WO2015192757A1 - 高亲和力hcv t细胞受体 - Google Patents

高亲和力hcv t细胞受体 Download PDF

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Publication number
WO2015192757A1
WO2015192757A1 PCT/CN2015/081491 CN2015081491W WO2015192757A1 WO 2015192757 A1 WO2015192757 A1 WO 2015192757A1 CN 2015081491 W CN2015081491 W CN 2015081491W WO 2015192757 A1 WO2015192757 A1 WO 2015192757A1
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tcr
seq
amino acid
variable domain
chain variable
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PCT/CN2015/081491
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English (en)
French (fr)
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李懿
陈磊
张剑冰
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广州市香雪制药股份有限公司
李懿
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Priority to CN201580024400.9A priority Critical patent/CN106459176B/zh
Publication of WO2015192757A1 publication Critical patent/WO2015192757A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Definitions

  • the present invention relates to the field of biotechnology, and more particularly to a T cell receptor (TCR) sufficient to specifically recognize a polypeptide derived from an HCV NS3 protein.
  • TCR T cell receptor
  • the invention also relates to the preparation and use of said receptors.
  • TCR T cell receptor
  • TCR On the T cell membrane, the TCR binds to the constant protein CD3 involved in signaling to form a complex.
  • TCR exists in many forms and is structurally similar, however T cells expressing these TCRs may exist in different anatomical locations and may have different functions.
  • the extracellular portion of the TCR consists of two near-membrane constant domains and two distal membrane variable domains with variable loops similar to the complementarity determining regions (CDRs) of the antibody. It is these loops that form the antigen (pMHC) binding site of the T cell receptor molecule and determine the specificity of this TCR.
  • CDRs complementarity determining regions
  • MHC class I and class II molecular ligands corresponding to TCR are also proteins of the immunoglobulin superfamily but are specific for antigen presentation, and different individuals have different MHCs, thereby presenting different shortness of one protein antigen Peptides to the surface of the respective APC cells.
  • Hepatitis C virus is a major cause of acute hepatitis and long-term liver diseases including cirrhosis and hepatocellular carcinoma.
  • Hepatocytes infected with HCV can degrade viral proteins into short peptides, wherein the KLVALGINAV peptide is derived from the non-structural protein NS3 of HCV, which is loaded by HLA-A2 and presented to the surface of HCV-infected cells to form HCV. Epitope.
  • the KLVALGINAV-HLA A2 complex provides a marker for TCR targeting HCV.
  • TCRs that are capable of targeting HCV markers can be used to deliver cytotoxic or immunostimulatory agents to infected cells, or to T cells, enabling them to destroy cells infected with HCV for what is known as adoptive immunotherapy.
  • the patient is given during the treatment.
  • the ideal TCR has a higher affinity so that the TCR can reside on the targeted cells for a long period of time.
  • a medium affinity TCR because a sharp increase in affinity is associated with loss of antigen specificity in TCR gene-modified CD8 T cells, resulting in non-specific activation of these TCR-transfected CD8 T cells (see Zhao et al. (2007) J Immunol. 179: 5845-54; Robbins et al., (2008) J Immunol. 180: 6116-31; see also WO 2008/038002).
  • Those skilled in the art are therefore committed to developing TCRs that target HCV markers that can be used to meet different purposes.
  • the complex has a higher affinity TCR.
  • TCR T cell receptor
  • the TCR is mutated in its ⁇ chain variable domain shown in SEQ ID NO: 2 and/or in its ⁇ chain variable domain amino acid residue shown in SEQ ID NO: 3;
  • the affinity and/or binding half-life of the TCR to the KLVALGINAV-HLA-A2 complex is at least twice that of wild-type HCV NS3 TCR;
  • amino acid sequence before the ⁇ chain variable domain mutation of the TCR is as shown in SEQ ID NO.: 2.
  • amino acid sequence before the ⁇ chain variable domain mutation of the TCR is as shown in SEQ ID NO.: 3.
  • the alpha chain variable domain of the TCR has at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO.: 2, and/or the beta chain variable domain of the TCR
  • the amino acid sequence set forth in SEQ ID NO.: 3 has at least 80% sequence identity
  • the dissociation equilibrium constant K D of the TCR to the KLVALGINAV-HLA-A2 complex is ⁇ 3.3 ⁇ M.
  • the dissociation equilibrium constant of the TCR to the KLVALGINAV-HLA-A2 complex is 0.5 ⁇ M ⁇ K D ⁇ 3.3 ⁇ M; preferably, 1 ⁇ M ⁇ K D ⁇ 3.3 ⁇ M; more preferably, 1 ⁇ M ⁇ K D ⁇ 2 ⁇ M.
  • the TCR dissociation equilibrium constant K D ⁇ 500nM for the solution KLVALGINAV-HLA-A2 complex preferably, 10pM ⁇ K D ⁇ 500nM.
  • the mutation occurs in the CDR regions of the alpha chain and/or beta chain.
  • the mutation occurs in CDR1 and/or CDR2 and/or CDR3 of the ⁇ chain, and/or in CDR1 and/or CDR2 and/or CDR3 of the ⁇ chain; preferably, said mutation occurs In CDR1 and/or CDR2 and/or CDR3 of the alpha chain, and/or in the CDR3 of the beta strand; more preferably, the mutation occurs in the CDR1 of the alpha chain and/or in the CDR3 of the beta strand.
  • the mutation occurs at one or more amino acid residue positions selected from the group consisting of the ⁇ chain variable domain of SEQ ID NO.: 2: 30S, 31D, 33Y, 53A, 54Y, 55K, 56Q, 57Q, 58N, 96E, 98D, 99K, 100I and 101I.
  • amino acid residue numbering is the number shown in SEQ ID NO.: 2.
  • the mutation occurs at one or more amino acid residue positions selected from the group consisting of the ⁇ -chain variable domain of SEQ ID NO.: 3: 29H, 50Y, 53N, 54S, 97P, 98Y, 99E, 100Q and 101Y.
  • amino acid residue numbering is given by the number shown in SEQ ID NO.: 3.
  • the mutated TCR alpha chain variable domain comprises one or more amino acid residues selected from the group consisting of: 30N; 31E; 33I; 53S; 54D, 54W or 54A; 55N, 55S or 55Y ; 56T, 56K, 56M, 56P or 56N; 57H, 57E, 57D, 57I or 57N; 58S or 58I; 96D; 98N, 98T, 98Q or 98V; 99S, 99Y or 99P; 100L and 101T; The numbering uses the number shown in SEQ ID NO.: 2.
  • the mutated TCR ⁇ chain variable domain comprises one or more amino acid residues selected from the group consisting of 29Y; 50L; 53G; 54H; 97S; 98A, 98L, 98E, 98S or 98G. 99A or 99P; 100L, 100M or 100V; 101V, 101I, 101F or 101S; wherein the amino acid residue number is SEQ ID NO.: The number shown in 3.
  • the amino acid sequence of the alpha chain variable domain of the TCR is selected from the group consisting of: SEQ ID NO.: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 64, 66, 67, 68, 69, 70, 71 and 72.
  • the ⁇ chain variable domain amino acid sequence of the TCR is selected from the group consisting of: SEQ ID NO.: 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 84, 85, 86, 87 and 88.
  • the TCR comprises a combination of alpha and beta chain variable domains as shown in the table below:
  • the TCR is an alpha beta heterodimeric TCR having alpha and beta chain constant domain sequences, the cysteine residues of which form a second between the alpha and beta chain constant domains of the TCR. Sulfur bond.
  • a cysteine residue forms an artificial disulfide bond between the alpha and beta chain constant domains of the TCR.
  • a cysteine residue forming an artificial disulfide bond replaces one or more sets of sites selected from the group consisting of:
  • the hydrophobic core of the TCR alpha chain variable domain and / or beta chain variable domain is mutated.
  • the TCR is a single-chain TCR consisting of an alpha variable domain and a beta variable domain, the alpha variable domain and the beta variable domain being joined by a flexible short peptide sequencer.
  • the hydrophobic core mutation occurs at one or more amino acid residue positions selected from the group consisting of the alpha chain variable domain of SEQ ID NO.: 2: 11M, 21L, 48V and 110I, wherein the amino acid residue numbering is the number shown in SEQ ID NO.: 2.
  • the hydrophobic core mutation occurs at one or more amino acid residue sites selected from the group consisting of the ⁇ -chain variable domain of SEQ ID NO.: 3, wherein: 78L and 81A, wherein Amino acid residue numbering using SEQ ID NO.: The number shown in 3.
  • the alpha chain variable domain of the TCR after the hydrophobic core mutation comprises one or more amino acid residues selected from the group consisting of 11L, 21I, 48L and 110V, wherein the amino acid residue numbering is employed The number shown in SEQ ID NO.: 2.
  • the ⁇ chain variable domain of the TCR after the hydrophobic core mutation comprises one or more amino acid residues selected from the group consisting of 78I and 81L, wherein the amino acid residue numbering is SEQ ID NO. : The number shown in 3.
  • amino acid sequence of the alpha chain variable domain of the TCR is selected from the group consisting of: SEQ ID NO.: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30 and 31.
  • the ⁇ chain variable domain amino acid sequence of the TCR is selected from the group consisting of: SEQ ID NO.: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 46 and 47.
  • the TCR comprises a combination of alpha and beta chain variable domains as shown in the table below:
  • the alpha chain and/or beta chain of the TCR incorporates a conjugate.
  • the conjugate is selected from one or more of the group consisting of:
  • the detectable label comprises: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (electron computed tomography) contrast agent, or an enzyme capable of producing a detectable product.
  • MRI magnetic resonance imaging
  • CT electron computed tomography
  • the therapeutic agent comprises: a radionuclide, a biotoxin, a cytokine (such as IL-2, etc.), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle/nanorod, a virus particle, a liposome, Nanomagnetic particles, prodrug activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (eg, cisplatin) or any form of nanoparticles, and the like.
  • a radionuclide e.g, a biotoxin, a cytokine (such as IL-2, etc.)
  • an antibody an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle/nanorod, a virus particle, a liposome, Nanomagnetic particles, prodrug activating enzymes (eg, DT-diaphorase
  • the therapeutic agent is an anti-CD3 antibody.
  • the conjugate is attached to the C- or N-terminus of the alpha and/or beta chains of the TCR.
  • the amino acid sequence of the ⁇ chain variable domain of the TCR bound to the conjugate is selected from the group consisting of: SEQ ID NO.: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 64, 66, 67, 68, 69, 70, 71 and 72; and/or the beta strand variable domain amino acid sequence of the TCR bound to the conjugate is selected from the group consisting of: SEQ ID NO.: 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 84, 85, 86, 87 and 88.
  • the amino acid sequence of the TCR ⁇ chain fused to the anti-CD3 antibody is selected from the group consisting of SEQ ID NO.: 95, 96 and 97.
  • the alpha chain variable domain amino acid sequence of the TCR bound to the conjugate is selected from the group consisting of: SEQ ID NO.: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30 and 31; and/or the beta strand variable domain amino acid sequence of the TCR bound to the conjugate is selected from the group consisting of: SEQ ID NO.: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 46 and 47.
  • the amino acid sequence after fusion of the TCR with an anti-CD3 antibody is selected from the group consisting of SEQ ID NO.: 89, 90, 91, 92, 93 and 94.
  • a multivalent TCR complex comprising at least two TCR molecules, and And at least one of the TCR molecules is the TCR of any of the first aspects of the invention.
  • a third aspect of the invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule of the first aspect of the invention or a complement thereof;
  • a vector comprising the nucleic acid molecule of the third aspect of the invention is provided.
  • a host cell comprising the vector of the fourth aspect of the invention or the nucleic acid molecule of the third aspect of the invention integrated with exogenous in the chromosome is provided.
  • an isolated cell expressing the TCR of the first aspect of the invention in a sixth aspect of the invention, there is provided an isolated cell expressing the TCR of the first aspect of the invention.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier, a TCR according to the first aspect of the invention, a TCR complex according to the second aspect of the invention, or The cell of the sixth aspect of the invention.
  • a method for treating a disease comprising administering an appropriate amount of the TCR according to the first aspect of the present invention, the TCR complex of the second aspect of the present invention, and the present invention to a subject in need of treatment
  • the cell of the sixth aspect, or the pharmaceutical composition of the seventh aspect of the invention comprising administering an appropriate amount of the TCR according to the first aspect of the present invention, the TCR complex of the second aspect of the present invention, and the present invention.
  • a use of the T cell receptor of the first aspect of the invention for the preparation of a medicament for treating a tumor or an HCV virus infection.
  • a method for the preparation of the T cell receptor of the first aspect of the invention comprising the steps of:
  • Figure 1a and Figure 1b show the wt-NS3 TCR alpha chain variable domain amino acid sequence (SEQ ID NO: 2) and the beta chain variable domain amino acid sequence (SEQ ID NO: 3), respectively.
  • Figure 2a and Figure 2b show the wt-NS3 TCR alpha chain variable domain DNA sequence (SEQ ID NO: 4) and the beta chain variable domain DNA sequence (SEQ ID NO: 5), respectively.
  • Figure 3a shows the amino acid sequence of wt-NS3sTv (SEQ ID NO: 6) with the hydrophobic core mutation site in bold letters;
  • Figure 3b shows the DNA sequence encoding wt-NS3sTv (SEQ ID NO: 7).
  • Figure 4a and Figure 4b show the alpha chain variable domain amino acid sequence (SEQ ID NO: 8) and the beta chain variable domain amino acid sequence (SEQ ID NO: 9), respectively, of wt-NS3sTv.
  • Figure 5 shows the linked short linker sequence of wt-NS3sTv (SEQ ID NO: 10).
  • Figure 6 shows the results of ELISA of HCV NS3sTv monoclonal phage after 3 rounds of screening.
  • Figure 7a-s shows high affinity for the HCV NS3KLVALGINAV-HLA-A2 complex, respectively
  • the HCV NS3sTv alpha chain variable domain amino acid sequence SEQ ID NOS: 11-22 and 25-31
  • the mutated residues are shown in bold and underlined.
  • Figure 8a-o shows the HCV NS3sTv ⁇ chain variable domain amino acid sequence (SEQ ID NOS: 32-41 and 43-47) with high affinity for the HCV NS3KLVALGINAV-HLA-A2 complex, with the mutated residues in bold and underlined. display.
  • Figure 9a and Figure 9b show the ⁇ -chain extracellular amino acid sequence of wt-NS3 TCR (SEQ ID NO: 48) and the ⁇ -chain extracellular amino acid sequence (SEQ ID NO: 49), respectively, with a cysteine residue introduced by the constant region.
  • the base is indicated by italics and underlined.
  • Figure 10a and Figure 10b show the alpha-strand extracellular DNA sequence of wt-NS3 TCR (SEQ ID NO: 50) (constant region containing introduced cysteine) and beta-strand extracellular DNA sequence (SEQ ID NO: 51), respectively. (The constant region contains the introduced cysteine).
  • Figure 11a-s shows the HCV NS3 TCR alpha chain variable domain amino acid sequence (SEQ ID NO: 52-61, 63, 64, 66-72) with high affinity for the HCV NS3KLVALGINAV-HLA-A2 complex, mutated residues Displayed in bold and underlined.
  • Figure 12a-o shows the HCV NS3 TCR ⁇ chain variable domain amino acid sequence (SEQ ID NOS: 73-82 and 84-88) with high affinity for the HCV NS3KLVALGINAV-HLA-A2 complex, with the mutated residues in bold and underlined display.
  • Figure 13 shows a BIAcore map of the affinity determination of wt-NS3 TCR for the KLVALGINAV-HLA-A2 complex.
  • Figures 14a and 14b show the amino acid sequences of the N- and C-terminal fusion anti-CD3 scFv, respectively, in the sTv molecule (alpha chain variable domain SEQ ID NO: 8 and beta chain variable domain SEQ ID NO: 32).
  • Figures 15a and 15b show the amino acid sequences of the N- and C-terminal fusion anti-CD3 scFv, respectively, in the sTv molecule (alpha chain variable domain SEQ ID NO: 17 and beta chain variable domain SEQ ID NO: 33).
  • Figures 16a and 16b show the amino acid sequences of the N- and C-terminal fusion anti-CD3 scFv, respectively, in the sTv molecule (alpha chain variable domain SEQ ID NO: 14 and beta chain variable domain SEQ ID NO: 32).
  • Figures 17a and 17b show the fusion of the N-terminal and C-terminal fusion anti-CD3 scFv of the sTv molecule ( ⁇ chain variable domain SEQ ID NO: 8 and ⁇ chain variable domain SEQ ID NO: 32) to KLVALGINAV-HLA-A2, respectively.
  • Figures 18a and 18b show the fusion molecule of the N-terminal and C-terminal fusion anti-CD3 scFv of the sTv molecule ( ⁇ chain variable domain SEQ ID NO: 17 and ⁇ chain variable domain SEQ ID NO: 33) to KLVALGINAV-HLA-A2, respectively.
  • Figures 19a and 19b show the fusion of the N-terminal and C-terminal fusion anti-CD3 scFv of the sTv molecule ( ⁇ chain variable domain SEQ ID NO: 14 and ⁇ chain variable domain SEQ ID NO: 32) to KLVALGINAV-HLA-A2, respectively.
  • Figure 20 shows the amino acid sequence of the anti-CD3 scFv and the TCR ⁇ -chain fusion molecule.
  • Figures 21a and 21b show the results of experiments with T2 cells stained with high affinity sTv molecules of the invention.
  • Figure 22 is a result of Elisa experiment of a fusion molecule-activated T cell of a high affinity HCV NS3sTv and an anti-CD3 antibody of the present invention.
  • Figure 23 is a result of Elispot experiment of fusion molecule-activated T cells of high affinity HCV NS3 TCR and anti-CD3 antibody of the present invention.
  • Figure 24 is a graph showing the results of cell killing experiments of fusion molecules of high affinity HCV NS3sTv and anti-CD3 antibodies of the present invention.
  • Figure 25 is a graph showing the results of cell killing experiments of fusion molecules of high affinity HCV NS3 TCR and anti-CD3 antibodies of the present invention.
  • the inventors obtained a recognition of KLVALGINAV peptide (derived from extensive and intensive research) High affinity T cell receptor (TCR) of the HCV NS3 protein) presented as a peptide-HLA-A2 complex.
  • TCR High affinity T cell receptor
  • the TCR of the present invention is mutated in its ⁇ chain variable domain and/or ⁇ chain variable domain relative to wild-type HCV NS3 TCR, and the affinity and/or binding half-life of the TCR of the present invention to the above KLVALGINAV-HLA A2 complex is wild type. At least twice the HCV NS3TCR.
  • TCR T cell receptor
  • the International Immunogenetics Information System can be used to describe TCR.
  • the native alpha-beta heterodimeric TCR has an alpha chain and a beta chain.
  • each strand comprises a variable region, a junction region, and a constant region
  • the beta strand typically also contains a short polymorphic region between the variable region and the junction region, but the polymorphic region is often considered part of the junction region.
  • the TCR junction region was determined by the unique IMGT TRAJ and TRBJ nomenclature, and the constant region was determined by IMGT TRAC and TRBC nomenclature.
  • Each variable region comprises three CDRs (complementarity determining regions) chimeric in a framework sequence, one of which is a CDR3, which is recombined from a variable region and a junction region, and is referred to as a hypervariable region.
  • CDR3 complementarity determining regions
  • the alpha chain variable regions (V ⁇ ) can be divided into several classes, and the beta chain variable regions (V ⁇ ) are also classified into several classes.
  • the types of different V ⁇ types and V ⁇ are referred to by the numbers of TRAV and TRBV, 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 ⁇ -chain constant domain has the following symbols: TRBC1*01 or TRBC2*01, where “TR” represents a T cell receptor gene; “B” represents a ⁇ chain gene; C represents a constant region; “*01” represents an allele 1.
  • TRBC1*01 or TRBC2*01 where “TR” represents a T cell receptor gene; “B” represents a ⁇ chain gene; C represents a constant region; “*01” represents an allele 1.
  • C1 and C2 there are two possible constant region genes "C1" and "C2".
  • TCR ⁇ variable domain refers to a linked TRAV and TRAJ region
  • TCR alpha constant domain refers to an extracellular TRAC region or a C-terminally truncated TRAC sequence
  • TCR[beta] variable domain refers to the ligated TRBV and TRBD/TRBJ regions
  • the TCR[beta] constant domain refers to the extracellular TRBC region or the C-terminally truncated TRBC sequence.
  • the present invention improves the above wt-NS3TCR to obtain a TCR having high affinity for the KLVALGINAV-HLA A2 complex.
  • a heterodimeric TCR specific for the HCV NS3:1406-1415 (KLVALGINAV)/HLA-A2 complex is referred to as HCV NS3 TCR; a flexible peptide chain will be linked to TCR ⁇ and
  • the TCR composed of the ⁇ variable domain is referred to as sTv; the sTv specific for the HCV NS3:1406-1415 (KLVALGINAV)/HLA-A2 complex is referred to as HCV NS3sTv.
  • the present invention provides a T cell receptor (TCR) comprising a TCR ⁇ variable domain and/or a TCR ⁇ variable domain having the properties of a KLAVALGINAV (SEQ ID NO: 1) HLA-A2 complex, wherein (i) the TCR is SEQ ID NO: 2 has its alpha chain variable domain amino acid and/or a mutation in its beta chain variable domain amino acid as shown in SEQ ID NO: 3; and (ii) said TCR to said KLVALGINAV (SEQ ID NO: 1)
  • the affinity of the HLA-A2 complex is at least twice that of wild-type HCV NS3 TCR.
  • the mutation in the alpha chain variable domain and/or the beta chain variable domain amino acid residue in (i) above occurs in one or more complementarity determining regions (CDRs) of the alpha and/or beta variable domains.
  • CDRs complementarity determining regions
  • the mutations enable a higher interaction force and/or a slower dissociation rate between the TCR of the invention and the KLVALGINAV (SEQ ID NO: 1) HLA-A2 complex.
  • Binding may be measured by any suitable method, the affinity (dissociation equilibrium constant and inversely proportional to K D) and half-life of binding (expressed as T 1/2). It should be understood that doubling the affinity of the TCR will result in a halving of K D . T 1/2 is calculated as In2 divided by the dissociation rate (K off ). Therefore, doubling T 1/2 will cause K off to be halved.
  • the same test protocol is used to detect the binding affinity or binding half-life of a given TCR several times, for example 3 or more times, and the average of the results is taken. In a preferred embodiment, these measurements are performed using the surface plasmon resonance (BIAcore) method of the examples herein.
  • the method detects wt-NS3 TCR (the artificial disulfide bond is introduced between the constant domains, and the extracellular alpha and beta chain amino acid sequences are SEQ ID NO: 48 and SEQ ID NO: 49, respectively) to KLVALGINAV (SEQ ID NO: 1).
  • the HLA-A2 complex had a K D of 6.7 ⁇ M and a K off of 5.1 ⁇ 10 -1 s -1 (i.e., T 1/2 was about 1.4 s). It should be understood that the introduction of this artificial disulfide bond does not affect the affinity of the TCR.
  • the affinity of the wild-type HCV NS3 TCR for the KLVALGINAV-HLA-A2 complex is identical to that of the wt-NS3 TCR for the KLVALGINAV-HLA-A2 complex.
  • Mutations can be carried out by any suitable method, including but not limited to those based on polymerase chain reaction (PCR), restriction enzyme-based cloning or linkage-independent cloning (LIC) methods. Many standard molecular biology textbooks detail these methods. For more details on polymerase chain reaction (PCR) mutagenesis and cloning based on restriction enzymes, see 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 linkage-independent cloning
  • the method of producing the TCR of the present invention may be, but is not limited to, screening for a TCR having a high affinity for the KLVALGINAV-HLA-A2 complex from a diverse library of phage particles displaying such TCR, which is well known to those skilled in the art. Library and filtering methods.
  • genes comprising amino acids similar to the alpha and beta chain variable domain amino acids of wt-NS3 TCR and the genes of the alpha and beta chain variable domain amino acids of the modified wt-NS3 TCR can be used to prepare template TCRs.
  • the changes required to produce the mutant high affinity TCR of the invention are then introduced into the DNA encoding the variable domain of the template TCR.
  • the desired mutation can be introduced by a number of methods, such as site-directed mutagenesis.
  • the TCR of the invention comprises its alpha chain variable domain having at least 80%, preferably at least 85%, or more preferably at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 (ie, less than 20) %, preferably less than 15%, or more preferably less than 10% of the TCR alpha chain variable domain amino acid residues shown in SEQ ID NO: 2 are mutated) and/or the TCR of the invention comprises a variable beta chain thereof
  • the domain has at least 80%, preferably at least 85%, or more preferably at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 3 (ie, less than 20%, preferably less than 15%, or More preferably, less than 10% of the TCR ⁇ chain variable domain amino acid residues shown in SEQ ID NO: 3 are mutated).
  • Sequence identity can be determined by, for example, manual alignment using a computer program.
  • the TCR of the invention comprises the alpha chain variable domain set forth in SEQ ID NO: 2 but at amino acid residues 30S, 31D, 33Y, 53A, 54Y, 55K, 56Q, 57Q, 58N thereof , one or more mutations in 96E, 98D, 99K, 100I and 101I (using the numbering shown in SEQ ID NO: 2), and/or comprising the beta chain variable domain shown in SEQ ID NO: 3 but in One or more mutations (using the numbering shown in SEQ ID NO: 3) are included in amino acid residues 29H, 50Y, 53N, 54S, 97P, 98Y, 99E, 100Q, 101Y.
  • a TCR of the invention may have an alpha chain variable domain amino acid residue 30N; 31E; 33I; 53S; 54D, 54W or 54A; 55N, 55S or 55Y; 56T, 56K, 56M, 56P or 56N; 57H, 57E, 57D , 57I or 57N; 58S or 58I; 96D; 98N, 98T, 98Q or 98V; 99S, 99Y or 99P; one or more of 100L and 101T (using the numbering shown in SEQ ID NO: 2) and/or ⁇ chain variable domain amino acid residue 29Y; 50L; 53G; 54H; 97S; 98A, 98L, 98E, 98S or 98G; 99A or 99P; 100L, 100M or 100V; and a group of 101V, 101I, 101F or 101S Or multiple groups (using the number shown in SEQ ID NO: 3).
  • the high affinity TCR of the present invention comprises the alpha chain variable domain amino acid sequences SEQ ID NO: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 64, 66, 67, 68, One of 69, 70, 71, 72 and/or the ⁇ chain variable domain amino acid sequence SEQ ID NO: 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, One of 84, 85, 86, 87, 88.
  • the TCR alpha chain of the alpha chain variable domain amino acid sequence (SEQ ID NO: 2) containing wt-NS3 TCR can comprise SEQ ID NO: 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 TCR ⁇ chain binding of one of 84, 85, 86, 87, 88.
  • the TCR ⁇ chain of the ⁇ variable domain amino acid sequence (SEQ ID NO: 3) containing wt-NS3 TCR may comprise SEQ ID NO: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
  • the TCR alpha chain of one of 63, 64, 66, 67, 68, 69, 70, 71, 72 is bound.
  • TCR alpha chain variable domain amino acid sequence SEQ ID NO: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 64, 66, 67, 68, 69, 70, 71
  • the TCR alpha chain of one of 72 may be SEQ ID NO: 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 84, 85, 86, 87, 88 comprising the TCR ⁇ chain variable domain amino acid sequence.
  • One of the TCR beta chains binds.
  • a TCR of the invention is a moiety having at least one TCR alpha and/or TCR beta chain variable domain. They usually comprise both a TCR alpha chain variable domain and a TCR beta chain variable domain. They may be alpha beta heterodimers or single stranded forms or any other form that is stable. In adoptive immunotherapy, the full length strand of the alpha beta heterodimeric TCR (including the cytoplasmic and transmembrane domains) can be transfected.
  • the TCR of the present invention can be used as a targeting agent for delivering a therapeutic agent to an antigen presenting cell or in combination with other molecules to prepare a bifunctional polypeptide to direct effector cells, in which case the TCR is preferably in a soluble form.
  • the TCR of the invention may be a TCR that introduces an artificial disulfide bond between the residues of its alpha and beta chain constant domains, as in the literature (Boulter et al. (2003) Protein Engineering (Protein Engineering) Said in 16:707-711).
  • the cysteine residue forms an artificial disulfide bond between the alpha and beta chain constant domains of the TCR.
  • a Thr248 residue of the exon 1 of TRAC*01 and a cysteine residue of Ser57 of the exon 1 of TRBC1*01 or TRBC2*01 are substituted to form a disulfide bond.
  • Other sites for introducing a cysteine residue to form a disulfide bond may also be: Thr45 of TRAC*01 exon 1 and Ser77 of TRBC1*01 or TRBC2*01 exon 1; TRAC*01 exon 1 of Tyr10 and TRBC1*01 or TRBC2*01 exon 1 of Ser17; TRAC*01 exon 1 of Thr45 and TRBC1*01 or TRBC2*01 exon 1 of Asp59; TRAC*01 exon 1 Ser15 and TRBC1*01 or TRBC2*01 exon 1 of Glu15.
  • a maximum of 15, or a maximum of 10, or a maximum of 8 or fewer amino acids may be truncated at one or more C-termini of the TCR constant domain of the invention such that it does not include a cysteine residue to achieve deletion of the native
  • the purpose of the disulfide bond can also be achieved by mutating a suitable cysteine residue to another amino acid.
  • the TCR of the present invention may comprise an artificial disulfide bond introduced between residues of its ⁇ and ⁇ chain constant domains.
  • the constant domains may or may not contain the introduced artificial disulfide bonds as described above, and the TCRs of the present invention may each contain a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence.
  • the TRAC constant domain sequence of TCR and the TRBC1 or TRBC2 constant domain sequence can be joined by a native disulfide bond present in the TCR.
  • the TCR of the present invention further comprises a TCR having a mutation in its hydrophobic core region, and the mutation of these hydrophobic core regions is preferably a mutation capable of increasing the stability of the TCR of the present invention, as in the publication number It is described in the patent document of WO2014/206304.
  • Such a TCR can be mutated at its position in the following variable domain hydrophobic core: (alpha and/or beta chain) variable region amino acids 11, 13, 19, 21, 53, 76, 89, 91, 94, and / Or the ⁇ -chain J gene (TRAJ) short peptide amino acid position reciprocal position 3, 5, 7 and/or ⁇ chain J gene (TRBJ) short peptide amino acid position reciprocal position 2, 4, 6 where the amino acid sequence position number The location number listed in the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • the TCR in which the hydrophobic core region is mutated in the present invention may be a super-stable TCR variable domain (sTv) composed of a flexible peptide chain connecting the variable domains of the ⁇ and ⁇ chains of the TCR.
  • sTv super-stable TCR variable domain
  • the flexible peptide chain of the present invention may be any peptide chain suitable for linking the TCR alpha to the beta variable domain.
  • the template strand constructed in Example 1 of the present invention for screening high-affinity HCV NS3sTv contains a mutation in the hydrophobic core region. It should be noted that mutations in the hydrophobic core region do not affect the binding affinity and binding half-life of sTv.
  • the alpha variable domain hydrophobic core amino acid residue 11M of the TCR of the invention (ie, the 11th position of the alpha variable region listed in IMGT), using the numbering set forth in SEQ ID NO: 2, 21L (ie, the 21st position of the alpha variable region listed in IMGT), 48V (ie, the 53rd position of the alpha variable region listed in IMGT) or 110I (ie, the amino acid reciprocal of the alpha chain J gene short peptide listed in IMGT) One or more mutations in position 3) and/or the numbering shown in SEQ ID NO: 3, the TCR ⁇ chain variable domain hydrophobic core amino acid residue 78L (ie, the ⁇ chain variable region listed in IMGT) Mutation occurred in one or more of position 91) or 81A (ie, position 94 of the beta variable region listed in IMGT).
  • the alpha variable domain hydrophobic core of the invention comprises one or more of amino acid residues 11L, 21I, 48L or 110V and/or employed, using the numbering set forth in SEQ ID NO: The numbering set forth in SEQ ID NO: 3, the TCR ⁇ variable domain comprising one or more of amino acid residues 78I or 81L.
  • the high affinity TCR of the invention further comprises an alpha chain variable domain amino acid sequence of SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 27 , 28, 29, 30, 31 and/or ⁇ chain variable domain amino acid sequences SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, One of 46, 47.
  • the TCR alpha chain of the alpha chain variable domain amino acid sequence (SEQ ID NO: 8) containing wt-NS3sTv can comprise SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41
  • the TCR ⁇ chain of one of 43, 43, 44, 46, 47 is bound.
  • the TCR ⁇ chain of the ⁇ chain variable domain amino acid sequence (SEQ ID NO: 9) containing wt-NS3sTv may comprise SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20
  • the TCR alpha chain of one of 21, 22, 25, 26, 27, 28, 29, 30, 31 is bound.
  • the TCR alpha chain of one of 31 may be SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 46, 47 comprising the ⁇ chain variable domain amino acid sequence.
  • One of the TCR beta chains binds.
  • 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, such as a tetrameric domain of p53 to produce a tetramer, or more A complex formed by combining a TCR of the invention with another molecule.
  • the TCR complexes of the invention can be used to track or target cells that present a particular antigen in vitro or in vivo, as well as intermediates that produce other multivalent TCR complexes for such applications.
  • the TCR of the present invention may be used singly or in combination with the conjugate in a covalent or other manner, preferably in a covalent manner.
  • the conjugate comprises a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of a cell presenting a KLVALGINAV HLA-A2 complex), a therapeutic agent, a PK (protein kinase) modified moiety or any of these substances The combination is combined or coupled.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (electron computed tomography) contrast agents, or capable of producing detectable products Enzyme.
  • Therapeutic agents that can be combined or coupled to the TCRs of the 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 51, 565); 3. Cytokines such as IL-2, etc.
  • Liposomes (Mamot et al., 2005, Cancer research 65, 11631); 9. Nanomagnetic particles; 10. Prodrug activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. Chemotherapeutic agents (eg, cisplatin) or any form Nanoparticles, etc.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • Chemotherapeutic agents eg, cisplatin or any form Nanoparticles, etc.
  • the antibody or fragment thereof to be combined with the TCR of the present invention includes an anti-T cell or an NK-cell determining antibody, such as an anti-CD3 or an anti-CD28 or an anti-CD16 antibody, and the binding of the above antibody or a fragment thereof to the TCR can effect the effector cell. Targeting better targets target cells.
  • a preferred embodiment is the binding of a TCR of the invention to an anti-CD3 antibody or a functional fragment or variant of the anti-CD3 antibody.
  • the TCR-anti-CD3 single-chain antibody fusion of the present invention comprises a TCR alpha chain variable domain amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30, 31, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 64, 66, 67, 68, 69, 70, 71, 72 and a TCR ⁇ chain variable domain amino acid sequence selected from the group consisting of SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 46, 47, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 84, 85, 86, 87, 88.
  • TCR alpha chain variable domain amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30, 31, 52, 53
  • amino acid sequence of the TCR and anti-CD3 single chain antibody fusions of the invention may be selected from one of the following amino acid sequences: SEQ ID NOs: 89, 90, 91, 92, 93 and 94. More specifically, the amino acid sequence of the TCR ⁇ chain of the present invention and the anti-CD3 single chain antibody fusion may be selected from one of the following amino acid sequences: 95, 96 and 97.
  • the invention also relates to nucleic acid molecules encoding the TCRs of the invention.
  • the nucleic acid molecule of the invention may be in the form of DNA or in the form of RNA.
  • the DNA can be a coding strand or a non-coding strand.
  • a nucleic acid sequence encoding a TCR of the invention may be the same or a degenerate variant of the nucleic acid sequence set forth in the Figures of the invention.
  • a "degenerate variant" in the present invention refers to a nucleic acid sequence which encodes a protein sequence having SEQ ID NO: 2 but differs from the sequence of SEQ ID NO: 4.
  • 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, recombinant methods or synthetic methods. At present, it has been possible to obtain a DNA sequence encoding the TCR (or a fragment thereof, or a derivative thereof) of the present invention completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • the invention also relates to vectors comprising the nucleic acid molecules of the invention, as well as host cells genetically engineered using the vectors or coding sequences of the invention.
  • the invention also encompasses isolated cells, particularly T cells, which express the TCR of the invention.
  • isolated cells particularly T cells, which express the TCR of the invention.
  • T cells There are a number of methods suitable for T cell transfection with DNA or RNA encoding the high affinity TCR of the invention (e.g., Robbins et al., (2008) J. Immunol. 180: 6116-6131).
  • T cells expressing the high affinity TCR of the invention can be used in adoptive immunotherapy.
  • Those skilled in the art will be aware of many suitable methods for performing adoptive therapy (e.g., 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 a TCR of the present invention, or a TCR complex of the present invention, or a cell which presents the TCR of the present invention.
  • the invention also provides a method of treating a disease comprising administering to a subject in need of treatment an appropriate amount of a TCR of the invention, or a TCR complex of the invention, or a cell presenting a TCR of the invention, or a pharmaceutical composition of the invention.
  • amino acid names in this article are identified by the international common single letter, and the corresponding amino acid names are abbreviated as: 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);
  • the TCR of the invention further comprises up to 5, preferably up to 3, more preferably up to 2, optimally 1 amino acid (especially an amino acid located outside the CDR regions) of the TCR of the invention, which is similar in nature Replace the amino acid with a similar amino acid and still be able to maintain its functionality.
  • the replacement of these amino acids of similar or similar nature is preferably carried out according to Table A.
  • the present invention also encompasses a TCR slightly modified for the TCR of the present invention.
  • Modifications include: chemically derivatized forms of the TCRs of the invention, such as acetylation or carboxylation.
  • Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of the TCRs of the invention or in further processing steps. Such modification can be accomplished by exposing the TCR to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme.
  • Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine.
  • TCRs that have been modified to enhance their anti-proteolytic properties or to optimize solubility properties.
  • the TCR, TCR complexes of the invention or TCR transfected T cells of the invention can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the TCR, multivalent TCR complex or cell of the invention is typically provided as part of a sterile pharmaceutical composition, which typically comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be in any suitable form (depending on the method desired for administration to a patient). It can be provided in 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 can include a plurality of said unit dosage forms.
  • the TCRs of the invention may be used alone or in combination or in combination with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent.
  • pharmaceutical carriers which do not themselves induce the production of antibodies harmful to the individual receiving the composition and which are not excessively toxic 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 a liquid 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.
  • the therapeutic compositions can be formulated as injectables, such as liquid solutions or suspensions; solid forms such as liquid carriers, which may be formulated in solution or suspension prior to injection.
  • composition of the invention can be administered by conventional routes including, but not limited to, intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably gastrointestinal.
  • External includes subcutaneous, intramuscular or intravenous.
  • the subject to be prevented or treated may be an animal; especially a human.
  • a pharmaceutical composition of various dosage forms may be employed depending on the use.
  • an injection, an oral preparation, or 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, disintegrating agents, binders, lubricants, diluents, buffers, isotonicity Isotonicities, preservatives, wetting agents, emulsifiers, dispersing agents, stabilizers and solubilizers, and the formulation process can be carried out in a customary manner depending on the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrating agents, binders, lubricants, diluents, buffers, isotonicity Isotonicities, preservatives, wetting agents, emulsifiers, dispersing agents, stabilizers and solubilizers, and the formulation process can be carried out in a customary manner depending on the dosage form.
  • compositions of the invention may also be administered in the form of sustained release agents.
  • the TCR of the present invention can be incorporated into a pill or microcapsule in which the sustained release polymer is used as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated.
  • the sustained-release polymer include ethylene-vinyl acetate copolymer, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, and lactic acid polymer.
  • a lactic acid-glycolic acid copolymer or the like is preferably exemplified by a biodegradable polymer such as a lactic acid polymer and a lactic acid-glycolic acid copolymer.
  • the TCR or TCR complex of the present invention as an active ingredient or the cell presenting the TCR of the present invention may be based on the body weight, age, sex, and degree of symptoms of each patient to be treated. And reasonable to determine, and ultimately the doctor determines the reasonable amount.
  • the present invention uses a hydrophobic core-mutated high-stability sTv molecule as a template to screen a TCR having high affinity for the KLVALGINAV-HLA-A2 complex, and has antigen specificity and cell killing efficacy.
  • the affinity and/or binding half-life of the TCR of the present invention to the KLVALGINAV-HLA-A2 complex is at least twice that of the wild-type HCV NS3 TCR.
  • the affinity and/or binding half-life of the high affinity TCR of the present invention to the KLVALGINAV-HLA-A2 complex can reach 10 3 -10 5 times or more of the wild type HCV NS3 TCR.
  • the fusion molecule of the high affinity HCV NS3TCR of the present invention and the anti-CD3 antibody can activate T cells well, and the fusion molecule of the wild type TCR and the anti-CD3 antibody has substantially no activation effect on T cells.
  • a fusion molecule comprising the high-intensity TCR of the present invention and an anti-CD3 antibody is capable of redirecting T cells, thereby killing target cells.
  • E. coli DH5 ⁇ is purchased from Tiangen, E. coli BL21 (DE3) and purchased from Tiangen, E. coli Tuner (DE3). From Novagen, plasmid pET 28a was purchased from Novagen.
  • TCR T cell receptor specific for the HCV NS3KLVALGINAV-HLA A2 complex
  • wt-NS3 T cell receptor specific for the HCV NS3KLVALGINAV-HLA A2 complex
  • TCR TCR
  • the variable domain amino acid sequences of its alpha and beta chains SEQ ID NOS: 2 and 3
  • Figures 1a and 1b respectively
  • the corresponding DNA sequences SEQ ID NOS: 4 and 5
  • Figure 2a A platform technique for constructing a highly stable sTv by mutating a hydrophobic core is disclosed in the patent document WO 2014/206304.
  • the core site in the high stability sTv platform technology was introduced into the variable domains of the ⁇ and ⁇ chains of wt-NS3TCR, and a flexible short peptide was used to link the variable domains of the ⁇ and ⁇ chains to construct a high screening.
  • Affinity The template strand of HCV NS3sTv, which is abbreviated as wt-NS3sTv.
  • the introduction of the above-mentioned core sites employs a site-directed mutagenesis method well known to those skilled in the art, that is, introduction of primers for overlap PCR to introduce mutations.
  • Figure 3a shows the amino acid sequence of the template strand wt-NS3sTv (SEQ ID NO: 6), wherein the mutation sites of the hydrophobic core region are indicated by bold letters. Its corresponding DNA sequence is (SEQ ID NO: 7) as shown in Figure 3b.
  • the CDR regions of the wt-NS3sTv ⁇ and ⁇ chains are identical to the CDR regions of the ⁇ and ⁇ chains of wt-NS3 TCR, respectively.
  • the variable domain amino acid sequences of the alpha and beta chains of wt-NS3sTv are shown in Figures 4a (SEQ ID NO: 8) and 4b (SEQ ID NO: 9), respectively, and the flexible short peptide sequence (SEQ ID NO: 10) is as Figure 5 shows.
  • the gene of interest carrying wt-NS3sTv was digested with NcoI and NotI and ligated with the pET 28a vector digested with NcoI and NotI.
  • the ligation product was transformed into E. coli DH5 ⁇ , coated with kanamycin-containing LB plate, inverted culture at 37 ° C overnight, and the positive clones were picked for PCR screening.
  • the positive recombinants were sequenced to determine the correct sequence and the recombinant plasmid was extracted.
  • E. coli BL21 (DE3) for expression.
  • the BL21(DE 3) colonies containing the recombinant plasmid pET28a-sTv prepared in Example 1 were all inoculated into LB medium containing kanamycin, cultured at 37 ° C until the OD 600 was 0.6-0.8, and IPTG was added to the final concentration. The culture was continued at 37 ° C for 4 h at 0.5 mM.
  • 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 fraction, centrifuged at 6000 rpm for 15 min, and collected. body.
  • the inclusion body was dissolved in a buffer (20 mM Tris-HCl pH 8.0, 8 M urea), and the insoluble matter was removed by high-speed centrifugation. The supernatant was fractionated by the BCA method, and then stored at -80 ° C until use.
  • the above-treated wt-NS3sTv was added dropwise to a 125 mL refolding buffer (100 mM Tris-HCl pH 8.1, 0.4 M L-arginine, 5 M urea, 2 mM EDTA, 6.5 mM ⁇ -mercapthoethylamine, 1.87 mM Cystamine) with a syringe.
  • a refolding buffer 100 mM Tris-HCl pH 8.1, 0.4 M L-arginine, 5 M urea, 2 mM EDTA, 6.5 mM ⁇ -mercapthoethylamine, 1.87 mM Cystamine
  • the dialysate was changed to 1 L of pre-cooled buffer (20 mM Tris-HCl pH 8.0), dialysis was continued for 8 h at 4 ° C, and the dialysate was replaced with the same fresh buffer to continue dialysis overnight.
  • the sample was filtered through a 0.45 ⁇ m filter, and the protein was purified by vacuum degassing through an anion exchange column (HiTrap Q HP, GE Healthcare) using a linear gradient of 0-mM NaCl prepared in 20 mM Tris-HCl pH 8.0.
  • the collected fractions were subjected to SDS-PAGE analysis, and the fractions containing wt-NS3sTv were concentrated and further purified by a gel filtration column (Superdex 75 10/300, GE Healthcare), and the target components were also subjected to SDS-PAGE analysis.
  • the eluted fraction for BIAcore analysis was further tested for purity using gel filtration.
  • the conditions were as follows: column Agilent Bio SEC-3 (300A, ⁇ 7.8 ⁇ 300 mm), mobile phase 150 mM phosphate buffer, flow rate 0.5 mL/min, column temperature 25 ° C, UV detection wavelength 214 nm.
  • HCV NS3sTv The binding activity of HCV NS3sTv to the HCV NS3KLVALGINAV-HLA-A2 complex was tested using a BIAcore T200 real-time assay system.
  • the anti-streptavidin antibody (GenScript) was added to a coupling buffer (10 mM sodium acetate buffer, pH 4.77), and then the antibody was passed through a CM5 chip previously activated with EDC and NHS to immobilize the antibody on the surface of the chip. Finally sealed with hydrochloric acid in hydrochloric acid The unreacted activated surface completes the coupling process with a coupling level of approximately 15,000 RU.
  • HCV NS3: 1406-1415 KLVALGINAV
  • HLA A2 complex is flowed through the detection channel, and the other channel is used as a reference channel, and then 0.05 mM biotin was flowed through the chip at a flow rate of 10 ⁇ L/min for 2 min to block the remaining binding sites of streptavidin.
  • the affinity was determined by single-cycle kinetic analysis.
  • the sTv was diluted to 5 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 bonding time of each injection is 120s, and let it dissociate for 600s after the last injection.
  • the chip was regenerated with 10 mM Gly-HCl, pH 1.75, after each round of assay.
  • Kinetic parameters were calculated using BIAcore Evaluation software.
  • E. coli bacterial solution inducing expression of heavy or light chain 100 ml of E. coli bacterial solution inducing expression of heavy or light chain was collected, and the cells were washed once with 8000 g of PBS at 10 ° C for 10 min, and then resuspended by vigorous shaking with 5 ml of BugBuster Master Mix Extraction Reagents (Merck). Incubate for 20 min at room temperature, then centrifuge at 6000 g for 15 min at 4 ° C, discard the supernatant, and collect inclusion bodies.
  • the above-mentioned inclusion weight was suspended in 5 ml BugBuster Master Mix, and incubated at room temperature for 5 min; 30 ml of BugBuster diluted 10 times, mixed, centrifuged at 6000 g for 15 min at 4 ° C; the supernatant was discarded, and 30 ml of BugBuster resuspended inclusion body was diluted 10 times.
  • the synthesized short peptide (NS3: 1408-1415 KLVALGINAV) was dissolved in DMSO to a concentration of 20 mg/ml.
  • the inclusion bodies of the light and heavy chains were dissolved with 8 M urea, 20 mM Tris pH 8.0, 10 mM DTT, and further denatured by adding 3 M guanidine hydrochloride, 10 mM sodium acetate, 10 mM EDTA before renaturation.
  • the NS3:1408-1415KLVALGINAV peptide was added to the refolding buffer at 25 mg/L (final concentration) (0.4 M L-arginine, 100 mM Tris pH 8.3, 2 mM EDTA, 0.5 mM oxidized glutathione, 5 mM reduced valley) Glutathione, 0.2 mM PMSF, cooled to 4 ° C), then added 20 mg / L light chain and 90 mg / L heavy chain (final concentration, heavy chain added three times, 8h / time), renaturation at 4 ° C After at least 3 days to completion, SDS-PAGE can be used to confirm the renaturation.
  • the renaturation buffer was replaced with 10 volumes of 20 mM Tris pH 8.0 for dialysis, and at least two buffers were exchanged to substantially reduce the ionic strength of the solution.
  • the protein solution was filtered through a 0.45 ⁇ m cellulose acetate filter and then loaded onto a HiTrap Q HP (GE General Electric Company) anion exchange column (5 ml bed volume).
  • the protein was eluted using a linear gradient of 0-400 mM NaCl prepared by an Akta Purifier (GE General Electric Company), 20 mM Tris pH 8.0, pMHC was eluted at approximately 250 mM NaCl, peak fractions were collected, and purity was determined by SDS-PAGE.
  • the purified pMHC molecule was concentrated using a Millipore ultrafiltration tube while the buffer was replaced with 20 mM Tris pH 8.0, followed by biotinylation reagent 0.05M Bicine pH 8.3, 10 mM ATP, 10 mM MgOAc, 50 ⁇ M D-Biotin, 100 ⁇ g/ml BirA
  • the enzyme (GST-BirA) was incubated overnight at room temperature and SDS-PAGE was used to determine if biotinylation was complete.
  • the biotinylated labeled pMHC molecule was concentrated to 1 ml using a Millipore ultrafiltration tube, biotinylated pMHC was purified by gel filtration chromatography, and HiPrep was pre-equilibrated with filtered PBS using an Akta Purifier (GE General Electric Company).
  • Akta Purifier GE General Electric Company
  • a TM 16/60 S200 HR column (GE General Electric Company) was loaded with 1 ml of concentrated biotinylated pMHC molecules and then eluted with PBS at a flow rate of 1 ml/min.
  • the biotinylated pMHC molecule appeared as a single peak elution at about 55 ml.
  • the protein-containing fractions were combined, concentrated using a Millipore ultrafiltration tube, protein concentration was determined by BCA method (Thermo), and biotinylated pMHC molecules were dispensed at -80 °C by adding protease inhibitor cocktail (Roche).
  • Phage display technology is a means of generating TCR high affinity variant libraries to identify 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 wt-NS3sTv in Example 1.
  • a library of high affinity sTvs was constructed and panned by mutating the CDR regions in the wt-NS3sTv.
  • Those skilled in the art are familiar with the above methods of building and screening. That is, it is achieved by using a primer having one or more codon changes required and a plasmid containing the relevant sTv strand DNA as a template.
  • the phage library specifically binds to the corresponding antigen, and the ELISA results are shown in Fig. 6. Pick a single clone and perform sequence analysis.
  • the ⁇ chain variable domains of these high-affinity sTv mutants are mutated in amino acids at one or more of the following positions 30S, 31D, 33Y, 53A, 54Y, 55K , 56Q, 57Q, 58N, 96E, 98D, 99K, 100I, 101I and/or using the numbering shown in SEQ ID NO: 3, the ⁇ -chain variable domains of these high-affinity sTv mutants are in one or more of the following positions Amino acid mutations at points 29H, 50Y, 53N, 54S, 97P, 98Y, 99E, 100Q, 101Y.
  • these high affinity sTv mutants have one or more of the following alpha chain variable domain amino acid residues 30N, 31E, 33I, 53S, 54D, 54W, 54A, 55N, 55S, 55Y, 56T, 56K, 56M, 56P, 56N, 57H, 57E, 57D, 57I, 57N, 58S, 58I, 96D, 98N, 98T, 98Q, 98V, 99S, 99Y, 99P, 100L, 101T and / or using the number shown in SEQ ID NO: 3, these high affinity sTv mutants have one or more of the following beta chain variable domain amino acids 29Y, 50L, 53G, 54H, 97S, 98A, 98L, 98E, 98S, 98G, 99A, 99P, 100L, 100M, 100V, 101V, 101I, 101F, 101S.
  • ⁇ -chain variable domains of high affinity HCV NS3sTv (SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30)
  • Specific examples of amino acid sequences of 31, and ⁇ chain variable domains (SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 46, 47) See Figures 7a-u and 8a-p, respectively.
  • the high affinity CDR region mutations screened in Example 4 were introduced into the corresponding sites of the variable domain of wt-NS3 TCR and their affinity for the HCV NS3KLVALGINAV-HLA-A2 complex was detected by BIAcore.
  • the above method of introducing a high affinity mutation site employs a method of site-directed mutagenesis well known to those skilled in the art.
  • the ⁇ -chain and ⁇ -strand extracellular amino acid sequences of the above wt-NS3TCR are shown in Figure 9a (SEQ ID).
  • the extracellular sequence genes of the TCR ⁇ and ⁇ chains to be expressed are synthesized and inserted into the respective methods by the standard methods described in the Molecular Cloning a Laboratory Manual (3rd edition, Sambrook and Russell).
  • the expression vector 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 sequenced to confirm that it was correct.
  • TCR ⁇ and ⁇ chain were transformed into expression plasmid BL21(DE3) by chemical transformation, respectively, and the bacteria were grown in LB medium.
  • the resulting inclusion bodies were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution.
  • the inclusion bodies were finally dissolved in 6 M guanidine hydrochloride, 10 mM dithiothreitol (DTT), 10 mM ethylenediaminetetraacetic acid (EDTA). ), in 20 mM Tris (pH 8.1).
  • the dissolved TCR ⁇ and ⁇ chains were rapidly mixed in 5 M urea, 0.4 M arginine, 20 mM Tris (pH 8.1), 3.7 mM cystamine, 6.6 mM ⁇ -mercapoethylamine (4 ° C) at a final concentration of 1:1. 60 mg/mL. After mixing, the solution was dialyzed against 10 volumes of deionized water (4 ° C), and after 12 hours, deionized water was exchanged for buffer (20 mM Tris, pH 8.0) and dialysis was continued at 4 ° C for 12 hours.
  • the solution after completion of dialysis was filtered through a 0.45 ⁇ M filter, and then purified by an anion exchange column (HiTrap Q HP, 5 ml, GE Healthcare).
  • the TCR containing the refolding successful alpha and beta dimers was confirmed by SDS-PAGE gel.
  • the TCR was then further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare).
  • the purified TCR purity was determined by SDS-PAGE to be greater than 90%, and the concentration was determined by the BCA method.
  • the CDR region mutations in the high affinity HCV NS3sTv are introduced into the corresponding positions of SEQ ID NO: 2 and/or SEQ ID NO: 3 to obtain new TCR alpha and beta chain variable domain amino acid sequences, as shown in Figures 11 and 12, respectively. Show.
  • the expression vector was constructed by the method described in Example 5, and the above-described HCV NS3 TCR with high affinity mutation was expressed, renatured and purified by the method described in Example 6, and then determined with BIAcore T200 and HLA-A2/HCV NS3. : Affinity of the 1406-1415 (KLVALGINAV) complex.
  • Example 3 using a multi-cycle kinetics of wt-NS3TCR measuring HLA-A2 / HCV NS3: Affinity 1406-1415 (KLVALGINAV) complex which K D value of 6.668 ⁇ M, which was shown in BIAcore 13 is shown.
  • the data for the interaction of the HCV NS3 TCR with the high affinity mutation and the HLA-A2/HCV NS3:1406-1415 (KLVALGINAV) complex are shown in Table 2.
  • the affinity is much higher than that of wt-NS3TCR versus HLA-A2/HCV NS3: Affinity of the 1406-1415 (KLVALGINAV) complex.
  • HCVNS3 TCR heterodimer with the same CDR mutation as the high affinity HCV NS3sTv has higher affinity for the HLA-A2/HCV NS3:1406-1415 (KLVALGINAV) complex.
  • the affinity of the high-affinity HCV NS3sTv for the HLA-A2/HCV NS3:1406-1415 (KLVALGINAV) complex therefore, it can be inferred that all high-affinity mutation sites screened in the HCV NS3sTv are introduced into the HCV NS3 TCR heterogeneity After the dimer, the heterodimeric TCR can also satisfy the requirement that the affinity and/or binding half-life of the HLA-A2/HCV NS3:1406-1415 (KLVALGINAV) complex is at least twice that of the wild-type HCV NS3TCR, ie high affinity TCR mutants hetero dimer solution binding of HLA-A2-peptide complexes KLVALGINAV was dissociation equilibrium constant K D or less of wild-type HCV NS3TCR solution binding of HLA-A2-peptide complexes KLVALGINAV was dissociation equilibrium constant K D bipartite one.
  • Example 8 Expression, renaturation and purification of anti-CD3 antibody and high affinity HCV NS3sTv fusion
  • High-affinity sTv and anti-CD3 antibody single-chain antibodies (scFv) against HLA-A2/HCV NS3:1406-1415 were fused at two levels at the genetic level: one is anti-CD3 scFv
  • the fusion is fused to the N-terminus of sTv, and the other is to fuse the anti-CD3 scFv to the C-terminus of sTv.
  • both molecules are linked by a flexible linker GGGGS.
  • the choice of polypeptide linker is not unique and any suitable linker sequence can be used in the fusion molecule.
  • the TCR can be a full length heterodimeric TCR or a single chain sTv.
  • Selected high affinity sTv molecules include: (1) an sTv molecule consisting of the alpha chain variable domain SEQ ID NO: 8 and the beta chain variable domain SEQ ID NO: 32, which is associated with an anti-CD3 scFv at sTv
  • the amino acid sequences of the fusion molecules fused at the N and C ends of the molecule are shown in Figures 14a and 14b, respectively.
  • the expression plasmid was transformed into E. coli strain BL21 (DE3), and LB plate (Kanamycin 50 ⁇ g/ml) was applied and cultured at 37 ° C overnight. On the next day, the clones were inoculated into 10 ml of LB liquid medium (kanamycin 50 ⁇ g/ml) for 2-3 h, and inoculated into 1 L of LB medium (kanamycin 50 ⁇ g/ml) at a volume ratio of 1:100. The culture was carried out until the OD 600 was 0.5-0.8, and then the expression of the protein of interest was induced using IPTG at a final concentration of 0.5 mM.
  • the cells were harvested by centrifugation at 6000 rpm for 10 min.
  • the cells were washed once in PBS buffer, and the cells were dispensed, and the cells corresponding to 200 ml of the bacterial culture were lysed with 5 ml of BugBuster Master Mix (Novagen), and the inclusion bodies were collected by centrifugation at 6000 g for 15 minutes.
  • a detergent wash was then performed 4 times to remove cell debris and membrane components.
  • the inclusion bodies are then washed with a buffer such as PBS to remove detergent and salt.
  • the inclusion bodies were dissolved in a Tris buffer solution containing 8 M urea, and the inclusion body concentration was measured, and the package was divided and stored at -80 ° C for cryopreservation.
  • inclusion bodies were taken out from the -80 ° C ultra-low temperature freezer and thawed, and dithiothreitol (DTT) was added to a final concentration of 10 mM, and incubated at 37 ° C for 30 minutes to 1 hour to ensure complete opening of the disulfide bond. Then, the inclusion body sample solution was separately dropped into 200 ml of 4 ° C pre-cooled refolding buffer (100 mM Tris pH 8.1, 400 mM L-arginine, 2 mM EDTA, 5 M urea, 6.5 mM ⁇ -mercapthoethylamine, 1.87 mM Cystamine), and the temperature was slow at 4 ° C. Stir for about 30 minutes.
  • 4 ° C pre-cooled refolding buffer 100 mM Tris pH 8.1, 400 mM L-arginine, 2 mM EDTA, 5 M urea, 6.5 mM ⁇ -mercapthoethylamine, 1.
  • the renaturation solution was dialyzed against 8 volumes of pre-cooled H 2 O for 16-20 hours. It was further dialyzed twice with 8 volumes of 10 mM Tris pH 8.0, and dialysis was continued at 4 ° C for about 8 hours. After dialysis, the sample was filtered and subjected to the following purification.
  • the dialyzed heavy fold (10 mM Tris pH 8.0) was eluted with a gradient of 0-600 mM NaCl using an POROS HQ/20 anion exchange chromatography prepacked column (Applied Biosystems) on an AKTA Purifier (GE Healthcare). Each component was analyzed by Coomassie brilliant blue stained SDS-PAGE and then combined.
  • the first step of the purified sample solution was concentrated for purification in this step, and the fusion protein was purified by Coomassie blue staining using a Superdex 7510/300GL gel filtration chromatography prepacked column (GE Healthcare) pre-equilibrated in PBS buffer. The fractions of the peaks were analyzed by SDS-PAGE and then combined.
  • the affinity of the three sets of fusion molecules in Example 8 for the HLA-A2/HCV NS3:1406-1415 (KLVALGINAV) complex was determined by the method described in Example 3 to evaluate the affinity of the sTv and anti-CD3 molecules for sTv affinity. influences.
  • the (1) group fusion molecule is an sTv molecule consisting of the ⁇ chain variable domain SEQ ID NO: 8 and the ⁇ chain variable domain SEQ ID NO: 32
  • the anti-CD3 scFv is in the N of the sTv molecule and C-terminal fusion molecules fused BIAcore profiles are shown in Figure 17a and 17b, which K D values of 9.6nM and 4.0 nM, whereas the ⁇ chain variable domain SEQ ID NO: 8 and SEQ ⁇ chain variable domain
  • the K D value of the sTv molecule constituted by ID NO: 32 was 7.0 nM.
  • the (2) group fusion molecule is an sTv molecule consisting of the ⁇ chain variable domain SEQ ID NO: 17 and the ⁇ chain variable domain SEQ ID NO: 33, and the anti-CD3 scFv at the N and C of the sTv molecule end of the fusion molecule fused BIAcore profiles are shown in FIG. 18a and 18b, which are 227.6pM K D values and 75.4pM, whereas the ⁇ chain variable domain SEQ ID NO: 17 and the ⁇ chain variable domain SEQ ID NO
  • the sTv molecule composed of :33 has a K D value of 0.6 nM.
  • the (3) group fusion molecule is an sTv molecule consisting of the ⁇ chain variable domain SEQ ID NO: 14 and the ⁇ chain variable domain SEQ ID NO: 32, and the anti-CD3 scFv at the N and C of the sTv molecule end of the fusion molecule fused BIAcore profiles are shown in Figure 19a and 19b, which are 881.2pM K D value and 536.4pM, but the ⁇ chain variable domain SEQ ID NO: 14 and the ⁇ chain variable domain SEQ ID NO : K D 32 sTv molecule is composed of 1.8nM.
  • Example 10 Expression, renaturation and purification of anti-CD3 antibody and high affinity HCV NS3 TCR fusion
  • a fusion molecule is prepared by fusing an anti-CD3 single-chain antibody (scFv) with a heterodimeric TCR containing an extracellular constant domain.
  • the anti-CD3 scFv is fused to the ⁇ chain of the TCR, and the TCR ⁇ chain may comprise the ⁇ chain variable domain of any of the above high affinity HCV NS3 TCRs, and SEQ ID NO: 73, SEQ ID NO: 74 and SEQ are used in this embodiment.
  • the TCR alpha chain of the fusion molecule may comprise the alpha chain variable domain of any of the above high affinity HCV NS3 TCRs, and the alpha chain variable domains set forth in SEQ ID NO: 2 and SEQ ID NO: 58 are used in this example.
  • the amino acid sequences of the anti-CD3 scFv and the TCR ⁇ -chain fusion molecule are shown in 20a, 20b and 20c, respectively, and the amino acid sequence contains a leading methionine introduced for efficient expression in bacteria.
  • the vector carrying the HCV NS3TCR ⁇ chain gene was digested with NcoI and NotI, and ligated with the pET 28a vector digested with NcoI and NotI.
  • the ligation product was transformed into E. coli DH5 ⁇ , plated on LB plate containing kanamycin, and cultured overnight at 37 ° C.
  • the positive clones were picked for PCR screening, and the positive recombinants were sequenced to determine the correct sequence and the recombinant plasmid was extracted. Transformed to E. coli Tuner (DE3) for expression.
  • the primers were designed to ligate the anti-CD3 scFv and the high-affinity HCV NS3 TCR ⁇ chain gene by overlapping PCR, the intermediate linker was GGGGS, and the anti-CD3 scFv was highly affinity.
  • the gene fragment of the fusion protein of the HCV NS3TCR ⁇ chain carries the restriction enzyme sites NcoI (CCATGG) and NotI (GCGGCCGC).
  • the PCR amplification product was digested with NcoI and NotI and ligated with the pET28a vector digested with NcoI and NotI. The ligation product was transformed into E.
  • coli DH5 ⁇ competent cells coated with kanamycin-containing LB plates, inverted culture at 37 ° C overnight, and positive clones were picked for PCR screening.
  • the positive recombinants were sequenced, and the correct sequence was determined, and the recombinant plasmid was extracted and transformed into E. coli Tuner (DE3) competent cells for expression.
  • the expression plasmids were separately transformed into E. coli Tuner (DE3) competent cells, and LB plates (kanamycin 50 ⁇ g/mL) were applied and cultured at 37 ° C overnight. On the next day, the clones were inoculated into 10 mL LB liquid medium (kanamycin 50 ⁇ g/mL) for 2-3 h, inoculated into 1 L LB medium at a volume ratio of 1:100, and the culture was continued until the OD600 was 0.5-0.8. The final concentration of 1 mM IPTG induced the expression of the protein of interest. After 4 hours of induction, the cells were harvested by centrifugation at 6000 rpm for 10 min.
  • the cells were washed once in PBS buffer, and the cells were dispensed, and the cells corresponding to 200 mL of the bacterial culture were lysed with 5 mL of BugBuster Master Mix (Merck), and the inclusion bodies were collected by centrifugation at 6000 g for 15 minutes. A detergent wash was then performed 4 times to remove cell debris and membrane components. The inclusion bodies are then washed with a buffer such as PBS to remove detergent and salt.
  • a buffer such as PBS to remove detergent and salt.
  • inclusion bodies were dissolved in a buffer solution containing 6 M guanidine hydrochloride, 10 mM dithiothreitol (DTT), 10 mM ethylenediaminetetraacetic acid (EDTA), 20 mM Tris, pH 8.1, and the inclusion body concentration was determined and dispensed. It was then stored frozen at -80 °C.
  • the dissolved TCR ⁇ chain and anti-CD3(scFv)- ⁇ chain were rapidly mixed in a mass ratio of 2:5 to 5M urea (urea), 0.4M L-arginine (L-arginine), 20mM Tris pH 8.1, 3.7 mM cystamine, 6.6 mM ⁇ -mercapoethylamine (4 ° C), final concentration ⁇ chain and anti-CD3 (scFv)- ⁇ chain were 0.1 mg/mL, 0.25 mg/mL, respectively.
  • the solution was dialyzed against 10 volumes of deionized water (4 ° C), and after 12 hours, deionized water was exchanged for buffer (10 mM Tris, pH 8.0) and dialysis was continued at 4 ° C for 12 hours.
  • the solution after completion of dialysis was filtered through a 0.45 ⁇ M filter, and then purified by an anion exchange column (HiTrap Q HP 5 ml, GE healthcare).
  • the TCR of the eluted peak containing the reconstituted TCR alpha chain and the anti-CD3 (scFv)-beta chain dimer was confirmed by SDS-PAGE gel.
  • the TCR fusion molecule was then further purified by size exclusion chromatography (S-10016/60, GE healthcare) and anion exchange column (HiTrap Q HP 5 ml, 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.
  • the affinity of the fusion molecule for the HLA-A2/HCV NS3: 1406-1415 (KLVALGINAV, SEQ ID NO.: 1) complex was determined by the method described in Example 3. The results showed that the affinity of the fusion molecule for the HLA-A2/HCV NS3:1406-1415 (KLVALGINAV) complex did not differ significantly from the affinity of the corresponding original TCR molecule for the HLA-A2/HCV NS3:1406-1415 (KLVALGINAV) complex. .
  • the high affinity HCV NS3sTv variant of the invention was biotinylated according to the method described in Example 3.
  • test medium 10% FBS (heat inactivated, Gibco, catalog number 10108-165); 1640 medium without phenol red: (Life, catalog number: 11835-030); 1% penicillin/ Streptomycin: (Invitrogen, Cat. No. 15070063); Washing solution: PBS pH 7.4w/o Ca Mg (Invitrogen, cat#10010015) and 5% FBS; Culture plate: Nunc 8-well plate (Fisher, Cat#TKT- 210-051T); culture plate coating solution: poly-L-lysine solution (SIGMA, Cat# P8920-100ML).
  • the poly-L-lysine solution was diluted 1:100 in PBS containing Ca, Mg, and added to an 8-well plate at 200 ⁇ l/well, and incubated at 37 ° C for 30 min to absorb poly-L-lysine.
  • the solution was washed 3 times with PBS buffer and air-dried at room temperature.
  • 1*10 6 T2 cells incubated with the relevant short peptide for 1 hour were suspended in 1 ml of washing solution, and divided into 4 groups.
  • the cells were washed with a 75004250 centrifuge (Thermo) at 500 g, centrifuged once for 5 minutes, and removed. The supernatant.
  • the biotinylated high affinity sTv molecules were each diluted to a final concentration of 10-7, 10-8, 10-9, 10-10 M with wash solution.
  • Four groups of T2 cells after suspension centrifugation were incubated on ice for 20 min, and the cells were washed with a 75004250 centrifuge (Thermo) at 500 g for 2 times by centrifugation for 5 minutes.
  • Streptavidin-PE (BD) was diluted 1:100 with a washing solution, and the centrifuged cells were suspended, incubated on ice for 20 min, and washed with a 75004250 centrifuge (Thermo) at 500 g for 2 minutes by centrifugation twice.
  • the centrifuged cells were suspended in 500 ⁇ l of washing solution, and 200 ⁇ l of the cell suspension was added to the dried 8-well plate.
  • the Zeiss microscope Observer Z.01PE channel was observed under a microscope at 63 times and photographed by Z-axis scanning. The Z-axis image was superimposed and the amount of surface fluorescence of the cells was calculated. A three-dimensional image of the cells was obtained by Z-stack acquisition (21 plane, 1 um interval).
  • Example 12 Activation of T cells by fusion molecules of high affinity HCV NS3sTv and anti-CD3 antibodies
  • This example demonstrates by the Elisa experiment that the fusion molecule of the high affinity HCV NS3sTv and anti-CD3 antibody of the present invention can activate T cells well.
  • the activation effect was verified by the release amount of IL-2.
  • Several fusion proteins of the high affinity HCV NS3sTv of the present invention and anti-CD3 antibodies were selected, including fusion protein 1 (SEQ ID NO: 91), fusion protein 2 (SEQ ID NO: 92), and fusion protein 3 (SEQ ID NO: 89). And the fusion protein 4 (SEQ ID NO: 90) was subjected to the Elisa experiment.
  • the main materials used in the Elisa experiment were: U-bottom 96-well plate (Nuk, catalog number 163320), bovine serum (heat-inactivated, Gibbco, catalog number 10108-165), RPMI 1640 without phenol red (Invitrogen, catalog number 32240014), L-glutamine (Invitrogen, catalog number 25030024), penicillin/streptomycin (Invitrogen, catalog number 15070063) PBS pH 7.4 (Invitrogen, catalog number 10010015), anti-human Interleukin-2 (IL-2) (e-Bioscience Cat. No. 14-7029-85), anti-human IL-2-biotin (e-Bioscience Cat. No.
  • IL-2 standard eBioscience catalog number 39-8021
  • streptavidin-HRP Invitrogen, catalog number 43-4323
  • chromogenic substrate Sigma, catalog number 54827-17-7
  • flat-bottom 96-well plate Nuk
  • target cells effector cells
  • fusion molecules of high affinity HCV NS3sTv and anti-CD3 antibodies prepared as described in Example 8, which can be diluted with the test medium
  • 1 M H2SO4 1 M H2SO4.
  • the solvent used includes: culture medium: RPMI 1640 phenol red free, containing 10% fetal bovine serum and 1% L-glutamine and 1% penicillin/streptomycin; washing solution: PBS containing 0.5% Tween; and blocking solution : PBS containing 5% milk powder.
  • the target cells used in this example were K562 cells transfected with HLA-A2 and NS3. A sufficient number of target cells (100,000 cells/well) were washed with a 75004250 centrifuge (Thermo Corporation) at 500 g, once every 5 minutes. The cells were then resuspended in test medium at 2 x 106 cells/ml.
  • T cells The effector cells (T cells) used in this example were CD8-positive T cells (obtained from PBL by negative selection (using CD8 negative isolation kit, MACS, catalog number 130-094-156)). The effector cells were thawed, placed in a test medium, and then washed with a 75004250 centrifuge (Thermo Corporation) at 500 g for 5 minutes by centrifugation. The cells were then resuspended in test medium at 4 times the desired final concentration.
  • the specific experimental procedure was as follows: Anti-human IL-2 coated antibody was diluted 1:250 with PBS. 100 ul of diluted antibody per well was added to a 96-well flat bottom plate. The plate was placed on a shaker and shaken overnight at 4 degrees. The coated antibody was removed from the plate, 200 ul of washing solution was added to each well, and the reaction was repeated, and this step was repeated 6 times. 100 ul of blocking solution was added to each well and shaken at room temperature for 2 h. The blocking solution in the plate was removed, 200 ul of washing solution was added to each well, and the reaction was repeated, and this step was repeated 6 times.
  • the culture supernatant was added to the blocked plate at 100 ul per well, and the human IL-2 standard was diluted to 10, 5, 2.5, 1.25, 0.625, 0.31, 0.16, and 0.08 ng/ml for use as a standard curve. Place on a shaker and shake at room temperature for 2 h. The supernatant in the plate was removed, 200 ul of washing solution was added to each well, and dried, and this step was repeated 6 times.
  • Anti-human gamma interferon-biotin was diluted with a blocking solution at a ratio of 1:500, and 100 ul per well was added. The antibody in the plate was removed, 200 ul of washing solution was added to each well, and the reaction was repeated, and this procedure was repeated 6 times.
  • the fusion molecule of the high affinity HCV NS3sTv and anti-CD3 antibody of the present invention can activate T cells well.
  • Example 13 Activation of T cells by fusion molecules of high affinity HCV NS3 TCR and anti-CD3 antibody
  • This example compares the fusion of several high-affinity HCV NS3TCR and anti-CD3 antibodies of the present invention and the fusion molecule of wild-type TCR and anti-CD3 antibody to a tumor cell line presenting the HCV short peptide-HLA-A2 complex. Thereby the ability to activate cytotoxic T lymphocytes (CTLs). The production of IFN- ⁇ was measured by ELISPOT assay as a readout value for T cell activation.
  • CTLs cytotoxic T lymphocytes
  • High affinity HCV NS3 TCR molecules of the invention are selected comprising: high affinity TCR1 (V ⁇ SEQ ID NO: 2; V ⁇ SEQ ID NO: 73), high affinity TCR2 (V ⁇ SEQ ID NO: 2; V ⁇ SEQ ID NO: 75), high affinity TCR3 (V ⁇ SEQ) ID NO: 2; V ⁇ SEQ ID NO: 74), high affinity TCR4 (V ⁇ SEQ ID NO: 58; V ⁇ SEQ ID NO: 74), high affinity TCR5 (V ⁇ SEQ ID NO: 58; V ⁇ SEQ ID NO: 87) and anti-CD3 antibody
  • the fusion molecule, and the fusion molecule of wild-type TCR (V ⁇ SEQ ID NO: 2; V ⁇ SEQ ID NO: 3) and an anti-CD3 antibody were subjected to the above ELISPOT assay.
  • the reagents used in the ELISPOT assay were as follows: test medium, wash buffer, PBS and human IFN- ⁇ ELISPOT PVDF-enzyme kit, target cells, effector cells, and fusion molecules of high affinity HCV NS3 TCR and anti-CD3 antibody (as in Example 10).
  • the preparation can be diluted with the test medium).
  • the preparation process of the target cells was as follows: The target cells used in this example were K562 cells transfected with HLA-A2 and NS3. A sufficient number of target cells (20,000 cells/well) were washed with a 75004250 centrifuge (Thermo Corporation) at 500 g, once every 5 minutes. The cells were then resuspended in test medium at 4 x 105 cells/ml.
  • the preparation process of effector cells is as follows:
  • the effector cells (T cells) used in this example are CD8-positive T cells (obtained from PBL by negative selection (using CD8 negative isolation kit, MACS, catalog number 130-094-156)) .
  • the effector cells were thawed, placed in a test medium, and then washed with a 75004250 centrifuge (Thermo Corporation) at 500 g for 5 minutes by centrifugation. The cells were then resuspended in test medium at 4 times the desired final concentration.
  • the ELISPOT plate preparation process was as follows: 50 ⁇ l of 35% alcohol was added to each well to pre-wet the bottom of the plate, and 100 ⁇ l of anti-IFN ⁇ capture antibody was diluted with 10 ml of sterile PBS per plate. An aliquot of 100 microliters of diluted capture antibody was then added to each well. The plates were incubated overnight at 4 °C. After incubation, the plates were washed (Procedure 1, Plate Type 2, 96-well plate washer; BioTech) to remove capture antibodies. 1640 medium containing 10% serum was then added to each well at 100 ⁇ l/well and the plate was incubated for 2 hours at room temperature to block the plate. The medium was then washed from the plate (Procedure 1, Plate Type 2, 96-well plate washer; BioTech) and any remaining wash buffer was removed by flicking and tapping the ELISPOT plate on a paper towel.
  • the ELISPOT assay procedure was as follows: The individual components of the assay were added to the ELISPOT plate in the following order: 50 ⁇ l target cells 4 ⁇ 105 cells/ml (total 20,000 target cells/well), 50 ⁇ l reagent (high affinity TCR-anti-CD3 fusion) Molecules; different concentrations), 50 ⁇ l of medium (test medium) and 50 ⁇ l of effector cells (1000 CD8+ cells/well), then incubated overnight (37 ° C, 5% CO 2 ). The plates were then washed and subjected to secondary detection and development, and the plates were dried for 1 hour, and spots formed on the membrane were counted using an immuno spot plate reader (ELISPOT READER system; AID Corporation).
  • ELISPOT READER system immuno spot plate reader
  • Example 14 Redirecting of T cells by fusion molecules of high affinity T cell receptor and anti-CD3 antibody
  • This example demonstrates that the fusion molecule of the high affinity HCV NS3sTv of the present invention and the anti-CD3 antibody and the fusion molecule of the high affinity HCV NS3 TCR and the anti-CD3 antibody are capable of redirecting T cells, thereby killing the target cells.
  • This example uses a non-radioactive cytotoxicity assay to verify killing.
  • the test is 51Cr release fine Colorimetric substitution test for cytotoxicity assay to quantify milk released after cell lysis
  • LDH Acid dehydrogenase
  • CytoTox96 non-radioactive cytotoxicity test (Promega) (G1780) containing substrate mixture, assay buffer, lysis solution and stop solution; test medium: 10% FBS (heat-inactivated, Gibb Company, catalog number 10108-165), phenol red-free 1640 medium (Life), catalog number: 11835-030); 1% penicillin/streptomycin (Invitrogen, catalog number 15070063); Nunc microporous circle Bottom 96-well tissue culture plates (Nunc, Cat. No. 163320); Nunc-Immune Plate Maxisorb (Nuk, Inc., Cat. No. 442404).
  • test medium 10% FBS (heat-inactivated, Gibb Company, catalog number 10108-165), phenol red-free 1640 medium (Life), catalog number: 11835-030); 1% penicillin/streptomycin (Invitrogen, catalog number 15070063); Nunc microporous circle Bottom 96-well tissue culture plates (Nunc, Cat. No. 163320); Nun
  • the target cells used in this example were derived from K562 cells in which the tumor cell lines were transfected with HLA-A2 and NS3 proteins. Target cells were prepared in the test medium; the target cell concentration was adjusted to 2 x 106 cells/ml, thereby obtaining 1 x 105 cells/well, 50 ⁇ l.
  • T cells The effector cells (T cells) used in this example were CD8-positive T cells (obtained from PBL by negative selection (using CD8 negative isolation kit, MACS, catalog number 130-094-156)). The effector cells were thawed, placed in a test medium, and then washed with a 75004250 centrifuge (Thermo Corporation) at 500 g for 5 minutes by centrifugation. The cells were then resuspended in test medium at 2 x 106/ml.
  • Fusion molecules of affinity HCV NS3sTv and anti-CD3 antibody and fusion molecules of high affinity HCV NS3 TCR and anti-CD3 antibody were prepared as described in Example 8 and Example 10, respectively, and diluted to different concentrations by test medium ( 10nM to 0.001pM).
  • the components of the assay were added to the plates in the following order: 50 ⁇ l of target cells (prepared as described above) were added to each well; 50 ⁇ l of effector cells (prepared as described above) were added to each well; 100 ⁇ l of fusion protein was added to each well.
  • the substrate mixture was reconstituted using assay buffer (12 ml). 50 ⁇ l of the reconstituted substrate mixture was then added to each well of the plate. The plate was covered with aluminum foil and incubated for 30 minutes at room temperature. 50 ⁇ l of the stop solution was added to each well of the plate to terminate the reaction. The absorbance at 490 nm was recorded on an Elisa plate reader within 1 hour after the addition of the stop solution.
  • Figure 24 and Figure 25 are the results of cell killing experiments of fusion molecules of high affinity HCV NS3sTv and anti-CD3 antibodies and fusion molecules of high affinity HCV NS3 TCR and anti-CD3 antibodies, respectively, which show the two fusions of the present invention. Molecules are able to redirect T cells, thereby killing target cells.

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Abstract

提供了一种T细胞受体（TCR），其结合KLVALGINAV-HLA-A 2复合物且包含TCR α可变域和/或TCR β可变域。还提供了所述TCR与治疗剂的融合体。所述TCR可以单独使用，也可与治疗剂联用，以靶向呈递KLVALGINAV-HLA-A 2复合物的HCV感染细胞。

Description

高亲和力HCV T细胞受体 技术领域
本发明涉及生物技术领域，更具体地涉及够特异性识别衍生自HCV NS3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。
在T细胞膜上，TCR与参与信号传导的恒定蛋白CD3结合而形成复合物。TCR以多种形式存在并在结构上相似，然而表达这些TCR的T细胞可存在于不同的解剖学位置并可能具有不同的功能。TCR的胞外部分由两个近膜的恒定结构域和两个远膜的可变结构域组成，所述可变结构域具有与抗体的互补决定区(CDRs)相似的可变环。正是这些环形成了T细胞受体分子的抗原(pMHC)结合位点以及决定了该TCR的特异性。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。
丙型肝炎病毒(HCV)是急性肝炎和包括肝硬化及肝细胞癌等长期肝脏疾病的主要起因。受HCV感染的肝细胞可将病毒蛋白降解成短肽，其中KLVALGINAV肽衍生自HCV的非结构蛋白NS3，该肽由HLA-A2负载，并呈递到被HCV感染的细胞表面从而形成HCV的一种抗原表位。
因此，KLVALGINAV-HLA A2复合物提供了一种TCR可靶向HCV的标记。例如，能够靶向HCV标记的TCR可用于将细胞毒性剂或免疫刺激剂递送到感染细胞，或被转化入T细胞，使它们能够破坏感染了HCV的细胞，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，因为亲和力的急剧增加与TCR基因修饰的CD8T细胞中抗原特异性丧失相关，从而导致这些TCR-转染CD8T细胞的非特异性激活(参见Zhao等.,(2007)J Immunol.179:5845-54；Robbins等.,(2008)J Immunol.180:6116-31；还可参见WO2008/038002)。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HCV标记的TCR。
发明内容
本发明的目的在于提供一种对HLA-A2/HCV NS3：1406-1415 (KLVALGINAV，SEQ ID NO:1)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KLVALGINAV(SEQ ID NO:1)HLA-A2复合物的特性，并包含TCRα链可变域和/或TCRβ链可变域，其中：
(ⅰ)所述TCR在SEQ ID NO:2所示其α链可变域和/或在SEQ ID NO:3所示其β链可变域氨基酸残基中发生突变；和
(ⅱ)所述TCR对所述KLVALGINAV-HLA-A2复合物的亲和力和/或结合半衰期是野生型HCV NS3TCR的至少两倍；或者
所述TCR结合所述KLVALGINAV-HLA-A2复合物的解离平衡常数KD小于或等于野生型HCV NS3TCR结合所述KLVALGINAV-HLA-A2复合物的解离平衡常数KD的二分之一。
在另一优选例中，所述TCR的α链可变域突变前的氨基酸序列如SEQ ID NO.:2所示。
在另一优选例中，所述TCR的β链可变域突变前的氨基酸序列如SEQ ID NO.:3所示。
在另一优选例中，所述TCR的α链可变域与SEQ ID NO.:2所示的氨基酸序列有至少80％的序列相同性，和/或所述TCR的β链可变域与SEQ ID NO.:3所示的氨基酸序列有至少80％的序列相同性；
在另一优选例中，所述TCR对所述KLVALGINAV-HLA-A2复合物的解离平衡常数KD≤3.3μM。
在另一优选例中，所述TCR对所述KLVALGINAV-HLA-A2复合物的解离平衡常数0.5μM≤KD≤3.3μM；优选地，1μM≤KD≤3.3μM；更优选地，1μM≤KD≤2μM。
在另一优选例中，所述TCR对所述KLVALGINAV-HLA-A2复合物的解离平衡常数KD≤500nM；优选地，10pM≤KD≤500nM。
在另一优选例中，所述突变发生在α链和/或β链的CDR区。
在另一优选例中，所述突变发生在α链的CDR1和/或CDR2和/或CDR3中，和/或β链的CDR1和/或CDR2和/或CDR3中；优选地，所述突变发生在α链的CDR1和/或CDR2和/或CDR3中，和/或β链的CDR3中；更优选地，所述突变发生在α链的CDR1中和/或β链的CDR3中。
在另一优选例中，所述突变发生在SEQ ID NO.:2所示其α链可变域的选自下组的一个或多个氨基酸残基位点：30S、31D、33Y、53A、54Y、55K、56Q、57Q、58N、96E、98D、99K、100I和101I。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，所述突变发生在SEQ ID NO.:3所示其β链可变域的选自下组的一个或多个氨基酸残基位点：29H、50Y、53N、54S、97P、98Y、99E、100Q和101Y。其中，氨基酸残基编号采用SEQ ID NO.:3所示的编号。
在另一优选例中，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：30N；31E；33I；53S；54D、54W或54A；55N、55S或55Y；56T、56K、56M、56P或56N；57H、57E、57D、57I或57N；58S或58I；96D；98N、98T、98Q或98V；99S、99Y或99P；100L和101T；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，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：29Y；50L；53G；54H；97S；98A、98L、98E、98S或98G；99A或99P；100L、100M或100V；101V、101I、101F或101S；其中，氨基酸残基编号采用SEQ  ID NO.:3所示的编号。
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.:52、53、54、55、56、57、58、59、60、61、63、64、66、67、68、69、70、71和72。
在另一优选例中，所述TCR的β链可变域氨基酸序列选自：SEQ ID NO.:73、74、75、76、77、78、79、80、81、82、84、85、86、87和88。
在另一优选例中，所述TCR包括下表所示的α和β链可变域组合：
Figure PCTCN2015081491-appb-000001
在另一优选例中，所述TCR是αβ异质二聚TCR，其具有α和β链恒定域序列，其半胱氨酸残基在所述TCR的α和β链恒定域之间形成二硫键。
在另一优选例中，在所述TCR中，半胱氨酸残基在所述TCR的α和β链恒定域之间形成人工二硫键。
在另一优选例中，在所述TCR中，形成人工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：
取代TRAC*01外显子1的Thr48和TRBC1*01或TRBC2*01外显子1的Ser57；
取代TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；
取代TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；
取代TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；和
取代TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15。
在另一优选例中，所述TCRα链可变域和/或β链可变域的疏水芯发生突变。
在另一优选例中，所述TCR是由α可变域和β可变域组成的单链TCR，所述α可变域和β可变域由一柔性短肽序列(linker)连接。
在另一优选例中，所述疏水芯突变发生在SEQ ID NO.:2所示其α链可变域的选自下组的一个或多个氨基酸残基位点：11M、21L、48V和110I，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，所述疏水芯突变发生在SEQ ID NO.:3所示其β链可变域的选自下组的一个或多个氨基酸残基位点：78L和81A，其中，氨基酸残基编号采用SEQ  ID NO.:3所示的编号。
在另一优选例中，疏水芯突变后的所述TCR的α链可变域包括选自下组的一个或多个氨基酸残基：11L、21I、48L和110V，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，疏水芯突变后的所述TCR的β链可变域包括选自下组的一个或多个氨基酸残基：78I和81L，其中，氨基酸残基编号采用SEQ ID NO.:3所示的编号。
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.:11、12、13、14、15、16、17、18、19、20、21、22、25、26、27、28、29、30和31。
在另一优选例中，所述TCR的β链可变域氨基酸序列选自：SEQ ID NO.:32、33、34、35、36、37、38、39、40、41、43、44、45、46和47。
在另一优选例中，所述TCR包括下表所示的α和β链可变域组合：
Figure PCTCN2015081491-appb-000002
Figure PCTCN2015081491-appb-000003
在另一优选例中，所述TCR的α链和/或β链结合有偶联物。
在另一优选例中，所述偶联物选自下组的一个或多个：
(1)可检测标记物；
(2)治疗剂；和/或
(3)PK修饰部分。
优选地，所述可检测标记物包括：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。
优选地，所述治疗剂包括：放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、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))、化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
在另一优选例中，所述治疗剂为抗-CD3的抗体。
在另一优选例中，所述偶联物连接于所述TCR的α和/或β链的C-或N-末端。
在另一优选例中，所述与偶联物结合的TCR的α链可变域氨基酸序列选自：SEQ ID NO.:52、53、54、55、56、57、58、59、60、61、63、64、66、67、68、69、70、71和72；和/或所述与偶联物结合的TCR的β链可变域氨基酸序列选自：SEQ ID NO.:73、74、75、76、77、78、79、80、81、82、84、85、86、87和88。
在另一优选例中，所述TCRβ链与抗-CD3抗体融合后的氨基酸序列选自下组SEQ ID NO.:95、96和97。
在另一优选例中，所述与偶联物结合的TCR的α链可变域氨基酸序列选自：SEQ ID NO.:11、12、13、14、15、16、17、18、19、20、21、22、25、26、27、28、29、30和31；和/或所述与偶联物结合的TCR的β链可变域氨基酸序列选自：SEQ ID NO.:32、33、34、35、36、37、38、39、40、41、43、44、45、46和47。
在另一优选例中，所述TCR与抗-CD3抗体融合后的氨基酸序列选自下组：SEQ ID NO.:89、90、91、92、93和94。
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中任一所述的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、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、本发明第二方面所述的TCR复合物、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，提供了一种本发明第一方面所述的T细胞受体的用途，用于制备治疗肿瘤或HCV病毒感染的药物。
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wt-NS3TCRα链可变域氨基酸序列(SEQ ID NO:2)和β链可变域氨基酸序列(SEQ ID NO:3)。
图2a和图2b分别显示了编码wt-NS3TCRα链可变域DNA序列(SEQ ID NO:4)和β链可变域DNA序列(SEQ ID NO:5)。
图3a显示了wt-NS3sTv的氨基酸序列(SEQ ID NO:6)，其疏水芯突变位点以黑体字母表示；图3b显示了编码wt-NS3sTv的DNA序列(SEQ ID NO:7)。
图4a和图4b分别显示了wt-NS3sTv的α链可变域氨基酸序列(SEQ ID NO:8)和β链可变域氨基酸序列(SEQ ID NO:9)。
图5显示了wt-NS3sTv的连接短肽(linker)序列(SEQ ID NO:10)。
图6显示了经3轮筛选后HCV NS3sTv单克隆噬菌体的ELISA结果。
图7a-s分别显示了对HCV NS3KLVALGINAV-HLA-A2复合物具有高亲和力 的HCV NS3sTvα链可变域氨基酸序列(SEQ ID NO:11-22和25-31)，突变的残基以黑体和下划线显示。
图8a-o分别显示了对HCV NS3KLVALGINAV-HLA-A2复合物具有高亲和力的HCV NS3sTvβ链可变域氨基酸序列(SEQ ID NO:32-41和43-47)，突变的残基以黑体和下划线显示。
图9a和图9b分别显示了wt-NS3TCR的α链胞外氨基酸序列(SEQ ID NO:48)和β链胞外氨基酸序列(SEQ ID NO:49)，其恒定区引入的半胱氨酸残基以斜体和下划线表示。
图10a和图10b分别显示了wt-NS3TCR的α链胞外DNA序列(SEQ ID NO:50)(恒定区含有引入的半胱氨酸)和β链胞外DNA序列(SEQ ID NO:51)(恒定区含有引入的半胱氨酸)。
图11a-s分别显示了对HCV NS3KLVALGINAV-HLA-A2复合物具有高亲和力的HCV NS3TCRα链可变域氨基酸序列(SEQ ID NO:52-61，63，64，66-72)，突变的残基以黑体和下划线显示。
图12a-o分别显示了对HCV NS3KLVALGINAV-HLA-A2复合物具有高亲和力的HCV NS3TCRβ链可变域氨基酸序列(SEQ ID NO:73-82和84-88)，突变的残基以黑体和下划线显示。
图13显示了wt-NS3TCR对KLVALGINAV-HLA-A2复合物的亲和力测定的BIAcore图谱。
图14a和14b分别显示了在sTv分子(α链可变域SEQ ID NO:8和β链可变域SEQ ID NO：32)N和C端融合抗-CD3scFv的氨基酸序列。
图15a和15b分别显示了在sTv分子(α链可变域SEQ ID NO:17和β链可变域SEQ ID NO：33)N和C端融合抗-CD3scFv的氨基酸序列。
图16a和16b分别显示了在sTv分子(α链可变域SEQ ID NO:14和β链可变域SEQ ID NO：32)N和C端融合抗-CD3scFv的氨基酸序列。
图17a和17b分别显示了sTv分子(α链可变域SEQ ID NO:8和β链可变域SEQ ID NO：32)N端和C端融合抗-CD3scFv的融合分子对KLVALGINAV-HLA-A2复合物的亲和力测定的BIAcore图谱。
图18a和18b分别显示了sTv分子(α链可变域SEQ ID NO:17和β链可变域SEQ ID NO：33)N端和C端融合抗-CD3scFv的融合分子对KLVALGINAV-HLA-A2复合物的亲和力测定的BIAcore图谱。
图19a和19b分别显示了sTv分子(α链可变域SEQ ID NO:14和β链可变域SEQ ID NO：32)N端和C端融合抗-CD3scFv的融合分子对KLVALGINAV-HLA-A2复合物的亲和力测定的BIAcore图谱。
图20显示了抗-CD3的scFv与TCR的β链融合分子的氨基酸序列。
图21a和21b显示了本发明的高亲和力sTv分子对T2细胞染色的实验结果。
图22为本发明高亲和力HCV NS3sTv与抗-CD3抗体的融合分子激活T细胞的Elisa实验结果。
图23为本发明高亲和力HCV NS3TCR与抗-CD3抗体的融合分子激活T细胞的Elispot实验结果。
图24为本发明的高亲和力HCV NS3sTv与抗-CD3抗体的融合分子的细胞杀伤实验结果。
图25为本发明的高亲和力HCV NS3TCR与抗-CD3抗体的融合分子的细胞杀伤实验结果。
具体实施方式
本发明人通过广泛而深入的研究，获得一种识别KLVALGINAV肽(衍生自 HCV NS3蛋白)的高亲和性T细胞受体(TCR)，所述KLVALGINAV肽以肽-HLA-A2复合物的形式呈递。相对于野生型HCV NS3TCR，本发明TCR在其α链可变域和/或β链可变域发生突变，并且本发明TCR对上述KLVALGINAV-HLA A2复合物的亲和力和/或结合半衰期是野生型HCV NS3TCR的至少两倍。
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α-β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 TRAJ和TRBJ命名法确定TCR的连接区，通过IMGT TRAC和TRBC命名法确定恒定区。各可变区包含嵌合在框架序列中的3个CDR(互补决定区)，其中一个是CDR3，由可变区和连接区重组而成，被称为超变区。根据框架、CDR1和CDR2序列以及部分确定的CDR3序列，α链可变区(Vα)可分成几类，β链可变区(Vβ)也分成几类。在IMGT命名法中，通过TRAV和TRBV的编号分别指代不同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。在β链的形式中，存在两个可能的恒定区基因“C1”和“C2”。
TCR的α和β链一般看作各有两个“结构域”即可变域和恒定域。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可变域”指连接的TRAV和TRAJ区，TCRα恒定域指胞外TRAC区或C-末端截短的TRAC序列。同样地，“TCRβ可变域”指连接的TRBV和TRBD/TRBJ区，TCRβ恒定域指胞外TRBC区或C-末端截短的TRBC序列。
Callender等于2006年报道了针对HCV NS3：1406-1415(KLVALGINAV)/HLA-A2特异性的TCR(Callender,et al.,(2006),Hepatology43(5):981-973)，其α及β链分别为AV38s2/AJ30/AC和BV11s1/BD2s1/BJ2s7/BC2，为方便描述，将该克隆称为wt-NS3TCR。该wt-NS3TCR的α和β链的可变域氨基酸序列分别如SEQ ID NO:2和SEQ ID NO:3所示。本发明对上述wt-NS3TCR进行改进，得到了对KLVALGINAV-HLA A2复合物具有高亲和性的TCR。同样地，为方便描述，在本文中，将对HCV NS3：1406-1415(KLVALGINAV)/HLA-A2复合物具有特异性的异源二聚TCR称为HCV NS3TCR；将由一柔性肽链连接TCRα和β可变结构域构成的TCR称为sTv；将对HCV NS3：1406-1415(KLVALGINAV)/HLA-A2复合物具有特异性的sTv称为HCV NS3sTv。
发明详述
本发明提供具有结合KLVALGINAV(SEQ ID NO：1)HLA-A2复合物特性的包含TCRα可变域和/或TCRβ可变域的T细胞受体(TCR)，其中，(ⅰ)所述TCR在SEQ ID NO:2所示其α链可变域氨基酸和/或在SEQ ID NO:3所示其β链可变域氨基酸中发生突变；和(ⅱ)所述TCR对所述KLVALGINAV(SEQ ID NO:1)HLA-A2复合物的亲和力是野生型HCV NS3TCR的至少两倍。
其中，在上述(ⅰ)中α链可变域和/或β链可变域氨基酸残基中的突变发生在α和/或β可变域的一个或多个互补决定区(CDR)中，所述突变能够使本发明TCR与KLVALGINAV(SEQ ID NO：1)HLA-A2复合物之间的相互作用力更高和/或解离速率更慢。
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)。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)方法进行这些检测。该方法检测到wt-NS3TCR(恒定域间引入了人工二硫键，其胞外α和β链氨基酸序列分别为SEQ ID NO:48和SEQ ID NO：49)对KLVALGINAV(SEQ ID NO：1)HLA-A2复合物的KD为6.7μM，Koff为5.1×10-1s-1(即T1/2约为1.4s)。应理解，该人工二硫键的引入并不会对TCR的亲和力产生影响。因此，野生型HCV NS3TCR对KLVALGINAV-HLA-A2复合物的亲和力与wt-NS3TCR对KLVALGINAV-HLA-A2复合物的亲和力相同。
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KLVALGINAV-HLA-A2复合物具有高亲和性的TCR，本领域技术人员熟知上述建库和筛选方法。
应理解，包含与wt-NS3TCR的α和β链可变域氨基酸相似的基因使用和略作修饰的wt-NS3TCR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突变型高亲和力TCR所需的改变。本领域技术人员熟知，可通过许多方法，如定点突变来引入所需的突变。
本发明的TCR包括其α链可变域与SEQ ID NO:2所示的氨基酸序列具有至少80％，优选地至少85％，或更优选地至少90％的序列相同性(即，不到20％，优选地不到15％，或更优选地不到10％的SEQ ID NO:2所示的TCRα链可变域氨基酸残基发生突变)和/或本发明的TCR包括其β链可变域与SEQ ID NO:3所示的氨基酸序列具有至少80％，优选地至少85％，或更优选地至少90％的序列相同性(即，不到20％，优选地不到15％，或更优选地不到10％的SEQ ID NO:3所示的TCRβ链可变域氨基酸残基发生突变)。可通过，例如手工或利用计算机程序进行序列比对来测定序列相同性。
在本发明的一些优选实施方式中，本发明TCR包含SEQ ID NO:2所示的α链可变域但在其氨基酸残基30S、31D、33Y、53A、54Y、55K、56Q、57Q、58N、96E、98D、99K、100I和101I中具有一个或多个突变(采用SEQ ID NO:2所示的编号)，和/或包含SEQ ID NO:3所示的β链可变域但在其氨基酸残基29H、50Y、53N、54S、97P、98Y、99E、100Q、101Y中具有一个或多个突变(采用SEQ ID NO:3所示的编号)。例如，本发明TCR可具有α链可变域氨基酸残基30N；31E；33I；53S；54D、54W或54A；55N、55S或55Y；56T、56K、56M、56P或56N；57H、57E、57D、57I或57N；58S或58I；96D；98N、98T、98Q或98V；99S、99Y或99P；100L和101T中的一组或多组(采用SEQ ID NO:2所示的编号)和/或β链可变域氨基酸残基29Y；50L；53G；54H；97S；98A、98L、98E、98S或98G；99A或99P；100L、100M或100V；和101V、101I、101F或101S中的一组或多组(采用SEQ ID NO:3所示的编号)。
本发明的高亲和性TCR包含α链可变域氨基酸序列SEQ ID NO:52、53、54、55、56、57、58、59、60、61、63、64、66、67、68、69、70、71、72之一和/或β链可变域氨基酸序列SEQ ID NO:73、74、75、76、77、78、79、80、81、82、 84、85、86、87、88之一。因此，含有wt-NS3TCR的α链可变域氨基酸序列(SEQ ID NO:2)的TCRα链可与包含SEQ ID NO:73、74、75、76、77、78、79、80、81、82、84、85、86、87、88之一的TCRβ链结合。或者，含有wt-NS3TCR的β可变域氨基酸序列(SEQ ID NO:3)的TCRβ链可与包含SEQ ID NO:52、53、54、55、56、57、58、59、60、61、63、64、66、67、68、69、70、71、72之一的TCRα链结合。又或者，包含TCRα链可变域氨基酸序列SEQ ID NO:52、53、54、55、56、57、58、59、60、61、63、64、66、67、68、69、70、71、72之一的TCRα链可与包含TCRβ链可变域氨基酸序列SEQ ID NO:73、74、75、76、77、78、79、80、81、82、84、85、86、87、88之一的TCRβ链结合。
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，一方面，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二硫键的TCR，如在文献(Boulter等(2003)蛋白质工程(Protein Engineering)16:707-711)中所述。半胱氨酸残基在所述TCR的α和β链恒定域间形成人工二硫键。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的半胱氨酸残基来形成二硫键。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。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二硫键的目的，也可通过将适宜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中的天然二硫键连接。
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的TCR氨基酸序列及不同TCR的氨基酸残基在IMGT中的位置编号。
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超稳定性TCR可变域(sTv)。应注意，本发明中柔性肽链可以是任何适合连接TCRα与β可变域的肽链。
为能更方便地评估本发明TCR的结合亲和力和结合半衰期，本发明实施例1中构建的用于筛选高亲和性HCV NS3sTv的模板链中含有疏水芯区域的突变。应注意，疏水芯区域的突变并不会对sTv的结合亲和力和结合半衰期产生影响。
在本发明的一些实施例中，采用SEQ ID NO:2所示的编号，本发明TCR的α可变域疏水芯氨基酸残基11M(即IMGT中列出的α可变区第11位)、21L(即IMGT中列出的α可变区第21位)、48V(即IMGT中列出的α可变区第53位)或110I(即IMGT中列出的α链J基因短肽氨基酸倒数第3位)中一个或多个发生突变和/或采用SEQ ID NO:3所示的编号，所述TCRβ链可变域疏水芯氨基酸残基78L(即IMGT中列出的β链可变区第91位)或81A(即IMGT中列出的β可变区第94位)中一个或多个发生突变。
在本发明的一些优选实施例中，采用SEQ ID NO:2所示的编号，本发明α可变域疏水芯包含氨基酸残基11L、21I、48L或110V中的一个或多个和/或采用SEQ ID NO:3所示的编号，所述TCRβ可变域包含氨基酸残基78I或81L中的一个或多个。
本发明的高亲和性TCR还包含α链可变域氨基酸序列SEQ ID NO:11、12、13、14、15、16、17、18、19、20、21、22、25、26、27、28、29、30、31之一和/或β链可变域氨基酸序列SEQ ID NO:32、33、34、35、36、37、38、39、40、41、43、44、45、46、47之一。因此，含有wt-NS3sTv的α链可变域氨基酸序列(SEQ ID NO:8)的TCRα链可与包含SEQ ID NO:32、33、34、35、36、37、38、39、40、41、43、44、45、46、47之一的TCRβ链结合。或者，含有wt-NS3sTv的β链可变域氨基酸序列(SEQ ID NO:9)的TCRβ链可与包含SEQ ID NO:11、12、13、14、15、16、17、18、19、20、21、22、25、26、27、28、29、30、31之一的TCRα链结合。又或者，包含α链可变域氨基酸序列SEQ ID NO:11、12、13、14、15、16、17、18、19、20、21、22、25、26、27、28、29、30、31之一的TCRα链可与包含β链可变域氨基酸序列SEQ ID NO:32、33、34、35、36、37、38、39、40、41、43、44、45、46、47之一的TCRβ链结合。
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KLVALGINAV HLA-A2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-抗CD3单链抗体融合体包括选自下组的TCRα链可变域氨基酸序列SEQ ID NO:11、12、13、14、15、16、17、18、19、20、21、22、25、26、27、28、29、30、31、52、53、54、55、56、57、58、59、60、61、63、64、66、67、68、69、70、71、72和选自下组的TCRβ链可变域氨基酸序列SEQ ID NO:32、33、34、35、36、37、38、39、40、41、43、44、45、46、47、73、74、75、76、77、78、79、80、81、82、84、85、86、87、88。更具体地，本发明TCR与抗-CD3单链抗体融合体的氨基酸序列可以选自下列氨基酸序列之一SEQ ID NO:89、90、91、92、93和94。更具体地，本发明TCRβ链与抗-CD3单链抗体融合体的氨基酸序列可选自下列氨基酸序列之一：95、96和97。
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。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:2的蛋白序列，但与SEQ ID NO:4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 Cancer8(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)；
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。这些性质相似或相近的氨基酸的替换优选根据表A进行。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
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)本发明以疏水芯突变的高稳定性sTv分子为模板筛选出了对所述KLVALGINAV-HLA-A2复合物具有高亲和力的TCR，同时具有抗原特异性和细胞杀伤效力。
(2)本发明的TCR对所述KLVALGINAV-HLA-A2复合物的亲和力和/或结合半衰期是野生型HCV NS3TCR的至少两倍。
(3)本发明的高亲和力的TCR对所述KLVALGINAV-HLA-A2复合物的亲和力和/或结合半衰期可以达到野生型HCV NS3TCR的103-105倍以上。
(4)本发明的高亲和力HCV NS3TCR与抗-CD3抗体的融合分子能够很好地激活T细胞，而野生型TCR与抗-CD3抗体的融合分子对T细胞的基本没有激活作用。
(5)包含本发明的高亲力TCR以及抗-CD3抗体的融合分子能够重定向T细胞，进而杀伤靶细胞。
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pET 28a购自Novagen。
实施例1疏水芯突变的稳定性HCV NS3sTv的产生
文献(Callender,et al.,(2006),Hepatology43(5):981-973)公开了对HCV NS3KLVALGINAV-HLA A2复合物具有特异性的T细胞受体(本文中简称为wt-NS3 TCR)，其α链和β链的可变域氨基酸序列(SEQ ID NO:2和3)分别如图1a和图1b所示，对应的DNA序列(SEQ ID NO:4和5)如图2a和图2b所示。专利文献WO2014/206304中公开了通过突变疏水芯来构建高稳定性sTv的平台技术。将该高稳定性sTv平台技术中的核心位点引入wt-NS3TCR的α与β链的可变域，并用一个柔性短肽(linker)连接该α与β链的可变域，来构建筛选高亲和性HCV NS3sTv的模板链，该模板链简称为wt-NS3sTv。上述核心位点的引入采用本领域技术人员熟知的定点突变方法，即通过设计引物进行重叠PCR(overlap PCR)来引入突变。图3a显示了该模板链wt-NS3sTv的氨基酸序列(SEQ ID NO:6)，其中疏水芯区域的突变位点以黑体字母表示。其对应的DNA序列为(SEQ ID NO:7)如图3b所示。wt-NS3sTvα与β链的CDR区分别与wt-NS3TCR的α与β链的CDR区相同。wt-NS3sTv的α链和β链的可变域氨基酸序列分别如图4a(SEQ ID NO:8)和4b(SEQ ID NO:9)所示，柔性短肽序列(SEQ ID NO:10)如图5所示。
将携带wt-NS3sTv的目的基因经NcoⅠ和NotⅠ双酶切，与经过NcoⅠ和NotⅠ双酶切的pET 28a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疏水芯突变的稳定性wt-NS3sTv表达、复性和纯化
将实施例1中制备的含有重组质粒pET28a-sTv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wt-NS3sTv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 pH8.1,0.4M L-精氨酸,5M尿素,2mM EDTA,6.5mMβ-mercapthoethylamine,1.87mM Cystamine)中滴加上述处理后的wt-NS3sTv，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。次日早上，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。次日早上，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wt-NS3sTv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，φ7.8×300mm)，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HCV NS3sTv与HCV NS3KLVALGINAV-HLA-A2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，pH 4.77)，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。
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HCV NS3：1406-1415(KLVALGINAV)/HLA A2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sTv用HEPES-EP缓冲液(10mM HEPES,150mM NaCl，3mM EDTA,0.005％P20，pH 7.4)稀释成5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HLA-A2/HCV NS3：1406-1415(KLVALGINAV)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30ml20mM 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(NS3：1408-1415KLVALGINAV)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NS3：1408-1415KLVALGINAV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 16/60 S200HR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HCV NS3sTv变体的产生
噬菌体展示技术是产生TCR高亲和力变体文库以鉴别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wt-NS3sTv。通过突变该wt-NS3sTv中的CDR区来建立高亲和性sTv的文库并进行淘选。本领域技术人员熟知上述建库及筛选方法。即通过使用具有所需的一个或多个密码子变化的引物和作为模板的含相关sTv链DNA的质粒来实现。经过3轮淘选后的噬菌体文库均和相应抗原有特异性结合，其ELISA结果如图6所示。从中挑取单克隆，并进行序列分析。
采用实施例3中BIAcore方法分析HCV NS3sTv与肽KLVALGINAV-HLA-A2复合物的相互作用，筛选出了亲和力和/或结合半衰期是野生型HCV NS3TCR的至少两倍的高亲和力sTv突变体，即筛选出的高亲和力sTv突变体结合肽KLVALGINAV-HLA-A2复合物的解离平衡常数KD小于等于野生型HCV NS3TCR结合肽KLVALGINAV-HLA-A2复合物的解离平衡常数KD的二分之一，结果如下表1所示。利用上述方法未检测到wt-NS3sTv与肽KLVALGINAV-HLA-A2复合物的相互作用。
具体地，采用SEQ ID NO:2中所示的编号，这些高亲和力sTv突变体的α链可变域在下列一个或多个位点的氨基酸发生突变30S、31D、33Y、53A、54Y、55K、56Q、57Q、58N、96E、98D、99K、100I、101I和/或采用SEQ ID NO:3中所示的编号，这些高亲和力sTv突变体的β链可变域在下列一个或多个位点的氨基酸发生突变29H、50Y、53N、54S、97P、98Y、99E、100Q、101Y。
更具体地，采用SEQ ID NO:2中所示的编号，这些高亲和力sTv突变体具有一个或多个下列α链可变域氨基酸残基30N、31E、33I、53S、54D、54W、54A、55N、55S、55Y、56T、56K、56M、56P、56N、57H、57E、57D、57I、57N、58S、58I、96D、98N、98T、98Q、98V、99S、99Y、99P、100L、101T和/或采用SEQ ID NO:3中所示的编号，这些高亲和力sTv突变体具有一个或多个下列β链可变域氨基酸29Y、50L、53G、54H、97S、98A、98L、98E、98S、98G、99A、99P、100L、100M、100V、101V、101I、101F、101S。
高亲和力HCV NS3sTv的α链可变域(SEQ ID NO:11、12、13、14、15、16、17、18、19、20、21、22、25、26、27、28、29、30、31)和β链可变域(SEQ ID NO:32、33、34、35、36、37、38、39、40、41、43、44、45、46、47)的氨基酸序列的具体例子分别如图7a-u和图8a-p所示。
表1
Figure PCTCN2015081491-appb-000004
Figure PCTCN2015081491-appb-000005
实施例5高亲和力HCV NS3TCR变体的产生
将实施例4中筛选到的高亲和力的CDR区突变点引入到wt-NS3TCR的可变域的相应位点中，并通过BIAcore来检测其与HCV NS3KLVALGINAV-HLA-A2复合物的亲和力。上述引入高亲和力突变点的方法采用本领域技术人员熟知的定点突变的方法。上述wt-NS3TCR的α链与β链胞外氨基酸序列分别如图9a(SEQ ID  NO:48)和9b(SEQ ID NO:49)所示，不含为在细菌中有效启动表达而引入的前导甲硫氨酸，其对应的DNA序列分别如图10a(SEQ ID NO:50)和10b(SEQ ID NO:51)所示。应注意，上述α和β链的恒定区中引入了半胱氨酸残基，以便在重折叠时形成人工的链间二硫键。引入的半胱氨酸残基在氨基酸序列中以加下划线的斜体字母表示。wt-NS3TCRα链和β链的可变域氨基酸序列(SEQ ID NO:2和3)分别如图1a和图1b所示。
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6HCV NS3TCR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,6.6mMβ-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7BIAcore分析结果
采用实施例3中所述方法检测引入高亲和力突变点的HCV NS3TCR与HLA-A2/HCV NS3：1406-1415(KLVALGINAV)复合物的亲和力。
将高亲和力HCV NS3sTv中的CDR区突变引入到SEQ ID NO:2和/或SEQ ID NO:3的相应位置，得到新的TCRα和β链可变域氨基酸序列，分别如图11和图12所示。采用实施例5中所述方法构建表达载体，采用实施例6中所述方法对上述引入高亲和力突变的HCV NS3TCR进行表达、复性和纯化，然后利用BIAcore T200测定其与HLA-A2/HCV NS3：1406-1415(KLVALGINAV)复合物的亲和力。采用实施例3中所述方法，利用多循环动力学测定wt-NS3TCR对HLA-A2/HCV NS3：1406-1415(KLVALGINAV)复合物的亲和力，其KD值为6.668μM，其BIAcore图谱如图13所示。引入高亲和力突变的HCV NS3TCR与HLA-A2/HCV NS3：1406-1415(KLVALGINAV)复合物的相互作用的数据如表2所示,其亲和力远高于wt-NS3TCR对HLA-A2/HCV NS3：1406-1415(KLVALGINAV)复合物的亲和力。
表2
Figure PCTCN2015081491-appb-000006
Figure PCTCN2015081491-appb-000007
由上表18组数据可得知，与高亲和性HCV NS3sTv具有相同CDR突变的HCVNS3TCR异质二聚体对HLA-A2/HCV NS3：1406-1415(KLVALGINAV)复合物的亲和力均要高于高亲和性HCV NS3sTv对HLA-A2/HCV NS3：1406-1415(KLVALGINAV)复合物的亲和力，因此，可以推知在HCV NS3sTv中筛选出的所有高亲和力的突变位点被引入到HCV NS3TCR异质二聚体中后，该异质二聚TCR同样能够满足对HLA-A2/HCV NS3：1406-1415(KLVALGINAV)复合物的亲和力和/或结合半衰期是野生型HCV NS3TCR至少两倍的要求，即高亲和力异质二聚TCR突变体结合肽KLVALGINAV-HLA-A2复合物的解离平衡常数KD小于等于野生型HCV NS3TCR结合肽KLVALGINAV-HLA-A2复合物的解离平衡常数KD的二分之一。
同时，我们还检测了上述高亲和性异质二聚TCR与其他10种非特异性抗原有无结合活性，结果如下表3所示。高亲和性异质二聚TCR与其他10种非特异性抗原均没有结合，说明其具有很强的特异性。
表3
Figure PCTCN2015081491-appb-000008
Figure PCTCN2015081491-appb-000009
实施例8抗-CD3抗体与高亲和性HCV NS3sTv融合体的表达、复性和纯化
针对HLA-A2/HCV NS3：1406-1415(KLVALGINAV)的高亲和力sTv和抗CD3抗体的单链抗体(scFv)在基因水平上通过两种方式进行了融合：一种是将抗-CD3的scFv融合在sTv的N端，另一种是将抗-CD3的scFv融合在sTv的C端。在两种融合方式中两种分子均用一个柔性短肽(linker)GGGGS连接，多肽接头的选择并不唯一，任何适合的接头序列都可用在该融合分子中。在这类分子中，TCR可以是全长异二聚体TCR也可以是单链的sTv。
我们选择了几种高亲和性sTv分子与抗CD3抗体的单链抗体(scFv)融合，构建了上述融合分子。选择的高亲和性sTv分子包括：(1)由α链可变域SEQ ID NO:8和β链可变域SEQ ID NO：32而构成的sTv分子，其与抗-CD3的scFv在sTv分子的N和C端融合的融合分子的氨基酸序列分别如图14a和14b所示。(2)由α链可变域SEQ ID NO:17和β链可变域SEQ ID NO：33而构成的sTv分子，其与抗-CD3的scFv在sTv分子的N和C端融合的融合分子的氨基酸序列分别如图15a和15b所示。(3)由α链可变域SEQ ID NO:14和β链可变域SEQ ID NO：32而构成的sTv分子，其与抗-CD3的scFv在sTv分子的N和C端融合的融合分子的氨基酸序列分别如图16a和16b所示。上述融合分子的氨基酸序列中含有为在细菌中有效表达而引入的前导甲硫氨酸。融合分子的制备过程如下：
融合蛋白的表达
将表达质粒转化入大肠杆菌菌株BL21(DE3)中，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(卡那霉素50μg/ml)中，继续培养至OD600为0.5-0.8，然后使用终浓度为0.5mM的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(Novagen)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8M尿素的Tris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。
融合蛋白的重折叠
从-80℃超低温冰箱中取出约10mg包涵体解冻，加二硫苏糖醇(DTT)至终浓度为10mM，在37℃中温育30min到1小时以确保二硫键完全打开。然后将包涵体样品溶液分别滴入200ml 4℃预冷重折叠缓冲液(100mM Tris pH 8.1，400mM L-精氨酸，2mM EDTA，5M尿素，6.5mMβ-mercapthoethylamine,1.87mM Cystamine)，4℃缓慢搅拌约30分钟。复性溶液用8倍体积预冷的H2O透析16-20小时。再用8倍体积的10mM Tris pH 8.0透析两次，4℃继续透析约8小时，透析后样品过滤后进行以下纯化。
融合蛋白的第一步纯化
经过透析的重折叠物(10mM Tris pH 8.0中)使用POROS HQ/20阴离子交换层析预装柱(Applied Biosystems)，在AKTA纯化仪(GE Healthcare)用0-600mM NaCl进行梯度洗脱。通过考马斯亮蓝染色的SDS-PAGE分析各个组分，然后合并。
融合蛋白的第二步纯化
将第一步纯化合并的样品溶液浓缩以供此步纯化，利用在PBS缓冲液中预平衡的Superdex 7510/300GL凝胶过滤层析预装柱(GE Healthcare)纯化融合蛋白，考马斯亮蓝染色的SDS-PAGE分析出峰的组分，然后合并。
实施例9sTv融合分子的亲和力测定
采用实施例3中所述方法测定实施例8中3组融合分子对HLA-A2/HCV NS3：1406-1415(KLVALGINAV)复合物的亲和力，以评估sTv和抗-CD3分子的融合对sTv亲和力的影响。其中，第(1)组融合分子即由α链可变域SEQ ID NO:8和β链可变域SEQ ID NO：32而构成的sTv分子，其与抗-CD3的scFv在sTv分子的N和C端融合的融合分子的BIAcore图谱分别如图17a和17b所示，其KD值分别为9.6nM和4.0nM,而由α链可变域SEQ ID NO:8和β链可变域SEQ ID NO：32而构成的sTv分子的KD值为7.0nM。第(2)组融合分子即由α链可变域SEQ ID NO:17和β链可变域SEQ ID NO：33而构成的sTv分子，其与抗-CD3的scFv在sTv分子的N和C端融合的融合分子的BIAcore图谱分别如图18a和18b所示，其KD值分别为227.6pM和75.4pM,而由α链可变域SEQ ID NO:17和β链可变域SEQ ID NO：33而构成的sTv分子的KD值为0.6nM。第(3)组融合分子即由α链可变域SEQ ID NO:14和β链可变域SEQ ID NO：32而构成的sTv分子，其与抗-CD3的scFv在sTv分子的N和C端融合的融合分子的BIAcore图谱分别如图19a和19b所示，其KD值分别为881.2pM和536.4pM,而由α链可变域SEQ ID NO:14和β链可变域SEQ ID NO：32构成的sTv分子的KD值为1.8nM。可见，sTv分子与抗-CD3分子的融合基本没有对sTv分子的亲和力产生影响，能够保证sTv分子与HLA-A2/HCV NS3：1406-1415(KLVALGINAV)复合物的结合效力。
实施例10抗-CD3抗体与高亲和性HCV NS3TCR融合体的表达、复性和纯化
将抗-CD3的单链抗体(scFv)与含有胞外恒定域的异质二聚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HCV NS3TCR的β链可变域，本实施例选用SEQ ID NO:73、SEQ ID NO:74和SEQ ID NO:87中所示的高亲和性TCRβ链可变域。融合分子的TCRα链可以包含任一上述高亲和性HCV NS3TCR的α链可变域，本实施例选用SEQ ID NO:2和SEQ ID NO:58所示的α链可变域。抗-CD3的scFv与TCR的β链融合分子的氨基酸序列分别如20a、20b和20c所示，氨基酸序列中含有为在细菌中有效表达而引入的前导甲硫氨酸。
融合分子表达载体的构建
1.α链表达载体的构建
将携带HCV NS3TCRα链基因的载体经NcoⅠ和NotⅠ双酶切，与经过NcoⅠ和NotⅠ双酶切的pET 28a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-CD3scFv和高亲和性HCV NS3TCRβ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HCV NS3TCRβ链的融合蛋白的基因片段带上限制性内切酶位点NcoⅠ(CCATGG)和NotⅠ(GCGGCCGC)。将PCR扩增产物经NcoⅠ和NotⅠ双酶切，与经过NcoⅠ和NotⅠ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,6.6mMβ-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-10016/60，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。
通过实施例3中所述方法测定融合分子对HLA-A2/HCV NS3：1406-1415(KLVALGINAV，SEQ ID NO.:1)复合物的亲和力。结果显示融合分子对HLA-A2/HCV NS3：1406-1415(KLVALGINAV)复合物的亲和力与其对应的原TCR分子对HLA-A2/HCV NS3：1406-1415(KLVALGINAV)复合物的亲和力并无明显差异。
实施例11高亲和力HCV NS3sTv变体对T2细胞的染色实验
首先，根据实施例3中所述方法对本发明的高亲和力HCV NS3sTv变体进行生物素化。
随后利用显微束染色方法测定生物素化的高亲和力sTv分子对T2细胞的染色。所用试剂包括：试验培养基:10％FBS(热灭活的，Gibco，目录号10108-165)；不含酚红的1640培养基：(Life，目录号:11835-030)；1％青霉素/链霉素：(英杰公司，目录号15070063)；洗涤液:PBS pH 7.4w/o Ca Mg(Invitrogen,cat#10010015)和5％FBS；培养板：Nunc 8孔板(Fisher,Cat#TKT-210-051T)；培养板包被液：聚L-赖氨酸溶液(SIGMA,Cat#P8920-100ML)。
具体步骤如下：用含Ca,Mg的PBS以1：100比例将聚L-赖氨酸溶液稀释，并以200μl/孔加入8孔板中，37℃孵育30min，吸去聚L-赖氨酸溶液，PBS缓冲液洗3遍，室温晾干。取与相关短肽共孵育1小时的1*106T2细胞悬浮在1ml洗涤液中，分为4组，利用75004250离心机(Thermo公司)以500g，5分钟离心1次来洗涤细胞，移去上清。用洗涤液分别将生物素化的高亲和力sTv分子稀释至终浓度10-7、10-8、10-9、10-10M。分4组悬浮离心后的T2细胞，冰上孵育20min，利用75004250离心机(Thermo公司)以500g,5分钟离心2次来洗涤细胞。用洗涤液将Streptavidin-PE(BD公司)以1：100稀释，悬浮离心后的细胞，冰上孵育20min,利用75004250离心机(Thermo公司) 以500g,5分钟离心2次来洗涤细胞。将离心后的细胞用500μl洗涤液悬浮，在干燥的8孔板中加入200μl细胞悬液。
Zeiss显微镜Observer Z.01PE通道63倍镜下观察细胞并进行Z轴扫描拍照，利用Z轴图片叠加并计算细胞的表面荧光数量。通过Z-栈获取(21平面，1um间隔)得到细胞的三维图像。
实验结果如图21a和21b所示，该结果显示了本发明的高亲和力sTv分子能够成功结合负载有相关短肽的T2细胞。
实施例12高亲和力HCV NS3sTv与抗-CD3抗体的融合分子对T细胞的激活实验
本实施例通过Elisa实验验证了本发明高亲和力HCV NS3sTv与抗-CD3抗体的融合分子能够很好激活T细胞。利用IL-2的释放量验证激活效果。选择几种本发明高亲和力HCV NS3sTv与抗-CD3抗体的融合蛋白，包括融合蛋白1(SEQ ID NO:91)、融合蛋白2(SEQ ID NO:92)、融合蛋白3(SEQ ID NO:89)以及融合蛋白4(SEQ ID NO：90)进行Elisa实验。
Elisa实验所用的主要材料包括：U底96孔板(纽克公司，目录号163320)、牛血清(热一灭活的，吉布可公司，目录号10108-165)、RPMI 1640不含酚红(英杰公司，目录号32404014)、L-谷氨酰胺(英杰公司,目录号25030024)、青霉素/链霉素(英杰公司,目录号15070063)PBS pH 7.4(英杰公司,目录号10010015)、抗人白细胞介素-2(IL-2)(e-Bioscience目录号14-7029-85)、抗人IL-2-生物素(e-Bioscience目录号13-7028-85)、IL-2标准品(eBioscience目录号39-8021)、链霉亲和素–HRP(英杰公司，目录号43-4323)、显色底物(西格玛，目录号54827-17-7)、平底96孔板(纽克公司，目录号163320)、靶细胞、效应细胞、高亲和力HCV NS3sTv与抗-CD3抗体的融合分子(如实施例8所述制备，可以用试验培养基来对其进行稀释)、以及1M H2SO4。
所用溶剂包括：培养液:RPMI 1640无酚红，含有10％胎牛血清和1％L-谷氨酰胺和1％青霉素/链霉素；洗涤液:含有0.5％吐温的PBS；以及封闭液:含有5％奶粉的PBS。
本实施例中所用的靶细胞是转染了HLA-A2和NS3的K562细胞。利用75004250离心机(Thermo公司)以500g，5分钟离心1次来洗涤充足的靶细胞(100,000个细胞/孔)。然后将细胞以2×106个细胞/毫升重悬在试验培养基中。
本实施例中所用的效应细胞(T细胞)是CD8阳性T细胞(通过负选择(利用CD8负分离试剂盒，MACS，目录号130-094-156)从PBL获得)。解冻效应细胞，置于试验培养基中，然后利用75004250离心机(Thermo公司)以500g，5分钟离心进行洗涤。然后将细胞以4倍所需终浓度重悬在试验培养基中。
具体实验步骤如下：用PBS以1:250稀释抗人IL-2包被抗体。在96孔平底培养板中加入每孔100ul稀释后的抗体。将该培养板置于摇床，4度摇晃过夜。甩去板中的包被抗体，每孔加入200ul洗涤液，甩干，重复这个步骤6次。每孔加入100ul封闭液，室温下摇晃2h。甩去板中的封闭液，每孔加入200ul洗涤液，甩干，重复这个步骤6次。
在封闭过的板中加入培养上清，每孔100ul,将人IL-2标准品稀释至10、5、2.5、1.25、0.625、0.31、0.16、0.08ng/ml用于作标准曲线，将板置于摇床常温摇晃2h。甩去板中的上清液，每孔加入200ul洗涤液，甩干，重复这个步骤6次。以1:500的比例用封闭液稀释抗人伽玛干扰素-生物素，每孔加入100ul。甩去板中的抗体，每孔加入200ul洗涤液，甩干，重复这个步骤6次。1:2500用封闭液稀释链霉亲和素–HRP，每孔加入100ul。甩去板中的抗体，每孔加入200ul洗涤液，甩干，重复这个步骤6次.每孔加入显色底物，显色5-10min。每孔加入1M H2SO4终止反应，45nm读取吸光值。所有实验组都设定3组平行对照组，并且最终体积为200ul。
实验结果如图22所示，本发明高亲和力HCV NS3sTv与抗-CD3抗体的融合分子能够很好激活T细胞。
实施例13高亲和力HCV NS3TCR与抗-CD3抗体的融合分子对T细胞的激活实验
本实施例比较几种本发明高亲和力HCV NS3TCR与抗-CD3抗体的融合分子以及野生型TCR与抗-CD3抗体的融合分子对呈递HCV短肽-HLA-A2复合物的肿瘤细胞系的反应，从而激活细胞毒性T淋巴细胞(CTL)的能力。利用ELISPOT实验检测IFN-γ的产量作为T细胞激活的读出值。
选择本发明的高亲和力HCV NS3TCR分子包括：高亲和力TCR1(VαSEQ ID NO:2；VβSEQ ID NO:73)、高亲和力TCR2(VαSEQ ID NO:2；VβSEQ ID NO:75)、高亲和力TCR3(VαSEQ ID NO:2；VβSEQ ID NO:74)、高亲和力TCR4(VαSEQ ID NO:58；VβSEQ ID NO:74)、高亲和力TCR5(VαSEQ ID NO:58；VβSEQ ID NO:87)与抗-CD3抗体的融合分子，以及野生型TCR(VαSEQ ID NO:2；VβSEQ ID NO:3)与抗-CD3抗体的融合分子，进行上述ELISPOT实验。
ELISPOT实验所用试剂如下：试验培养基、洗涤缓冲液、PBS和人IFN-γELISPOT PVDF-酶促试剂盒、靶细胞、效应细胞以及高亲和力HCV NS3TCR与抗-CD3抗体的融合分子(如实施例10所述制备，可以用试验培养基来对其进行稀释)。
靶细胞的制备过程如下：本实施例所用的靶细胞是转染了HLA-A2和NS3的K562细胞。利用75004250离心机(Thermo公司)以500g，5分钟离心1次来洗涤充足的靶细胞(20,000个细胞/孔)。然后将细胞以4×105个细胞/毫升重悬在试验培养基中。
效应细胞的制备过程如下：本实施例中所用的效应细胞(T细胞)是CD8阳性T细胞(通过负选择(利用CD8负分离试剂盒，MACS，目录号130-094-156)从PBL获得)。解冻效应细胞，置于试验培养基中，然后利用75004250离心机(Thermo公司)以500g，5分钟离心进行洗涤。然后将细胞以4倍所需终浓度重悬在试验培养基中。
ELISPOT平板准备过程如下：每孔加入35％酒精50微升以预湿润板底，以每块平板10毫升无菌PBS稀释100微升抗-IFNγ捕捉抗体。然后将100微升的稀释捕捉抗体等份加入各孔。4℃下温育平板过夜。温育后，洗涤平板(程序1，平板类型2,96孔板洗涤仪；BioTech)以除去捕捉抗体。然后将含有10％血清的1640培养基以100μl/孔加入各孔并在室温下温育平板2小时以封闭平板。然后从平板中洗去培养基(程序1，平板类型2,96孔板洗涤仪；BioTech)，通过在纸巾上轻弹和轻拍ELISPOT平板以除去任何剩余的洗涤缓冲液。
ELISPOT实验步骤如下：按以下顺序将试验的各个组分加入ELISPOT平板:50μl靶细胞4×105个细胞/毫升(得到总共20,000个靶细胞/孔)、50μl试剂(高亲和力TCR-抗-CD3融合分子；不同浓度)、50μl培养基(试验培养基)以及50μl效应细胞(1000个CD8+细胞/孔)，然后温育平板过夜(37℃，5％CO2)。随后洗涤平板并进行二级检测和显影，干燥平板1小时，再利用免疫斑点平板读数计(ELISPOT READER system；AID公司)计数膜上形成的斑点。
实验结果如图23所示，该结果显示野生型TCR与抗-CD3抗体的融合分子对T细胞的激活作用甚微，而本发明的高亲和力HCV NS3TCR与抗-CD3抗体的融合分子能够很好地激活T细胞。
实施例14高亲和力T细胞受体与抗-CD3抗体的融合分子对T细胞的重定向实验
本实施例验证了本发明的高亲和力HCV NS3sTv与抗-CD3抗体的融合分子以及高亲和力HCV NS3TCR与抗-CD3抗体的融合分子都能够重定向T细胞，从而使其杀伤靶细胞。
本实施例采用非放射性细胞毒性试验来验证杀伤作用。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乳
酸脱氢酶(LDH)。采用30-分钟偶联的酶试验检测培养上清液中释放的LDH，其将四唑盐(INT)转化成红色甲暨产物。形成的颜色量与裂解细胞的数量成比例。利用标准96孔平板读数计收集490nm的吸光度数据。
所用材料如下：CytoTox96非放射性细胞毒性试验(普罗迈格公司)(G1780)含有底物混合物、试验缓冲液、裂解溶液和终止溶液；试验培养基:10％FBS(热一灭活的，吉布可公司，目录号10108-165)、不含酚红的1640培养基(Life)，目录号:11835-030)；1％青霉素/链霉素(英杰公司，目录号15070063)；Nunc微孔圆底96孔组织培养板(纽克公司(Nunc)，目录号163320)；Nunc-免疫平板Maxisorb(纽克公司，目录号442404)。
本实施例所用的靶细胞来自肿瘤细胞系转染了HLA-A2和NS3蛋白的K562细胞。在试验培养基中制备靶细胞；靶细胞浓度调节至2x106个细胞/毫升，从而得到1x105个细胞/孔，50μl。
本实施例中所用的效应细胞(T细胞)是CD8阳性T细胞(通过负选择(利用CD8负分离试剂盒，MACS，目录号130-094-156)从PBL获得)。解冻效应细胞，置于试验培养基中，然后利用75004250离心机(Thermo公司)以500g，5分钟离心进行洗涤。然后将细胞以2×106/ml重悬在试验培养基中。
分别按照实施例8及实施例10中所述方式制备亲和力HCV NS3sTv与抗-CD3抗体的融合分子以及高亲和力HCV NS3TCR与抗-CD3抗体的融合分子，并通过试验培养基稀释至不同的浓度(10nM到0.001pM)。
采用以下顺序将试验的诸组分加入平板:50μl靶细胞(如上所述制备)加入各孔；50μl效应细胞(如上所述制备)加入各孔；100μl融合蛋白加入各孔。
如下所述制备几个对照:靶细胞自发释放:仅有200μl靶细胞；靶细胞最大释放:仅有200μl靶细胞。所有孔一式三份制备，终体积为200μl。以250x g离心平板4分钟，然后在37℃温育24小时。将15μl裂解溶液加入靶细胞最大释放对照孔，45分钟后收集上清液。以250x g离心平板4分钟。将试验平板各孔的50μl清液转移至平底96孔Nunc Vlnxisorb板的相应孔。利用试验缓冲液(12m1)重建底物混合物。然后将50μl重建的底物混合物加入平板的各孔。平板盖上铝箔，室温下温育30分钟。将50μl终止溶液加入平板的各孔以终止反应。加入终止溶液后1小时内用Elisa平板读数计记录490nm的吸光度。
结果计算:从实验、靶细胞自发释放吸光度值中扣除背景的平均吸光度值,从靶细胞最大释放对照获得的吸光度值中扣除体积校正对照的平均吸光度值。前两步骤中获得的校正值用于下式以计算细胞毒性百分比:％细胞毒性＝100x(实验－靶细胞自发/(靶细胞最大－靶细胞自发)。
图24和图25分别为本发明的高亲和力HCV NS3sTv与抗-CD3抗体的融合分子以及高亲和力HCV NS3TCR与抗-CD3抗体的融合分子的细胞杀伤实验结果，该结果显示本发明的两种融合分子都能够重定向T细胞，从而使其杀伤靶细胞。
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 (32)

  1. 一种T细胞受体(TCR)，其具有结合KLVALGINAV HLA-A2复合物的特性，并包含TCRα链可变域和/或TCRβ链可变域，其特征在于：
    (ⅰ)所述TCR在SEQ ID NO.:2所示其α链可变域和/或在SEQ ID NO.:3所示其β链可变域氨基酸残基中发生突变；和
    (ⅱ)所述TCR结合所述KLVALGINAV-HLA-A2复合物的解离平衡常数KD小于或等于野生型HCV NS3TCR结合所述KLVALGINAV-HLA-A2复合物的解离平衡常数KD的二分之一。
  2. 如权利要求1所述的T细胞受体，其特征在于，所述TCR的α链可变域与SEQ ID NO.:2所示的氨基酸序列有至少80％的序列相同性，和/或所述TCR的β链可变域与SEQ ID NO.:3所示的氨基酸序列有至少80％的序列相同性。
  3. 如权利要求1或2所述的T细胞受体，其特征在于，所述突变发生在α链的CDR1和/或CDR2和/或CDR3中，和/或β链的CDR1和/或CDR2和/或CDR3中；优选地，所述突变发生在α链的CDR1和/或CDR2和/或CDR3中，和/或β链的CDR3中；更优选地，所述突变发生在α链的CDR1中和/或β链的CDR3中。
  4. 如以上任一权利要求所述的T细胞受体，其特征在于，所述突变发生在SEQ ID NO.:2所示其α链可变域的选自下组的一个或多个氨基酸残基位点：30S、31D、33Y、53A、54Y、55K、56Q、57Q、58N、96E、98D、99K、100I和101I；其中，氨基酸残基编号采用SEQ ID NO.:2所示的编号；和/或
    所述突变发生在SEQ ID NO.:3所示其β链可变域的选自下组的一个或多个氨基酸残基位点：29H、50Y、53N、54S、97P、98Y、99E、100Q和101Y，其中，氨基酸残基编号采用SEQ ID NO.:3所示的编号。
  5. 如以上任一权利要求所述的T细胞受体，其特征在于，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：30N；31E；33I；53S；54D、54W或54A；55N、55S或55Y；56T、56K、56M、56P或56N；57H、57E、57D、57I或57N；58S或58I；96D；98N、98T、98Q或98V；99S、99Y或99P；100L和101T；其中，氨基酸残基编号采用SEQ ID NO.:2所示的编号；和/或
    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：29Y；50L；53G；54H；97S；98A、98L、98E、98S或98G；99A或99P；100L、100M或100V；101V、101I、101F或101S；其中，氨基酸残基编号采用SEQ ID NO.:3所示的编号。
  6. 如以上任一权利要求所述的T细胞受体，其特征在于，所述TCR的α链可变域氨基酸序列选自：SEQ ID NO.:52、53、54、55、56、57、58、59、60、61、63、64、66、67、68、69、70、71和72；和/或
    所述TCR的β链可变域氨基酸序列选自：SEQ ID NO.:73、74、75、76、77、78、79、80、81、82、84、85、86、87和88。
  7. 如以上任一权利要求所述的T细胞受体，其特征在于，所述TCR包括下表所示的α和β链可变域组合：
    Figure PCTCN2015081491-appb-100001
    Figure PCTCN2015081491-appb-100002
  8. 如以上任一权利要求所述的T细胞受体，其特征在于，所述TCR是αβ异质二聚TCR，其具有α和β链恒定域序列，其半胱氨酸残基在所述TCR的α和β链恒定域之间形成二硫键。
  9. 如权利要求8所述的T细胞受体，其特征在于，半胱氨酸残基在所述TCR的α和β链恒定域之间形成人工二硫键。
  10. 如权利要求9所述的T细胞受体，其特征在于，在所述TCR中，形成人工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，
    取代TRAC*01外显子1的Thr48和TRBC1*01或TRBC2*01外显子1的Ser57；
    取代TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；
    取代TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；
    取代TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；和
    取代TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15。
  11. 如权利要求1-7中任一所述的T细胞受体，其特征在于，所述TCRα链可变域和/或β链可变域的疏水芯发生突变。
  12. 如权利要求11所述的T细胞受体，其特征在于，所述TCR是由α可变域和β可变域组成的单链TCR，所述α可变域和β可变域由一柔性短肽序列(linker)连接。
  13. 如权利要求12所述的T细胞受体，其特征在于，所述疏水芯突变发生在SEQ ID NO.:2所示α链可变域的选自下组的一个或多个氨基酸残基位点：11M、21L、48V和110I；其中，氨基酸残基编号采用SEQ ID NO.:2所示的编号；和/或
    所述疏水芯突变发生在SEQ ID NO.:3所示β链可变域的选自下组的一个或多个氨基酸残基位点：78L和81A，其中，氨基酸残基编号采用SEQ ID NO.:3所示的编号。
  14. 如权利要求13所述的T细胞受体，其特征在于，疏水芯突变后的所述TCR的α链可变域包括选自下组的一个或多个氨基酸残基：11L、21I、48L和110V，其中，氨基酸残基编号采用SEQ ID NO.:2所示的编号；和/或
    疏水芯突变后的所述TCR的β链可变域包括选自下组的一个或多个氨基酸残基：78I和81L，其中，氨基酸残基编号采用SEQ ID NO.:3所示的编号。
  15. 如权利要求14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选自：SEQ ID NO.:11、12、13、14、15、16、17、18、19、20、21、22、25、26、27、28、29、30和31；和/或
    所述TCR的β链可变域氨基酸序列选自：SEQ ID NO.:32、33、34、35、36、37、38、39、40、41、43、44、45、46和47。
  16. 如权利要求15所述的T细胞受体，其特征在于，所述TCR包括下表所示的 α和β链可变域组合：
    Figure PCTCN2015081491-appb-100003
    Figure PCTCN2015081491-appb-100004
  17. 如以上任一权利要求所述的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。
  18. 如权利要求17所述的T细胞受体，其特征在于，与所述T细胞受体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。
  19. 如权利要求18所述的T细胞受体，其特征在于，与所述T细胞受体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。
  20. 如权利要求19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选自：SEQ ID NO.:52、53、54、55、56、57、58、59、60、61、63、64、66、67、68、69、70、71和72；和/或所述TCR的β链可变域氨基酸序列选自：SEQ ID NO.:73、74、75、76、77、78、79、80、81、82、84、85、86、87和88。
  21. 如权利要求20所述的T细胞受体，其特征在于，所述TCRβ链与抗-CD3抗体结合后的氨基酸序列选自下组SEQ ID NO.:95、96和97。
  22. 如权利要求19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选自：SEQ ID NO.:11、12、13、14、15、16、17、18、19、20、21、22、25、26、27、28、29、30和31；和/或所述TCR的β链可变域氨基酸序列选自：SEQ ID NO.:32、33、34、35、36、37、38、39、40、41、43、44、45、46和47。
  23. 如权利要求22所述的T细胞受体，其特征在于，所述TCR与抗-CD3抗体融合后的氨基酸序列选自下组：SEQ ID NO.:89、90、91、92、93和94。
  24. 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分子为上述权利要求中任一项所述的TCR。
  25. 一种核酸分子，其特征在于，所述核酸分子包含编码上述任一权利要求所述的TCR分子的核酸序列或其互补序列；
  26. 一种载体，其特征在于，所述的载体含有权利要求25中所述的核酸分子。
  27. 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26中所述的载体或染色体中整合有外源的权利要求25中所述的核酸分子。
  28. 一种分离的细胞，其特征在于，所述细胞表达权利要求1-24中任一项所述的TCR；
  29. 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1-23中任一项所述的TCR、或权利要求24中所述的TCR复合物、或权利要求28中所述的细胞。
  30. 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适量的权利要求1-23中任一所述的TCR、或权利要求24中所述的TCR复合物、或权利要求28中所述的细胞、或权利要求29中所述的药物组合物。
  31. 权利要求1-23任一项所述的T细胞受体、权利要求24中所述的TCR复合物或权利要求28中所述细胞的用途，其特征在于，用于制备治疗肿瘤或HCV病毒感染的药物。
  32. 一种制备权利要求1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27所述的宿主细胞，从而表达权利要求1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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