WO2022022696A1 - 一种识别afp的高亲和力tcr - Google Patents

一种识别afp的高亲和力tcr Download PDF

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WO2022022696A1
WO2022022696A1 PCT/CN2021/109705 CN2021109705W WO2022022696A1 WO 2022022696 A1 WO2022022696 A1 WO 2022022696A1 CN 2021109705 W CN2021109705 W CN 2021109705W WO 2022022696 A1 WO2022022696 A1 WO 2022022696A1
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tcr
chain
iqssqre
dsaiyn
sghdt
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French (fr)
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李懿
陈少沛
孙含丽
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香雪生命科学技术（广东）有限公司
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    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to the field of biotechnology, and more particularly to a T cell receptor (TCR) capable of recognizing a polypeptide derived from an AFP protein.
  • TCR T cell receptor
  • the present invention also relates to the preparation and use of said receptors.
  • TCR T cell receptor
  • TCR is the only receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC), and this exogenous or endogenous peptide may be the only sign of abnormal cells.
  • MHC major histocompatibility complex
  • APCs antigen-presenting cells
  • the MHC class I and class II molecular ligands corresponding to TCR are also proteins of the immunoglobulin superfamily, but they are specific for the presentation of antigens. Different individuals have different MHCs, which can present a protein antigen. Peptides to the respective APC cell surfaces.
  • the human MHC is often referred to as the HLA gene or HLA complex.
  • AFP ( ⁇ Fetoprotein), also known as ⁇ -fetoprotein, is a protein expressed during embryonic development and is the main component of embryonic serum. During development, AFP is expressed at relatively high levels in the yolk sac and liver, and is subsequently suppressed. In liver cancer, the expression of AFP is activated. AFP is processed intracellularly into antigenic peptides, and combined with MHC (major histocompatibility complex) molecules to form complexes, which are presented on the surface of tumor cells.
  • KWVESIFLIF is a short peptide derived from the AFP antigen and is a target for the treatment of AFP-related diseases.
  • the KWVESIFLIF-HLA A2402 complex provides a TCR-targetable marker for tumor cells.
  • the TCR that can bind to the KWVESIFLIF-HLA A2402 complex has a high application value for tumor therapy.
  • a TCR capable of targeting this tumor cell marker can be used to deliver cytotoxic or immunostimulatory agents to target cells, or be transformed into T cells so that T cells expressing the TCR can destroy tumor cells for Administered to patients during the course of treatment with adoptive immunotherapy.
  • the ideal TCR is one that has a high affinity so that the TCR can reside on the targeted cells for a long time.
  • the purpose of the present invention is to provide a kind of TCR with higher affinity to KWVESIFLIF-HLA A2402 complex.
  • Another object of the present invention is to provide a preparation method of the above-mentioned type of TCR and the use of the above-mentioned type of TCR.
  • the first aspect of the present invention provides a T cell receptor (TCR) comprising an ⁇ chain variable domain and a ⁇ chain variable domain, which has the activity of binding to KWVESIFLIF-HLA A2402 complex, and the TCR ⁇ chain
  • TCR T cell receptor
  • the amino acid sequence of the variable domain has at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence of the variable domain of the TCR beta chain has at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO: 2. 90% sequence homology.
  • the amino acid sequence of the TCR ⁇ chain variable domain and the amino acid sequence of the TCR ⁇ chain variable domain are not both the amino acid sequence of the wild-type TCR ⁇ chain variable domain and the amino acid sequence of the wild-type TCR ⁇ chain variable domain. sequence.
  • the amino acid sequence of the variable domain of the TCR ⁇ chain is not the amino acid sequence shown in SEQ ID NO:1, and/or the amino acid sequence of the variable domain of the TCR ⁇ chain is not the amino acid sequence shown in SEQ ID NO:2 amino acid sequence.
  • the ⁇ chain variable domain of the TCR comprises at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences with 98% or 99% sequence homology.
  • the ⁇ chain variable domain of the TCR has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences with 98%, 99% or 100% sequence homology.
  • the amino acid sequence of the variable domain of the TCR ⁇ chain has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO: 1.
  • amino acid sequence of the variable domain of the TCR beta chain has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO: 2.
  • the affinity of the TCR to the KWVESIFLIF-HLA A2402 complex is at least 2 times that of the wild-type TCR.
  • amino acid sequence of the ⁇ chain variable domain of the wild-type TCR is shown in SEQ ID NO: 1; the amino acid sequence of the ⁇ chain variable domain is shown in SEQ ID NO: 2.
  • the reference sequences of the three CDR regions (complementarity determining regions) of the variable domain of the TCR ⁇ chain are as follows,
  • CDR3 ⁇ AVRRRNYGQNFV, and CDR3 ⁇ contains at least one of the following mutations:
  • amino acid mutation in the CDR3 ⁇ comprises:
  • the number of amino acid mutations in the variable domain of the TCR ⁇ chain is 1-6, preferably 1-4.
  • the number of amino acid mutations in the variable domain of the TCR ⁇ chain is 2, 3 or 4.
  • amino acid mutation site of the variable domain of the TCR ⁇ chain is positions 1-4 of CDR3 ⁇ , wherein:
  • amino acid mutation sites of the variable domain of the TCR ⁇ chain are positions 3, 4 and 6 of CDR3 ⁇ , wherein:
  • amino acid mutation sites of the variable domain of the TCR ⁇ chain are the 3rd and 4th positions of CDR3 ⁇ , wherein:
  • amino acid mutation sites of the variable domain of the TCR ⁇ chain are positions 6 and 7 of CDR3 ⁇ , wherein:
  • the CDR1 ⁇ in the variable domain of the TCR ⁇ chain is: DSAIYN, and the CDR2 ⁇ is IQSSQRE.
  • the CDR3 ⁇ of the variable domain of the TCR ⁇ chain is: ASSLIAQNHNEQF.
  • the three CDRs of the variable domain of the TCR ⁇ chain are:
  • amino acid sequence of CDR3 ⁇ in the variable domain of the TCR ⁇ chain is selected from the group consisting of: ASSLIAQNHNEQF, ASSLIAQNHALWT and ASSLIAQNHAMWT.
  • amino acid sequence of the variable domain of the TCR ⁇ chain is SEQ ID NO:2.
  • the CDR3 ⁇ of the variable domain of the TCR ⁇ chain is selected from GGVYRNYGQNFV, AVTYRQYGQNFV and AVIYRNYGQNFV.
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , wherein the amino acid sequence of CDR1 ⁇ is DSAIYN, the amino acid sequence of CDR2 ⁇ is IQSSQRE, and the amino acid sequence of CDR3 ⁇ is: [3 X1][3 X2 ][3X3][3X4][3X5][3X6][3X7]GQNFV, where [3X1] is A or G or S, and/or [3X2] is V or A or G or I or L or S or T, and/or [3 X3] is R or I or T or V, and/or [3 X4] is R or W or Y or N or M, and/or [3 X5] is R or Y, and/or [3 X6] is N or E or H or Q or R or T or I or V or M, and/or [3 X7] is Y or L or P or W or V
  • the TCR is mutated in the ⁇ chain variable domain shown in SEQ ID NO: 1, and the mutation is selected from A91G/S, V92A/G/I/L/S/T, R93I / One or more groups of T/V, R94W/Y/N/M, R95Y, N96E/H/Q/R/T/I/V/M and Y97L/P/W/V in which amino acid residues
  • the numbering adopts the numbering shown in SEQ ID NO:1.
  • the TCR has a CDR selected from the group consisting of:
  • the TCR is soluble.
  • the TCR is an ⁇ heterodimeric TCR, comprising the ⁇ chain TRAC constant region sequence and the ⁇ 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) each comprise the variable domain and at least a portion of the constant domain of the TCR chain.
  • an artificial interchain disulfide bond is contained between the constant region of the ⁇ chain and the constant region of the ⁇ chain of the TCR.
  • cysteine residue that forms an artificial interchain disulfide bond between the constant region of the TCR ⁇ and the ⁇ chain is substituted for one or more groups of sites selected from the following:
  • the amino acid sequence of the ⁇ chain variable domain of the TCR is one of SEQ ID NOs: 1 and 13-39; and/or the amino acid sequence of the ⁇ chain variable domain of the TCR is SEQ ID NO: One of 2, 40, 41.
  • 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, preferably, the conjugate is a detectable label, a therapeutic agent, or a PK modification some or a combination of any of these substances.
  • the therapeutic agent bound to the TCR is an anti-CD3 antibody linked to the C- or N-terminus of the ⁇ or ⁇ chain of the TCR.
  • a 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 any one of the preceding claims.
  • the third aspect of the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule described in the first aspect of the present invention or the multivalent TCR complex described in the second aspect of the present invention or its complement sequence.
  • the fourth aspect of the present invention provides a vector, the vector contains 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 exogenous 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, the cell expressing the TCR described in the first aspect of the present invention, preferably, the isolated cell T cell, NK cell, NKT cell, most preferably, The isolated cells are T cells.
  • the seventh aspect of the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the TCR described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, or the cell according to the sixth aspect of the present invention.
  • the eighth aspect of the present invention provides a method for treating a disease, comprising administering an appropriate amount of the TCR described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, or the present invention to a subject in need of treatment.
  • the disease is an AFP-positive tumor, more preferably, the tumor is liver cancer.
  • the ninth aspect of the present invention provides the use of the TCR described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, or the cell described in the sixth aspect of the present invention, for preparing and treating tumors
  • the disease is an AFP-positive tumor, more preferably, the tumor is liver cancer.
  • a tenth aspect of the present invention provides a method for preparing the T cell receptor described in the first aspect of the present invention, comprising the steps of:
  • Figure 1a and Figure 1b show the amino acid sequences of wild-type TCR ⁇ and ⁇ chain variable domains that can specifically bind to the KWVESIFLIF-HLA A2402 complex, respectively.
  • Figure 2a and Figure 2b are respectively the amino acid sequence of the alpha chain variable domain and the amino acid sequence of the beta chain variable domain of the single-chain template TCR constructed by the present invention.
  • Fig. 3a and Fig. 3b are respectively the DNA sequence of the variable domain of the ⁇ chain and the DNA sequence of the variable domain of the ⁇ chain of the single-chain template TCR constructed by the present invention.
  • Figure 4a and Figure 4b are respectively the amino acid sequence and DNA sequence of the linker of the single-chain template TCR constructed by the present invention.
  • Figure 5a and Figure 5b are respectively the amino acid sequence and DNA sequence of the single-chain template TCR constructed by the present invention.
  • Figure 6a and Figure 6b are the amino acid sequences of the soluble reference TCR ⁇ chain and ⁇ chain in the present invention, respectively.
  • Figures 7(1)-(27) show the alpha chain variable domain amino acid sequences of heterodimeric TCRs with high affinity for the KWVESIFLIF-HLA A2402 complex, respectively, with mutated residues underlined.
  • Figures 8(1)-(2) show the ⁇ -chain variable domain amino acid sequences of heterodimeric TCRs with high affinity for the KWVESIFLIF-HLA A2402 complex, respectively, and mutated residues are underlined.
  • Figure 9a and Figure 9b show the extracellular amino acid sequences of wild-type TCR ⁇ chain and ⁇ chain that can specifically bind to the KWVESIFLIF-HLA A2402 complex, respectively.
  • Figure 10a and Figure 10b show the amino acid sequences of the wild-type TCR alpha chain and beta chain that can specifically bind to the KWVESIFLIF-HLA A2402 complex, respectively.
  • Figure 11 is a binding curve of a soluble reference TCR, ie, wild-type TCR, to KWVESIFLIF-HLA A2402 complex.
  • Fig. 12a-Fig. 12c are the results of the activation function experiment of effector cells transfected with the high-affinity TCR of the present invention for T2 cells loaded with short peptides.
  • Figures 13a-13c are the results of the activation function experiment of effector cells transfected with the high-affinity TCR of the present invention for tumor cell lines.
  • Figure 14 shows the experimental results of the killing function of the effector cells transfected with the high-affinity TCR of the present invention against T2 cells loaded with short peptides in a gradient.
  • Figures 15a-15c are the results of LDH experiments on the killing function of effector cells transfected with the high-affinity TCR of the present invention against tumor cell lines.
  • Figure 16a and Figure 16b are the results of IncuCyte experiments on the killing function of effector cells transfected with the high-affinity TCR of the present invention against tumor cell lines.
  • the present invention obtains a high-affinity T cell receptor (TCR) that recognizes KWVESIFLIF short peptide (derived from AFP protein) through extensive and in-depth research, and the KWVESIFLIF short peptide is in the form of peptide-HLA A2402 complex. presented.
  • TCR high-affinity T cell receptor
  • the high-affinity TCR has 3 CDR regions in its alpha chain variable domain:
  • CDR3 ⁇ mutated in AVRRRNYGQNFV; and/or in the 3 CDR regions of its ⁇ -chain variable domain:
  • CDR3 ⁇ mutated in ASSLIAQNHNEQF; and the affinity and/or binding half-life of the TCR of the present invention to the above-mentioned KWVESIFLIF-HLA A2402 complex after the mutation is at least 2 times that of the wild-type TCR.
  • TCR T cell receptor
  • TCRs can be described using the International Information System for Immunogenetics (IMGT).
  • IMGT International Information System for Immunogenetics
  • Native ⁇ heterodimeric TCRs have ⁇ and ⁇ chains. Broadly speaking, each chain contains a variable region, a linker region and a constant region, and the beta chain typically also contains a short variable region between the variable region and the linker region, but the variable region is often considered part of the linker region.
  • the junctional region of the TCR is determined by TRAJ and TRBJ of the unique IMGT, and the constant region of the TCR is determined by the TRAC and TRBC of the IMGT.
  • Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, chimerically incorporated in a framework sequence.
  • the different numbers of TRAV and TRBV refer to different V ⁇ types and V ⁇ types, 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 the allele Gene 1.
  • the beta chain constant domain has the following symbols: TRBC1*01 or TRBC2*01, where "TR” denotes the T cell receptor gene; "B” denotes the beta chain gene; C denotes the constant region; "*01” denotes the allele 1.
  • TR denotes the T cell receptor gene
  • B denotes the beta chain gene
  • C denotes the constant region
  • *01 denotes the allele 1.
  • the constant region of the alpha chain is uniquely defined, and in the form of the beta chain, there are two possible constant region genes "C1" and "C2". Those skilled in the art can obtain the constant region gene sequences of TCR ⁇ and ⁇ chains through the public IMGT database.
  • TCR alpha chain variable domain refers to linked TRAV and TRAJ regions
  • TCR beta chain variable domain refers to linked TRBV and TRBD/TRBJ regions.
  • the three CDRs of the variable domain of the TCR ⁇ chain are CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , respectively; the three CDRs of the variable domain of the TCR ⁇ chain are CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , respectively.
  • the framework sequences of the TCR variable domains of the present invention may be of murine or human origin, preferably human.
  • the constant domains of TCRs comprise an intracellular portion, a transmembrane region and an extracellular portion.
  • the amino acid sequences of the ⁇ and ⁇ chain variable domains of the wild-type TCR capable of binding to the KWVESIFLIF-HLA A2402 complex are SEQ ID NO: 1 and SEQ ID NO: 2, respectively, as shown in Figure 1a and Figure 1b.
  • the ⁇ chain amino acid sequence and ⁇ chain amino acid sequence of the soluble "reference TCR" described in the present invention are SEQ ID NO: 11 and SEQ ID NO: 12, respectively, as shown in Figure 6a and Figure 6b.
  • the ⁇ -chain extracellular amino acid sequence and ⁇ -chain extracellular amino acid sequence of the "wild-type TCR" in the present invention are SEQ ID NO: 42 and SEQ ID NO: 43, respectively, as shown in Figure 9a and Figure 9b.
  • the TCR sequences used in the present invention are of human origin.
  • the ⁇ chain amino acid sequence and ⁇ chain amino acid sequence of the "wild-type TCR" described in the present invention are SEQ ID NO: 44 and SEQ ID NO: 45, respectively, as shown in Figures 10a and 10b.
  • the terms "polypeptide of the present invention”, “TCR of the present invention”, “T cell receptor of the present invention” are used interchangeably.
  • the position numbers of the amino acid sequences of TRAC*01 and TRBC1*01 or TRBC2*01 in the present invention are numbered sequentially from the N-terminus to the C-terminus.
  • the 60th amino acid in the sequence from end to C end is P (proline), then it can be described as Pro60 of exon 1 of TRBC1*01 or TRBC2*01 in the present invention, and it can also be expressed as TRBC1* 01 or the 60th amino acid of TRBC2*01 exon 1, and in TRBC1*01 or TRBC2*01, the 61st amino acid is Q (glutamine) in the sequence from the N-terminus to the C-terminus, then this In the invention, it can be described as Gln61 of TRBC1*01 or TRBC2*01 exon 1, and it can also be described 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 the variable regions TRAV and TRBV are numbered according to the position numbers listed in IMGT.
  • the position number listed in IMGT is 46, then it is described as the 46th amino acid of TRAV in the present invention, and so on.
  • the special instructions are followed.
  • tumor is meant to encompass all types of cancer cell growth or oncogenic processes, metastatic or malignantly transformed cells, tissues or organs, regardless of the type of pathology or stage of infection.
  • tumors include, without limitation, solid tumors, soft tissue tumors, and metastatic lesions.
  • solid tumors include: malignancies of various organ systems such as sarcomas, lung squamous cell carcinomas and cancers. For example: infected prostate, lung, breast, lymph, gastrointestinal (eg: colon), and genitourinary tract (eg: kidney, epithelial cells), pharynx.
  • Squamous cell carcinoma of the lung includes malignant tumors, for example, most colon cancers, rectal cancers, renal cell carcinomas, liver cancers, non-small cell carcinomas of the lungs, small bowel cancers and esophageal cancers. Metastatic lesions of the aforementioned cancers can likewise be treated and prevented with the methods and compositions of the present invention.
  • the ⁇ chain variable domain amino acid sequence and the ⁇ chain variable domain amino acid sequence of the wild-type TCR capable of binding the antigenic short peptide KWVESIFLIF and HLA A2402 complex are SEQ ID NO: 1 and SEQ ID NO: 1 and SEQ ID NO: 1 respectively. ID NO: 2, this sequence was discovered by the inventor for the first time. It has the following CDR regions:
  • the present invention obtains a high-affinity TCR whose affinity with the KWVESIFLIF-HLA A2402 complex is at least 2 times that of the wild-type TCR and the KWVESIFLIF-HLA A2402 complex through mutation screening of the above-mentioned CDR region.
  • the TCR of the present invention is an ⁇ heterodimeric TCR
  • the ⁇ chain variable domain of the TCR comprises at least 85% of the amino acid sequence shown in SEQ ID NO: 1; preferably, at least 90%; more preferably Preferably, at least 92%; more preferably, at least 94% (eg, can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% %, 99% sequence homology) amino acid sequence
  • the ⁇ chain variable domain of the TCR comprises at least 90% with the amino acid sequence shown in SEQ ID NO: 2, preferably , at least 92%; more preferably, at least 94% (eg, can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence identity homology to the amino acid sequence of the sequence homology.
  • the TCR of the present invention is a single-chain TCR
  • the ⁇ -chain variable domain of the TCR comprises at least 85%, preferably, at least 90%, of the amino acid sequence shown in SEQ ID NO: 3; more preferably, at least 92%; most preferably, at least 94% (eg, can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% % sequence homology) amino acid sequence of sequence homology
  • the ⁇ chain variable domain of the TCR comprises at least 85%, preferably at least 90%, with the amino acid sequence shown in SEQ ID NO: 4 %; more preferably, at least 92%; most preferably, at least 94% (eg, can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) amino acid sequences of sequence homology.
  • 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 91-102 of SEQ ID NO: 1, respectively .
  • the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 1, and 91A is the first position A of CDR3 ⁇ , 92V is the second position V of CDR3 ⁇ , and 93R is the third position R and 94R of CDR3 ⁇ are The 4th R, 95R of CDR3 ⁇ is the 5th R of CDR3 ⁇ , 96N is the 6th N of CDR3 ⁇ , and 97Y is the 7th Y of CDR3 ⁇ .
  • the present invention provides a TCR having the property of binding to the KWVESIFLIF-HLA A2402 complex, and comprising an alpha chain variable domain and a beta chain variable domain, wherein the TCR is in the alpha chain variable domain shown in SEQ ID NO: 1 Mutation occurs, and the mutated amino acid residue positions include one or more of 91A, 92V, 93R, 94R, 95R, 96N and 97Y, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 1.
  • the mutated TCR ⁇ chain variable domain comprises one or more amino acid residues selected from the group consisting of: 91G or 91S; 92A or 92G or 92I or 92L or 92S or 92T; 93I or 93T or 93V; 94W or 94Y or 94N or 94M; 95Y; 96E or 96H or 96Q or 96R or 96T or 96I or 96V or 96M; 97L or 97P or 97W or 97V, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO:1.
  • specific forms of the mutation in the alpha chain variable domain include A91G/S, V92A/G/I/L/S/T, R93I/T/V, R94W/Y/N/M, R95Y, N96E One or more of /H/Q/R/T/I/V/M and Y97L/P/W/V.
  • amino acids in this article are identified by the international single English letter, and the corresponding three English letter abbreviations of the amino acid names are: Ala(A), Arg(R), Asn(N), Asp(D), Cys (C), Gln(Q), Glu(E), Gly(G), His(H), Ile(I), Leu(L), Lys(K), Met(M), Phe(F), Pro (P), Ser(S), Thr(T), Trp(W), Tyr(Y), Val(V);
  • both Pro60 or 60P represent proline at position 60.
  • the expression of the specific form of the mutation in the present invention such as "A91G/S”, represents that A at position 91 is substituted by G or substituted by S, and so on.
  • cysteine substitutions enable the formation of artificial interchain disulfide bonds between the constant regions of the ⁇ and ⁇ chains of the reference TCR to form a more stable soluble TCR, allowing for easier assessment of TCR complexing with KWVESIFLIF-HLA A2402 Binding affinity and/or binding half-life between substances. It should be understood that the CDR regions of the TCR variable region determine the affinity between it and the pMHC complex, therefore, the cysteine substitutions in the TCR constant region described above have no effect on the binding affinity and/or binding half-life of the TCR.
  • the measured binding affinity between the reference TCR and the KWVESIFLIF-HLA A2402 complex is considered to be the binding affinity between the wild-type TCR and the KWVESIFLIF-HLA A2402 complex.
  • the binding affinity between the TCR of the invention and the KWVESIFLIF-HLA A2402 complex is measured to be at least 10 times the binding affinity between the reference TCR and the KWVESIFLIF-HLA A2402 complex, it is equivalent to the TCR of the invention and KWVESIFLIF -
  • the binding affinity between the HLA A2402 complex is at least 10 times the binding affinity between the wild-type TCR and the KWVESIFLIF-HLA A2402 complex.
  • Binding affinity (inversely proportional to the dissociation equilibrium constant KD) and binding half-life (expressed as T1/2) can be determined by any suitable method, such as by surface plasmon resonance techniques. It will be appreciated that doubling the affinity of the TCR will result in a halving of the KD. T1/2 was calculated as In2 divided by the dissociation rate (Koff). Therefore, doubling T1/2 results in a halving of Koff.
  • the same assay protocol is used to measure the binding affinity or binding half-life of a given TCR several times, eg, 3 times or more, and the results are averaged.
  • the surface plasmon resonance (BIAcore) method in the examples herein is used to detect the affinity of soluble TCR, and the conditions are: the temperature is 25°C, and the pH value is 7.1-7.5.
  • This method detects that the dissociation equilibrium constant KD of the reference TCR to the KWVESIFLIF-HLA A2402 complex is 1.06E-04M, that is, 106 ⁇ M.
  • the dissociation equilibrium constant KD of the wild-type TCR to the KWVESIFLIF-HLA A2402 complex is Also 106 ⁇ M.
  • the affinity of the -HLA A2402 complex is 10 times that of the wild-type TCR for the KWVESIFLIF-HLA A2402 complex.
  • the affinity of the TCR to the KWVESIFLIF-HLA A2402 complex is at least 2 times that of the wild-type TCR.
  • Mutagenesis can be performed using any suitable method, including but not limited to those based on polymerase chain reaction (PCR), restriction enzyme based cloning or ligation independent cloning (LIC) methods. These methods are detailed in many standard molecular biology textbooks. More details on polymerase chain reaction (PCR) mutagenesis and restriction enzyme-based cloning can be found in Sambrook and Russell, (2001) Molecular Cloning-A Laboratory Manual (Third Edition) CSHL publishing house. More information on the LIC method can be found (Rashtchian, (1995) Curr Opin Biotechnol 6(1):30-6).
  • PCR polymerase chain reaction
  • LIC ligation independent cloning
  • the method of generating the TCRs of the present invention can be, but is not limited to, screening TCRs with high affinity for the KWVESIFLIF-HLA-A2402 complex from a diverse library of phage particles displaying such TCRs, as described in the literature (Li, et al. (2005) Nature Biotech 23(3):349-354).
  • a gene expressing the alpha and beta chain variable domain amino acids of a wild-type TCR or a gene expressing a slightly modified wild-type TCR's alpha and beta chain variable domain amino acids can be used to prepare a template TCR.
  • the changes required to generate the high affinity TCRs of the invention are then introduced into the DNA encoding the variable domains of the template TCR.
  • the high-affinity TCR of the present invention comprises an ⁇ chain variable domain amino acid sequence of one of SEQ ID NO: 1, 13-39; and/or the ⁇ chain variable domain amino acid sequence of the TCR is SEQ ID NO: 2, One of 40 and 41.
  • the amino acid sequences of the ⁇ -chain variable domain and the ⁇ -chain variable domain forming the heterodimeric TCR molecule are preferably selected from the following table 1:
  • a TCR of the present invention is a portion having at least one variable domain of the TCR ⁇ and/or TCR ⁇ chain. They usually contain both the TCR ⁇ chain variable domain and the TCR ⁇ chain variable domain. They can be alpha beta heterodimers or single-chain forms or any other form that can be stably present.
  • the full-length chain of ⁇ heterodimeric TCRs (comprising cytoplasmic and transmembrane domains) can be transfected.
  • the TCRs of the present invention can be used as targeting agents for delivering therapeutic agents to antigen-presenting cells or in combination with other molecules to produce bifunctional polypeptides to target effector cells, in which case the TCRs are preferably in a soluble form.
  • the TCR of the present invention may be one in which artificial interchain disulfide bonds are introduced between residues of its alpha and beta chain constant domains.
  • Cysteine residues form artificial interchain disulfide bonds between the constant domains of the alpha and beta chains of the TCR. Cysteine residues can be substituted for other amino acid residues at appropriate sites in the native TCR to form artificial interchain disulfide bonds.
  • substitution of Thr48 in exon 1 of TRAC*01 and substitution of Ser57 in exon 1 of TRBC1*01 or TRBC2*01 to form a disulfide bond can also be: Thr45 in exon 1 of TRAC*01 and Ser77 in exon 1 of TRBC1*01 or TRBC2*01; exon 1 of TRAC*01 1 Tyr10 and TRBC1*01 or Ser17 of 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 in exon 1 of TRBC1*01 or TRBC2*01; Arg53 in exon 1 of TRAC*01 and Ser54 in exon 1 of TRBC1*01 or TRBC2*01; Pro89 and exon 1 of TRAC*01 Ala19 of exon 1 of TRBC1*01 or TRBC2*01;
  • cysteine residues are substituted for any set of sites in the constant domains of the alpha and beta chains described above.
  • One or more C-termini of the TCR constant domains of the invention may be truncated by up to 15, or up to 10, or up to 8 or less amino acids so that they do not include cysteine residues to achieve deletion of native
  • 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 TCRs of the present invention may comprise artificial interchain disulfide bonds introduced between residues of their alpha and beta chain constant domains.
  • the TCRs of the invention may contain both a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, with or without the artificial disulfide bonds introduced above between the constant domains.
  • the TRAC constant domain sequence of the TCR and the TRBC1 or TRBC2 constant domain sequence may 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 TCR can significantly improve the stability of TCR. Therefore, the high-affinity TCR of the present invention may also contain artificial interchain disulfide bonds between the variable region of the ⁇ chain and the constant region of the ⁇ chain.
  • cysteine residue that forms an artificial interchain disulfide bond between the ⁇ chain variable region and the ⁇ chain constant region of the TCR is substituted for: amino acid 46 of TRAV and TRBC1*01 or TRBC2* 01 amino acid 60 of exon 1; TRAV amino acid 47 and TRBC1*01 or TRBC2*01 exon 1 amino acid 61; TRAV 46 amino acid and TRBC1*01 or TRBC2*01 exon Amino acid 61 of exon 1; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01.
  • such a TCR may comprise (i) all or part of the TCR alpha chain excluding its transmembrane domain, and (ii) all or part of the TCR beta chain excluding its transmembrane domain, wherein (i) and (ii) ) both contain 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.
  • such a TCR may contain an alpha chain variable domain and a beta chain variable domain and all or part of the beta chain constant domain except the transmembrane domain, but it does not contain the alpha chain constant domain, the alpha chain of the TCR.
  • the chain variable domains form heterodimers with beta chains.
  • the TCRs of the present invention also include TCRs with mutations in their hydrophobic core regions, and the mutations in these hydrophobic core regions are preferably mutations that can improve the stability of the TCRs of the present invention, as disclosed in Publication No. Described in the patent document of WO2014/206304.
  • Such a TCR may be mutated at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable domain amino acids 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or Or the alpha chain J gene (TRAJ) short peptide amino acid position 3,5,7 from the bottom, and/or the beta chain J gene (TRBJ) short peptide amino acid position 2,4,6 from the bottom, where the position number of the amino acid sequence Numbered by position as listed in the International Information System on Immunogenetics (IMGT).
  • IMGT International Information System on Immunogenetics
  • the mutated TCR in the hydrophobic core region can be a highly stable single-chain TCR composed of a flexible peptide chain connecting the variable domains of the ⁇ chain and the ⁇ chain of the TCR.
  • the CDR region of the TCR variable region determines its affinity with the short peptide-HLA complex, and the mutation of the hydrophobic core can make the TCR more stable, but does 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 linking the variable domains of TCR ⁇ and ⁇ chain.
  • the template chain constructed in Example 1 of the present invention for screening high-affinity TCRs is the above-mentioned high-stability single-chain TCRs containing hydrophobic core mutations. Using a TCR with higher stability can more conveniently evaluate the affinity between the TCR and the KWVESIFLIF-HLA-A2402 complex.
  • the CDR regions of the ⁇ -chain variable domain and the ⁇ -chain variable domain of the single-chain template TCR are identical to those of the wild-type TCR. That is, the three CDRs of the ⁇ chain variable domain are CDR1 ⁇ : DSAIYN; CDR2 ⁇ : IQSSQRE; CDR3 ⁇ : AVRRRNYGQNFV and the three CDRs of the ⁇ chain variable domain are CDR1 ⁇ : SGHDT; CDR2 ⁇ : YYEEEE; CDR3 ⁇ : ASSLIAQNHNEQF.
  • 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. In this way, a single-chain TCR composed of ⁇ -chain variable domain and ⁇ -chain variable domain with high affinity to KWVESIFLIF-HLA A2402 complex was screened out.
  • the ⁇ heterodimer with high affinity for the KWVESIFLIF-HLA-A2402 complex of the present invention is obtained by transferring the CDR regions of the variable domains of the ⁇ and ⁇ chains of the screened 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 TCRs of the present invention may also be provided in the form of multivalent complexes.
  • the multivalent TCR complexes of the present invention comprise two, three, four or more multimers formed by combining the TCRs of the present invention, for example, the tetramerization domain of p53 can be used to generate tetramers, or multiple A complex formed by combining a TCR of the present invention with another molecule.
  • the TCR complexes of the present invention can be used to track or target cells presenting specific antigens in vitro or in vivo, as well as to generate intermediates for other multivalent TCR complexes with such applications.
  • the TCR of the present invention can be used alone, or can be combined with the conjugate in a covalent or other manner, preferably in a covalent manner.
  • the conjugate includes a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the KWVESIFLIF-HLA-A2402 complex), a therapeutic agent, a PK (protein kinase) modification moiety, or any of the above Combination binding or coupling of substances.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing detectable products enzyme.
  • Therapeutic agents that can be bound or conjugated to 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 (Nature) 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy (Cancer Immunology and Immunotherapy) 51, 565); 3. Cytokines such as IL-2, etc.
  • Viral particles (Peng et al, 2004, Gene Gene therapy 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research 65, 11631); 9. Nanomagnetic particles; 10. Prodrug-activating enzymes (eg, DT-myocardial flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. chemotherapeutic agents (eg, cisplatin) or nanoparticles in any form, etc.
  • DTD DT-myocardial flavinase
  • BPHL biphenyl hydrolase-like protein
  • 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 binding of such antibodies or fragments thereof to TCR is capable of effecting effector cells. directed to better target target cells.
  • a preferred embodiment is that the TCR of the present invention binds to an anti-CD3 antibody or a functional fragment or variant of said anti-CD3 antibody.
  • the fusion molecule of TCR and anti-CD3 single-chain antibody of the present invention comprises a variable domain amino acid sequence selected from the TCR ⁇ chain variable domain as one of SEQ ID NOs: 1, 13-39; and/or the ⁇ chain variable of the TCR
  • the domain amino acid sequence is one of SEQ ID NOs: 2, 40, 41.
  • the present invention also relates to nucleic acid molecules encoding the TCRs of the present invention.
  • the nucleic acid molecules of the present invention may be in the form of DNA or RNA.
  • DNA can be the coding or non-coding strand.
  • the nucleic acid sequences encoding the TCRs of the present invention may be identical or degenerate variants of the nucleic acid sequences shown in the figures of the present invention.
  • degenerate variant refers to encoding a protein sequence having SEQ ID NO:3, but with the sequence of SEQ ID NO:5 Different nucleic acid sequences.
  • the full-length sequence of the nucleic acid molecule of the present invention or a fragment thereof can generally be obtained by, but not limited to, PCR amplification method, recombinant method or artificial synthesis method.
  • the DNA sequences encoding the TCRs of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • the present invention also relates to vectors comprising the nucleic acid molecules of the present invention, as well as host cells genetically engineered with the vectors or coding sequences of the present invention.
  • the present invention also includes isolated cells, particularly T cells, expressing the TCRs of the present invention.
  • isolated cells particularly T cells, expressing the TCRs of the present invention.
  • T cells expressing the high affinity TCRs of the invention.
  • T cells expressing the high-affinity TCR of the present invention can be used for adoptive immunotherapy.
  • Those skilled in the art are aware of 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
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the TCR of the present invention, or the TCR complex of the present invention, or a cell that presents the TCR of the present invention.
  • the present invention also provides a method of treating a disease, comprising administering an appropriate amount of the TCR of the present invention, or the TCR complex of the present invention, or a cell presenting the TCR of the present invention, or the pharmaceutical composition of the present invention to a subject in need of treatment.
  • the TCR of the present invention also includes up to 5, preferably up to 3, more preferably up to 2, and optimally 1 amino acid (especially the amino acid outside the CDR region) of the TCR of the present invention, which are similar in nature or similar amino acids, and still retain its functional TCR.
  • the present invention also includes slightly modified TCRs of the present invention.
  • Modified (generally without altering the primary structure) forms include: chemically derivatized forms of the TCRs of the invention such as acetylated or carboxylated.
  • Modifications also include glycosylation, such as those TCRs resulting from glycosylation modifications in the synthesis and processing of the TCRs of the invention or in further processing steps. This modification can be accomplished by exposing the TCR to enzymes that perform glycosylation, such as mammalian glycosylases or deglycosylases.
  • Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are TCRs that have been modified to increase their resistance to proteolysis or to optimize solubility.
  • the TCRs of the present invention, TCR complexes or TCR-transfected T cells of the present invention can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the TCRs, multivalent TCR complexes or cells of the present invention are typically provided as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be in any suitable form (depending on the desired method of administration to the patient). It may be provided in unit dosage form, usually in a sealed container, as part of a kit. Such kits, but not necessarily, include instructions for use. It may comprise a plurality of such unit dosage forms.
  • TCRs of the present invention can be used alone, or in combination or conjugation with other therapeutic agents (eg, formulated in the same pharmaceutical composition).
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent.
  • pharmaceutical carriers that do not themselves induce the production of antibodies detrimental to the individual receiving the composition, and are not undue toxicity upon 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., NJ 1991).
  • Such carriers include, but are not limited to: Saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • Pharmaceutically acceptable carriers in therapeutic compositions can contain liquids such as water, saline, glycerol and ethanol.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • therapeutic compositions can be prepared as injectables, eg, liquid solutions or suspensions; solid forms suitable for solution or suspension, liquid carriers, prior to injection can also be prepared.
  • compositions of the present invention may be administered by conventional routes including, but not limited to: intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably gastrointestinal External includes subcutaneous, intramuscular or intravenous.
  • the subject to be prevented or treated can be an animal; especially a human.
  • compositions of the present invention When the pharmaceutical composition of the present invention is used for actual treatment, various pharmaceutical compositions in different dosage forms can be adopted according to the usage. Preferably, injections, oral preparations and the like can be exemplified.
  • compositions can be formulated according to conventional methods by mixing, diluting or dissolving, with occasional addition of suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicity isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers, and the formulation process can be carried out in a conventional manner according to the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicity isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers, and the formulation process can be carried out in a conventional manner according to the dosage form.
  • compositions of the present invention can also be administered in the form of sustained release formulations.
  • the TCRs of the present invention can be incorporated into pellets or microcapsules in a slow release polymer carrier, which are then surgically implanted into the tissue to be treated.
  • sustained-release polymers ethylene-vinyl acetate copolymer, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, 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 the TCR complex of the present invention or the cell presenting the TCR of the present invention as an active ingredient can be used according to the weight, age, sex, degree of symptoms of each patient to be treated And be reasonably determined, and ultimately by the physician to decide a reasonable dosage.
  • the affinity and/or binding half-life of the high-affinity TCR of the present invention to the KWVESIFLIF-HLA-A2402 complex is at least 2 times that of the wild-type TCR.
  • the high-affinity TCR of the present invention can specifically bind to the KWVESIFLIF-HLA A2402, and the cells transfected with the high-affinity TCR of the present invention can be specifically activated.
  • the effector cells transfected with the high-affinity TCR of the present invention have a strong specific killing effect.
  • E.coli DH5 ⁇ is purchased from Tiangen
  • E.coli BL21 (DE3) is purchased from Tiangen
  • E.coli Tuner (DE3) is purchased from Tiangen.
  • plasmid pET28a was purchased from Novagen.
  • the present invention utilizes the method of site-directed mutagenesis, according to the patent document WO2014/206304, to construct a stable single-chain TCR molecule composed of a flexible short peptide (linker) connecting 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. And use the single-chain TCR molecule as a template to screen high-affinity TCR molecules.
  • the amino acid sequences of the ⁇ chain variable domain (SEQ ID NO: 3) and ⁇ chain variable domain (SEQ ID NO: 4) of the template chain are shown in Figures 2a and 2b; the corresponding DNA sequences are SEQ ID NO: : 5 and SEQ ID NO: 6, as shown in Figures 3a and 3b; the amino acid sequence and DNA sequence of the flexible short peptide (linker) are respectively SEQ ID NO: 7 and 8, as shown in Figures 4a and 4b.
  • the target gene carrying the template chain was double digested with Nco I and Not I, and ligated with the pET28a vector that was double digested with Nco I and Not I.
  • the ligation product was transformed into E.coli DH5 ⁇ , coated with LB plate containing kanamycin, and cultured overnight at 37°C by inversion. Positive clones were picked for PCR screening, and the positive recombinants were sequenced. After confirming that the sequences were correct, the recombinant plasmids were extracted and transformed. to E. coli BL21(DE3) for expression.
  • Example 2 Expression, renaturation and purification of the stable single-chain TCR constructed in Example 1
  • the cell pellet was harvested by centrifugation at 5000 rpm for 15 min, the cell pellet was lysed with Bugbuster Master Mix (Merck), the inclusion bodies were recovered by centrifugation at 6000 rpm for 15 min, and then washed with Bugbuster (Merck) to remove cell debris and membrane components, and the inclusion bodies were collected by centrifugation at 6000 rpm for 15 min. body.
  • the inclusion bodies were dissolved in buffer (20mM Tris-HCl pH 8.0, 8M urea), and insoluble matter was removed by high-speed centrifugation. The supernatant was quantified by BCA method and then packaged and stored at -80°C for later use.
  • renaturation buffer 100mM Tris-HCl pH 8.1, 0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mM ⁇ -mercapthoethylamine, 1.87mM Cystamine
  • the sample was filtered through a 0.45 ⁇ m filter membrane, degassed in vacuo and passed through an anion exchange column (HiTrap Q HP, GE Healthcare), and the protein was purified with a linear gradient of 0-1 M NaCl prepared with 20 mM Tris-HCl pH 8.0, The collected elution fractions were analyzed by SDS-PAGE, the fractions containing single-chain TCR were concentrated and further purified by gel filtration column (Superdex 75 10/300, GE Healthcare), and the target fractions were also analyzed by SDS-PAGE.
  • the eluted fractions for BIAcore analysis were further tested for purity by gel filtration.
  • the conditions are: Column Agilent Bio SEC-3 (300A, ), the mobile phase was 150 mM phosphate buffer, the flow rate was 0.5 mL/min, the column temperature was 25 °C, and the UV detection wavelength was 214 nm.
  • the binding activity of TCR molecules to KWVESIFLIF-HLA-A2402 complex was detected using BIAcore T200 real-time analysis system.
  • Anti-streptavidin antibody (GenScript) was added to coupling buffer (10 mM sodium acetate buffer, pH 4.77), and the antibody was then flowed through a CM5 chip preactivated with EDC and NHS to immobilize the antibody on the chip. surface, and finally blocked the unreacted activated surface with ethanolamine hydrochloric acid solution to complete the coupling process with a coupling level of about 15,000 RU.
  • the conditions are: the temperature is 25°C, and the pH value is 7.1-7.5.
  • the low concentration of streptavidin was flowed over the surface of the chip coated with the antibody, then KWVESIFLIF-HLA-A2402 complex was flowed through the detection channel, and the other channel was used as a reference channel, and 0.05mM biotin was added to 10 ⁇ L/ The flow rate of min flows through the chip for 2 min, blocking the remaining binding sites of streptavidin. Affinity was determined using a single-cycle kinetic assay. TCR was diluted to several different concentrations with HEPES-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, pH 7.4) at a flow rate of 30 ⁇ L/min.
  • the binding time of each injection is 120s, and the dissociation is 600s after the last injection.
  • the chip was regenerated with 10 mM Gly-HCl, pH 1.75, after each round of measurements.
  • Kinetic parameters were calculated using BIAcore Evaluation software.
  • KWVESIFLIF peptide was added to renaturation 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 added 20mg/L light chain and 90mg/L heavy chain in sequence (final concentration, heavy chain was added in three times, 8h/time), and renatured at 4°C for at least 3 days To complete, SDS-PAGE test whether the renaturation is successful.
  • renaturation 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
  • Biotinylation Concentrate the purified pMHC molecules with Millipore ultrafiltration tubes while replacing the buffer with 20mM Tris pH 8.0, then add biotinylation reagents 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, and check whether the biotinylation is complete by SDS-PAGE.
  • biotinylated complex The biotinylated pMHC molecules were concentrated to 1 ml with a Millipore ultrafiltration tube, and the biotinylated pMHC was purified by gel filtration chromatography. Company), a HiPrepTM 16/60S200HR column (GE) was pre-equilibrated with filtered PBS, loaded with 1 ml of concentrated biotinylated pMHC molecules, and then eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules eluted as a single peak at about 55 ml.
  • the fractions containing protein were combined, concentrated with Millipore ultrafiltration tube, the protein concentration was determined by 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 of generating TCR high-affinity variant libraries for screening high-affinity variants.
  • the TCR phage display and screening method described by Li et al. ((2005) Nature Biotech 23(3):349-354) was applied to the single-chain TCR template in Example 1.
  • a library of high affinity TCRs is created and panned by mutating the CDR regions of the template strand. After several rounds of panning, the phage library has specific binding to the corresponding antigen, and single clones are picked and analyzed.
  • the CDR region of the screened high-affinity single-chain TCR was mutated into the corresponding site of the variable domain of ⁇ heterodimeric TCR, and its affinity with KWVESIFLIF-HLA-A2402 complex was detected by BIAcore.
  • the introduction of the high-affinity mutation points in the above-mentioned CDR regions adopts the method of site-directed mutagenesis well known to those skilled in the art.
  • the amino acid sequences of the ⁇ chain and ⁇ 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 was introduced in the region to form the TCR of the artificial interchain disulfide bond, and the amino acid sequences of the TCR ⁇ and ⁇ chains were respectively shown in Figure 6a (SEQ ID NO. : 11) and 6b (SEQ ID NO: 12), the introduced cysteine residues are shown in bold letters.
  • the extracellular sequence genes for the TCR alpha and beta chains to be expressed were synthesized and inserted into expression vectors, respectively, by standard methods described in Molecular Cloning a Laboratory Manual (Third Edition, Sambrook and Russell) pET28a+ (Novagene), the upstream and downstream cloning sites are NcoI and NotI, respectively. Mutations in the CDR regions are introduced by overlapping PCR (overlap PCR) well known to those skilled in the art. The insert was confirmed by sequencing.
  • the formed inclusion bodies 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 were rapidly 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, the final concentration is 60 mg/mL.
  • the solution was dialyzed in 10 times the volume of deionized water (4°C). After 12 hours, the deionized water was replaced with a buffer (20 mM Tris, pH 8.0), and the dialysis was continued at 4°C for 12 hours.
  • the solution after dialysis was filtered through a 0.45 ⁇ M filter membrane, and then purified through an anion exchange column (HiTrap Q HP, 5 ml, GE Healthcare).
  • the eluted peaks of TCR containing successfully renatured ⁇ and ⁇ dimers were confirmed by SDS-PAGE gel.
  • TCR was then further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was more than 90% determined by SDS-PAGE, and the concentration was determined by BCA method.
  • Example 3 The method described in Example 3 was used to test the affinity of the ⁇ heterodimeric TCR introduced with the high affinity CDR region to the KWVESIFLIF-HLA-A2402 complex.
  • the present invention obtains novel amino acid sequences of TCR ⁇ chain and ⁇ chain variable domains, as shown in Fig. 7(1)-(27) and Fig. 8(1)-(2) respectively. shown. Since the CDR region of the TCR molecule determines its affinity to the corresponding pMHC complex, those skilled in the art can expect that the ⁇ heterodimeric TCR introduced with the high-affinity mutation point also has a high affinity for the KWVESIFLIF-HLA-A2402 complex .
  • the expression vector was constructed by the method described in Example 4, and the ⁇ heterodimeric TCR introduced with high-affinity mutation was expressed, renatured and purified by the method described in Example 5, and then BIAcore T200 was used to determine its interaction with KWVESIFLIF-
  • Table 2 The affinity of the HLA-A2402 complex is shown in Table 2 below.
  • the affinity of the heterodimeric TCR is at least 2 times that of the wild-type TCR for the KWVESIFLIF-HLA-A2402 complex.
  • Example 7 Expression, renaturation and purification of fusion of anti-CD3 antibody and high-affinity ⁇ heterodimeric TCR
  • Fusion molecules were prepared by fusing anti-CD3 single chain antibody (scFv) to ⁇ heterodimeric TCR.
  • the anti-CD3 scFv is fused to the ⁇ chain of the TCR
  • the TCR ⁇ chain may comprise any of the above-mentioned high-affinity ⁇ heterodimeric TCR ⁇ -chain variable domains
  • the TCR ⁇ chain of the fusion molecule may comprise any of the above-mentioned high-affinity ⁇ chain variable domains
  • ⁇ chain expression vector The target gene of the ⁇ chain carrying the ⁇ heterodimeric TCR was double digested with Nco I and Not I, and then connected to the pET28a vector that was double digested with Nco I and Not I.
  • the ligation product was transformed into E.coli DH5 ⁇ , spread on LB plate containing kanamycin, and cultured overnight at 37°C upside down, positive clones were picked for PCR screening, positive recombinants were sequenced, and the recombinant plasmids were extracted after confirming that the sequences were correct. Transform into E.coli Tuner (DE3) for expression.
  • anti-CD3 (scFv)- ⁇ chain expression vector by overlapping PCR method, design primers to connect anti-CD3 scFv and high-affinity heterodimeric TCR ⁇ chain gene, the middle connection
  • the short peptide (linker) is GGGGS (SEQ ID NO: 31)
  • the gene fragment of the fusion protein of anti-CD3 scFv and high-affinity heterodimeric TCR ⁇ chain is provided with restriction endonuclease site Nco I (CCATGG (SEQ ID NO:32)) and Not I (GCGGCCGC (SEQ ID NO:33)).
  • the PCR amplification product was double digested with Nco I and Not I, and ligated with the pET28a vector that was double digested with Nco I and Not I.
  • the ligation product was transformed into E.coli DH5 ⁇ competent cells, coated with LB plate containing kanamycin, and cultured overnight at 37°C upside down.
  • the positive clones were picked for PCR screening, and the positive recombinants were sequenced to confirm that the sequence was correct and then extracted.
  • the recombinant plasmid was transformed into E. coli Tuner (DE3) competent cells for expression.
  • the expression plasmids were transformed into E.coli Tuner (DE3) competent cells respectively, coated on LB plates (kanamycin 50 ⁇ g/mL) and cultured at 37°C overnight. The next day, pick the clone and inoculate it into 10mL LB liquid medium (kanamycin 50 ⁇ g/mL) for 2-3h, inoculate it into 1L LB medium by volume ratio of 1:100, continue to cultivate to OD600 of 0.5-0.8, add The final concentration was 1 mM IPTG to induce the expression of the target protein. After 4 hours of induction, cells were harvested by centrifugation at 6000 rpm for 10 min. The cells were washed once with PBS buffer, and the cells were subpackaged.
  • the cells equivalent to 200 mL of bacterial culture were taken to lyse the bacteria with 5 mL of BugBuster Master Mix (Merck), and the inclusion bodies were collected by centrifugation at 6000 g for 15 min. Four detergent washes were then performed to remove cellular debris and membrane components. The inclusion bodies are then washed with a buffer such as PBS to remove detergents and salts.
  • a buffer such as PBS
  • inclusion bodies were dissolved with a buffer solution containing 6M guanidine hydrochloride, 10mM dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA), 20mM Tris, pH 8.1, and the concentration of the inclusion bodies was determined, and it was aliquoted It was then stored at -80°C frozen.
  • DTT dithiothreitol
  • EDTA ethylenediaminetetraacetic acid
  • the dissolved TCR ⁇ chain and anti-CD3(scFv)- ⁇ chain were rapidly mixed in 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, 6.6 mM ⁇ -mercapoethylamine (4°C), final concentrations of ⁇ chain and anti-CD3(scFv)- ⁇ chain were 0.1 mg/mL and 0.25 mg/mL, respectively.
  • the solution was dialyzed in 10 times the volume of deionized water (4°C). After 12 hours, the deionized water was changed to buffer (10 mM Tris, pH 8.0) and the dialysis was continued at 4°C for 12 hours. The solution after dialysis was filtered through a 0.45 ⁇ M filter membrane and purified by an anion exchange column (HiTrap Q HP 5ml, GE healthcare). The eluted peaks containing TCR ⁇ chain of successfully renatured and anti-CD3(scFv)- ⁇ chain dimer were confirmed by SDS-PAGE gel.
  • the TCR fusion molecule was then further purified by size exclusion chromatography (S-100 16/60, GE healthcare) and again by anion exchange column (HiTrap Q HP 5ml, GE healthcare).
  • the purity of the purified TCR fusion molecule was determined by SDS-PAGE to be more than 90%, and the concentration was determined by BCA method.
  • Example 8 Activation function experiment of effector cells transfected with the high-affinity TCR of the present invention for target cells loaded with short peptides
  • IFN- ⁇ is a powerful immunoregulatory factor produced by activated T lymphocytes. Therefore, in this example, the number of IFN- ⁇ was detected by the ELISPOT assay well known to those skilled in the art to verify the activation of cells transfected with the high-affinity TCR of the present invention. Function and antigen specificity.
  • the high-affinity TCR of the present invention was transfected into CD3+ T cells isolated from the blood of healthy volunteers as effector cells, and CD3+ T cells transfected with other TCR (A6) from the same volunteer were used as controls.
  • the target cells used were T2-A24 loaded with AFP antigen short peptide KWVESIFLIF, loaded with irrelevant peptide or empty (refers to T2 cells transfected with HLA-A2402, the same below).
  • the high-affinity TCR can be learned from Table 2, which are respectively
  • ELISPOT plates were ethanol activated and coated overnight at 4°C. 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 components of the test to the ELISPOT plate: target cells are 1*10 4 cells/well, effector cells are 2* 10 3 cells/well (calculated according to the positive rate of transfection), and set up two duplicate wells. The corresponding short peptides were added so that the final concentration of the short peptides in the ELISPOT plate was 1 ⁇ 10 -6 M. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, the plate was washed for secondary detection and color development, the plate was dried, and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID20 company).
  • ELISPOT READER system AID20 company
  • the ELISPOT plate First prepare the ELISPOT plate, add 2 ⁇ 10 4 cells/well of target cells and 2 ⁇ 10 3 cells/well of effector cells (calculated according to the positive rate of transfection) into the corresponding wells, and then add AFP antigen peptides to the experimental group KWVESIFLIF, and the final concentration of the short peptide in the ELISPOT plate is 1 ⁇ 10 -13 M to 1 ⁇ 10 -6 M, a total of 8 gradients; add irrelevant peptides to the control group, and make the final concentration of the short peptide is 1 ⁇ 10 -6 M.
  • the remaining steps are as described in this embodiment (I).
  • the effector cells transfected with the high-affinity TCR of the present invention have a significant activation effect, and respond when the concentration of the specific antigen peptide is low.
  • the effector cells transfected with other TCRs are inactive; meanwhile, the effector cells transfected with the TCR of the present invention are basically inactive to target cells loaded with irrelevant peptides or empty.
  • Example 9 Activation function experiment of effector cells transfected with high-affinity TCR of the present invention for tumor cell lines
  • tumor cell lines were used to verify the activation function and specificity of effector cells transfected with the high-affinity TCR of the present invention again. It is also detected by the ELISPOT assay well known to those skilled in the art.
  • the high-affinity TCR of the present invention was transfected into CD3+ T cells isolated from the blood of healthy volunteers as effector cells, and the same volunteer was transfected with other TCR (A6) or wild-type TCR (WT-TCR). ) CD3+ T cells as a negative control.
  • the following experiments were carried out in two batches (I), (II) and (III) successively:
  • the high-affinity TCR can be learned from Table 2, which are respectively
  • AFP-positive tumor cell lines used in this batch were HepG2, SK-HEP-1-AFP (AFP overexpressed), and the negative tumor cell lines were SK-HEP-1, NCI-H226, NCI-H1299, and HUH-1.
  • AFP-positive tumor cell lines used in this batch were HepG2, SK-HEP-1-AFP (AFP overexpressed), and the negative tumor cell lines were SK-HEP-1, NCI-H226, NCI-H1299, HUH-1, MKN7 .
  • AFP positive tumor cell lines used in this batch were HepG2, SK-HEP-1-AFP (AFP overexpression), and the negative tumor cell lines were SK-HEP-1, NCI-H226, SW480, HUH-1.
  • the ELISPOT plate was prepared. ELISPOT plates were ethanol activated and coated overnight at 4°C. On the first day of the experiment, remove the coating solution, wash and block, incubate at room temperature for two hours, remove the blocking solution, and add each component of the test to the ELISPOT plate: target cells are 2*10 4 cells/well, effector cells are 2* 10 3 cells/well (calculated according to the positive rate of transfection), and set up two duplicate wells. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, the plate was washed for secondary detection and color development, the plate was dried, and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID20 company).
  • ELISPOT READER system an immunospot plate reader
  • the effector cells transfected with the high-affinity TCR of the present invention have a more obvious activation effect than the effector cells transfected with the wild-type, while the effector cells transfected with other TCRs have a more obvious activation effect.
  • the effector cells are inactive; meanwhile, the effector cells transfected with the high-affinity TCR of the present invention are basically inactive against AFP-negative tumor cell lines.
  • Example 10 Killing function experiment of effector cells transfected with high-affinity TCR of the present invention for T2 cells loaded with gradient short peptides
  • Lactate dehydrogenase is abundant in the cytoplasm and cannot pass through the cell membrane under normal conditions, but can be released to the outside of the cell when the cell is damaged or died. At this time, the activity of LDH in the cell culture medium is proportional to the number of cells dying.
  • non-radioactive cytotoxicity experiments well known to those skilled in the art are used to measure the release of LDH, thereby verifying the killing function of cells transfected with the TCR of the present invention. This assay is a colorimetric alternative to the 51Cr release cytotoxicity assay and quantifies the LDH released after cell lysis.
  • LDH released in the medium was detected using a 30 min coupled enzymatic reaction in which LDH converts a tetrazolium salt (INT) to red formazan.
  • the amount of red product produced is proportional to the number of cells lysed.
  • the high-affinity TCR of the present invention was transfected into CD3+ T cells isolated from the blood of healthy volunteers as effector cells, and the same volunteer was empty-transfected (NC) or transfected with other TCRs (A6) of CD3+ T cells served as a negative control.
  • the high-affinity TCR and its number are learned from Table 2, which are TCR1 (alpha chain variable domain SEQ ID NO: 13, beta chain variable domain SEQ ID NO: 2), TCR3 (alpha chain variable domain SEQ ID NO: 2), TCR3 (alpha chain variable domain SEQ ID NO: 2) NO: 15, beta chain variable domain SEQ ID NO: 2) and TCR6 (alpha chain variable domain SEQ ID NO: 18, beta chain variable domain SEQ ID NO: 2).
  • Target cells were KWVESIFLIF peptide-loaded, irrelevant peptide-loaded, or empty T2-A24.
  • the LDH plate First prepare the LDH plate, add 3 ⁇ 10 4 cells/well of target cells and 3 ⁇ 10 4 cells/well of effector cells (calculated according to the positive rate of transfection) into the corresponding wells, and then add AFP antigen peptides to the experimental group KWVESIFLIF, and the final concentration of the short peptide in the LDH well plate was 1 ⁇ 10 -15 M to 1 ⁇ 10 -8 M, a total of 8 gradients; other short peptides were added to the control group, and the short peptide was finalized. The concentrations were 1 ⁇ 10 -10 M to 1 ⁇ 10 -6 M in sequence, with a total of 5 gradients, and three replicate wells were set for each.
  • effector cell spontaneous wells were set. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, color development was detected, and the absorbance value was recorded at 490 nm with a microplate reader (Bioteck) after the reaction was terminated.
  • the experimental results are shown in Figure 14.
  • the effector cells transfected with the high-affinity TCR of the present invention showed a strong killing effect, and when the concentration of the above-mentioned specific short peptide was low, it was effective.
  • T cells transfected with other TCRs or empty-transfected basically have no killing effect; meanwhile, effector cells transfected with the high-affinity TCR of the present invention have no killing effect on target cells loaded with irrelevant peptides or empty.
  • Example 11 Killing function experiment of effector cells transfected with the high-affinity TCR of the present invention for tumor cell lines
  • the release of LDH is also measured by non-radioactive cytotoxicity experiments well known to those skilled in the art, so as to verify the killing function of the cells transfected with the TCR of the present invention.
  • 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 other TCR (A6) or wild-type TCR (WT-TCR) CD3+ T cells as a negative control.
  • the AFP-positive tumor cell line used in this batch was HepG2, and the negative tumor cell lines were NCI-H226 and SK-HEP-1.
  • the experimental steps are as follows: first prepare the LDH plate, and add the components of the test to the plate in the following order: target cells 3 ⁇ 10 4 cells/well, effector cells 3 ⁇ 10 4 cells/well (calculated according to the transfection positive rate) Add to corresponding wells, and set up three duplicate wells. At the same time, effector cell spontaneous wells, target cell spontaneous wells, target cell maximum wells, volume correction control wells and medium background control wells were set. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, color development was detected, and the absorbance value was recorded at 490 nm with a microplate reader (Bioteck) after the reaction was terminated.
  • TCR21 ⁇ chain variable domain SEQ ID NO: 31, ⁇ chain variable domain SEQ ID NO: 2
  • TCR8 ⁇ chain variable domain SEQ ID NO:20, beta chain variable domain SEQ ID NO:2
  • TCR7 alpha chain variable domain SEQ ID NO:19, beta chain variable domain SEQ ID NO: 2.
  • the AFP-positive tumor cell line used in this batch was HepG2, and the negative tumor cell lines were T84, Hs578T, and NCI-H526.
  • the experimental procedure is the same as in this example (I).
  • TCR2 ⁇ chain variable domain SEQ ID NO: 14, ⁇ chain variable domain SEQ ID NO: 2
  • TCR9 ⁇ chain variable domain SEQ ID NO: 2 SEQ ID NO: 21, beta chain variable domain SEQ ID NO: 2.
  • the AFP-positive tumor cell lines used in this batch were HepG2, SK-HEP-1-AFP (AFP overexpressed), and the negative tumor cell line was NCI-H226.
  • the components added to the plate are as follows: target cells 2.5 ⁇ 10 4 cells/well, effector cells 2.5 ⁇ 10 4 cells/well (calculated according to the positive rate of transfection), and other experimental steps are the same as in Example (I).
  • IncuCyte is a functional analysis system that can automatically analyze images at different time points and quantify real-time apoptosis through real-time microphotography in an incubator.
  • TCR1 alpha chain variable domain SEQ ID NO:13, beta chain variable domain SEQ ID NO:2
  • TCR3 alpha chain variable domain SEQ ID NO:15 , ⁇ chain variable domain SEQ ID NO: 2
  • HepG2 was a positive tumor cell line
  • SK-HEP-1 was a negative tumor cell line as a control.
  • the target cells were digested and centrifuged; resuspended in complete medium of RPMI1640+10% FBS without phenol red, and the target cells were evenly plated in a 96-well plate: 2*10 4 cells/cell Well; put it back into the incubator at 37 degrees, 5% CO 2 and incubate overnight; the next day, the medium in the 96-well plate was discarded and replaced with phenol red-free RPMI1640+10% FBS containing dye caspase3/7 reagent medium so that the dye concentration is 2 drops/ml.
  • cells transfected with the high-affinity TCR of the present invention can show a strong and effective killing effect in a short period of time, while effector cells transfected with other TCRs have no killing effect; Meanwhile, the cells transfected with the high-affinity TCR of the present invention basically do not kill negative tumor cells.

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Abstract

本发明提供了一种T细胞受体(TCR)，其具有结合KWVESIFLIF-HLA A2402复合物的特性；并且所述TCR对所述KWVESIFLIF-HLA A2402复合物的结合亲和力是野生型TCR对KWVESIFLIF-HLA A2402复合物的结合亲和力的至少2倍。本发明还提供了此类TCR与治疗剂的融合分子。此类TCR可以单独使用，也可与治疗剂联用，以靶向呈递KWVESIFLIF-HLA A2402复合物肿瘤细胞。

Description

一种识别AFP的高亲和力TCR
本申请要求申请日为2020年7月30日的中国专利申请202010752773.0的优先权。
技术领域
本发明涉及生物技术领域，更具体地涉及能够识别衍生自AFP蛋白多肽的T细胞受体(T cell receptor，TCR)。本发明还涉及所述受体的制备和用途。
背景技术
仅仅有两种类型的分子能够以特异性的方式识别抗原。其中一种是免疫球蛋白或抗体；另一种是T细胞受体(TCR)，它是由α链/β链或者γ链/δ链以异二聚体形式存在的细胞膜表面的糖蛋白。免疫系统的TCR总谱的组成是在胸腺中通过V(D)J重组，然后进行阳性和阴性选择而产生的。在外周环境中，TCR介导了T细胞对主组织相容性复合体-肽复合物(pMHC)的特异性识别，因此其对免疫系统的细胞免疫功能是至关重要的。
TCR是呈递在主组织相容性复合体(MHC)上的特异性抗原肽的唯一受体，这种外源肽或内源肽可能会是细胞出现异常的唯一迹象。在免疫系统中，通过抗原特异性的TCR与pMHC复合物的结合引发T细胞与抗原呈递细胞(APC)直接的物理接触，然后T细胞及APC两者的其他细胞膜表面分子就发生相互作用，这就引起了一系列后续的细胞信号传递和其他生理反应，从而使得不同抗原特异性的T细胞对其靶细胞发挥免疫效应。
与TCR相对应的MHC I类和II类分子配体也是免疫球蛋白超家族的蛋白质但对于抗原的呈递具有特异性，不同的个体有不同的MHC，从而能呈递一种蛋白抗原中不同的短肽到各自的APC细胞表面。人类的MHC通常称为HLA基因或HLA复合体。
AFP(αFetoprotein)也称α胎蛋白，是胚胎发育过程中表达的一种蛋白，是胚胎血清的主要成分。在发育过程中，AFP在卵黄囊及肝脏中有比较高的表达水平，随后被抑制。在肝癌中，AFP的表达被激活。AFP在细胞内加工处理成抗原肽，并与MHC(主组织相容性复合体)分子结合形成复合物，被呈递到肿瘤细胞表面。KWVESIFLIF是衍生自AFP抗原的短肽，是AFP相关疾病治疗的一种靶标。
因此，KWVESIFLIF-HLA A2402复合物提供了一种TCR可靶向肿瘤细胞的标记。能够结合KWVESIFLIF-HLA A2402复合物的TCR对肿瘤的治疗具有很高的应用价值。例如，能够靶向该肿瘤细胞标记的TCR可用于将细胞毒性剂或免疫刺激剂递送到靶细胞，或被转化入T细胞，使表达该TCR的T细胞能够破坏肿瘤细胞，以便在被称为过继免疫治疗的治疗过程中给予患者。对于前一目的，理想的TCR是具有较高的亲和力的，从而使该TCR能够长期驻留在所靶向的细胞上面。对于后一目的，则优选使用中等亲和力的TCR。因此，本领域技术人员致力于开发可用于满足不同目的的靶向肿瘤细胞标记的TCR。
发明内容
本发明的目的在于提供一种对KWVESIFLIF-HLA A2402复合物具有较高亲和力的TCR。
本发明的再一目的是提供一种上述类型TCR的制备方法及上述类型TCR的用途。
本发明的第一方面，提供了一种包含了α链可变域和β链可变域的T细胞受体(TCR)，其具有结合KWVESIFLIF-HLA A2402复合物的活性，并且所述TCRα链可变域的氨基酸序列与SEQ ID NO:1所示的氨基酸序列有至少90％的序列同源性和所述TCRβ链可变域的氨基酸序列与SEQ ID NO:2所示的氨基酸序列有至少90％的序列同源性。
在一优选例中，所述TCRα链可变域的氨基酸序列和所述TCRβ链可变域的氨基酸序列不同时为野生型TCRα链可变域的氨基酸序列和野生型TCRβ链可变域的氨基酸序列。
在进一步的优选例中，所述TCRα链可变域的氨基酸序列不是SEQ ID NO:1所示的氨基酸序列，和/或所述TCRβ链可变域的氨基酸序列不是SEQ ID NO:2所示的氨基酸序列。
在另一优选例中，所述TCR的α链可变域包含与SEQ ID NO:1所示的序列有至少91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性的氨基酸序列。
在另一优选例中，所述TCR的β链可变域为与SEQ ID NO:2所示的序列有至少91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同源性的氨基酸序列。
在另一优选例中，所述TCRα链可变域的氨基酸序列与SEQ ID NO:1所示的氨基酸序列有至少95％的序列同源性。
在另一优选例中，所述TCRβ链可变域的氨基酸序列与SEQ ID NO:2所示的氨基酸序列有至少95％的序列同源性。
在另一优选例中，所述TCR与KWVESIFLIF-HLA A2402复合物的亲和力是野生型TCR的至少2倍。
在另一优选例中，所述野生型TCR的α链可变域的氨基酸序列如SEQ ID NO:1所示；β链可变域的氨基酸序列如SEQ ID NO:2所示。
在另一优选例中，所述TCRα链可变域的3个CDR区(互补决定区)的基准序列如下，
CDR1α：DSAIYN
CDR2α：IQSSQRE
CDR3α：AVRRRNYGQNFV，并且CDR3α含有至少一个下列突变：
突变前的残基 突变后的残基
CDR3α的第1位A G或S
CDR3α的第2位V A或G或I或L或S或T
CDR3α的第3位R I或T或V
CDR3α的第4位R W或Y或N或M
CDR3α的第5位R Y
CDR3α的第6位N E或H或Q或R或T或I或V或M
CDR3α的第7位Y L或P或W或V
在另一优选例中，所述CDR3α中氨基酸突变包含：
突变前的残基 突变后的残基
CDR3α的第4位R Y
在另一优选例中，所述TCRα链可变域的氨基酸突变个数为1-6个，优选地为1-4个。
在另一优选例中，所述TCRα链可变域的氨基酸突变个数为2个或3个或4个。
在另一优选例中，所述TCRα链可变域的氨基酸突变位点为CDR3α的第1-4位，其中：
突变前的残基 突变后的残基
CDR3α的第1位A G或S
CDR3α的第2位V A或G或I或L或S或T
CDR3α的第3位R I或V
CDR3α的第4位R W或Y
在另一优选例中，所述TCRα链可变域的氨基酸突变位点为CDR3α的第3位、第4位和第6位，其中：
突变前的残基 突变后的残基
CDR3α的第3位R I或T
CDR3α的第4位R W或Y
CDR3α的第6位N Q或T
在另一优选例中，所述TCRα链可变域的氨基酸突变位点为CDR3α的第3位和第4位，其中：
突变前的残基 突变后的残基
CDR3α的第3位R I
CDR3α的第4位R W或Y
在另一优选例中，所述TCRα链可变域的氨基酸突变位点为CDR3α的第6位和第7位，其中：
突变前的残基 突变后的残基
CDR3α的第6位N E或H或Q或T或M
CDR3α的第7位Y L或P或W或V
在另一优选例中，所述TCRα链可变域中CDR1α为：DSAIYN，和CDR2α为IQSSQRE。
在另一优选例中，所述TCRβ链可变域的CDR3β为：ASSLIAQNHNEQF。
在另一优选例中，所述TCRβ链可变域的3个CDR为：
CDR1β：SGHDT；
CDR2β：YYEEEE；和
CDR3β：ASSLIAQNHNEQF。
在另一优选例中，所述TCRβ链可变域中CDR3β的氨基酸序列选自：ASSLIAQNHNEQF、ASSLIAQNHALWT和ASSLIAQNHAMWT。
在另一优选例中，所述TCRα链可变域的氨基酸序列为SEQ ID NO:2。
在另一优选例中，所述TCRα链可变域的CDR3α选自GGVYRNYGQNFV、AVTYRQYGQNFV和AVIYRNYGQNFV。
在另一优选例中，所述TCRα链可变域包含CDR1α、CDR2α和CDR3α，其中CDR1α的氨基酸序列为DSAIYN，CDR2α的氨基酸序列为IQSSQRE，并且CDR3α的氨基酸序列 为：[3 X1][3 X2][3 X3][3 X4][3 X5][3 X6][3 X7]GQNFV，其中[3 X1]为A或G或S，和/或[3 X2]为V或A或G或I或L或S或T，和/或[3 X3]为R或I或T或V，和/或[3 X4]为R或W或Y或N或M，和/或[3 X5]为R或Y，和/或[3 X6]为N或E或H或Q或R或T或I或V或M，和/或[3 X7]为Y或L或P或W或V。
在另一优选例中，所述TCR在SEQ ID NO：1所示的α链可变域中发生突变，所述突变选自A91G/S、V92A/G/I/L/S/T、R93I/T/V、R94W/Y/N/M、R95Y、N96E/H/Q/R/T/I/V/M和Y97L/P/W/V中的一组或几组，其中氨基酸残基编号采用SEQ ID NO:1所示的编号。
在另一优选例中，所述TCR具有选自下组的CDR:
Figure PCTCN2021109705-appb-000001
在另一优选例中，所述TCR是可溶的。
在另一优选例中，所述TCR为αβ异质二聚TCR，包含α链TRAC恒定区序列和β链TRBC1或TRBC2恒定区序列。
在另一优选例中，所述TCR包含(i)除其跨膜结构域以外的全部或部分TCRα链，和(ii)除其跨膜结构域以外的全部或部分TCRβ链，其中(i)和(ii)均包含TCR链的可变域和至少一部分恒定域。
在另一优选例中，所述TCR的α链恒定区与β链恒定区之间含有人工链间二硫键。
在另一优选例中，在所述TCRα与β链的恒定区之间形成人工链间二硫键的半胱氨酸残 基取代了选自下列的一组或多组位点：
TRAC*01外显子1的Thr48和TRBC1*01或TRBC2*01外显子1的Ser57；
TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；
TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；
TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；
TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；
TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；
TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；
和TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。
在另一优选例中，所述TCR的α链可变域氨基酸序列为SEQ ID NO:1、13-39之一；和/或所述TCR的β链可变域氨基酸序列为SEQ ID NO:2、40、41之一。
在另一优选例中，所述TCR选自下组：
Figure PCTCN2021109705-appb-000002
在另一优选例中，所述TCR为单链TCR。
在另一优选例中，所述TCR是由α链可变域和β链可变域组成的单链TCR，所述α链可变域和β链可变域由一柔性短肽序列(linker)连接。
在另一优选例中，所述TCR的α链和/或β链的C-或N-末端结合有偶联物，优选地，所 述偶联物为可检测标记物、治疗剂、PK修饰部分或任何这些物质的组合。
在另一优选例中，与所述TCR结合的治疗剂为连接于所述TCR的α或β链的C-或N-末端的抗-CD3抗体。
本发明的第二方面，提供了一种多价TCR复合物，其包含至少两个TCR分子，并且其中的至少一个TCR分子为上述权利要求中任一项所述的TCR。
本发明的第三方面，提供了一种核酸分子，所述核酸分子包含编码本发明第一方面所述的TCR分子或者本发明第二方面所述的多价TCR复合物的核酸序列或其互补序列。
本发明的第四方面，提供了一种载体，所述的载体含有本发明第三方面所述的所述的核酸分子。
本发明的第五方面，提供了一种宿主细胞，所述的宿主细胞中含有本发明第四方面所述的载体或染色体中整合有外源的本发明第三方面所述的核酸分子。
本发明的第六方面，提供了一种分离的细胞，所述细胞表达本发明第一方面所述的TCR，优选地，所述分离的细胞T细胞、NK细胞、NKT细胞，最优选地，所述分离的细胞是T细胞。
本发明的第七方面，提供了一种药物组合物，所述组合物含有药学上可接受的载体以及本发明第一方面所述的TCR、或本发明第二方面所述的TCR复合物、或本发明第六方面所述的细胞。
本发明的第八方面，提供了一种治疗疾病的方法，包括给需要治疗的对象施用适量的本发明第一方面所述的TCR、或本发明第二方面所述的TCR复合物、或本发明第六方面所述的细胞、或本发明第七方面所述的药物组合物,优选地，所述疾病为AFP阳性肿瘤，更优选地，所述肿瘤为肝癌。
本发明的第九方面，提供了本发明第一方面所述的TCR、或本发明第二方面所述的TCR复合物、或本发明第六方面所述的细胞的用途，用于制备治疗肿瘤的药物，优选地，所述疾病为AFP阳性肿瘤，更优选地，所述肿瘤为肝癌。
本发明的第十方面，提供了一种制备本发明第一方面所述的T细胞受体的方法，包括步骤：
(i)培养本发明第五方面所述的宿主细胞，从而表达本发明第一方面所述的T细胞受体；
(ii)分离或纯化出所述的T细胞受体。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1a和图1b分别显示了对KWVESIFLIF-HLA A2402复合物能够特异性结合的野生型TCRα与β链可变域氨基酸序列。
图2a和图2b分别为本发明构建的单链模板TCR的α链可变域的氨基酸序列和β链可变域的氨基酸序列。
图3a和图3b分别为本发明构建的单链模板TCR的α链可变域的DNA序列和β链可变域的DNA序列。
图4a和图4b分别为本发明构建的单链模板TCR的连接短肽(linker)的氨基酸序列和DNA序列。
图5a、图5b分别为本发明构建的单链模板TCR的氨基酸序列和DNA序列。
图6a和图6b分别为本发明中可溶性参比TCRα链与β链的氨基酸序列。
图7(1)-(27)分别显示了对KWVESIFLIF-HLA A2402复合物具有高亲和力的异质二聚TCR的α链可变域氨基酸序列，突变的残基以加下划线表示。
图8(1)-(2)分别显示了对KWVESIFLIF-HLA A2402复合物具有高亲和力的异质二聚TCR的β链可变域氨基酸序列，突变的残基以加下划线表示。
图9a和图9b分别显示了对KWVESIFLIF-HLA A2402复合物能够特异性结合的野生型TCRα链与β链的胞外氨基酸序列。
图10a和图10b分别显示了对KWVESIFLIF-HLA A2402复合物能够特异性结合的野生型TCRα链与β链的氨基酸序列。
图11为可溶性参比TCR即野生型TCR与KWVESIFLIF-HLA A2402复合物的结合曲线。
图12a-图12c为针对负载短肽的T2细胞，转染本发明高亲和力TCR的效应细胞的激活功能实验结果。
图13a-图13c为针对肿瘤细胞系，转染本发明高亲和力TCR的效应细胞的激活功能实验结果。
图14为针对梯度负载短肽的T2细胞，转染本发明高亲和力TCR的效应细胞的杀伤功能实验结果。
图15a-图15c为针对肿瘤细胞系，转染本发明高亲和力TCR的效应细胞的杀伤功能LDH实验结果。
图16a和图16b为针对肿瘤细胞系，转染本发明高亲和力TCR的效应细胞的杀伤功能IncuCyte实验结果。
具体实施方式
本发明通过广泛而深入的研究，获得一种识别KWVESIFLIF短肽(衍生自AFP蛋白)的高亲和性T细胞受体(TCR)，所述KWVESIFLIF短肽以肽-HLA A2402复合物的形式被呈递。所述高亲和性TCR在其α链可变域的3个CDR区：
CDR1α：DSAIYN
CDR2α：IQSSQRE
CDR3α：AVRRRNYGQNFV中发生突变；和/或在其β链可变域的3个CDR区：
CDR1β：SGHDT
CDR2β：YYEEEE
CDR3β：ASSLIAQNHNEQF中发生突变；并且，突变后本发明TCR对上述KWVESIFLIF-HLA A2402复合物的亲和力和/或结合半衰期是野生型TCR的至少2倍。
在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且其意图不是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。
虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。
术语
T细胞受体(T cell receptor,TCR)
可以采用国际免疫遗传学信息系统(IMGT)来描述TCR。天然αβ异源二聚TCR具有α链和β链。广义上讲，各链包含可变区、连接区和恒定区，β链通常还在可变区和连接区之间含有短的多变区，但该多变区常视作连接区的一部分。通过独特的IMGT的TRAJ和TRBJ确定TCR的连接区，通过IMGT的TRAC和TRBC确定TCR的恒定区。
各可变区包含嵌合在框架序列中的3个CDR(互补决定区)，CDR1、CDR2和CDR3。在IMGT命名法中，TRAV和TRBV的不同编号分别指代不同Vα类型和Vβ的类型。在IMGT系统中，α链恒定结构域具有以下的符号：TRAC*01，其中“TR”表示T细胞受体基因；“A”表示α链基因；C表示恒定区；“*01”表示等位基因1。β链恒定结构域具有以下的符号：TRBC1*01或TRBC2*01，其中“TR”表示T细胞受体基因；“B”表示β链基因；C表示恒定区；“*01”表示等位基因1。α链的恒定区是唯一确定的，在β链的形式中，存在两个可能的恒定区基因“C1”和“C2”。本领域技术人员通过公开的IMGT数据库可以获得TCRα与β链的恒定区基因序列。
TCR的α和β链一般看作各有两个“结构域”即可变域和恒定结构域。可变域由连接的可变区和连接区构成。因此，在本申请的说明书和权利要求书中，“TCRα链可变域”指连接的TRAV和TRAJ区，同样地，“TCRβ链可变域”指连接的TRBV和TRBD/TRBJ区。TCRα链可变域的3个CDR分别为CDR1α、CDR2α和CDR3α；TCRβ链可变域的3个CDR分别为CDR1β、CDR2β和CDR3β。本发明TCR可变域的框架序列可以为鼠源的或人源的，优选为人源的。TCR的恒定结构域包含胞内部分、跨膜区和胞外部分。
本发明中，能够结合KWVESIFLIF-HLA A2402复合物的野生型TCR的α与β链可变域氨基酸序列分别为SEQ ID NO:1和SEQ ID NO:2，如图1a和图1b所示。本发明中所述可溶性“参比TCR”的α链氨基酸序列及β链氨基酸序列分别为SEQ ID NO:11和SEQ ID NO: 12，如图6a和图6b所示。本发明中所述“野生型TCR”的α链胞外氨基酸序列及β链胞外氨基酸序列分别为SEQ ID NO：42和SEQ ID NO：43，如图9a和图9b所示。本发明中所用的TCR序列为人源的。本发明中所述“野生型TCR”的α链氨基酸序列及β链氨基酸序列分别为SEQ ID NO:44和SEQ ID NO:45，如图10a和10b所示。在本发明中，术语“本发明多肽”、“本发明的TCR”、“本发明的T细胞受体”可互换使用。
天然链间二硫键与人工链间二硫键
在天然TCR的近膜区Cα与Cβ链间存在一组二硫键，本发明中称为“天然链间二硫键”。在本发明中，将人工引入的，位置与天然链间二硫键的位置不同的链间共价二硫键称为“人工链间二硫键”。
为方便描述，本发明中TRAC*01与TRBC1*01或TRBC2*01氨基酸序列的位置编号按从N端到C端依次的顺序进行位置编号，如TRBC1*01或TRBC2*01中，按从N端到C端依次的顺序第60个氨基酸为P(脯氨酸)，则本发明中可将其描述为TRBC1*01或TRBC2*01外显子1的Pro60，也可将其表述为TRBC1*01或TRBC2*01外显子1的第60位氨基酸，又如TRBC1*01或TRBC2*01中，按从N端到C端依次的顺序第61个氨基酸为Q(谷氨酰胺)，则本发明中可将其描述为TRBC1*01或TRBC2*01外显子1的Gln61，也可将其表述为TRBC1*01或TRBC2*01外显子1的第61位氨基酸，其他以此类推。本发明中，可变区TRAV与TRBV的氨基酸序列的位置编号，按照IMGT中列出的位置编号。如TRAV中的某个氨基酸，IMGT中列出的位置编号为46，则本发明中将其描述为TRAV第46位氨基酸，其他以此类推。本发明中，其他氨基酸的序列位置编号有特殊说明的，则按特殊说明。
肿瘤
术语“肿瘤”指包括所有类型的癌细胞生长或致癌过程，转移性组织或恶性转化细胞、组织或器官，不管病理类型或侵染的阶段。肿瘤的实施例非限制性地包括：实体瘤，软组织瘤，和转移性病灶。实体瘤的实施例包括：不同器官系统的恶性肿瘤，例如肉瘤，肺鳞癌和癌症。例如：感染的前列腺，肺，乳房，淋巴，肠胃(例如：结肠)，和生殖泌尿道(例如：肾脏，上皮细胞)，咽头。肺鳞癌包括恶性肿瘤，例如，多数的结肠癌，直肠癌，肾细胞癌，肝癌，肺部的非小细胞癌，小肠癌和食道癌。上述癌症的转移性病变可同样用本发明的方法和组合物来治疗和预防。
发明详述
众所周知，TCR的α链可变域与β链可变域各含有3个CDR，类似于抗体的互补决定区。CDR3与抗原短肽相互作用，CDR1和CDR2与HLA相互作用。因此，TCR分子的CDR决定了其与抗原短肽-HLA复合物的相互作用。能够结合抗原短肽KWVESIFLIF与HLA A2402复合物(即，KWVESIFLIF-HLA A2402复合物)的野生型TCR的α链可变域氨基酸序列与β 链可变域氨基酸序列分别为SEQ ID NO:1和SEQ ID NO:2,该序列为本发明人首次发现。其具有下列CDR区：
α链可变域CDR CDR1α：DSAIYN
CDR2α：IQSSQRE
CDR3α：AVRRRNYGQNFV
和β链可变域CDR CDR1β：SGHDT
CDR2β：YYEEEE和
CDR3β：ASSLIAQNHNEQF。
本发明通过对上述CDR区进行突变筛选，获得了与KWVESIFLIF-HLA A2402复合物的亲和力是野生型TCR与KWVESIFLIF-HLA A2402复合物亲和力至少2倍的高亲和力TCR。
进一步，本发明所述TCR是αβ异质二聚TCR，所述TCR的α链可变域包含与SEQ ID NO:1所示的氨基酸序列有至少85％；优选地，至少90％；更优选地，至少92％；更优选地，至少94％(如，可以是至少88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同源性)的序列同源性的氨基酸序列；和/或所述TCR的β链可变域包含与SEQ ID NO:2所示的氨基酸序列有至少90％，优选地，至少92％；更优选地，至少94％(如，可以是至少91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同源性)的序列同源性的氨基酸序列。
进一步，本发明所述TCR是单链TCR，所述TCR的α链可变域包含与SEQ ID NO:3所示的氨基酸序列有至少85％，优选地，至少90％；更优选地，至少92％；最优选地，至少94％(如，可以是至少88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同源性)的序列同源性的氨基酸序列；和/或所述TCR的β链可变域包含与SEQ ID NO:4所示的氨基酸序列有至少85％，优选地，至少90％；更优选地，至少92％；最优选地，至少94％(如，可以是至少91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同源性)的序列同源性的氨基酸序列。
本发明中野生型TCRα链可变域SEQ ID NO:1的3个CDR即CDR1、CDR2和CDR3分别位于SEQ ID NO:1的第27-32位、第50-56位和第91-102位。据此，氨基酸残基编号采用SEQ ID NO:1所示的编号，91A即为CDR3α的第1位A、92V即为CDR3α的第2位V、93R即为CDR3α的第3位R、94R即为CDR3α的第4位R、95R即为CDR3α的第5位R、96N即为CDR3α的第6位N、97Y即为CDR3α的第7位Y。
本发明提供具有结合KWVESIFLIF-HLA A2402复合物的特性的TCR，并包含α链可变域和β链可变域，其中，所述TCR在SEQ ID NO:1所示的α链可变域中发生突变，所述突变的氨基酸残基位点包括91A、92V、93R、94R、95R、96N和97Y中的一个或多个，其中，氨基酸残基编号采用SEQ ID NO:1所示的编号。
优选地，突变后的所述TCRα链可变域包括选自下组的一个或多个氨基酸残基：91G或91S；92A或92G或92I或92L或92S或92T；93I或93T或93V；94W或94Y或94N或94M； 95Y；96E或96H或96Q或96R或96T或96I或96V或96M；97L或97P或97W或97V，其中，氨基酸残基编号采用SEQ ID NO:1所示的编号。
更具体地，α链可变域中所述突变的具体形式包括A91G/S、V92A/G/I/L/S/T、R93I/T/V、R94W/Y/N/M、R95Y、N96E/H/Q/R/T/I/V/M和Y97L/P/W/V中的一组或几组。
应理解，本文中氨基酸名称采用国际通用的单英文字母标识，与其相对应的氨基酸名称三英文字母简写分别是：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)；
本发明中，Pro60或者60P均表示第60位脯氨酸。另外，本发明中所述突变的具体形式的表述方式如“A91G/S”代表第91位的A被G取代或被S取代，其他以此类推。
根据本领域技术人员熟知的定点突变的方法，将野生型TCRα链恒定区TRAC*01外显子1的Thr48突变为半胱氨酸，β链恒定区TRBC1*01或TRBC2*01外显子1的Ser57突变为半胱氨酸，即得到参比TCR，其氨基酸序列分别为SEQ ID NO:11和SEQ ID NO:12，如图6a和图6b所示，突变后的半胱氨酸残基以加粗字母表示。上述半胱氨酸取代能使参比TCR的α与β链的恒定区之间形成人工链间二硫键，以形成更加稳定的可溶性TCR，从而能够更加方便地评估TCR与KWVESIFLIF-HLA A2402复合物之间的结合亲和力和/或结合半衰期。应理解，TCR可变区的CDR区决定了其与pMHC复合物之间的亲和力，因此，上述TCR恒定区的半胱氨酸取代并不会对TCR的结合亲和力和/或结合半衰期产生影响。所以，在本发明中，测得的参比TCR与KWVESIFLIF-HLA A2402复合物之间的结合亲和力即认为是野生型TCR与KWVESIFLIF-HLA A2402复合物之间的结合亲和力。同样地，如果测得本发明TCR与KWVESIFLIF-HLA A2402复合物之间的结合亲和力是参比TCR与KWVESIFLIF-HLA A2402复合物之间的结合亲和力的至少10倍，即等同于本发明TCR与KWVESIFLIF-HLA A2402复合物之间的结合亲和力是野生型TCR与KWVESIFLIF-HLA A2402复合物之间的结合亲和力的至少10倍。
可通过任何合适的方法测定结合亲和力(与解离平衡常数KD成反比)和结合半衰期(表示为T1/2)，如采用表面等离子共振技术进行检测。应了解，TCR的亲和力翻倍将导致KD减半。T1/2计算为In2除以解离速率(Koff)。因此，T1/2翻倍会导致Koff减半。优选采用相同的试验方案检测给定TCR的结合亲和力或结合半衰期数次，例如3次或更多，取结果的平均值。在优选的实施方式中，采用本文实施例中的表面等离振子共振(BIAcore)方法检测可溶性TCR的亲和力，条件为：温度25℃，PH值为7.1-7.5。该方法检测到参比TCR对KWVESIFLIF-HLA A2402复合物的解离平衡常数KD为1.06E-04M，即106μM，本发明中即认为野生型TCR对KWVESIFLIF-HLA A2402复合物的解离平衡常数KD也为106μM。由于TCR的亲和力翻倍将导致KD减半，所以若检测到高亲和力TCR对KWVESIFLIF-HLA A2402复合物的解离平衡常数KD为1.06E-05M，即10.6μM，则说明该高亲和力TCR对KWVESIFLIF-HLA A2402复合物的亲和力是野生型TCR对KWVESIFLIF-HLA A2402复合 物的亲和力的10倍。本领域技术人员熟知KD值单位间的换算关系，即1M＝10 6μM,,1μM＝1000nM，1nM＝1000pM。在本发明中，所述TCR与KWVESIFLIF-HLA A2402复合物的亲和力是野生型TCR的至少2倍。
可采用任何合适的方法进行突变，包括但不限于依据聚合酶链式反应(PCR)的那些、依据限制性酶的克隆或不依赖连接的克隆(LIC)方法。许多标准分子生物学教材详述了这些方法。聚合酶链式反应(PCR)诱变和依据限制性酶的克隆的更多细节可参见Sambrook和Russell,(2001)分子克隆-实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)CSHL出版社。LIC方法的更多信息可见(Rashtchian,(1995)Curr Opin Biotechnol 6(1):30-6)。
产生本发明的TCR的方法可以是但不限于从展示此类TCR的噬菌体颗粒的多样性文库中筛选出对KWVESIFLIF-HLA-A2402复合物具有高亲和性的TCR，如文献(Li,et al(2005)Nature Biotech 23(3):349-354)中所述。
应理解，表达野生型TCRα和β链可变域氨基酸的基因或者表达略作修饰的野生型TCR的α和β链可变域氨基酸的基因都可用来制备模板TCR。然后在编码该模板TCR的可变域的DNA中引入产生本发明的高亲和力TCR所需的改变。
本发明的高亲和性TCR包含α链可变域氨基酸序列为SEQ ID NO:1、13-39之一；和/或所述TCR的β链可变域氨基酸序列为SEQ ID NO:2、40、41之一。本发明中，形成异质二聚TCR分子的α链可变域与β链可变域的氨基酸序列优选自下表1：
表1
Figure PCTCN2021109705-appb-000003
Figure PCTCN2021109705-appb-000004
基于本发明的目的，本发明TCR是具有至少一个TCRα和/或TCRβ链可变域的部分。它们通常同时包含TCRα链可变域和TCRβ链可变域。它们可以是αβ异源二聚体或是单链形式或是其他任何能够稳定存在的形式。在过继性免疫治疗中，可将αβ异源二聚TCR的全长链(包含胞质和跨膜结构域)进行转染。本发明TCR可用作将治疗剂递送至抗原呈递细胞的靶向剂或与其他分子结合制备双功能多肽来定向效应细胞，此时TCR优选为可溶形式。
对于稳定性而言，现有技术中公开了在TCR的α与β链恒定域之间引入人工链间二硫键能够获得可溶且稳定的TCR分子，如专利文献PCT/CN2015/093806中所述。因此，本发明TCR可以是在其α和β链恒定域的残基之间引入人工链间二硫键的TCR。半胱氨酸残基在所述TCR的α和β链恒定域间形成人工链间二硫键。半胱氨酸残基可以取代在天然TCR中合适位点的其他氨基酸残基以形成人工链间二硫键。例如，取代TRAC*01外显子1的Thr48和取代TRBC1*01或TRBC2*01外显子1的Ser57来形成二硫键。引入半胱氨酸残基以形成二硫键的其他位点还可以是：TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；或TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。即半胱氨酸残基取代了上述α与β链恒定域中任一组位点。可在本发明TCR恒定域的一个或多个C末端截短最多15个、或最多10个、或最多8个或更少的氨基酸，以使其不包括半胱氨酸残基来达到缺失天然链间二硫键的目的，也可通过将形成天然链间二硫键的半胱氨酸残基突变为另一氨基酸来达到上述目的。
如上所述，本发明的TCR可以包含在其α和β链恒定域的残基间引入的人工链间二硫键。应注意，恒定域间含或不含上文所述的引入的人工二硫键，本发明的TCR均可含有TRAC恒定域序列和TRBC1或TRBC2恒定域序列。TCR的TRAC恒定域序列和TRBC1或TRBC2恒定域序列可通过存在于TCR中的天然链间二硫键连接。
另外，对于稳定性而言，专利文献PCT/CN2016/077680还公开了在TCR的α链可变区与β链恒定区之间引入人工链间二硫键能够使TCR的稳定性显著提高。因此，本发明的高亲和力TCR的α链可变区与β链恒定区之间还可以含有人工链间二硫键。具体地，在所述TCR的α链可变区与β链恒定区之间形成人工链间二硫键的半胱氨酸残基取代了：TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸；TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的61位氨基酸；TRAV的第46位氨基酸和 TRBC1*01或TRBC2*01外显子1的第61位氨基酸；或TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸。优选地，这样的TCR可以包含(i)除其跨膜结构域以外的全部或部分TCRα链，和(ii)除其跨膜结构域以外的全部或部分TCRβ链，其中(i)和(ii)均包含TCR链的可变域和至少一部分恒定域，α链与β链形成异质二聚体。更优选地，这样的TCR可以包含α链可变域和β链可变域以及除跨膜结构域以外的全部或部分β链恒定域，但其不包含α链恒定域，所述TCR的α链可变域与β链形成异质二聚体。
对于稳定性而言，另一方面，本发明TCR还包括在其疏水芯区域发生突变的TCR，这些疏水芯区域的突变优选为能够使本发明TCR的稳定性提高的突变，如在公开号为WO2014/206304的专利文献中所述。这样的TCR可在其下列可变域疏水芯位置发生突变：(α和/或β链)可变区氨基酸第11，13，19，21，53，76，89，91，94位，和/或α链J基因(TRAJ)短肽氨基酸位置倒数第3,5,7位,和/或β链J基因(TRBJ)短肽氨基酸位置倒数第2,4,6位，其中氨基酸序列的位置编号按国际免疫遗传学信息系统(IMGT)中列出的位置编号。本领域技术人员知晓上述国际免疫遗传学信息系统，并可根据该数据库得到不同TCR的氨基酸残基在IMGT中的位置编号。
更具体地，本发明中疏水芯区域发生突变的TCR可以是由一柔性肽链连接TCR的α链与β链的可变域而构成的高稳定性单链TCR。TCR可变区的CDR区决定了其与短肽-HLA复合物之间的亲和力，疏水芯的突变能够使TCR更加稳定，但并不会影响其与短肽-HLA复合物之间的亲和力。应注意，本发明中柔性肽链可以是任何适合连接TCRα与β链可变域的肽链。本发明实施例1中构建的用于筛选高亲和性TCR的模板链即为上述含有疏水芯突变的高稳定性单链TCR。采用稳定性较高的TCR，能够更方便的评估TCR与KWVESIFLIF-HLA-A2402复合物之间的亲和力。
该单链模板TCR的α链可变域及β链可变域的CDR区与野生型TCR的CDR区完全相同。即α链可变域的3个CDR分别为CDR1α：DSAIYN；CDR2α：IQSSQRE；CDR3α：AVRRRNYGQNFV和β链可变域的3个CDR分别为CDR1β：SGHDT；CDR2β：YYEEEE；CDR3β：ASSLIAQNHNEQF。该单链模板TCR的氨基酸序列(SEQ ID NO:9)及核苷酸序列(SEQ ID NO:10)分别如图5a和5b所示。以此筛选出对KWVESIFLIF-HLA A2402复合物具有高亲和性的由α链可变域和β链可变域构成的单链TCR。
本发明的对KWVESIFLIF-HLA-A2402复合物具有高亲和性的αβ异质二聚体的获得是通过将筛选出的高亲和性单链TCR的α与β链可变域的CDR区转移到野生型TCRα链可变域(SEQ ID NO:1)与β链可变域(SEQ ID NO:2)的相应位置而得到。
本发明的TCR也可以多价复合体的形式提供。本发明的多价TCR复合体包含两个、三个、四个或更多个本发明TCR相结合而形成的多聚物，如可以用p53的四聚结构域来产生四聚体，或多个本发明TCR与另一分子结合而形成的复合物。本发明的TCR复合物可用于体外或体内追踪或靶向呈递特定抗原的细胞，也可用于产生具有此类应用的其他多价TCR复合物的中间体。
本发明的TCR可以单独使用，也可与偶联物以共价或其他方式结合，优选以共价方式结合。所述偶联物包括可检测标记物(为诊断目的，其中所述TCR用于检测呈递KWVESIFLIF-HLA-A2402复合物的细胞的存在)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明TCR结合或偶联的治疗剂包括但不限于：1.放射性核素(Koppe等，2005，癌转移评论(Cancer metastasis reviews)24，539)；2.生物毒(Chaudhary等，1989，自然(Nature)339，394；Epel等，2002，癌症免疫学和免疫治疗(Cancer Immunology and Immunotherapy)51，565)；3.细胞因子如IL-2等(Gillies等，1992，美国国家科学院院刊(PNAS)89，1428；Card等，2004，癌症免疫学和免疫治疗(Cancer Immunology and Immunotherapy)53，345；Halin等，2003，癌症研究(Cancer Research)63，3202)；4.抗体Fc片段(Mosquera等，2005，免疫学杂志(The Journal Of Immunology)174，4381)；5.抗体scFv片段(Zhu等，1995，癌症国际期刊(International Journal of Cancer)62,319)；6.金纳米颗粒/纳米棒(Lapotko等，2005，癌症通信(Cancer letters)239，36；Huang等，2006，美国化学学会杂志(Journal of the American Chemical Society)128，2115)；7.病毒颗粒(Peng等，2004，基因治疗(Gene therapy)11，1234)；8.脂质体(Mamot等，2005，癌症研究(Cancer research)65，11631)；9.纳米磁粒；10.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；11.化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
与本发明TCR结合的抗体或其片段包括抗-T细胞或NK-细胞决定抗体，如抗-CD3或抗-CD28或抗-CD16抗体，上述抗体或其片段与TCR的结合能够对效应细胞进行定向来更好地靶向靶细胞。一个优选的实施方式是本发明TCR与抗-CD3抗体或所述抗-CD3抗体的功能片段或变体结合。具体地，本发明的TCR与抗CD3单链抗体的融合分子包括选自TCRα链可变域氨基酸序列为SEQ ID NO:1、13-39之一；和/或所述TCR的β链可变域氨基酸序列为SEQ ID NO:2、40、41之一。
本发明还涉及编码本发明TCR的核酸分子。本发明的核酸分子可以是DNA形式或RNA形式。DNA可以是编码链或非编码链。例如，编码本发明TCR的核酸序列可以与本发明附图中所示的核酸序列相同或是简并的变异体。举例说明“简并的变异体”的含义，如本文所用，“简并的变异体”在本发明中是指编码具有SEQ ID NO:3的蛋白序列，但与SEQ ID NO:5的序列有差别的核酸序列。
本发明的核酸分子全长序列或其片段通常可以用但不限于PCR扩增法、重组法或人工合成的方法获得。目前，已经可以完全通过化学合成来得到编码本发明TCR(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本发明也涉及包含本发明的核酸分子的载体，以及用本发明的载体或编码序列经基因工 程产生的宿主细胞。
本发明还包括表达本发明TCR的分离细胞，特别是T细胞。有许多方法适合于用编码本发明的高亲和力TCR的DNA或RNA进行T细胞转染(如，Robbins等.，(2008)J.Immunol.180:6116-6131)。表达本发明高亲和性TCR的T细胞可以用于过继免疫治疗。本领域技术人员能够知晓进行过继性治疗的许多合适方法(如，Rosenberg等.，(2008)Nat Rev Cancer 8(4):299-308)。
本发明还提供一种药物组合物，所述药物组合物含有药学上可接受的载体以及本发明TCR、或本发明TCR复合物、或呈递本发明TCR的细胞。
本发明还提供了一种治疗疾病的方法，包括给需要治疗的对象施用适量的本发明TCR、或本发明TCR复合物、或呈递本发明TCR的细胞、或本发明的药物组合物。
在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的结构和功能。因此，本发明TCR还包括本发明TCR的至多5个，较佳地至多3个，更佳地至多2个，最佳地1个氨基酸(尤其是位于CDR区之外的氨基酸)，被性质相似或相近的氨基酸所替换，并仍能够保持其功能性的TCR。
本发明还包括对本发明TCR略作修饰后的TCR。修饰(通常不改变一级结构)形式包括：本发明TCR的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在本发明TCR的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的TCR。这种修饰可以通过将TCR暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的TCR。
本发明的TCR、TCR复合物或本发明TCR转染的T细胞可与药学上可接受的载体一起在药物组合物中提供。本发明的TCR、多价TCR复合物或细胞通常作为无菌药物组合物的一部分提供，所述组合物通常包括药学上可接受的载体。该药物组合物可以是任何合适的形式(取决于给予患者的所需方法)。其可采用单位剂型提供，通常在密封的容器中提供，可作为试剂盒的一部分提供。此类试剂盒(但非必需)包括使用说明书。其可包括多个所述单位剂型。
此外，本发明的TCR可以单用，也可与其他治疗剂结合或偶联在一起使用(如配制在同一药物组合物中)。
药物组合物还可含有药学上可接受的载体。术语“药学上可接受的载体”指用于治疗剂给药的载体。该术语指这样一些药剂载体：它们本身不诱导产生对接受该组合物的个体有害的抗体，且给药后没有过分的毒性。这些载体是本领域普通技术人员所熟知的。在雷明顿药物科学(Remington”s Pharmaceutical Sciences(Mack Pub.Co.，N.J.1991))中可找到关于药学上可接受的赋形剂的充分讨论。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、佐剂、及其组合。
治疗性组合物中药学上可接受的载体可含有液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如润湿剂或乳化剂、pH缓冲物质等。
通常，可将治疗性组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液中、液体载体的固体形式。
一旦配成本发明的组合物，可将其通过常规途径进行给药，其中包括(但并不限于)：眼内、肌内、静脉内、皮下、皮内、或局部给药，优选为胃肠外包括皮下、肌肉内或静脉内。待预防或治疗的对象可以是动物；尤其是人。
当本发明的药物组合物被用于实际治疗时，可根据使用情况而采用各种不同剂型的药物组合物。较佳地，可以例举的有针剂、口服剂等。
这些药物组合物可根据常规方法通过混合、稀释或溶解而进行配制，并且偶尔添加合适的药物添加剂，如赋形剂、崩解剂、粘合剂、润滑剂、稀释剂、缓冲剂、等渗剂(isotonicities)、防腐剂、润湿剂、乳化剂、分散剂、稳定剂和助溶剂，而且该配制过程可根据剂型用惯常方式进行。
本发明的药物组合物还可以缓释剂形式给药。例如，本发明TCR可被掺入以缓释聚合物为载体的药丸或微囊中，然后将该药丸或微囊通过手术植入待治疗的组织。作为缓释聚合物的例子，可例举的有乙烯-乙烯基乙酸酯共聚物、聚羟基甲基丙烯酸酯(polyhydrometaacrylate)、聚丙烯酰胺、聚乙烯吡咯烷酮、甲基纤维素、乳酸聚合物、乳酸-乙醇酸共聚物等，较佳地可例举的是可生物降解的聚合物如乳酸聚合物和乳酸-乙醇酸共聚物。
当本发明的药物组合物被用于实际治疗时，作为活性成分的本发明TCR或TCR复合物或呈递本发明TCR的细胞，可根据待治疗的每个病人的体重、年龄、性别、症状程度而合理地加以确定，最终由医师决定合理的用量。
本发明的主要优点在于：
(1)本发明的高亲和力TCR对所述KWVESIFLIF-HLA-A2402复合物的亲和力和/或结合半衰期是野生型TCR的至少2倍。
(2)本发明的高亲和力TCR能够与所述KWVESIFLIF-HLA A2402特异性结合，同时转染了本发明高亲和力TCR的细胞能够被特异性激活。
(3)转染本发明的高亲和力TCR的效应细胞具有强的特异性杀伤作用。
下面的具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如(Sambrook和Russell等人，分子克隆：实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)(2001)CSHL出版社)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
材料和方法
本发明实施例中所用的实验材料如无特殊说明均可从市售渠道获得，其中，E.coli DH5α购自Tiangen、E.coli BL21(DE3)购自Tiangen、E.coli Tuner(DE3)购自Novagen、质粒pET28a购自Novagen。
实施例1 疏水芯突变的稳定性单链TCR模板链的产生
本发明利用定点突变的方法，根据专利文献WO2014/206304中所述，构建了以一个柔性短肽(linker)连接TCRα与β链可变域而构成的稳定性单链TCR分子，其氨基酸及DNA序列分别为SEQ ID NO:9和SEQ ID NO:10，如图5a和图5b所示。并以该单链TCR分子为模板进行高亲和性TCR分子的筛选。该模板链的α链可变域(SEQ ID NO:3)及β链可变域(SEQ ID NO:4)的氨基酸序列如图2a和2b所示；其对应的DNA序列分别为SEQ ID NO:5和SEQ ID NO:6，如图3a和3b所示；柔性短肽(linker)的氨基酸序列及DNA序列分别为SEQ ID NO:7和8，如图4a和4b所示。
将携带模板链的目的基因经Nco Ⅰ和Not Ⅰ双酶切，与经过Nco Ⅰ和Not Ⅰ双酶切的pET28a载体连接。连接产物转化至E.coli DH5α，涂布含卡那霉素的LB平板，37℃倒置培养过夜，挑取阳性克隆进行PCR筛选，对阳性重组子进行测序，确定序列正确后抽提重组质粒转化至E.coli BL21(DE3),用于表达。
实施例2 实施例1中构建的稳定性单链TCR的表达、复性和纯化
将实施例1中制备的含有重组质粒pET28a-模板链的BL21(DE 3)菌落全部接种于含有卡那霉素的LB培养基中,37℃培养至OD600为0.6-0.8，加入IPTG至终浓度为0.5mM，37℃继续培养4h。5000rpm离心15min收获细胞沉淀物，用Bugbuster Master Mix(Merck)裂解细胞沉淀物，6000rpm离心15min回收包涵体，再用Bugbuster(Merck)进行洗涤以除去细胞碎片和膜组分，6000rpm离心15min,收集包涵体。将包涵体溶解在缓冲液(20mM Tris-HCl pH 8.0,8M尿素)中，高速离心去除不溶物，上清液用BCA法定量后进行分装，于-80℃保存备用。
向5mg溶解的单链TCR包涵体蛋白中，加入2.5mL缓冲液(6M Gua-HCl，50mM Tris-HCl pH 8.1，100mM NaCl，10mM EDTA)，再加入DTT至终浓度为10mM,37℃处理30min。用注射器向125mL复性缓冲液(100mM Tris-HCl pH 8.1,0.4M L-精氨酸,5M尿素,2mM EDTA,6.5mM β-mercapthoethylamine,1.87mM Cystamine)中滴加上述处理后的单链TCR，4℃搅拌10min，然后将复性液装入截留量为4kDa的纤维素膜透析袋,透析袋置于1L预冷的水中，4℃缓慢搅拌过夜。17小时后，将透析液换成1L预冷的缓冲液(20mM Tris-HCl pH 8.0)，4℃继续透析8h，然后将透析液换成相同的新鲜缓冲液继续透析过夜。17小时后，样品经0.45μm滤膜过滤，真空脱气后通过阴离子交换柱(HiTrap Q HP,GE Healthcare)，用20mM Tris-HCl pH 8.0配制的0-1M NaCl线性梯度洗脱液纯化蛋白，收集的洗脱组分进行 SDS-PAGE分析，包含单链TCR的组分浓缩后进一步用凝胶过滤柱(Superdex 75 10/300,GE Healthcare)进行纯化，目标组分也进行SDS-PAGE分析。
用于BIAcore分析的洗脱组分进一步采用凝胶过滤法测试其纯度。条件为：色谱柱Agilent Bio SEC-3(300A，
Figure PCTCN2021109705-appb-000005
)，流动相为150mM磷酸盐缓冲液，流速0.5mL/min，柱温25℃，紫外检测波长214nm。
实施例3 结合表征
BIAcore分析
使用BIAcore T200实时分析系统检测TCR分子与KWVESIFLIF-HLA-A2402复合物的结合活性。将抗链霉亲和素的抗体(GenScript)加入偶联缓冲液(10mM醋酸钠缓冲液，pH4.77)，然后将抗体流过预先用EDC和NHS活化过的CM5芯片，使抗体固定在芯片表面，最后用乙醇胺的盐酸溶液封闭未反应的活化表面，完成偶联过程，偶联水平约为15，000RU。条件为：温度25℃，PH值为7.1-7.5。
使低浓度的链霉亲和素流过已包被抗体的芯片表面，然后将KWVESIFLIF-HLA-A2402复合物流过检测通道，另一通道作为参比通道，再将0.05mM的生物素以10μL/min的流速流过芯片2min，封闭链霉亲和素剩余的结合位点。采用单循环动力学分析方法测定其亲和力，将TCR用HEPES-EP缓冲液(10mM HEPES,150mM NaCl，3mM EDTA,0.005％P20，pH 7.4)稀释成几个不同的浓度，以30μL/min的流速，依次流过芯片表面，每次进样的结合时间为120s，最后一次进样结束后让其解离600s。每一轮测定结束后用pH 1.75的10mM Gly-HCl再生芯片。利用BIAcore Evaluation软件计算动力学参数。
上述KWVESIFLIF-HLA-A2402复合物的制备过程如下：
a.纯化：收集100ml诱导表达重链或轻链的E.coli菌液，于4℃8000g离心10min后用10ml PBS洗涤菌体一次，之后用5ml BugBuster Master Mix Extraction Reagents(Merck)剧烈震荡重悬菌体，并于室温旋转孵育20min，之后于4℃，6000g离心15min，弃去上清，收集包涵体。
将上述包涵体重悬于5ml BugBuster Master Mix中，室温旋转孵育5min；加30ml稀释10倍的BugBuster，混匀，4℃6000g离心15min；弃去上清，加30ml稀释10倍的BugBuster重悬包涵体，混匀，4℃6000g离心15min，重复两次，加30ml 20mM Tris-HCl pH 8.0重悬包涵体，混匀，4℃6000g离心15min，最后用20mM Tris-HCl 8M尿素溶解包涵体，SDS-PAGE检测包涵体纯度，BCA试剂盒测浓度。
b.复性：将合成的短肽KWVESIFLIF(北京赛百盛基因技术有限公司)溶解于DMSO至20mg/ml的浓度。轻链和重链的包涵体用8M尿素、20mM Tris pH 8.0、10mM DTT来溶解，复性前加入3M盐酸胍、10mM醋酸钠、10mM EDTA进一步变性。将KWVESIFLIF肽以25mg/L(终浓度)加入复性缓冲液(0.4M L-精氨酸、100mM Tris pH 8.3、2mM EDTA、0.5mM氧化性谷胱甘肽、5mM还原型谷胱甘肽、0.2mM PMSF，冷却至4℃)，然后依次 加入20mg/L的轻链和90mg/L的重链(终浓度，重链分三次加入，8h/次)，复性在4℃进行至少3天至完成，SDS-PAGE检测能否复性成功。
c.复性后纯化：用10体积的20mM Tris pH 8.0作透析来更换复性缓冲液，至少更换缓冲液两次来充分降低溶液的离子强度。透析后用0.45μm醋酸纤维素滤膜过滤蛋白质溶液，然后加载到HiTrap Q HP(GE通用电气公司)阴离子交换柱上(5ml床体积)。利用Akta纯化仪(GE通用电气公司)，20mM Tris pH 8.0配制的0-400mM NaCl线性梯度液洗脱蛋白，pMHC约在250mM NaCl处洗脱，收集诸峰组分，SDS-PAGE检测纯度。
d.生物素化：用Millipore超滤管将纯化的pMHC分子浓缩，同时将缓冲液置换为20mM Tris pH 8.0，然后加入生物素化试剂0.05M Bicine pH 8.3、10mM ATP、10mM MgOAc、50μM D-Biotin、100μg/ml BirA酶(GST-BirA)，室温孵育混合物过夜，SDS-PAGE检测生物素化是否完全。
e.纯化生物素化后的复合物：用Millipore超滤管将生物素化标记后的pMHC分子浓缩至1ml，采用凝胶过滤层析纯化生物素化的pMHC，利用Akta纯化仪(GE通用电气公司)，用过滤过的PBS预平衡HiPrepTM 16/60S200HR柱(GE通用电气公司)，加载1ml浓缩过的生物素化pMHC分子，然后用PBS以1ml/min流速洗脱。生物素化的pMHC分子在约55ml时作为单峰洗脱出现。合并含有蛋白质的组分，用Millipore超滤管浓缩，BCA法(Thermo)测定蛋白质浓度，加入蛋白酶抑制剂cocktail(Roche)将生物素化的pMHC分子分装保存在-80℃。
实施例4 高亲和力TCR的产生
噬菌体展示技术是产生TCR高亲和力变体文库以筛选高亲和力变体的一种手段。将Li等((2005)Nature Biotech 23(3):349-354)描述的TCR噬菌体展示和筛选方法应用于实施例1中的单链TCR模板。通过突变该模板链的CDR区来建立高亲和性TCR的文库并进行淘选。经过几轮淘选后的噬菌体文库均和相应抗原有特异性结合，从中挑取单克隆，并进行分析。
将筛选到的高亲和力的单链TCR的CDR区突变引入到αβ异质二聚TCR的可变域的相应位点中，并通过BIAcore来检测其与KWVESIFLIF-HLA-A2402复合物的亲和力。上述CDR区高亲和力突变点的引入采用本领域技术人员熟知的定点突变的方法。上述野生型TCR的α链与β链可变域氨基酸序列分别如图1a(SEQ ID NO:1)和1b(SEQ ID NO:2)所示。
应注意，为获得更加稳定的可溶性TCR，以便更方便地评估TCR与KWVESIFLIF-HLA A2402复合物之间的结合亲和力和/或结合半衰期，αβ异质二聚TCR可以是在α和β链的恒定区中分别引入了一个半胱氨酸残基以形成人工链间二硫键的TCR，本实施例中引入半胱氨酸残基后TCRα与β链的氨基酸序列分别如图6a(SEQ ID NO:11)和6b(SEQ ID NO:12)所示，引入的半胱氨酸残基以加粗字母表示。
通过《分子克隆实验室手册》(Molecular Cloning a Laboratory Manual)(第三版，Sambrook和Russell)中描述的标准方法将待表达的TCRα和β链的胞外序列基因经合成后分别插入到 表达载体pET28a+(Novagene)，上下游的克隆位点分别是NcoI和NotI。CDR区的突变通过本领域技术人员熟知的重叠PCR(overlap PCR)引入。插入片段经过测序确认无误。
实施例5 高亲和力TCR的表达、复性和纯化
将TCRα和β链的表达载体分别通过化学转化法转化进入表达细菌BL21(DE3),细菌用LB培养液生长，于OD600＝0.6时用终浓度0.5mM IPTG诱导，TCR的α和β链表达后形成的包涵体通过BugBuster Mix(Novagene)进行提取，并且经BugBuster溶液反复多次洗涤，包涵体最后溶解于6M盐酸胍,10mM二硫苏糖醇(DTT),10mM乙二胺四乙酸(EDTA),20mM Tris(pH 8.1)中。
溶解后的TCRα和β链以1:1的质量比快速混合于5M尿素,0.4M精氨酸,20mM Tris(pH 8.1),3.7mM cystamine,6.6mM β-mercapoethylamine(4℃)中，终浓度为60mg/mL。混合后将溶液置于10倍体积的去离子水中透析(4℃)，12小时后将去离子水换成缓冲液(20mM Tris,pH 8.0)继续于4℃透析12小时。透析完成后的溶液经0.45μM的滤膜过滤后，通过阴离子交换柱(HiTrap Q HP,5ml,GE Healthcare)纯化。洗脱峰含有复性成功的α和β二聚体的TCR通过SDS-PAGE胶确认。TCR随后通过凝胶过滤层析(HiPrep 16/60，Sephacryl S-100HR，GE Healthcare)进一步纯化。纯化后的TCR纯度经过SDS-PAGE测定大于90％，浓度由BCA法确定。
实施例6 BIAcore分析结果
采用实施例3中所述方法检测引入高亲和力CDR区的αβ异质二聚TCR与KWVESIFLIF-HLA-A2402复合物的亲和力。
本发明得到新的TCRα链和β链可变域氨基酸序列，分别如图7(1)-(27)和图8(1)-(2)所示。所示。由于TCR分子的CDR区决定了其与相应的pMHC复合物的亲和力，所以本领域技术人员能够预料引入高亲和力突变点的αβ异质二聚TCR也具有对KWVESIFLIF-HLA-A2402复合物的高亲和力。利用实施例4中所述方法构建表达载体，利用实施例5中所述方法对上述引入高亲和力突变的αβ异质二聚TCR进行表达、复性和纯化，然后利用BIAcore T200测定其与KWVESIFLIF-HLA-A2402复合物的亲和力，如下表2所示。
表2
Figure PCTCN2021109705-appb-000006
Figure PCTCN2021109705-appb-000007
由上表2可知，所述异质二聚TCR的亲和力是野生型TCR对KWVESIFLIF-HLA-A2402复合物的亲和力的至少2倍。
实施例7 抗-CD3抗体与高亲和性αβ异质二聚TCR的融合体的表达、复性和纯化
将抗-CD3的单链抗体(scFv)与αβ异质二聚TCR融合，制备融合分子。抗-CD3的scFv与TCR的β链融合，该TCRβ链可以包含任一上述高亲和性αβ异质二聚TCR的β链可变域，融合分子的TCRα链可以包含任一上述高亲和性αβ异质二聚TCR的α链可变域。
融合分子表达载体的构建
1.α链表达载体的构建：将携带αβ异质二聚TCR的α链的目的基因经Nco Ⅰ和Not Ⅰ双酶切，与经过Nco Ⅰ和Not Ⅰ双酶切的pET28a载体连接。连接产物转化至E.coli DH5α，涂布于含卡那霉素的LB平板，37℃倒置培养过夜，挑取阳性克隆进行PCR筛选，对阳性重组子进行测序，确定序列正确后抽提重组质粒转化至E.coli Tuner(DE3),用于表达。
2.抗-CD3(scFv)-β链表达载体的构建：通过重叠(overlap)PCR的方法，设计引物将抗-CD3 scFv和高亲和性异质二聚TCRβ链基因连接起来，中间的连接短肽(linker)为GGGGS(SEQ ID NO:31)，并且使抗-CD3的scFv与高亲和性异质二聚TCRβ链的融合蛋白的基因片段带上限制性内切酶位点Nco Ⅰ(CCATGG(SEQ ID NO:32))和Not Ⅰ(GCGGCCGC(SEQ ID NO:33))。将PCR扩增产物经Nco Ⅰ和Not Ⅰ双酶切，与经过Nco Ⅰ和Not Ⅰ双酶切的pET28a载体连接。连接产物转化至E.coli DH5α感受态细胞，涂布含卡那霉素的LB平板，37℃倒置培养过夜，挑取阳性克隆进行PCR筛选，对阳性重组子进行测序，确定序列正确后抽提重组质粒转化至E.coli Tuner(DE3)感受态细胞，用于表达。
融合蛋白的表达、复性及纯化
将表达质粒分别转化进入E.coli Tuner(DE3)感受态细胞，涂布LB平板(卡那霉素50 μg/mL)置于37℃培养过夜。次日，挑克隆接种至10mL LB液体培养基(卡那霉素50μg/mL)培养2-3h，按体积比1:100接种至1L LB培养基中，继续培养至OD600为0.5-0.8，加入终浓度为1mM IPTG诱导目的蛋白的表达。诱导4小时以后，以6000rpm离心10min收获细胞。PBS缓冲液洗涤菌体一次，并且分装菌体，取相当于200mL的细菌培养物的菌体用5mL BugBuster Master Mix(Merck)裂解细菌，以6000g离心15min收集包涵体。然后进行4次洗涤剂洗涤以去除细胞碎片和膜组分。然后，用缓冲液如PBS洗涤包涵体以除去洗涤剂和盐。最终，将包涵体用含6M盐酸胍,10mM二硫苏糖醇(DTT),10mM乙二胺四乙酸(EDTA),20mM Tris,pH 8.1缓冲溶液溶解，并测定包涵体浓度，将其分装后置于-80℃冷冻保存。
溶解后的TCRα链和抗-CD3(scFv)-β链以2：5的质量比快速混合于5M尿素(urea),0.4M L-精氨酸(L-arginine),20mM Tris pH 8.1,3.7mM cystamine,6.6mM β-mercapoethylamine(4℃)，终浓度α链和抗-CD3(scFv)-β链分别为0.1mg/mL，0.25mg/mL。
混合后将溶液置于10倍体积的去离子水中透析(4℃)，12小时后将去离子水换成缓冲液(10mM Tris,pH 8.0)继续于4℃透析12小时。透析完成后的溶液经0.45μM的滤膜过滤后，通过阴离子交换柱(HiTrap Q HP 5ml,GE healthcare)纯化。洗脱峰含有复性成功的TCRα链与抗-CD3(scFv)-β链二聚体的TCR通过SDS-PAGE胶确认。TCR融合分子随后通过尺寸排阻色谱法(S-100 16/60，GE healthcare)进一步纯化，以及阴离子交换柱(HiTrap Q HP 5ml,GE healthcare)再次纯化。纯化后的TCR融合分子纯度经过SDS-PAGE测定大于90％，浓度由BCA法测定。
实施例8 针对负载短肽的靶细胞，转染本发明高亲和力TCR的效应细胞的激活功能实验
IFN-γ是活化T淋巴细胞产生的一种强有力的免疫调节因子，因此本实施例通过本领域技术人员熟知的ELISPOT实验检测IFN-γ数以验证转染本发明高亲和力TCR的细胞的激活功能及抗原特异性。将本发明高亲和力TCR转染至从健康志愿者的血液中分离到的CD3+T细胞作为效应细胞，并以同一志愿者转染其他TCR(A6)的CD3+T细胞作为对照。所用的靶细胞为负载AFP抗原短肽KWVESIFLIF的、负载了无关肽的或空载的T2-A24(指转染了HLA-A2402的T2细胞，下同)。
以下分两个批次(I)、(II)、(III)先后进行实验：
(I)所述高亲和力TCR可从表2获悉，分别为
Figure PCTCN2021109705-appb-000008
首先准备ELISPOT平板。ELISPOT平板乙醇活化包被，4℃过夜。实验第1天，去掉包被液，洗涤封闭，室温下孵育两个小时，去除封闭液，将试验的各个组分加入ELISPOT平 板：靶细胞为1*10 4个/孔，效应细胞为2*10 3个/孔(按转染阳性率计算)，并设置二个复孔。加入对应短肽，使短肽在ELISPOT孔板中的终浓度为1×10 -6M。温育过夜(37℃，5％CO2)。实验第2天，洗涤平板并进行二级检测和显色，干燥平板，再利用免疫斑点平板读数计(ELISPOT READER system；AID20公司)计数膜上形成的斑点。
(II)所述高亲和力TCR可从表2获悉，分别为
Figure PCTCN2021109705-appb-000009
实验步骤如本实施例(I)所述。
(III)所述高亲和力TCR可从表2获悉，分别为
  TCR1 TCR3 TCR6
α链可变域SEQ ID 13 15 18
β链可变域SEQ ID 2 2 2
首先准备ELISPOT平板，先按靶细胞2×10 4个细胞/孔、效应细胞2×10 3个细胞/孔(按转染阳性率计算)加入对应孔中，然后在实验组加入AFP抗原短肽KWVESIFLIF，且使其短肽在ELISPOT孔板中的终浓度依次为1×10 -13M到1×10 -6M，共8个梯度；在对照组加入无关肽，且使其短肽终浓度为1×10 -6M。其余步骤如本实施例(I)所述。
实验结果如图12a-12c所示，针对负载了AFP抗原短肽KWVESIFLIF的靶细胞，转染本发明高亲和力TCR的效应细胞有明显的激活效应，且在特定抗原肽浓度较低时即起反应，而转染其他TCR的效应细胞无活性；同时，转染本发明TCR的效应细胞对于负载无关肽或空载的靶细胞基本无活性。
实施例9 针对肿瘤细胞系，转染本发明高亲和力TCR的效应细胞的激活功能实验
本实施例利用肿瘤细胞系再次验证转染本发明高亲和力TCR的效应细胞的激活功能及特异性。同样是通过本领域技术人员熟知的ELISPOT实验进行检测。将本发明高亲和力TCR转染至从健康志愿者的血液中分离到的CD3+T细胞作为效应细胞，并以同一志愿者转染其他TCR(A6)的或转染野生型TCR(WT-TCR)的CD3+T细胞作为阴性对照。以下分两个批次(I)、(II)、(III)先后进行实验：
(I)所述高亲和力TCR可从表2获悉，分别为
Figure PCTCN2021109705-appb-000010
该批次使用的AFP阳性肿瘤细胞系为HepG2、SK-HEP-1-AFP(AFP过表达)，阴性肿瘤细胞系为SK-HEP-1、NCI-H226、NCI-H1299、HUH-1。
(II)所述高亲和力TCR可从表2获悉，分别为
Figure PCTCN2021109705-appb-000011
该批次使用的AFP阳性肿瘤细胞系为HepG2、SK-HEP-1-AFP(AFP过表达)，阴性肿瘤细胞系为SK-HEP-1、NCI-H226、NCI-H1299、HUH-1、MKN7。
(III)所述高亲和力TCR可从表2获悉，分别为
Figure PCTCN2021109705-appb-000012
该批次使用的AFP阳性肿瘤细胞系为HepG2、SK-HEP-1-AFP(AFP过表达)，阴性肿瘤细胞系为SK-HEP-1、NCI-H226、SW480、HUH-1。
以上三个批次皆进行以下步骤：首先准备ELISPOT平板。ELISPOT平板乙醇活化包被，4℃过夜。实验第1天，去掉包被液，洗涤封闭，室温下孵育两个小时，去除封闭液，将试验的各个组分加入ELISPOT平板：靶细胞为2*10 4个/孔，效应细胞为2*10 3个/孔(按转染的阳性率计算)，并设置二个复孔。温育过夜(37℃，5％CO2)。实验第2天，洗涤平板并进行二级检测和显色，干燥平板，再利用免疫斑点平板读数计(ELISPOT READER system；AID20公司)计数膜上形成的斑点。
实验结果如图13a-13c所示，针对AFP阳性肿瘤细胞系，转染本发明高亲和力TCR的效应细胞相比于转染野生型的效应细胞起更明显的激活效应，而转染其他TCR的效应细胞无活性；同时，转染本发明高亲和力TCR的效应细胞对AFP阴性肿瘤细胞系基本无活性。
实施例10 针对梯度负载短肽的T2细胞，转染本发明高亲和力TCR的效应细胞的杀伤功能实验
乳酸脱氢酶(LDH)在胞浆内含量丰富，正常时不能通过细胞膜，当细胞受损伤或死亡时可释放到细胞外，此时细胞培养液中LDH活性与细胞死亡数目成正比。本实施例通过本领域技术人员熟知的非放射性细胞毒性实验，测定LDH的释放，从而验证转染本发明TCR的细胞的杀伤功能。该试验是51Cr释放细胞毒性试验的比色替代试验，定量测定细胞裂解后释放的LDH。采用30分钟偶联的酶反应来检测释放在培养基中的LDH，在酶反应中LDH可使一种四唑盐(INT)转化为红色的甲臜(formazan)。生成的红色产物的量与裂解的细胞数成正比。可以用标准的96孔读板计收集490nm可见光吸光值数据。计算公式:％细胞毒性＝100％×(实验-效应细胞自发-靶细胞自发)/(靶细胞最大-靶细胞自发)。
本实施例将转染本发明高亲和力TCR至从健康志愿者的血液中分离到的CD3+T细胞作为效应细胞，并以同一志愿者空转染(NC)的或转染其他TCR(A6)的CD3+T细胞作为阴性对照。其中所述高亲和力TCR以及其编号从表2获悉，分别为TCR1(α链可变域SEQ ID NO:13，β链可变域SEQ ID NO:2)、TCR3(α链可变域SEQ ID NO:15，β链可变域SEQ ID NO:2)和TCR6(α链可变域SEQ ID NO:18，β链可变域SEQ ID NO:2)。靶细胞为负载 KWVESIFLIF肽的、负载无关肽的或空载的T2-A24。
首先准备LDH平板，先按靶细胞3×10 4个细胞/孔、效应细胞3×10 4个细胞/孔(按转染阳性率计算)加入对应孔中，然后在实验组加入AFP抗原短肽KWVESIFLIF，且使其短肽在LDH孔板中的终浓度依次为1×10 -15M到1×10 -8M，共8个梯度；在对照组加入其他短肽，且使其短肽终浓度为依次为1×10 -10M到1×10 -6M，共5个梯度，并各设置三个复孔。同时设置效应细胞自发孔，靶细胞自发孔，靶细胞最大孔，体积校正对照孔及培养基背景对照孔。温育过夜(37℃，5％CO2)。实验第2天，检测显色，终止反应后用酶标仪(Bioteck)在490nm记录吸光值。
实验结果如图14所示，针对梯度负载AFP抗原短肽KWVESIFLIF的靶细胞，转染本发明高亲和力TCR的效应细胞表现出很强的杀伤作用，且在上述特定短肽浓度较低时即起反应，而转染其他TCR的或空转染的T细胞基本无杀伤作用；同时，转染本发明高亲和力TCR的效应细胞对负载无关肽或空载的靶细胞均无杀伤效应。
实施例11 针对肿瘤细胞系，转染本发明高亲和力TCR的效应细胞的杀伤功能实验
本实施例同样通过本领域技术人员熟知的非放射性细胞毒性实验，测定LDH的释放，从而验证转染本发明TCR的细胞的杀伤功能。本实施例LDH实验用从健康志愿者的血液中分离到的CD3+T细胞转染本发明高亲和力TCR作为效应细胞，并以同一志愿者转染其他TCR(A6)的或转染野生型TCR(WT-TCR)的CD3+T细胞作为阴性对照。
以下分三个批次(I)、(II)、(III)先后进行实验：
(I)所述高亲和力TCR以及其编号从表2获悉，分别为
Figure PCTCN2021109705-appb-000013
该批次使用的AFP阳性肿瘤细胞系为HepG2，阴性肿瘤细胞系为NCI-H226、SK-HEP-1。实验步骤如下：首先准备LDH平板，按以下顺序将试验的各个组分加入平板：靶细胞3×10 4个细胞/孔、效应细胞3×10 4个细胞/孔(按转染阳性率计算)加入对应孔中，并设置三个复孔。同时设置效应细胞自发孔，靶细胞自发孔，靶细胞最大孔，体积校正对照孔及培养基背景对照孔。温育过夜(37℃，5％CO2)。实验第2天，检测显色，终止反应后用酶标仪(Bioteck)在490nm记录吸光值。
(II)所述高亲和力TCR以及其编号从表2获悉，分别为TCR21(α链可变域SEQ ID NO:31、β链可变域SEQ ID NO:2)、TCR8(α链可变域SEQ ID NO:20、β链可变域SEQ ID NO:2)和TCR7(α链可变域SEQ ID NO:19、β链可变域SEQ ID NO:2)。该批次使用的AFP阳性肿瘤细胞系为HepG2，阴性肿瘤细胞系为T84、Hs578T和NCI-H526。实验步骤与本实施例(I)相同。
(III)所述高亲和力TCR以及其编号从表2获悉，分别为TCR2(α链可变域SEQ ID NO:14、 β链可变域SEQ ID NO:2)和TCR9(α链可变域SEQ ID NO:21、β链可变域SEQ ID NO:2)。该批次使用的AFP阳性肿瘤细胞系为HepG2、SK-HEP-1-AFP(AFP过表达)，阴性肿瘤细胞系为NCI-H226。加入平板的组分如下：靶细胞2.5×10 4个细胞/孔、效应细胞2.5×10 4个细胞/孔(按转染阳性率计算)，其余实验步骤与本实施例(I)相同。
实验结果如图15a-15c所示，针对AFP阳性肿瘤细胞系，转染本发明高亲和力TCR的效应细胞仍表现出强杀伤效力，且明显比转染野生型TCR的T细胞的杀伤功能更强，而转染其他TCR的T细胞基本不起反应；同时，转染本发明高亲和力TCR的T细胞对阴性肿瘤细胞系基本无杀伤，进一步体现了转染本发明高亲和力TCR的细胞的很好的特异性杀伤功能。
实施例12 针对肿瘤细胞系，转染本发明高亲和力TCR分子的效应细胞的杀伤功能验证(IncuCyte实验)
本实施例通过本领域技术人员熟知的IncuCyte实验进一步验证转染本发明高亲和力TCR的效应细胞对靶细胞的特异性杀伤作用及其灵敏性。IncuCyte是在培养箱中通过实时显微拍摄，能对不同时间点图像进行自动分析，量化实时的细胞凋亡数的功能分析系统。
随机选择本发明TCR转染从健康志愿者的血液中分离到的CD3+T细胞，作为效应细胞，并以同一志愿者转染其他TCR(A6)的或空转染(NC)的CD3+T细胞作为对照组。所述TCR以及其编号从表2获悉，分别为TCR1(α链可变域SEQ ID NO:13，β链可变域SEQ ID NO:2)和TCR3(α链可变域SEQ ID NO:15，β链可变域SEQ ID NO:2)。靶细胞系中，HepG2为阳性肿瘤细胞系；SK-HEP-1为阴性肿瘤细胞系，作为对照。
实验第一天，将靶细胞进行消化处理，离心；用无酚红的RPMI1640+10％FBS的完全培养基重悬，将靶细胞均匀的平铺在96孔板中：2*10 4个/孔；放回37度，5％CO 2的培养箱中，孵育过夜；第二天将96孔板中培养基弃掉，换成含有染料caspase3/7 reagent的无酚红的RPMI1640+10％FBS培养基，使染料浓度为2滴/ml。弃去旧的培养基，更换新的无酚红的RPMI1640+10％FBS的培养基，将效应细胞1*10 4个/孔(按转染的阳性率计算)和已铺有靶细胞的实验组进行共孵育；将板子放至Incucyte检测专用的实时动态活细胞成像分析仪-IncuCyte ZooM中，孵育半小时后；开始实时观察并拍照；采用IncuCyte ZooM 2016A对检测结果进行处理和数据分析、导出。
实验结果如图16a和图16b所示，针对AFP阳性肿瘤细胞系，转染本发明高亲和力TCR的细胞能够在短期内显示强有效的杀伤作用，而转染其他TCR的效应细胞无杀伤效应；同时，转染本发明高亲和力TCR的细胞对阴性肿瘤细胞基本无杀伤。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (24)

  1. 一种T细胞受体(TCR)，所述T细胞受体包含TCRα链可变域和TCRβ链可变域，其特征在于，其具有结合KWVESIFLIF-HLA A2402复合物的活性，并且所述TCRα链可变域的氨基酸序列与SEQ ID NO:1所示的氨基酸序列有至少90％的序列同源性和所述TCRβ链可变域的氨基酸序列与SEQ ID NO:2所示的氨基酸序列有至少90％的序列同源性。
  2. 如权利要求1所述的T细胞受体，其特征在于，所述TCR与KWVESIFLIF-HLA A2402复合物的亲和力是野生型TCR的至少2倍。
  3. 如权利要求1所述的T细胞受体，其特征在于，所述TCR是可溶的。
  4. 如权利要求1所述的T细胞受体，其特征在于，所述TCR为αβ异质二聚TCR；优选地，所述TCR具有α链恒定区序列TRAC*01和β链恒定区序列TRBC1*01或TRBC2*01。
  5. 如权利要求1所述的T细胞受体，其特征在于，所述TCRα链可变域的3个CDR区(互补决定区)的基准序列如下，
    CDR1α：DSAIYN
    CDR2α：IQSSQRE
    CDR3α：AVRRRNYGQNFV，并且CDR3α含有至少一个下列突变：
    突变前的残基 突变后的残基 CDR3α的第1位A G或S CDR3α的第2位V A或G或I或L或S或T CDR3α的第3位R I或T或V CDR3α的第4位R W或Y或N或M CDR3α的第5位R Y CDR3α的第6位N E或H或Q或R或T或I或V或M CDR3α的第7位Y L或P或W或V
    优选地，所述CDR3α中氨基酸突变包含：
    突变前的残基 突变后的残基 CDR3α的第4位R Y
  6. 如权利要求1所述的T细胞受体，其特征在于，所述TCRα链可变域的氨基酸序列与SEQ ID NO:1所示的氨基酸序列有至少95％的序列同源性；和所述TCRβ链可变域的氨基酸序列与SEQ ID NO:2所示的氨基酸序列有至少95％的序列同源性。
  7. 如权利要求1所述的TCR，其特征在于，所述TCRβ链可变域的3个CDR为：
    CDR1β：SGHDT；
    CDR2β：YYEEEE；和
    CDR3β：ASSLIAQNHNEQF。
  8. 如权利要求1所述的T细胞受体，其特征在于，所述TCRβ链可变域的氨基酸序列为SEQ ID NO:2。
  9. 如权利要求1所述的T细胞受体，其特征在于，所述TCRα链可变域的CDR3α选自GGVYRNYGQNFV、AVTYRQYGQNFV和AVIYRNYGQNFV。
  10. 如权利要求1所述的T细胞受体，其特征在于，所述TCR具有选自下组的CDR:
    CDR编号 α-CDR1 α-CDR2 α-CDR3 β-CDR1 β-CDR2 β-CDR3 1 DSAIYN IQSSQRE GGVYRNYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 2 DSAIYN IQSSQRE AVTYRQYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 3 DSAIYN IQSSQRE AVIYRNYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 4 DSAIYN IQSSQRE SAIWRNYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 5 DSAIYN IQSSQRE AVIWRNYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 6 DSAIYN IQSSQRE AVIWRQYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 7 DSAIYN IQSSQRE AVIYRQYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 8 DSAIYN IQSSQRE GSIYRNYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 9 DSAIYN IQSSQRE AVRRRTLGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 10 DSAIYN IQSSQRE AVRRREVGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 11 DSAIYN IQSSQRE AVRRREPGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 12 DSAIYN IQSSQRE AVRRRQVGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 13 DSAIYN IQSSQRE AVRRRNYGQNFV SGHDT YYEEEE ASSLIAQNHALWT 14 DSAIYN IQSSQRE GLIWRNYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 15 DSAIYN IQSSQRE GAIYRNYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 16 DSAIYN IQSSQRE AVRRRNYGQNFV SGHDT YYEEEE ASSLIAQNHAMWT 17 DSAIYN IQSSQRE AVINYRYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 18 DSAIYN IQSSQRE AVRRRHWGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 19 DSAIYN IQSSQRE GTIWRNYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 20 DSAIYN IQSSQRE GIIYRNYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 21 DSAIYN IQSSQRE GAIWRNYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 22 DSAIYN IQSSQRE GVIWRNYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 23 DSAIYN IQSSQRE AIIWRNYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 24 DSAIYN IQSSQRE ASIWRNYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 25 DSAIYN IQSSQRE AVIWRTYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 26 DSAIYN IQSSQRE GAVWRNYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 27 DSAIYN IQSSQRE AVRMRIYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 28 DSAIYN IQSSQRE AVRMRVYGQNFV SGHDT YYEEEE ASSLIAQNHNEQF 29 DSAIYN IQSSQRE AVRRRMWGQNFV SGHDT YYEEEE ASSLIAQNHNEQF
  11. 如权利要求1所述的T细胞受体，其特征在于，所述TCR包含(ⅰ)TCRα链可变域和除跨膜结构域以外的全部或部分TCRα链恒定区；和(ⅱ)TCRβ链可变域和除跨膜结构域以外的全部或部分TCRβ链恒定区。
  12. 如权利要求1所述的T细胞受体，其特征在于，所述TCR包含α链恒定区与β链恒定区，并且所述α链恒定区与β链恒定区之间含有人工链间二硫键；优选地，在所述TCRα与β链的恒定区之间形成人工链间二硫键的半胱氨酸残基取代了选自下列的一组或多组位点：
    TRAC*01外显子1的Thr48和TRBC1*01或TRBC2*01外显子1的Ser57；
    TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；
    TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；
    TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；
    TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；
    TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；
    TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；
    和TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。
  13. 如权利要求1所述的T细胞受体，其特征在于，所述TCR的α链可变域氨基酸序列为SEQ ID NO:1、13-39之一；和所述TCR的β链可变域氨基酸序列为SEQ ID NO:2、40、41之一；优选地，所述TCR选自下组：
    Figure PCTCN2021109705-appb-100001
  14. 如权利要求1所述的T细胞受体，其特征在于，所述TCR为单链TCR；优选地，所述TCR是由α链可变域和β链可变域组成的单链TCR，所述α链可变域和β链可变域 由一柔性短肽序列(linker)连接。
  15. 如权利要求1所述的T细胞受体，其特征在于，所述TCR的α链和/或β链的C-或N-末端结合有偶联物；优选地，所述偶联物为可检测标记物或治疗剂；
    更优选地，与所述TCR结合的治疗剂为连接于所述TCR的α或β链的C-或N-末端的抗-CD3抗体。
  16. 一种多价TCR复合物，其特征在于，包含至少两个TCR分子，并且其中的至少一个TCR分子为权利要求1中所述的TCR。
  17. 一种核酸分子，其特征在于，所述核酸分子包含编码权利要求1所述的TCR的核酸序列或其互补序列。
  18. 一种载体，其特征在于，所述的载体含有权利要求28中所述的核酸分子。
  19. 一种宿主细胞，其特征在于，所述的宿主细胞中含有权利要求18中所述的载体或染色体中整合有外源的权利要求17中所述的核酸分子。
  20. 一种分离的细胞，其特征在于，所述细胞表达权利要求1中所述的TCR，优选地所述分离的细胞为T细胞。
  21. 一种药物组合物，其特征在于，所述组合物含有药学上可接受的载体以及权利要求1中所述的TCR、或权利要求16中所述的TCR复合物、或权利要求20中所述的细胞。
  22. 一种治疗疾病的方法，其特征在于，包括给需要治疗的对象施用权利要求1中所述的TCR、或权利要求16中所述的TCR复合物、或权利要求20中所述的细胞、或权利要求21中所述的药物组合物，优选地，所述疾病为AFP阳性肿瘤，更优选地，所述肿瘤为肝癌。
  23. 权利要求1中所述的T细胞受体、权利要16中所述的TCR复合物或权利要求20中所述细胞的用途，其特征在于，用于制备治疗肿瘤的药物，优选地，所述肿瘤为AFP阳性肿瘤，更优选地，所述肿瘤为肝癌。
  24. 一种制备权利要求1中所述的T细胞受体的方法，其特征在于，包括步骤：
    (i)培养权利要求19中所述的宿主细胞，从而表达权利要求1中所述的T细胞受体；
    (ii)分离或纯化出所述的T细胞受体。
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