • TCR16 ⁇ chain variable domain SEQ ID NO: 28, ⁇ chain variable domain SEQ ID NO: 2
  • TCR5 ⁇ chain variable domain SEQ ID NO: 17, ⁇ chain variable domain SEQ ID NO: 2
  • TCR8 ⁇ chain variable domain SEQ ID NO: 20, ⁇ chain variable domain SEQ ID NO: 2).
  • A375-E7 HPV16 E7 overexpression
  • A375, HCCC9810, SK-MEL-1, and KATO-III cells are negative tumor cell lines, as controls.
  • ELISPOT plate was activated and coated with ethanol 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 the test components to the ELISPOT plate in the following order: target cells are 2*10 4 cells/well, effector cells 2*10 3 /well (calculated according to the positive rate of transfection), and set up two duplicate wells. Incubate overnight (37°C, 5% CO 2 ). On the second day of the experiment, the plate was washed and subjected to secondary detection and color development, the plate was dried, and then the spots formed on the membrane were counted with an immunospot plate reader (ELISPOT READER system; AID20 company).
  • ELISPOT READER system an immunospot plate reader
  • the experimental results are shown in Figure 13.
  • the effector cells transfected with the high-affinity TCR of the present invention can be well activated by the positive tumor cell lines, while for negative target cells, the effector cells transfected with the high-affinity TCR of the present invention basically have no activation effect; Effector cells transfected with other TCRs cannot be activated.
  • Example 10 The killing function experiment of the effector cells transfected with the high-affinity TCR of the present invention against T2 cells loaded with short peptides in a gradient
  • a non-radioactive cytotoxicity experiment well-known to those skilled in the art was used to measure the release of LDH, thereby verifying the killing function of the cells transfected with the TCR of the present invention.
  • This test is a colorimetric alternative to the 51Cr release cytotoxicity test, which quantitatively measures the lactate dehydrogenase (LDH) released after cell lysis.
  • LDH lactate dehydrogenase
  • a 30-minute coupled enzyme reaction is used to detect the LDH released in the culture medium.
  • LDH can convert a tetrazolium salt (INT) into red formazan (formazan).
  • the amount of red product produced is proportional to the number of cells lysed.
  • TCR CD3+ T cells isolated from the blood of healthy volunteers were used to transfect the high-affinity TCR of the present invention as effector cells, and the same volunteer was used to transfect wild-type TCR (WT-TCR) and other transfections.
  • WT-TCR wild-type TCR
  • the high-affinity TCR and its number are known from Table 2.
  • TCR1 ⁇ chain variable domain SEQ ID NO: 13, ⁇ chain variable domain SEQ ID NO: 2)
  • TCR3 ⁇ chain variable domain SEQ ID NO: 15, ⁇ chain variable domain SEQ ID NO: 2)
  • TCR4 ⁇ chain variable domain SEQ ID NO: 16, ⁇ chain variable domain SEQ ID NO: 2
  • TCR2 ⁇ chain variable domain SEQ ID NO: 14, ⁇ chain variable domain SEQ ID NO: 2).
  • the target cells are T2 cells loaded with YMLDLQPET peptide, and T2 cells loaded with other antigens and empty are used as controls.
  • the LDH plate First prepare the LDH plate, first press the target cells 3*10 4 cells/well, effector cells 3*10 4 cells/well and add the corresponding ones, and then add the HPV16 E7 antigen short peptide YMLDLQPET to the experimental group, and make it short peptide
  • the final concentration in the ELISPOT plate is from 1 ⁇ 10 -15 M to 1 ⁇ 10 -8 M, a total of 8 gradients; add other short peptides in the control group, and make the final concentration of the short peptides sequentially 1 ⁇ 10 -8 M to 1 ⁇ 10 -6 M, a total of 3 gradients, and three replicate holes.
  • the results of the experiment are shown in Figure 14.
  • the effector cells transfected with the high-affinity TCR of the present invention against T2 cells loaded with gradient HPV16 E7 antigen short peptide YMLDLQPET have a strong killing function, and react when the concentration of the above short peptide is low.
  • the effector cells transfected with other TCRs have no killing effect from the beginning; at the same time, the effector cells transfected with the high-affinity TCR of the present invention have no killing effect on target cells loaded with other short peptides.
  • Example 11 The killing function experiment of effector cells transfected with the high-affinity TCR of the present invention (LDH experiment) against tumor cell lines
  • the non-radioactive cytotoxicity test well-known to those skilled in the art was also used to measure the release of LDH, so as to verify the killing function of the cells transfected with the TCR of the present invention.
  • TCR CD3+ T cells isolated from the blood of healthy volunteers were used to transfect the high-affinity TCR of the present invention as effector cells, and the same volunteer was used to transfect wild-type TCR (WT-TCR) and other transfections.
  • WT-TCR wild-type TCR
  • the high-affinity TCR and its number are known from Table 2.
  • TCR1 ⁇ chain variable domain SEQ ID NO: 13, ⁇ chain variable domain SEQ ID NO: 2)
  • TCR3 ⁇ chain variable domain SEQ ID NO: 15, ⁇ chain variable domain SEQ ID NO: 2)
  • TCR4 ⁇ chain variable domain SEQ ID NO: 16, ⁇ chain variable domain SEQ ID NO: 2
  • TCR2 ⁇ chain variable domain SEQ ID NO: 14, ⁇ chain variable domain SEQ ID NO: 2.
  • CASKI is a positive cell line; while A375 and HCCC9810 are negative cell lines as controls.
  • the test components To prepare the LDH plate, add the test components to the plate in the following order: target cells 25,000 cells/well, effector cells 20,000 cells/well into the corresponding wells, and set up three multiple wells. At the same time, set the spontaneous hole for effector cells, the spontaneous hole for target cells, the largest hole for target cells, the volume correction control hole and the culture medium background control hole. Incubate overnight (37°C, 5% CO 2 ). On the second day of the experiment, the color development was detected. After the reaction was terminated, the absorbance value was recorded at 490nm with a microplate reader (Bioteck).
  • the experimental results are shown in Figure 15.
  • the cells transfected with wild-type TCR can kill positive tumor cell lines by 20%, and the cells transfected with the high-affinity TCR of the present invention have significantly enhanced killing effect on positive tumor cell lines.
  • the effector cells of other TCRs have basically no killing effect.
  • the cells transfected with the high-affinity TCR of the present invention have basically no killing effect on target cells that do not express the relevant antigen.
  • IncuCyte is a functional analysis system that can automatically analyze images at different time points through real-time micro-shooting in the incubator, and quantify the number of apoptosis in real-time.
  • CD3+T cells isolated from the blood of healthy volunteers were randomly selected to be transfected with the TCR of the present invention as effector cells, and CD3+T cells transfected with other TCR (A6) from the same volunteer were used as the control group.
  • the TCR and its number are learned from Table 2. They are TCR1 ( ⁇ chain variable domain SEQ ID NO: 13, ⁇ chain variable domain SEQ ID NO: 2), TCR2 ( ⁇ chain variable domain SEQ ID NO: 14 , ⁇ chain variable domain SEQ ID NO: 2).
  • A375-E7 HPV16 E7 overexpression
  • HCCC9810 is a negative cell line, as a control.
  • the target cells were digested and centrifuged; resuspended in phenol red-free RPMI1640+10% FBS complete medium, and evenly spread the target cells in a 96-well plate: 2*10 4 cells/ Well; put it back in a 37°C, 5% CO2 incubator and incubate overnight; the next day, discard the medium in the 96-well plate and replace it with phenol red-free RPMI1640+10% FBS medium containing the dye caspase3/7reagent , The dye concentration is 2 drops/ml.
  • IncuCyte ZooM a real-time dynamic live cell imaging analyzer dedicated to Incucyte detection, after half an hour of incubation; real-time observation and photos were started; IncuCyte ZooM 2016A was used to process the detection results, analyze and export the data .
  • cells transfected with the high-affinity TCR of the present invention can achieve a strong and effective killing effect in a short period of time, while the effector cells transfected with other TCRs have basically no killing effect; At the same time, the cells transfected with the high-affinity TCR of the present invention have basically no killing effect on target cells that do not express the relevant antigen.

Landscapes

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

Abstract

La présente invention concerne un récepteur de lymphocyte T (TCR) ayant une propriété de liaison au complexe YMLDLQPET-HLA A0201. L'affinité de liaison du TCR au complexe YMLDLQPET-HLA A0201 est au moins 2 fois celle d'un TCR de type sauvage au complexe YMLDLQPET-HLA A0201. L'invention concerne également une molécule de fusion de ce TCR et un agent thérapeutique. Un tel TCR peut être utilisé seul ou en association avec un agent thérapeutique pour cibler des cellules tumorales présentant le complexe YMLDLQPET-HLA A0201.
PCT/CN2021/086455 2020-04-10 2021-04-12 Tcr à haute affinité pour reconnaître hpv16 Ceased WO2021204287A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010280784.3 2020-04-10
CN202010280784.3A CN113512124A (zh) 2020-04-10 2020-04-10 一种识别hpv16的高亲和力tcr

Publications (1)

Publication Number Publication Date
WO2021204287A1 true WO2021204287A1 (fr) 2021-10-14

Family

ID=78022584

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/086455 Ceased WO2021204287A1 (fr) 2020-04-10 2021-04-12 Tcr à haute affinité pour reconnaître hpv16

Country Status (3)

Country Link
CN (1) CN113512124A (fr)
TW (1) TW202144403A (fr)
WO (1) WO2021204287A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2022268348B2 (en) * 2021-11-10 2024-01-18 Tscan Therapeutics, Inc. Binding proteins recognizing hpv16 e7 antigen and uses thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113072635B (zh) * 2020-01-06 2023-08-25 香雪生命科学技术（广东）有限公司 一种识别hpv抗原的t细胞受体及其编码序列

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103249430A (zh) * 2010-09-20 2013-08-14 生物技术公司 抗原特异性t细胞受体和t细胞表位
CN107428816A (zh) * 2015-03-13 2017-12-01 马克思-德布鲁克-分子医学中心亥姆霍兹联合会 针对肿瘤抗原ny‑eso‑1的mhc i和mhc ii‑限制表位的癌症的组合t细胞受体基因疗法
WO2019067243A1 (fr) * 2017-09-29 2019-04-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Récepteurs de lymphocytes t reconnaissant p53 mutée
WO2019195486A1 (fr) * 2018-04-05 2019-10-10 Juno Therapeutics, Inc. Récepteurs de lymphocytes t et cellules modifiées les exprimant

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103249430A (zh) * 2010-09-20 2013-08-14 生物技术公司 抗原特异性t细胞受体和t细胞表位
CN107428816A (zh) * 2015-03-13 2017-12-01 马克思-德布鲁克-分子医学中心亥姆霍兹联合会 针对肿瘤抗原ny‑eso‑1的mhc i和mhc ii‑限制表位的癌症的组合t细胞受体基因疗法
WO2019067243A1 (fr) * 2017-09-29 2019-04-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Récepteurs de lymphocytes t reconnaissant p53 mutée
WO2019195486A1 (fr) * 2018-04-05 2019-10-10 Juno Therapeutics, Inc. Récepteurs de lymphocytes t et cellules modifiées les exprimant

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
YANG YOUDAO, SHI JIANFENG: "Preliminary Study on the Effect of Specific Biological Therapy on the Immune Function of HPV Patients and its Curative Effect", SHIYONG YI-JI ZAZHI = JOURNAL OF PRACTICAL MEDICAL TECHNIQUES, ZHONGHUA YIXUEHUI SHANXI FENHUI, CN, vol. 13, no. 3, 28 February 2006 (2006-02-28), CN , pages 376 - 376, XP055856204, ISSN: 1671-5098 *
YU HUAHUI; YE CHUNMEI; LI JIEYU; PAN CHUNLI; LIN WANSONG; CHEN HUIJING; ZHOU ZHIFENG; YE YUNBIN: "An altered HLA-A0201-restricted MUC1 epitope that could induce more efficient anti-tumor effects against gastric cancer", EXPERIMENTAL CELL RESEARCH, ELSEVIER, AMSTERDAM, NL, vol. 390, no. 1, 7 March 2020 (2020-03-07), AMSTERDAM, NL , XP086125962, ISSN: 0014-4827, DOI: 10.1016/j.yexcr.2020.111953 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2022268348B2 (en) * 2021-11-10 2024-01-18 Tscan Therapeutics, Inc. Binding proteins recognizing hpv16 e7 antigen and uses thereof

Also Published As

Publication number Publication date
CN113512124A (zh) 2021-10-19
TW202144403A (zh) 2021-12-01

Similar Documents

Publication Publication Date Title
CN112759641B (zh) 一种识别Kras G12V的高亲和力TCR
US12275773B2 (en) High affinity cell receptor for recognizing AFP antigen
CN111662374B (zh) 一种识别afp抗原的高亲和力tcr
WO2021204287A1 (fr) Tcr à haute affinité pour reconnaître hpv16
WO2021185368A1 (fr) Tcr à haute affinité pour reconnaître un antigène afp
CN111187345B (zh) 一种识别afp的高亲和力tcr
WO2021043284A1 (fr) Récepteur de lymphocytes t à haute affinité qui reconnaît ssx2
WO2021032020A1 (fr) Récepteur de lymphocytes t à haute affinité pour la reconnaissance d'afp
WO2021036924A1 (fr) Tcr à haute affinité pour reconnaître un antigène ssx2
WO2024217538A1 (fr) Récepteur de lymphocytes t à haute affinité pour identifier une mutation kras et son utilisation
CN114057864B (zh) 一种识别afp的高亲和力tcr
WO2021254458A1 (fr) Récepteur de lymphocytes t à haute affinité pour reconnaître un antigène hpv
WO2023221959A1 (fr) Récepteurs de lymphocytes t à haute affinité pour la reconnaissance de mage et leur utilisation
WO2023179768A1 (fr) Tcr à haute affinité pour identifier un antigène mage-a4, séquence et utilisation de celui-ci
WO2021228255A1 (fr) Récepteur de lymphocytes t à haute affinité pouvant reconnaître un antigène afp
CN117659163A (zh) 一种识别afp的高亲和力t细胞受体及其应用
WO2021023116A1 (fr) Récepteur de slymphocytes t de haute affinité capable d'identifier l'antigène ny-eso-1
WO2022206860A1 (fr) Récepteur des lymphocytes t pour afp
WO2022166905A1 (fr) Tcr à haute affinité contre le hpv
WO2025051209A1 (fr) Tcr à haute affinité ciblant magea4
CN116135879A (zh) 针对afp的高亲和力tcr
WO2022262842A1 (fr) Récepteur de lymphocytes t à haute affinité pour antigène afp
CN115819555A (zh) 一种识别ssx2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: 21783887

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21783887

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 17/05/2023)

122 Ep: pct application non-entry in european phase

Ref document number: 21783887

Country of ref document: EP

Kind code of ref document: A1