WO2021043284A1 - 一种识别ssx2的高亲和力t细胞受体 - Google Patents

一种识别ssx2的高亲和力t细胞受体 Download PDF

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WO2021043284A1
WO2021043284A1 PCT/CN2020/113583 CN2020113583W WO2021043284A1 WO 2021043284 A1 WO2021043284 A1 WO 2021043284A1 CN 2020113583 W CN2020113583 W CN 2020113583W WO 2021043284 A1 WO2021043284 A1 WO 2021043284A1
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tcr
chain
variable domain
amino acid
exon
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French (fr)
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黄金花
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广东香雪精准医疗技术有限公司
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Priority to EP20859846.6A priority Critical patent/EP4026844A4/en
Priority to US17/640,601 priority patent/US20220402997A1/en
Publication of WO2021043284A1 publication Critical patent/WO2021043284A1/zh

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    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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Definitions

  • TCR T cell receptor
  • TCR is the only receptor for specific antigen peptides presented on the main histocompatibility complex (MHC). This exogenous or endogenous peptide may be the only sign of abnormal cells.
  • MHC main histocompatibility complex
  • APC antigen presenting cells
  • the KASEKIFYV-HLA A0201 complex provides a marker for TCR to target tumor cells.
  • the TCR that can be combined with KASEKIFYV-HLA A0201 complex has high application value for tumor treatment.
  • TCR that can target the tumor cell marker can be used to deliver cytotoxic or immunostimulant to target cells, or be transformed into T cells, so that T cells expressing the TCR can destroy tumor cells, so that they can be called Adoptive immunotherapy is given to patients during the course of treatment.
  • the ideal TCR has a higher affinity, so that the TCR can reside on the targeted cells for a long time.
  • the purpose of the present invention is to provide a TCR with higher affinity to the KASEKIFYV-HLA A0201 complex.
  • Another object of the present invention is to provide a method for preparing 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), which has the activity of binding to the KASEKIFYV-HLA A0201 complex.
  • the alpha chain variable domain of the TCR contains at least 90% of the amino acid sequence shown in SEQ ID NO:1; preferably, at least 92%; more preferably, at least 94% (e.g., It may be an amino acid sequence with sequence homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology); and/or
  • the ⁇ -chain variable domain of the TCR contains at least 90% of the amino acid sequence shown in SEQ ID NO: 2, preferably at least 92%; more preferably, at least 94% (e.g., may be at least 91%, 92%). %, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology).
  • the TCR has a mutation in the ⁇ chain variable domain shown in SEQ ID NO: 2, and the mutation is selected from D96V, E98I, L99P, L100E/I/N/P/V, F101D /H/N/Y, Y102A/K/N/P/V, N103A/D/E/P, E104L/V, Q105P/T, F106H, one or several groups, wherein the amino acid residue numbering adopts SEQ ID NO: the number shown in 2.
  • the TCR ⁇ chain variable domain comprises three CDR regions, wherein the amino acid sequence of CDR3 ⁇ is: AYRSGIIQGAQKLV.
  • the TCR ⁇ chain variable domain comprises 3 CDR regions, and the sequence of the 3 CDR regions is:
  • amino acid sequence of the variable domain of the TCR ⁇ chain is SEQ ID NO:1.
  • the TCR ⁇ chain variable domain comprises three CDR regions, wherein the amino acid sequence of CDR1 ⁇ is PRHDT; and the amino acid sequence of CDR2 ⁇ is: FYEKMQ.
  • the CDR3 ⁇ of the TCR ⁇ chain variable domain is selected from the group consisting of ASSDRIPPYYNEQF, ASSSDRELNDAPEQF, ASSSVRELNYVDEQF and ASSSDRELLFYNEQF.
  • the affinity of the TCR and KASEKIFYV-HLA A0201 complex is at least 2 times that of wild-type TCR; preferably, at least 5 times; more preferably, at least 10 times.
  • the affinity of the TCR and KASEKIFYV-HLA A0201 complex is at least 50 times that of wild-type TCR; preferably, at least 100 times; more preferably, at least 300 times.
  • the dissociation equilibrium constant KD of the KASEKIFYV-HLA A0201 complex of the TCR is ⁇ 50 ⁇ M;
  • the dissociation equilibrium constant of the TCR to the KASEKIFYV-HLA A0201 complex is 1 ⁇ M ⁇ KD ⁇ 50 ⁇ M.
  • the TCR is soluble.
  • the TCR is ⁇ heterodimeric TCR.
  • the TCR has an ⁇ -chain constant region sequence TRAC*01 and a ⁇ -chain constant region sequence TRBC1*01 or TRBC2*01.
  • the TCR comprises (i) all or part of the TCR ⁇ chain excluding its transmembrane domain, and (ii) all or part of the TCR ⁇ chain excluding its transmembrane domain, wherein (i) And (ii) both comprise the variable domain and at least a part of the constant domain of the TCR chain.
  • the alpha chain constant region and the beta chain constant region of the TCR contain artificial interchain disulfide bonds.
  • 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 )connection.
  • a conjugate is bound to the C- or N-terminus of the ⁇ chain and/or ⁇ chain of the TCR.
  • the conjugate that binds to the TCR is a detectable label, a therapeutic agent, a PK modified portion, or a combination of any of these substances.
  • the therapeutic agent that binds to the TCR is an anti-CD3 antibody linked to the C- or N-terminus of the ⁇ or ⁇ chain of the TCR.
  • the third aspect of the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule according to the first aspect of the present invention or the multivalent TCR complex according to the second aspect of the present invention or its complement sequence.
  • the fourth aspect of the present invention provides a vector containing the nucleic acid molecule described in the third aspect of the present invention.
  • the fifth aspect of the present invention provides a host cell containing the vector of the fourth aspect of the present invention or the nucleic acid molecule of the third aspect of the present invention integrated into the chromosome.
  • the sixth aspect of the present invention provides an isolated cell that expresses the TCR described in the first aspect of the present invention.
  • the seventh aspect of the present invention provides a pharmaceutical composition containing a pharmaceutically acceptable carrier and the TCR according to the first aspect of the present invention, or the TCR complex according to the second aspect of the present invention, Or the cell described in the sixth aspect of the present invention.
  • the eighth aspect of the present invention provides a method for treating diseases, comprising administering an appropriate amount of the TCR according to the first aspect of the present invention, or the TCR complex according to the second aspect of the present invention, or the present invention to a subject in need of treatment.
  • the ninth aspect of the present invention provides the use of the TCR according to the first aspect of the present invention, or the TCR complex according to the second aspect of the present invention, or the cell according to the sixth aspect of the present invention, for preparing and treating tumors
  • the tumor is an SSX2 positive tumor.
  • the T cell receptor according to the first aspect, the TCR complex according to the second aspect or the cells according to the sixth aspect are used as a drug for treating tumors.
  • the tumor is an SSX2 positive tumor.
  • the eleventh aspect of the present invention provides a method for preparing the T cell receptor according to the first aspect of the present invention, including the steps:
  • Figure 1a and Figure 1b respectively show the amino acid sequences of wild-type TCR ⁇ and ⁇ chain variable domains that can specifically bind to the KASEKIFYV-HLA A0201 complex.
  • Figures 2a and 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 in the present invention.
  • Figures 3a and 3b are respectively the DNA sequence of the ⁇ chain variable domain and the DNA sequence of the ⁇ chain variable domain of the single-stranded template TCR constructed in the present invention.
  • Figures 4a and 4b are respectively the amino acid sequence and nucleotide sequence of the linker of the single-stranded template TCR constructed in the present invention.
  • Figure 5a and Figure 5b are the amino acid sequence and DNA sequence of the single-stranded template TCR constructed in the present invention, respectively.
  • Figure 7 (1)- Figure 7 (12) respectively show the amino acid sequence of the ⁇ chain variable domain of a heterodimeric TCR with high affinity for the KASEKIFYV-HLA A0201 complex, and the mutated residues are underlined.
  • Figure 8a and Figure 8b respectively show the extracellular amino acid sequences of wild-type TCR ⁇ and ⁇ chains that can specifically bind to the KASEKIFYV-HLA A0201 complex.
  • Figures 9a and 9b respectively show the amino acid sequences of wild-type TCR ⁇ and ⁇ chains that can specifically bind to the KASEKIFYV-HLA A0201 complex.
  • Figure 10 is the binding curve of the soluble reference TCR, that is, the wild-type TCR and the KASEKIFYV-HLA A0201 complex.
  • FIG. 11 shows the results of the activation function experiment of effector cells transfected with the high-affinity TCR of the present invention.
  • the present invention obtains a high-affinity T cell receptor (TCR) that recognizes KASEKIFYV short peptide (derived from AFP protein), and the form of the KASEKIFYV-HLA A0201 complex is presented.
  • TCR high-affinity T cell receptor
  • the high-affinity TCR is in the 3 CDR regions of its ⁇ chain variable domain:
  • CDR3 ⁇ A mutation in AYRSGIIQGAQKLV; and/or in the 3 CDR regions of the ⁇ chain variable domain:
  • CDR3 ⁇ Mutation occurs in ASSSDRELLFYNEQF; and after the mutation, the affinity and/or binding half-life of the TCR of the present invention for the KASEKIFYV-HLA A0201 complex is at least twice that of the wild-type TCR.
  • TCR T cell receptor
  • the International Immunogenetics Information System can be used to describe TCR.
  • the natural ⁇ heterodimeric TCR has an ⁇ chain and a ⁇ chain. Broadly speaking, each chain includes a variable region, a connecting region, and a constant region.
  • the beta chain usually also contains a short variable region between the variable region and the connecting region, but the variable region is often regarded as a part of the connecting region.
  • the unique IMGT TRAJ and TRBJ are used to determine the TCR connection region, and the IMGT TRAC and TRBC are used to determine the TCR constant region.
  • the constant region of the ⁇ chain is uniquely determined.
  • TCR ⁇ chain variable domain refers to the connected TRAV and TRAJ regions
  • TCR ⁇ chain variable domain refers to the connected TRBV and TRBD/TRBJ regions.
  • the three CDRs of the variable domain of the TCR ⁇ chain are CDR1 ⁇ , CDR2 ⁇ , and CDR3 ⁇ ; the three CDRs of the variable domain of the TCR ⁇ chain are CDR1 ⁇ , CDR2 ⁇ , and CDR3 ⁇ , respectively.
  • the framework sequence of the TCR variable domain of the present invention can be of murine or human origin, and is preferably of human origin.
  • the constant domain of TCR contains an intracellular part, a transmembrane region and an extracellular part.
  • the TCR of the present invention preferably does not include a transmembrane region.
  • the amino acid sequence of the TCR of the present invention refers to the extracellular amino acid sequence of the TCR.
  • the TCR sequence used in the present invention is of human origin.
  • the alpha chain amino acid sequence and the beta chain amino acid sequence of the "wild-type TCR" in the present invention are SEQ ID NO: 27 and SEQ ID NO: 28, respectively, as shown in Figures 9a and 9b.
  • the alpha chain amino acid sequence and the beta chain amino acid sequence of the "reference TCR" in the present invention are SEQ ID NO: 11 and SEQ ID NO: 12, respectively, as shown in Figure 6a and Figure 6b.
  • the extracellular amino acid sequence of the alpha chain and the extracellular amino acid sequence of the beta chain of the "wild-type TCR" in the present invention are SEQ ID NO: 25 and SEQ ID NO: 26, respectively, as shown in Figure 8a and Figure 8b.
  • amino acid sequences of the alpha and beta chain variable domains of the wild-type TCR capable of binding to the KASEKIFYV-HLA A0201 complex are SEQ ID NO: 1 and SEQ ID NO: 2, respectively, as shown in Fig. 1a and Fig. 1b.
  • 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 sequence of TRAC*01 and TRBC1*01 or TRBC2*01 in the present invention are numbered in sequence from the N-terminus to the C-terminus.
  • TRBC1*01 or TRBC2*01 press from N
  • the 60th amino acid in the sequence from end to C end is P (proline)
  • Pro60 of TRBC1*01 or TRBC2*01 exon 1 in the present invention or it can be expressed as TRBC1* 01 or TRBC2*01 exon 1 of the 60th amino acid
  • the 61st amino acid is Q (glutamine) in sequence from N-terminal to C-terminal, then this
  • it can be described as Gln61 of TRBC1*01 or TRBC2*01 exon 1, or it can be expressed as the 61st amino acid of TRBC1*01 or TRBC2*01 exon 1, and so on.
  • the position numbers of the amino acid sequences of TRAV and TRBV in the variable regions are numbered according to the position numbers listed in IMGT.
  • the position number listed in IMGT is 46, it is described as the 46th amino acid of TRAV in the present invention, and the rest can be deduced by analogy.
  • the special instructions shall be followed.
  • the ⁇ chain variable domain and ⁇ chain variable domain of TCR each contain 3 CDRs, which are similar to the complementarity determining regions of antibodies.
  • CDR3 interacts with short antigen peptides
  • CDR1 and CDR2 interact with HLA. Therefore, the CDR of the TCR molecule determines its interaction with the antigen short peptide-HLA complex.
  • the amino acid sequence of the alpha chain variable domain and the amino acid sequence of the beta chain variable domain of the wild-type TCR capable of binding the antigen short peptide KASEKIFYV and HLA A0201 complex ie, KASEKIFYV-HLA A0201 complex
  • this sequence is the first discovery by the inventor. It has the following CDR regions:
  • the present invention provides a T cell receptor (TCR), which has the activity of binding KASEKIFYV-HLA A0201 complex.
  • the three CDRs of the wild-type TCR ⁇ chain variable domain SEQ ID NO:1, namely CDR1, CDR2, and CDR3 are located at positions 27-33, 51-58, and 93-106 of SEQ ID NO:1, respectively .
  • the three CDRs of the wild-type TCR ⁇ chain variable domain SEQ ID NO: 2, namely CDR1, CDR2, and CDR3 are located at positions 27-31, 49-54, and 92-106 of SEQ ID NO: 2, respectively . Therefore, the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 2, 96D is the fifth position D of CDR3 ⁇ , 98E is the seventh position E of CDR3 ⁇ , and 99L is the eighth position L and 100L of CDR3 ⁇ .
  • the 9th L and 101F of CDR3 ⁇ are the 10th F of CDR11 ⁇
  • 102Y is the 11th Y of CDR3 ⁇
  • 103N is the 12th N of CDR3 ⁇
  • 104E is the 13th E and 105Q of CDR3 ⁇ .
  • the 14th Q and 106F of CDR3 ⁇ are the 15th F of CDR3 ⁇ .
  • the mutated TCR ⁇ chain variable domain comprises one or more amino acid residues selected from the following group: 96V, 98I, 99P, 100E or 100I or 100N or 100P or 100V, 101D or 101H or 101N or 101Y , 102A or 102K or 102N or 102P or 102V, 103A or 103D or 103E or 103P, 104L or 104V, 105P or 105T, 106H, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 2.
  • the specific forms of the mutation in the ⁇ chain variable domain include D96V, E98I, L99P, L100E/I/N/P/V, F101D/H/N/Y, Y102A/K/N/P/V , N103A/D/E/P, E104L/V, Q105P/T, F106H one or several groups.
  • the TCR of the present invention is an ⁇ heterodimeric TCR
  • the ⁇ chain variable domain of the TCR contains at least 90% of the amino acid sequence shown in SEQ ID NO:1; preferably, at least 92%; more preferably Ground, at least 94% (eg, can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) sequence homology
  • the ⁇ -chain variable domain of the TCR contains at least 90%, preferably at least 92%, and more preferably at least 94% (such as , It can be an amino acid sequence with at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology).
  • % preferably, at least 92%; most preferably, at least 94% (eg, it can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Sequence homology) of the amino acid sequence of sequence homology.
  • the TCR comprises (i) all or part of the TCR ⁇ chain excluding its transmembrane domain, and (ii) all or part of the TCR ⁇ chain excluding its transmembrane domain, wherein (i) and (ii) Both contain the variable domain and at least a part of the constant domain of the TCR chain.
  • the above cysteine substitution can form artificial interchain disulfide bonds between the constant regions of the ⁇ and ⁇ chains of the reference TCR to form a more stable soluble TCR, which makes it easier to evaluate the complex of TCR and KASEKIFYV-HLA A0201
  • the binding affinity and/or binding half-life between substances It should be understood that the CDR region of the TCR variable region determines its affinity with the pMHC complex. Therefore, the above-mentioned cysteine substitution in the TCR constant region will not affect the binding affinity and/or binding half-life of the TCR.
  • the measured binding affinity between the reference TCR and the KASEKIFYV-HLA A0201 complex is considered to be the binding affinity between the wild-type TCR and the KASEKIFYV-HLA A0201 complex.
  • the binding affinity between the TCR of the present invention and the KASEKIFYV-HLA A0201 complex is at least 10 times that between the reference TCR and the KASEKIFYV-HLA A0201 complex, it is equivalent to the TCR of the present invention and KASEKIFYV.
  • the binding affinity between the -HLA A0201 complex is at least 10 times that between the wild-type TCR and the KASEKIFYV-HLA A0201 complex.
  • the surface plasmon resonance (BIAcore) method in the examples herein is used to detect the affinity of the soluble TCR, and the conditions are: a temperature of 25° C. and a pH of 7.1-7.5.
  • the method detects that the dissociation equilibrium constant K D of the reference TCR to the KASEKIFYV-HLA A0201 complex is 1.21E-04M, which is 121 ⁇ M, and the present invention considers the dissociation equilibrium constant of the wild-type TCR to the KASEKIFYV-HLA A0201 complex K D is also 121 ⁇ M.
  • the affinity of the TCR and KASEKIFYV-HLA A0201 complex is at least 2 times that of wild-type TCR; preferably, at least 5 times; more preferably, at least 10 times.
  • the affinity of the TCR and KASEKIFYV-HLA A0201 complex is at least 50 times that of wild-type TCR; preferably, at least 100 times; more preferably, at least 300 times.
  • PCR polymerase chain reaction
  • LIC ligation-independent cloning
  • genes expressing wild-type TCR alpha and beta chain variable domain amino acids or genes expressing slightly modified wild-type TCR alpha and beta chain variable domain amino acids can be used to prepare template TCRs.
  • the DNA encoding the variable domain of the template TCR then introduces the changes required to produce the high-affinity TCR of the present invention.
  • the TCR of the present invention is a part having at least one TCR ⁇ and/or TCR ⁇ chain variable domain. They usually contain both the TCR ⁇ chain variable domain and the TCR ⁇ chain variable domain. They can be ⁇ heterodimers or single-stranded forms or any other forms that can exist stably. In adoptive immunotherapy, the full-length chain of ⁇ heterodimeric TCR (including cytoplasmic and transmembrane domains) can be transfected.
  • the TCR of the present invention can be used as a targeting agent for delivering therapeutic agents to antigen-presenting cells or combined with other molecules to prepare bifunctional polypeptides to target effector cells. In this case, the TCR is preferably in a soluble form.
  • Thr48 in TRAC*01 exon 1 and replacing Ser57 in TRBC1*01 or TRBC2*01 exon 1 to form a disulfide bond can also be: Thr45 of TRAC*01 exon 1 and TRBC1*01 or Ser77 of TRBC2*01 exon 1; TRAC*01 exon Tyr10 of 1 and Ser17 of TRBC1*01 or TRBC2*01 exon 1; Thr45 of TRAC*01 exon 1 and Asp59 of TRBC1*01 or TRBC2*01 exon 1; TRAC*01 exon 1 Ser15 and TRBC1*01 or TRBC2*01 exon 1 Glu15; TRAC*01 exon 1 Arg53 and TRBC1*01 or TRBC2*01 exon 1 Ser54; TRAC*01 exon 1 Pro89 and Ala19 of TRBC1*01 or TRBC2*01 exon 1; or Tyr10 of TRAC*01 exon 1 and Glu20
  • cysteine residues replace any set of positions in the constant domains of the ⁇ and ⁇ chains.
  • a maximum of 15, or a maximum of 10, or a maximum of 8 or less amino acids can be truncated at one or more C-termini of the TCR constant domain of the present invention so that it does not include cysteine residues to achieve the deletion of natural
  • the purpose of interchain disulfide bonds can also be achieved by mutating the cysteine residues that form natural interchain disulfide bonds to another amino acid.
  • the TCR of the present invention may contain artificial interchain disulfide bonds introduced between the residues of the constant domains of its ⁇ and ⁇ chains. It should be noted that, with or without the introduced artificial disulfide bonds between the constant domains, the TCR of the present invention can contain the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence.
  • the TRAC constant domain sequence of TCR and the TRBC1 or TRBC2 constant domain sequence can be linked by natural interchain disulfide bonds present in the TCR.
  • patent document PCT/CN2016/077680 also discloses that the introduction of artificial interchain disulfide bonds between the ⁇ chain variable region and the ⁇ chain constant region of the TCR can significantly improve the stability of the TCR. Therefore, the high-affinity TCR of the present invention may also contain artificial interchain disulfide bonds between the ⁇ chain variable region and the ⁇ chain constant region.
  • the TCR of the present invention also includes TCRs with mutations in the hydrophobic core region. These mutations in the hydrophobic core region are preferably mutations that can improve the stability of the TCR of the present invention, as described in Publication No.
  • TCR can be mutated at the following variable domain hydrophobic core positions: ( ⁇ and/or ⁇ chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94 and/or The 3rd, 5th and 7th positions of the short peptide of ⁇ chain J gene (TRAJ) and/or the 2nd, 4th and 6th positions of the short peptide of ⁇ chain J gene (TRBJ).
  • the position numbers of the amino acid sequence are according to the international The position number listed in the Immunogenetics Information System (IMGT).
  • IMGT Immunogenetics Information System
  • the template chain constructed in Example 1 of the present invention for screening high-affinity TCRs is the above-mentioned high-stability single-chain TCR containing a hydrophobic core mutation. Using TCR with higher stability can more conveniently evaluate the affinity between TCR and KASEKIFYV-HLA-A0201 complex.
  • the CDR regions of the ⁇ -chain variable domain and ⁇ -chain variable domain of the single-chain template TCR are exactly the same as the CDR regions of the wild-type TCR. That is, the three CDRs of the ⁇ chain variable domain are CDR1 ⁇ : TSESDYY, CDR2 ⁇ : QEAYKQQN, CDR3 ⁇ : AYRSGIIQGAQKLV, and the three CDRs of the ⁇ chain variable domain are CDR1 ⁇ : PRHDT, CDR2 ⁇ : FYEKMQ, CDR3 ⁇ : ASSSDRELLFYNEQF.
  • the amino acid sequence (SEQ ID NO: 9) and nucleotide sequence (SEQ ID NO: 10) of the single-stranded template TCR are shown in Figure 5a and Figure 5b, respectively. Based on this, a single-chain TCR composed of ⁇ -chain variable domain and ⁇ -chain variable domain with high affinity to the KASEKIFYV-HLA A0201 complex was screened out.
  • the three CDRs of the single-stranded template TCR ⁇ chain variable domain SEQ ID NO: 3, namely CDR1, CDR2 and CDR3 are located at positions 27-33, 51-58 and 93- of SEQ ID NO:1, respectively. 106.
  • the three CDRs of the single-stranded template TCR ⁇ chain variable domain SEQ ID NO: 4, namely CDR1, CDR2 and CDR3 are located at positions 27-31, 49-54 and 92-th of SEQ ID NO: 2, respectively. 106. Therefore, the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 4, 96D is the fifth position D of CDR3 ⁇ , 98E is the seventh position E of CDR3 ⁇ , and 99L is the eighth position L and 100L of CDR3 ⁇ .
  • the 9th L and 101F of CDR3 ⁇ are the 10th F of CDR11 ⁇
  • 102Y is the 11th Y of CDR3 ⁇
  • 103N is the 12th N of CDR3 ⁇
  • 104E is the 13th E and 105Q of CDR3 ⁇ .
  • the 14th Q and 106F of CDR3 ⁇ are the 15th F of CDR3 ⁇ .
  • the ⁇ heterodimer with high affinity to the KASEKIFYV-HLA-A0201 complex of the present invention is obtained by transferring the CDR regions of the ⁇ and ⁇ chain variable domains of the selected high-affinity single-chain TCR To the corresponding positions of the wild-type TCR ⁇ chain variable domain (SEQ ID NO: 1) and ⁇ chain variable domain (SEQ ID NO: 2).
  • the TCR of the present invention can also be provided in the form of a multivalent complex.
  • the multivalent TCR complex of the present invention comprises a polymer formed by combining two, three, four or more TCRs of the present invention.
  • the tetramerization domain of p53 can be used to generate a tetramer, or more A complex formed by combining the TCR of the present invention with another molecule.
  • the TCR complex of the present invention can be used to track or target cells presenting a specific antigen in vitro or in vivo, and can also be used to produce intermediates of other multivalent TCR complexes with such applications.
  • the TCR of the present invention can be used alone, or can be combined with the conjugate in a covalent or other manner, preferably in a covalent manner.
  • the conjugate includes a detectable marker (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the KASEKIFYV-HLA-A0201 complex), a therapeutic agent, a PK (protein kinase) modified portion, or any of the above Combination of substances combined or coupled.
  • Detectable markers used for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computed tomography technology) contrast agents, or capable of producing detectable products Of enzymes.
  • Therapeutic agents that can be combined or coupled with the TCR of the present invention include but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539); 2. Biotoxicity (Chaudhary et al., 1989) , Nature 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy (Cancer Immunology and Immunotherapy 51, 565); 3.
  • Cytokines such as IL-2 (Gillies et al., 1992, National Academy of Sciences) Journal (PNAS) 89, 1428; Card et al., 2004, Cancer Immunology and Immunotherapy (Cancer Immunology and Immunotherapy) 53, 345; Halin et al., 2003, Cancer Research (Cancer Research) 63, 3202); 4. Antibody Fc Fragments (Mosquera et al., 2005, The Journal of Immunology 174, 4381); 5. Antibody scFv fragments (Zhu et al., 1995, International Journal of Cancer (International Journal of Cancer) 62,319); 6.
  • Gold nanoparticles/ Nanorods (Lapotko et al., 2005, Cancer letters 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115); 7. Virus particles (Peng et al., 2004, Gene therapy (Genetherapy) 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research (Cancer research) 65, 11631); 9. Nano magnetic particles; 10. Prodrug activating enzymes (for example, DT- Diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. chemotherapeutics (for example, cisplatin) or any form of nanoparticles, etc.
  • DTD DT- Diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the antibodies or fragments thereof that bind to the TCR of the present invention include anti-T cell or NK-cell determining antibodies, such as anti-CD3 or anti-CD28 or anti-CD16 antibodies.
  • the combination of the above-mentioned antibodies or fragments with TCR can affect effector cells. Orientation to better target target cells.
  • a preferred embodiment is that the TCR of the present invention is combined with an anti-CD3 antibody or a functional fragment or variant of the anti-CD3 antibody.
  • the fusion molecule of TCR and anti-CD3 single-chain antibody of the present invention includes the amino acid sequence of the variable domain of the TCR ⁇ chain: SEQ ID NO: 1 and/or the amino acid sequence of the variable domain of the TCR ⁇ chain: one of SEQ ID NO: 13-24 .
  • the invention also relates to a nucleic acid molecule encoding the TCR of the invention.
  • the nucleic acid molecule of the present invention may be in the form of DNA or RNA.
  • DNA can be a coding strand or a non-coding strand.
  • the nucleic acid sequence encoding the TCR of the present invention may be the same as the nucleic acid sequence shown in the drawings of the present invention or a degenerate variant.
  • degenerate variant refers to a protein sequence that encodes SEQ ID NO: 9, but is similar to the sequence of SEQ ID NO: 10 Different nucleic acid sequences.
  • the full-length sequence of the nucleic acid molecule of the present invention or its fragments can usually be obtained by but not limited to PCR amplification method, recombination method or artificial synthesis method.
  • the DNA sequence encoding the TCR (or a fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • the present invention also relates to a vector containing the nucleic acid molecule of the present invention, and a host cell produced by genetic engineering using the vector or coding sequence of the present invention.
  • the present invention also includes isolated cells expressing the TCR of the present invention, especially T cells.
  • T cells There are many methods suitable for T cell transfection with DNA or RNA encoding the high-affinity TCR of the present invention (eg, Robbins et al., (2008) J. Immunol. 180: 6116-6131).
  • T cells expressing the high-affinity TCR of the present invention can be used for adoptive immunotherapy.
  • Those skilled in the art can know many suitable methods for adoptive therapy (eg, Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).
  • the TCR of the present invention also includes at most 5 of the TCR of the present invention, preferably at most 3, more preferably at most 2, and most preferably 1 amino acid (especially the amino acid located outside the CDR region), which are similar in nature. Or similar amino acids are replaced, and still maintain its functional TCR.
  • the present invention also includes a TCR slightly modified from the TCR of the present invention.
  • Modified (usually not changing the primary structure) forms include: chemically derived forms of the TCR of the present invention such as acetylation or carboxylation.
  • Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of the TCR of the present invention or in further processing steps. This modification can be accomplished by exposing the TCR to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase).
  • Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, and phosphothreonine). It also includes TCRs that have been modified to improve their resistance to proteolysis or optimize their solubility.
  • the TCR, TCR complex of the present invention or T cells transfected with the TCR of the present invention can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the TCR, multivalent TCR complex or cell of the present invention is usually provided as part of a sterile pharmaceutical composition, which usually includes a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be in any suitable form (depending on the desired method of administration to the patient). It can be provided in a unit dosage form, usually in a sealed container, and can be provided as part of a kit. Such kits (but not required) include instructions for use. It may include a plurality of such unit dosage forms.
  • the TCR of the present invention can be used alone, or can be combined or coupled with other therapeutic agents (for example, formulated in the same pharmaceutical composition).
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent.
  • pharmaceutical carriers that do not themselves induce the production of antibodies that are harmful to the individual receiving the composition, and do not have excessive toxicity after administration. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
  • Such carriers include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • the pharmaceutically acceptable carrier in the therapeutic composition may contain liquids such as water, saline, glycerol and ethanol.
  • these carriers may also contain auxiliary substances, such as wetting or emulsifying agents, and pH buffering substances.
  • the therapeutic composition can be made into an injectable, such as a liquid solution or suspension; it can also be made into a solid form suitable for being formulated into a solution or suspension in a liquid carrier before injection.
  • an injectable such as a liquid solution or suspension
  • it can also be made into a solid form suitable for being formulated into a solution or suspension in a liquid carrier before injection.
  • composition of the present invention can be administered by conventional routes, including (but not limited to): intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably gastrointestinal External includes subcutaneous, intramuscular or intravenous.
  • routes including (but not limited to): intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably gastrointestinal External includes subcutaneous, intramuscular or intravenous.
  • the objects to be prevented or treated can be animals; especially humans.
  • composition of the present invention When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition can be used according to the use situation. Preferably, injections, oral preparations and the like can be exemplified.
  • compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic (Isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a customary manner according to the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic (Isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a customary manner according to the dosage form.
  • the pharmaceutical composition of the present invention can also be administered in the form of a sustained-release dosage form.
  • the TCR of the present invention can be incorporated into a pill or microcapsule with a sustained-release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated.
  • sustained-release polymers ethylene-vinyl acetate copolymers, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, Lactic acid-glycolic acid copolymers and the like are preferably exemplified by biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.
  • the TCR or TCR complex of the present invention as the active ingredient or the cells presenting the TCR of the present invention can be based on the weight, age, sex, and degree of symptoms of each patient to be treated. It is determined reasonably, and the doctor finally decides the reasonable dosage.
  • the affinity and/or binding half-life of the TCR of the present invention for the KASEKIFYV-HLA-A0201 complex is at least 2 times, preferably at least 10 times, that of the wild-type TCR.
  • the affinity and/or binding half-life of the TCR of the present invention for the KASEKIFYV-HLA-A0201 complex is at least 50 times, preferably at least 100 times, and more preferably up to 300 times that of the wild-type TCR.
  • the effector cells that transduce the high-affinity TCR of the present invention have a strong activating function on the target cells.
  • E. coli DH5 ⁇ was purchased from Tiangen
  • E. coli BL21 (DE3) was purchased from Tiangen
  • E. coli Tuner (DE3) was purchased.
  • plasmid pET28a was purchased from Novagen.
  • Example 1 Stability of hydrophobic core mutations. Generation of single-stranded TCR template strands
  • the present invention uses the method of site-directed mutagenesis, according to the patent document WO2014/206304, to construct a stable single-stranded TCR molecule composed of a flexible short peptide (linker) connecting TCR ⁇ and ⁇ chain variable domains, its amino acids and DNA
  • the sequences are SEQ ID NO: 9 and SEQ ID NO: 10, respectively, as shown in Figure 5a and Figure 5b.
  • the inclusion bodies were dissolved in a buffer (20mM Tris-HCl pH 8.0, 8M urea), centrifuged at a high speed to remove insoluble materials, and the supernatant was quantified by the BCA method and then aliquoted and stored at -80°C for later use.
  • refolding buffer 100mM Tris-HCl pH 8.1, 0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mM ⁇ -mercapthoethylamine, 1.87mM Cystamine
  • the refolding solution was put into a cellulose membrane dialysis bag with a cutoff of 4kDa. The dialysis bag was placed in 1L of pre-cooled water and stirred slowly at 4°C overnight.
  • the collected elution fractions were analyzed by SDS-PAGE, and the fractions containing single-stranded TCR were concentrated and further purified with a gel filtration column (Superdex 75 10/300, GE Healthcare), and the target fractions were also analyzed by SDS-PAGE.
  • the eluted fractions used for BIAcore analysis were further tested for purity by gel filtration.
  • the conditions are: Column Agilent Bio SEC-3 (300A, ), the mobile phase is 150mM phosphate buffer, the flow rate is 0.5mL/min, the column temperature is 25°C, and the UV detection wavelength is 214nm.
  • BIAcore T200 real-time analysis system was used to detect the binding activity of TCR molecule and KASEKIFYV-HLA-A0201 complex.
  • the conditions are: the temperature is 25°C and the PH value is 7.1-7.5.
  • the binding time of each injection is 120s, and let it dissociate for 600s after the last injection.
  • the chip was regenerated with 10mM Gly-HCl pH 1.75. Use BIAcore Evaluation software to calculate kinetic parameters.
  • the synthetic short peptide KASEKIFYV (Beijing Saibaisheng Gene Technology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg/ml.
  • the inclusion bodies of the light chain and the heavy chain were dissolved with 8M urea, 20mM Tris pH 8.0, 10mM DTT, and 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA were added before renaturation to further denature.
  • the protein-containing fractions were combined, concentrated with a Millipore ultrafiltration tube, and the protein concentration was determined by the BCA method (Thermo).
  • the protease inhibitor cocktail (Roche) was added to store the biotinylated pMHC molecules at -80°C.
  • the screened high-affinity single-chain TCR mutations in the CDR region were introduced into the corresponding positions of the variable domains of the ⁇ heterodimeric TCR, and the affinity with the KASEKIFYV-HLA-A0201 complex was detected by BIAcore.
  • the introduction of the above-mentioned high-affinity mutation points in the CDR region adopts a site-directed mutation method well known to those skilled in the art.
  • the amino acid sequences of the alpha chain and beta chain variable domains of the wild-type TCR are shown in Figure 1a (SEQ ID NO: 1) and 1b (SEQ ID NO: 2), respectively.
  • the ⁇ heterodimeric TCR can be constant in the ⁇ and ⁇ chains.
  • a cysteine residue is introduced into the regions to form the TCR of the artificial inter-chain disulfide bond.
  • the amino acid sequences of the TCR ⁇ and ⁇ chains after the introduction of cysteine residues are shown in Figure 6a (SEQ ID NO : 11) and shown in 6b (SEQ ID NO: 12), the introduced cysteine residues are indicated in bold letters.
  • TCR ⁇ and ⁇ chains were respectively transformed into expressing bacteria BL21(DE3) by chemical transformation method.
  • the ⁇ and ⁇ chains of TCR were expressed
  • the inclusion bodies formed later were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution.
  • the inclusion bodies were finally dissolved in 6M guanidine hydrochloride, 10mM dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA). ), 20mM Tris (pH 8.1).
  • the dissolved TCR ⁇ and ⁇ chains are quickly mixed with 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 to the final concentration It is 60mg/mL.
  • After mixing place the solution in 10 times the volume of deionized water for dialysis (4°C). After 12 hours, change the deionized water to a buffer (20mM Tris, pH 8.0) and continue dialysis at 4°C for 12 hours. After the dialysis, the solution is filtered through a 0.45 ⁇ M filter membrane and purified by an anion exchange column (HiTrap Q HP, 5ml, GE Healthcare).
  • the eluted peak contains the successfully renatured ⁇ and ⁇ dimer TCRs, which are confirmed by SDS-PAGE gel.
  • the TCR is then further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was determined by SDS-PAGE to be greater than 90%, and the concentration was determined by the BCA method.
  • Example 3 The method described in Example 3 was used to detect the affinity of the ⁇ heterodimeric TCR introduced into the high-affinity CDR region and the KASEKIFYV-HLA-A0201 complex.
  • the affinity of the high-affinity TCR obtained in the present invention is at least twice that of the wild-type TCR for the KASEKIFYV-HLA-A0201 complex.
  • Example 7 Expression, renaturation and purification of the fusion of anti-CD3 antibody and high-affinity ⁇ heterodimeric TCR
  • the anti-CD3 single-chain antibody (scFv) is fused with ⁇ heterodimeric TCR to prepare a fusion molecule.
  • the anti-CD3 scFv is fused with the ⁇ chain of the TCR.
  • the TCR ⁇ chain may include any of the above-mentioned high-affinity ⁇ heterodimeric TCR ⁇ -chain variable domains
  • the TCR ⁇ chain of the fusion molecule may include any of the above-mentioned high affinity
  • primers are designed to link the anti-CD3 scFv and the high-affinity heterodimeric TCR ⁇ chain gene, the linker in the middle is GGGGS, and the anti-CD3 scFv is connected with
  • the gene fragment of the high-affinity heterodimeric TCR ⁇ chain fusion protein has restriction endonuclease sites Nco I (CCATGG) and Not I (GCGGCCGC).
  • CCATGG restriction endonuclease sites
  • Not I GCGGCCGC
  • the ligation product was transformed into E.coli DH5 ⁇ competent cells, spread on LB plates containing kanamycin, incubated overnight at 37°C, picked positive clones for PCR screening, sequenced the positive recombinants, and extracted after confirming the correct sequence
  • the recombinant plasmid is transformed into E. coli Tuner (DE3) competent cells for expression.
  • the dissolved TCR ⁇ chain and anti-CD3 (scFv)- ⁇ chain are quickly mixed with 5M urea (urea), 0.4M L-arginine (L-arginine), 20mM Tris pH 8.1, 3.7 at a mass ratio of 2:5 mM cystamine, 6.6mM ⁇ -mercapoethylamine (4°C), the final concentration of ⁇ chain and anti-CD3 (scFv)- ⁇ chain are 0.1 mg/mL and 0.25 mg/mL, respectively.
  • the TCR fusion molecule is then further purified by size exclusion chromatography (S-100 16/60, GE healthcare), and again purified by anion exchange column (HiTrap Q HP 5ml, GE healthcare).
  • the purity of the purified TCR fusion molecule was determined by SDS-PAGE to be greater than 90%, and the concentration was determined by the BCA method.
  • Example 8 Activation function experiment of effector cells transfected with high-affinity TCR of the present invention
  • the CD3+ T cells isolated from the blood of healthy volunteers were randomly selected to be transfected with the TCR of the present invention as effector cells.
  • the TCR and its number are learned from Table 2.
  • TCR5 ⁇ chain variable domain SEQ ID NO:1, ⁇ chain variable domain SEQ ID NO: 17
  • TCR6 ⁇ chain variable domain SEQ ID NO:1 , ⁇ -chain variable domain SEQ ID NO: 18
  • TCR7 ⁇ -chain variable domain SEQ ID NO: 1, ⁇ -chain variable domain SEQ ID NO: 19
  • TCR1 ⁇ -chain variable domain SEQ ID NO: 1 , ⁇ -chain variable domain SEQ ID NO: 13
  • TCR9 ⁇ -chain variable domain SEQ ID NO: 1, ⁇ -chain variable domain SEQ ID NO: 21
  • TCR10 ⁇ -chain variable domain SEQ ID NO: 1 , ⁇ -chain variable domain SEQ ID NO: 22
  • TCR2 ⁇ -chain variable domain SEQ ID NO: 1, ⁇ -chain variable domain SEQ ID NO: 14
  • TCR8 ⁇ -chain variable domain SEQ ID NO: 1 , ⁇ chain variable domain SEQ ID NO: 20
  • the control effector cells are labeled wild-type TCR (cells trans
  • the target cell lines were K562-A2 (A2 overexpression), SW620-SSX2 (SSX2 overexpression) and K562-A11 (A11 overexpression) cells. Among them, the target cell lines K562-A2 and SW620-SSX2 are used as positive tumor cell lines; K562-A11 is a negative tumor cell line as controls.
  • ELISPOT tablet was activated and coated with ethanol at 4°C overnight. On the first day of the experiment, remove the coating solution, wash and block, incubate for two hours at room temperature, remove the blocking solution, and add the test components to the ELISPOT plate: target cells are 2 ⁇ 10 4 cells/well, and effector cells are 10 3 Pcs/well (calculated based on the positive rate of transfection), and set up two duplicate wells. Incubate overnight (37°C, 5% CO 2 ).
  • the plate was washed and subjected to secondary detection and color development, and the plate was dried, and then the spots formed on the membrane were counted with an immunospot plate reader (ELISPOT READER system; AID20 company).
  • ELISPOT READER system AID20 company
  • the effector cells transfected with the high-affinity TCR of the present invention have a very good specific activation effect, and the function is much better than that of the effector cells transfected with the wild-type TCR; and Cells transduced with other TCRs basically had no activation effect.

Abstract

本发明提供了一种识别SSX2的高亲和力T细胞受体(TCR)，其具有结合KASEKIFYV-HLA A0201复合物的特性；并且所述TCR对所述KASEKIFYV-HLA A0201复合物的结合亲和力是野生型TCR对KASEKIFYV-HLA A0201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KASEKIFYV-HLA A0201复合物肿瘤细胞。

Description

一种识别SSX2的高亲和力T细胞受体 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SSX2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。
SSX2是滑膜肉瘤X断点，也被称为HOM-MEL-40。SSX2是SSX家族十种高度同源的核酸蛋白之一。SSX蛋白是肿瘤睾丸抗原，只在肿瘤细胞以及没有MHC表达的睾丸胚细胞中表达。SSX2在多种人类癌细胞中表达，包括但不限于，肝癌、肺癌、纤维肉瘤、乳腺癌、结肠癌、前列腺癌。SSX2在细胞内生成后被降解成小分子多肽，并与MHC(主组织相容性复合体)分子结合形成复合物，被呈递到细胞表面。KASEKIFYV是衍生自SSX2抗原的短肽，是SSX2相关疾病治疗的一种靶标。
因此，KASEKIFYV-HLA A0201复合物提供了一种TCR可靶向肿瘤细胞的标记。能够结合KASEKIFYV-HLA A0201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KASEKIFYV-HLA A0201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T细胞受体(TCR)，其具有结合KASEKIFYV-HLA A0201复合物的活性。
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％的序列同源性)的序列同源性的氨基酸序列。
在另一优选例中，所述TCR在SEQ ID NO:2所示的β链可变域中发生突变，所述突变选自D96V、E98I、L99P、L100E/I/N/P/V、F101D/H/N/Y、Y102A/K/N/P/V、N103A/D/E/P、E104L/V、Q105P/T、F106H中的一组或几组，其中，氨基酸残基编号采用SEQ ID NO:2所示的编号。
在另一优选例中，所述TCRα链可变域包含3个CDR区，其中CDR3α的氨基酸序列为：AYRSGIIQGAQKLV。
在另一优选例中，所述TCRα链可变域包含3个CDR区，3个CDR区的序列为：
CDR1α：TSESDYY
CDR2α：QEAYKQQN
CDR3α：AYRSGIIQGAQKLV
在另一优选例中，所述TCRα链可变域的氨基酸序列为SEQ ID NO:1。
在另一优选例中，所述TCRβ链可变域包含3个CDR区，其中CDR1β的氨基酸序列为PRHDT；和CDR2β的氨基酸序列为：FYEKMQ。
在另一优选例中，所述TCRβ链可变域的CDR3β选自下组：ASSSDRIPPYYNEQF、ASSSDRELNDAPEQF、ASSSVRELNYVDEQF和ASSSDRELLFYNEQF。
在另一优选例中，所述TCR具有选自下组的CDR:
Figure PCTCN2020113583-appb-000001
在另一优选例中，所述TCR的α链可变域氨基酸序列选自：SEQ ID NO:1；和/或所述TCR的β链可变域氨基酸序列选自：SEQ ID NO:13-24。
在另一优选例中，所述TCR选自下组：
Figure PCTCN2020113583-appb-000002
在另一优选例中，所述TCR与KASEKIFYV-HLA A0201复合物的亲和力是野生型TCR的至少2倍。
在本发明的一个优选例中，所述TCR与KASEKIFYV-HLA A0201复合物的亲和力是野生型TCR的至少2倍；优选地，至少5倍；更优选地，至少10倍。
在另一优选例中，所述TCR与KASEKIFYV-HLA A0201复合物的亲和力是野生型TCR的至少50倍；优选地，至少100倍；更优选地，至少300倍。
具体地，所述TCR对KASEKIFYV-HLA A0201复合物的解离平衡常数KD≤50μM；
在另一优选例中，所述TCR对KASEKIFYV-HLA A0201复合物的解离平衡常数为1μM≤KD≤50μM。
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。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。
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。
在另一优选例中，与所述TCR结合的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，包含至少两个TCR分子，并且其中的至少一个TCR分子为本发明第一方面所述的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。
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。
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，所述肿瘤为SSX2阳性肿瘤。
本发明的第十方面，权利要求第一方面所述的T细胞受体、第二方面所述的TCR复合物或第六方面所述的细胞用作治疗肿瘤的药物。优选地，所述肿瘤为SSX2阳性肿瘤。
本发明的第十一方面，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KASEKIFYV-HLA A0201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)的氨基酸序列和核苷酸序列。
图5a和图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分别显示了本发明中可溶性参比TCRα与β链的氨基酸序列。
图7(1)-图7(12)分别显示了对KASEKIFYV-HLA A0201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8a和图8b分别显示了对KASEKIFYV-HLA A0201复合物能够特异性结合的野生型TCRα与β链胞外氨基酸序列。
图9a和图9b分别显示了对KASEKIFYV-HLA A0201复合物能够特异性结合的野生型TCRα与β链的氨基酸序列。
图10为可溶性参比TCR即野生型TCR与KASEKIFYV-HLA A0201复合物的结合曲线。
图11为转染本发明高亲和力TCR的效应细胞的激活功能实验结果。
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KASEKIFYV短肽(衍生自AFP蛋白)的高亲和性T细胞受体(TCR)，所述KASEKIFYV-HLA A0201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：TSESDYY
CDR2α：QEAYKQQN
CDR3α：AYRSGIIQGAQKLV中发生突变；和/或在其β链可变域的3个CDR区：
CDR1β：PRHDT
CDR2β：FYEKMQ
CDR3β：ASSSDRELLFYNEQF中发生突变；并且，突变后本发明TCR对上述KASEKIFYV-HLA A0201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异质二聚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。为获得可溶性TCR，以便测定TCR与KASEKIFYV-HLA A0201复合物之间的亲和力，本发明TCR优选地不包含跨膜区。更优选地，本发明TCR的氨基酸序列是指TCR的胞外氨基酸序列。
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:27和SEQ ID NO:28，如图9a和9b所示。本发明中所述“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:25和SEQ ID NO:26，如图8a和图8b所示。本发明中，能够结合KASEKIFYV-HLA A0201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。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KASEKIFYV与HLA A0201复合物(即，KASEKIFYV-HLA A0201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：TSESDYY
CDR2α：QEAYKQQN
CDR3α：AYRSGIIQGAQKLV
β链可变域CDR
CDR1β：PRHDT
CDR2β：FYEKMQ
CDR3β：ASSSDRELLFYNEQF
本发明通过对上述CDR区进行突变筛选，获得了与KASEKIFYV-HLA A0201复合物的亲和力是野生型TCR与KASEKIFYV-HLA A0201复合物亲和力至少2倍的高亲和力TCR。
本发明提供了一种T细胞受体(TCR)，其具有结合KASEKIFYV-HLA A0201复合物的活性。
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27-33位、第51-58位和第93-106位。
本发明中野生型TCRβ链可变域SEQ ID NO:2的3个CDR即CDR1、CDR2和CDR3分别位于SEQ ID NO:2的第27-31位、第49-54位和第92-106位。因此，氨基酸残基编号采用SEQ ID NO:2所示的编号，96D即为CDR3β的第5位D、98E即为CDR3β的第7位E、99L即为CDR3β的第8位L、100L即为CDR3β的第9位L、101F即为CDR11β的第10位F、102Y即为CDR3β的第11位Y、103N即为CDR3β的第12位N、104E即为CDR3β的第13位E、105Q即为CDR3β的第14位Q和106F即为CDR3β的第15位F。
优选地，突变后的所述TCRβ链可变域包括选自下组的一个或多个氨基酸残基：96V、98I、99P、100E或100I或100N或100P或100V、101D或101H或101N或101Y、102A或102K或102N或102P或102V、103A或103D或103E或103P、104L或104V、105P或105T、106H，其中，氨基酸残基编号采用SEQ ID NO:2所示的编号。
更具体地，β链可变域中所述突变的具体形式包括D96V、E98I、L99P、L100E/I/N/P/V、F101D/H/N/Y、Y102A/K/N/P/V、N103A/D/E/P、E104L/V、Q105P/T、F106H中的一组或几组。
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90％；优选地，至少92％；更优选地，至少94％(如，可以是至少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％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。
优选地，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。
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KASEKIFYV-HLA A0201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KASEKIFYV-HLA A0201复合物之间的结合亲和力即认为是野生型TCR与KASEKIFYV-HLA A0201复合物之间的结合亲和力。同样地，如果测得本发明TCR与KASEKIFYV-HLA A0201复合物之间的结合亲和力是参比TCR与KASEKIFYV-HLA A0201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KASEKIFYV-HLA A0201复合物之间的结合亲和力是野生型TCR与KASEKIFYV-HLA A0201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K D成反比)和结合半衰期(表示为T 1/2)。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K D减半。T 1/2计算为In2除以解离速率(K off)。因此，T 1/2翻倍会导致K off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KASEKIFYV-HLA A0201复合物的解离平衡常数K D为1.21E-04M，即121μM，本发明中即认为野生型TCR对KASEKIFYV-HLA A0201复合物的解离平衡常数K D也为121μM。由于TCR的亲和力翻倍将导致K D减半，所以若检测到高亲和力TCR对KASEKIFYV-HLA A0201复合物的解离平衡常数K D为1.21E-05M，即12.1μM，则说明该高亲和力TCR对KASEKIFYV-HLA A0201复合物的亲和力是野生型TCR对KASEKIFYV-HLA A0201复合物的亲和力的10倍。本领域技术人员熟知K D值单位间的换算关系，即1M＝10 6μM，1μM＝1000nM，1nM＝1000pM。
在本发明的一个优选例中，所述TCR与KASEKIFYV-HLA A0201复合物的亲和力是野生型TCR的至少2倍；优选地，至少5倍；更优选地，至少10倍。
在另一优选例中，所述TCR与KASEKIFYV-HLA A0201复合物的亲和力是野生型TCR的至少50倍；优选地，至少100倍；更优选地，至少300倍。
具体地，所述TCR对KASEKIFYV-HLA A0201复合物的解离平衡常数KD≤60μM；
在另一优选例中，所述TCR对KASEKIFYV-HLA A0201复合物的解离平衡常数为1μM≤KD≤50μM。
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KASEKIFYV-HLA-A0201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 PCTCN2020113583-appb-000003
Figure PCTCN2020113583-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异质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异质二聚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KASEKIFYV-HLA-A0201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：TSESDYY，CDR2α：QEAYKQQN，CDR3α：AYRSGIIQGAQKLV和β链可变域的3个CDR分别为CDR1β：PRHDT，CDR2β：FYEKMQ，CDR3β：ASSSDRELLFYNEQF。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和图5b所示。以此筛选出对KASEKIFYV-HLA A0201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。
本发明中单链模板TCRα链可变域SEQ ID NO:3的3个CDR，即CDR1、CDR2和CDR3分别位于SEQ ID NO:1的第27-33位、第51-58位和第93-106位。
本发明中单链模板TCRβ链可变域SEQ ID NO:4的3个CDR，即CDR1、CDR2和CDR3分别位于SEQ ID NO:2的第27-31位、第49-54位和第92-106位。因此，氨基酸残基编号采用SEQ ID NO:4所示的编号，96D即为CDR3β的第5位D、98E即为CDR3β的第7位E、 99L即为CDR3β的第8位L、100L即为CDR3β的第9位L、101F即为CDR11β的第10位F、102Y即为CDR3β的第11位Y、103N即为CDR3β的第12位N、104E即为CDR3β的第13位E、105Q即为CDR3β的第14位Q和106F即为CDR3β的第15位F。
本发明的对KASEKIFYV-HLA-A0201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KASEKIFYV-HLA-A0201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TCRα链可变域氨基酸序列：SEQ ID NO:1和/或TCRβ链可变域氨基酸序列：SEQ ID NO:13-24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:9的蛋白序列，但与SEQ ID NO:10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。
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“D96V”代表第96位的D被V取代，“E104L/V”代表第104位的E被L取代或被V取代。其他以此类推。
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TCR对所述KASEKIFYV-HLA-A0201复合物的亲和力和/或结合半衰期是野生型TCR的至少2倍，优选地，至少10倍。
(2)本发明的TCR对所述KASEKIFYV-HLA-A0201复合物的亲和力和/或结合半衰期是野生型TCR的至少50倍，优选地，至少100倍，更优选地，可达300倍。
(3)针对靶细胞，转导本发明的高亲和力TCR的效应细胞对靶细胞有很强的激活功能。
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，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可变域(SEQ ID NO:3)及β可变域(SEQ ID NO:4)的氨基酸序列如图2a和图2b所示；其对应的DNA序列分别为SEQ ID NO:5和6，如图3a和图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和图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OD 600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 PCTCN2020113583-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KASEKIFYV-HLA-A0201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KASEKIFYV-HLA-A0201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KASEKIFYV-HLA-A0201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KASEKIFYV(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KASEKIFYV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HiPrep TM16/60S200HR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，并进行序列分析。
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KASEKIFYV-HLA-A0201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KASEKIFYV-HLA A0201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所示(SEQ ID NO:12)，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OD 600＝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KASEKIFYV-HLA-A0201复合物的亲和力。
本发明得到高亲和力TCRβ链可变域氨基酸序列，如图7(1)-图7(12)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KASEKIFYV-HLA-A0201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KASEKIFYV-HLA-A0201复合物的亲和力，如下表2所示。
表2
Figure PCTCN2020113583-appb-000006
由表2可知，本发明得到的高亲和力TCR的亲和力是野生型TCR对KASEKIFYV-HLA-A0201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，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)和 Not Ⅰ(GCGGCCGC)。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50μg/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.转染本发明高亲和力TCR的效应细胞的激活功能实验
本实施例验证了转染本发明高亲和力TCR的效应细胞对靶细胞有很好的特异性激活作用。通过ELISPOT实验检测本发明高亲和力TCR在细胞中的功能及特异性。
本领域技术人员熟知利用ELISPOT实验检测细胞功能的方法。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。所述TCR以及其编号从表2获悉，分别为TCR5(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:17)、TCR6(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:18)、TCR7(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:19)、TCR1(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:13)、TCR9(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:21)、TCR10(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:22)、TCR2(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:14)和TCR8(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:20)，对照组效应细胞标号为野 生型TCR(转染野生型TCR的细胞)和A6(转染其他TCR的细胞)。靶细胞系为K562-A2(A2过表达)、SW620-SSX2(SSX2过表达)和K562-A11(A11过表达)细胞。其中，靶细胞系K562-A2和SW620-SSX2作为阳性肿瘤细胞系；K562-A11为阴性肿瘤细胞系，作为对照。
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10 3个/孔(按转染的阳性率计算)，并设置两个复孔。温育过夜(37℃，5％CO 2)。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11所示，针对阳性靶细胞系，转染本发明高亲和力TCR的效应细胞产生了很好的特异性激活作用，功能要远远好于转染野生型TCR的效应细胞；而转导其他TCR的细胞基本没有产生激活作用。
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 (28)

  1. 一种T细胞受体(TCR)，其特征在于，其具有结合KASEKIFYV-HLA A0201复合物的活性，并且所述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90％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％的序列同源性的氨基酸序列。
  2. 如权利要求1所述的TCR，其特征在于，所述TCR与KASEKIFYV-HLA A0201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TCR，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。
  4. 如权利要求1所述的TCR，其特征在于，所述TCR是可溶的。
  5. 如权利要求1所述的TCR，其中，所述TCRα链可变域包含3个CDR区,所述TCRβ链可变域包含3个CDR区,其特征在于，CDR3α的氨基酸序列为：AYRSGIIQGAQKLV；和/或CDR3β的氨基酸序列为ASSSDRIPPYYNEQF。
  6. 如权利要求1所述的TCR，其特征在于，所述TCRα链可变域包含3个CDR区,3个CDR区的序列为：
    CDR1α：TSESDYY；
    CDR2α：QEAYKQQN；
    CDR3α：AYRSGIIQGAQKLV。
  7. 如权利要求1所述的TCR，其特征在于，所述TCRα链可变域的氨基酸序列为SEQ ID NO:1。
  8. 如权利要求1所述的TCR，其特征在于，所述TCRβ链可变域包含3个CDR区,其中CDR1β的氨基酸序列为PRHDT；和CDR2β的氨基酸序列为：FYEKMQ。
  9. 如权利要求1所述的TCR，其中，所述TCRα链可变域包含3个CDR区,所述TCRβ链可变域包含3个CDR区,其特征在于，所述TCRα链可变域中3个CDR区的氨基酸序列为：
    CDR1α：TSESDYY；
    CDR2α：QEAYKQQN；
    CDR3α：AYRSGIIQGAQKLV；和/或所述TCRβ链可变域中3个CDR区的氨基酸序列为：
    CDR1β：PRHDT；
    CDR2β：FYEKMQ；
    CDR3β：ASSSDRELLFYNEQF；其中在β链的3个CDR中任选地有一个或多个突变。
  10. 如权利要求9所述的TCR，其特征在于，所述β链CDR区中的突变选自D96V、E98I、L99P、L100E/I/N/P/V、F101D/H/N/Y、Y102A/K/N/P/V、N103A/D/E/P、E104L/V、Q105P/T、F106H中的一组或几组，其中，氨基酸残基编号采用SEQ ID NO:2所示的编号。
  11. 如权利要求1所述的TCR，其特征在于，所述TCRβ链可变域的CDR3β选自下组： ASSSDRIPPYYNEQF、ASSSDRELNDAPEQF、ASSSVRELNYVDEQF和
    ASSSDRELLFYNEQF。
  12. 如权利要求1所述的TCR，其特征在于，所述TCR具有选自下组的CDR:
    Figure PCTCN2020113583-appb-100001
  13. 如权利要求1所述的TCR，其特征在于，所述TCR的α链可变域氨基酸序列为：SEQ ID NO:1；和/或所述TCR的β链可变域氨基酸序列选自：SEQ ID NO:13-24。
  14. 如权利要求1所述的TCR，其特征在于，所述TCR选自下组：
    Figure PCTCN2020113583-appb-100002
  15. 如权利要求1所述的TCR，其特征在于，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。
  16. 如权利要求15所述的TCR，其特征在于，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，优选地，所述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。
  17. 如权利要求1所述的TCR，其特征在于，所述TCR为单链TCR，优选地，所述单链TCR是α链可变域和β链可变域由一柔性短肽序列(linker)连接而成。
  18. 如以上任一权利要求所述的TCR，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，所述偶联物为抗-CD3抗体。
  19. 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分子为上述任一权利要求中所述的TCR。
  20. 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1-18中任一项所述的TCR的核酸序列或其互补序列。
  21. 一种载体，其特征在于，所述的载体含有权利要求20中所述的核酸分子。
  22. 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21中所述的载体或染色体中整合有外源的权利要求20中所述的核酸分子。
  23. 一种分离的细胞，其特征在于，所述细胞表达权利要求1-18中任一项所述的TCR；优选地，所述细胞为T细胞。
  24. 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1-18中任一项所述的TCR、或权利要求19中所述的TCR复合物、或权利要求23中所述的细胞。
  25. 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1-18中任一项所述的TCR、或权利要求19中所述的TCR复合物、或权利要求23中所述的细胞、或权利要求24中所述的药物组合物。
  26. 权利要求1-18中任一项所述的T细胞受体、权利要求19中所述的TCR复合物或权利要求23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SSX2阳性肿瘤。
  27. 权利要求1-18中任一项所述的T细胞受体、权利要求19中所述的TCR复合物或权利要求23中所述的细胞用作治疗肿瘤的药物。优选地，所述肿瘤为SSX2阳性肿瘤。
  28. 一种制备权利要求1-18中任一项所述的T细胞受体的方法，其特征在于，包括步骤：
    (i)培养权利要求22中所述的宿主细胞，从而表达权利要求1-18中任一项所述的T细胞受体；
    (ii)分离或纯化出所述的T细胞受体。
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