WO2020182082A1 - 一种识别afp抗原的高亲和力tcr - Google Patents

一种识别afp抗原的高亲和力tcr Download PDF

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WO2020182082A1
WO2020182082A1 PCT/CN2020/078271 CN2020078271W WO2020182082A1 WO 2020182082 A1 WO2020182082 A1 WO 2020182082A1 CN 2020078271 W CN2020078271 W CN 2020078271W WO 2020182082 A1 WO2020182082 A1 WO 2020182082A1
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tcr
variable domain
chain variable
chain
amino acid
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PCT/CN2020/078271
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English (en)
French (fr)
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李懿
李小林
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广东香雪精准医疗技术有限公司
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Priority to US17/437,212 priority Critical patent/US20220169697A1/en
Priority to CA3132743A priority patent/CA3132743A1/en
Priority to KR1020217032584A priority patent/KR20210142666A/ko
Priority to EP20771141.7A priority patent/EP3936520A4/en
Priority to JP2021553138A priority patent/JP2022524112A/ja
Priority to AU2020238796A priority patent/AU2020238796A1/en
Publication of WO2020182082A1 publication Critical patent/WO2020182082A1/zh

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Definitions

  • the present invention relates to the field of biotechnology, and more specifically to a T cell receptor (TCR) capable of recognizing a polypeptide derived from an AFP protein.
  • TCR T cell receptor
  • the invention also relates to the preparation and use of the receptor.
  • TCR T cell receptor
  • TCR is the only receptor for specific antigen peptides presented on the main histocompatibility complex (MHC). This exogenous or endogenous peptide may be the only sign of abnormal cells.
  • MHC main histocompatibility complex
  • APC antigen-presenting cells
  • the MHC class I and class II molecular ligands corresponding to TCR are also proteins of the immunoglobulin superfamily but have specificity for antigen presentation. Different individuals have different MHCs, which can present different shortcomings in a protein antigen. Peptides to the surface of the respective APC cells. Human MHC is usually called HLA gene or HLA complex.
  • AFP ( ⁇ Fetoprotein), also known as ⁇ fetoprotein, is a protein expressed during embryonic development and the main component of embryonic serum. During development, AFP has a relatively high expression level in the yolk sac and liver, and is subsequently inhibited. In hepatocellular carcinoma, the expression of AFP is activated. After AFP is produced in the cell, it is degraded into small molecule polypeptides, and combined with MHC (major histocompatibility complex) molecules to form a complex, which is presented to the cell surface.
  • FMNKFIYEI (SEQ ID NO: 25) is a short peptide derived from AFP antigen and a target for the treatment of AFP-related diseases.
  • the FMNKFIYEI-HLA A0201 complex provides a marker for TCR to target tumor cells.
  • the TCR that can be combined with FMNKFIYEI-HLA A0201 complex has high application value for tumor treatment.
  • TCR that can target the tumor cell marker can be used to deliver cytotoxic agents or immunostimulants to target cells, or be transformed into T cells, so that T cells expressing the TCR can destroy tumor cells, so as to be called Adoptive immunotherapy is given to patients during the course of treatment.
  • the ideal TCR has a high affinity, so that the TCR can reside on the targeted cells for a long time.
  • it is preferable to use a medium affinity TCR it is preferable to use a medium affinity TCR. Therefore, those skilled in the art devote themselves to developing TCRs that can be used for different purposes to target tumor cell markers.
  • the purpose of the present invention is to provide a TCR with higher affinity to the FMNKFIYEI-HLA A0201 complex.
  • Another object of the present invention is to provide a method for preparing the above type of TCR and use of the above type of TCR.
  • the first aspect of the present invention provides a T cell receptor (TCR), which has the activity of binding to the FMNKFIYEI-HLA A0201 complex.
  • the T cell receptor has the activity of binding FMNKFIYEI-HLA A0201 complex, and the T cell receptor comprises a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain, and the TCR ⁇
  • the chain variable domain contains 3 CDR regions, and the reference sequence of the 3 CDR regions of the TCR ⁇ chain variable domain is as follows:
  • CDR3 ⁇ AVNSGGSNYKLT
  • CDR3 ⁇ contains at least one of the following mutations:
  • the ⁇ chain variable domain of the TCR is an amino acid sequence having at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO: 2.
  • the ⁇ chain variable domain of the TCR is at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence shown in SEQ ID NO: 2 , 97%, 98%, 99% or 100% sequence homology amino acid sequence.
  • the number of CDR3 ⁇ mutations in the variable domain of the TCR ⁇ chain is 1 to 4.
  • the affinity of the TCR and the FMNKFIYEI-HLA A0201 complex is at least 5 times that of the wild-type TCR.
  • the ⁇ chain variable domain of the TCR contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence shown in SEQ ID NO:1. , 97%, 98% or 99% sequence homology amino acid sequence.
  • the TCR ⁇ chain variable domain comprises 3 CDR regions, and the amino acid sequences of the 3 CDR regions of the TCR ⁇ chain variable domain are as follows:
  • amino acid sequence of the variable domain of the TCR ⁇ chain is SEQ ID NO: 2.
  • the TCR comprises a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , wherein the amino acid sequence of CDR1 ⁇ is DSAIYN, and the amino acid sequence of CDR2 ⁇ is IQSSQRE
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , wherein the amino acid sequence of CDR1 ⁇ is SGHVS, the amino acid sequence of CDR2 ⁇ is FQNEAQ, and the amino acid sequence of CDR3 ⁇ is ASSLFGQGREKLF.
  • the TCR comprises a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇
  • the amino acid sequence of CDR1 ⁇ is DSAIYN
  • the amino acid sequence of CDR2 ⁇ is IQSSQRE
  • the amino acid sequence of CDR3 ⁇ is:
  • the [3 ⁇ X1] is N or D or E.
  • the [3 ⁇ X2] is S or D or G or A or W or T or H.
  • the [3 ⁇ X3] is G or Q or A or V or H or W or Y or M or I.
  • the [3 ⁇ X4] is G or D or R or P or Q or T or Y.
  • the [3 ⁇ X5] is S or G or D.
  • the [3 ⁇ X6] is N or G or D.
  • the TCR has a CDR selected from the following group:
  • the TCR is soluble.
  • the TCR is an ⁇ heterodimeric TCR, which comprises an ⁇ chain TRAC constant region sequence and a ⁇ chain TRBC1 or TRBC2 constant region sequence.
  • the TCR comprises (i) all or part of the TCR ⁇ chain except its transmembrane domain, and (ii) all or part of the TCR ⁇ chain except its transmembrane domain, wherein (i) And (ii) both contain the variable domain and at least a part of the constant domain of the TCR chain.
  • the ⁇ chain constant region and the ⁇ chain constant region of the TCR contain artificial interchain disulfide bonds.
  • cysteine residues forming artificial interchain disulfide bonds between the constant regions of the TCR ⁇ and ⁇ chains are substituted for one or more sets of sites selected from the following:
  • the amino acid sequence of the ⁇ chain variable domain of the TCR is selected from: SEQ ID NO: 11-24; and/or the amino acid sequence of the ⁇ chain variable domain of the TCR is SEQ ID NO: 2.
  • the TCR is selected from the following group:
  • the TCR is a single-chain TCR.
  • the TCR is a single-chain TCR composed of an ⁇ -chain variable domain and a ⁇ -chain variable domain, and the ⁇ -chain variable domain and ⁇ -chain variable domain are composed of a flexible short peptide sequence (linker )connection.
  • a conjugate is bound to the C- or N-terminus of the ⁇ chain and/or ⁇ chain of the TCR.
  • the conjugate that binds to the TCR is a detectable label, a therapeutic agent, a PK modified part or a combination of any of these substances.
  • the therapeutic agent that binds to the TCR is an anti-CD3 antibody linked to the C- or N-terminus of the ⁇ or ⁇ chain of the TCR.
  • the affinity of the TCR and FMNKFIYEI-HLA A0201 complex is at least 5 times that of the wild-type TCR; preferably, at least 10 times; more preferably, at least 50 times.
  • the affinity of the TCR and FMNKFIYEI-HLA A0201 complex is at least 100 times that of the wild-type TCR; preferably, at least 500 times; more preferably, at least 1000 times.
  • the dissociation equilibrium constant K D ⁇ 20 ⁇ M of the TCR to the FMNKFIYEI-HLA A0201 complex preferably, 5 ⁇ M ⁇ K D ⁇ 10 ⁇ M.
  • the dissociation equilibrium constant of the TCR to the FMNKFIYEI-HLA A0201 complex is 0.1 ⁇ M ⁇ K D ⁇ 1 ⁇ M; preferably, 1 nM ⁇ K D ⁇ 100 nM.
  • the T cell receptor has the activity of binding to the FMNKFIYEI-HLA A0201 complex, and comprises a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain.
  • the TCR A mutation occurs in the ⁇ chain variable domain shown in SEQ ID NO: 1, and the mutated amino acid residue positions include one or more of 93N, 94S, 95G, 96G, 97S and 98N, wherein the amino acid residues
  • the base number adopts the number shown in SEQ ID NO:1;
  • the TCR ⁇ chain variable domain after mutation includes one or more amino acid residues selected from the following group: 93D or 93E; 94D or 94G or 94A or 94W or 94T or 94H; 95Q or 95A or 95V or 95H Or 95W or 95Y or 95M or 95I; 96D or 96R or 96P or 96Q or 96T or 96Y; 97G or 97D; and 98G or 98D, wherein the amino acid residue numbering adopts the numbering shown in SEQ ID NO:1.
  • the second aspect of the present invention provides a multivalent TCR complex comprising at least two TCR molecules, and at least one of the TCR molecules is the TCR described in the first aspect of the present invention.
  • the third aspect of the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule described in the first aspect of the present invention or the multivalent TCR complex described in the second aspect of the present invention or its complement sequence.
  • the fourth aspect of the present invention provides a vector containing the nucleic acid molecule described in the third aspect of the present invention.
  • the fifth aspect of the present invention provides a host cell that contains the vector of the fourth aspect of the present invention or the nucleic acid molecule of the third aspect of the present invention integrated into the chromosome.
  • the sixth aspect of the present invention provides an isolated cell that expresses the TCR described in the first aspect of the present invention.
  • the seventh aspect of the present invention provides a pharmaceutical composition containing a pharmaceutically acceptable carrier and the TCR according to the first aspect of the present invention, or the TCR complex according to the second aspect of the present invention, Or the cell described in the sixth aspect of the present invention.
  • the eighth aspect of the present invention provides a method for treating diseases, comprising administering an appropriate amount of the TCR according to the first aspect of the present invention, or the TCR complex according to the second aspect of the present invention, or the present invention to a subject in need of treatment.
  • the disease is an AFP-positive tumor.
  • the AFP-positive tumor is liver cancer, breast cancer or germ cell tumor; more preferably, the AFP-positive tumor is hepatocellular carcinoma.
  • the ninth aspect of the present invention provides the use of the TCR according to the first aspect of the present invention, or the TCR complex according to the second aspect of the present invention, or the use of the cell according to the sixth aspect of the present invention, for preparing and treating tumors medicine.
  • the tumor is an AFP positive tumor.
  • the AFP-positive tumor is liver cancer, breast cancer or germ cell tumor; more preferably, the AFP-positive tumor is hepatocellular carcinoma.
  • the tenth aspect of the present invention provides a method for preparing the T cell receptor of the first aspect of the present invention, including the steps:
  • Figure 1a and Figure 1b respectively show the amino acid sequences of wild-type TCR ⁇ and ⁇ chain variable domains that can specifically bind to the FMNKFIYEI-HLA A0201 complex.
  • Figure 2a and Figure 2b are the amino acid sequence of the alpha variable domain and the amino acid sequence of the beta chain variable domain of the single-chain template TCR constructed in the present invention.
  • Figures 3a and 3b are respectively the DNA sequence of the ⁇ variable domain and the DNA sequence of the ⁇ chain variable domain of the single-stranded template TCR constructed in the present invention.
  • Figures 4a and 4b are respectively the amino acid sequence and nucleotide sequence of the linker of the single-stranded template TCR constructed in the present invention.
  • Figures 5a and 5b are the amino acid sequence and DNA sequence of the single-stranded template TCR constructed in the present invention, respectively.
  • Figures 6(1)-(14) respectively show the amino acid sequence of the ⁇ chain variable domain of a heterodimeric TCR with high affinity for the FMNKFIYEI-HLA A0201 complex, and the mutated residues are underlined.
  • Figures 7a and 7b respectively show the amino acid sequences of the reference TCR ⁇ and ⁇ chains of the present invention.
  • Figures 8a and 8b respectively show the amino acid sequences of wild-type TCR ⁇ and ⁇ chains that can specifically bind to the FMNKFIYEI-HLA A0201 complex.
  • Figure 9 is the binding curve of the reference TCR, that is, the wild-type TCR and the FMNKFIYEI-HLA A0201 complex.
  • Figures 10a-f are graphs showing the experimental results of the activation function of the effector cells transfected with the high-affinity TCR of the present invention against T2 cells loaded with specific short peptides.
  • Figure 11 is a graph showing the results of the activation function experiment of effector cells transfected with the high-affinity TCR of the present invention against tumor cell lines.
  • Figure 12 shows the results of the killing function experiment of the effector cells transfected with the high-affinity TCR of the present invention.
  • Figure 13 shows the results of in vivo efficacy experiments of T cells transfected with the high-affinity TCR of the present invention.
  • the present invention has obtained a high-affinity T cell receptor (TCR) that recognizes the FMNKFIYEI short peptide (derived from the AFP protein).
  • the FMNKFIYEI short peptide is in the form of a peptide-HLA A0201 complex. Submit.
  • the high-affinity TCR is in the 3 CDR regions of its ⁇ chain variable domain:
  • CDR3 ⁇ A mutation in AVNSGGSNYKLT
  • the affinity and/or binding half-life of the TCR of the present invention to the above-mentioned FMNKFIYEI-HLA A0201 complex is at least 5 times that of the wild-type TCR.
  • TCR T cell receptor
  • the International Immunogenetics Information System can be used to describe TCR.
  • the natural ⁇ heterodimeric TCR has an ⁇ chain and a ⁇ chain. Broadly speaking, each chain includes a variable region, a connecting region, and a constant region.
  • the beta chain usually also contains a short variable region between the variable region and the connecting region, but the variable region is often regarded as a part of the connecting region.
  • the unique IMGT TRAJ and TRBJ are used to determine the TCR connection region, and the IMGT TRAC and TRBC are used to determine the TCR constant region.
  • Each variable region contains 3 CDRs (complementarity determining regions), CDR1, CDR2, and CDR3, chimeric in the framework sequence.
  • the different numbers of TRAV and TRBV refer to different types of V ⁇ and V ⁇ respectively.
  • the alpha chain constant domain has the following symbols: TRAC*01, where "TR” represents the T cell receptor gene; "A” represents the alpha chain gene; C represents the constant region; "*01” represents alleles Gene 1.
  • the ⁇ chain constant domain has the following symbols: TRBC1*01 or TRBC2*01, where "TR" represents the T cell receptor gene; "B” represents the ⁇ chain gene; C represents the constant region; "*01” represents the allele 1.
  • the constant region of the ⁇ chain is uniquely determined.
  • TCR ⁇ chain variable domain refers to the connected TRAV and TRAJ regions
  • TCR ⁇ chain variable domain refers to the connected TRBV and TRBD/TRBJ regions.
  • the three CDRs of the variable domain of the TCR ⁇ chain are CDR1 ⁇ , CDR2 ⁇ , and CDR3 ⁇ ; the three CDRs of the variable domain of the TCR ⁇ chain are CDR1 ⁇ , CDR2 ⁇ , and CDR3 ⁇ , respectively.
  • the framework sequence of the TCR variable domain of the present invention can be murine or human, preferably human.
  • the constant domain of TCR contains an intracellular part, a transmembrane region and an extracellular part.
  • the alpha chain amino acid sequence and the beta chain amino acid sequence of the "wild-type TCR” in the present invention are SEQ ID NO: 28 and SEQ ID NO: 29, respectively, as shown in Figures 8a and 8b.
  • the alpha chain amino acid sequence and beta chain amino acid sequence of the "reference TCR” in the present invention are SEQ ID NO: 26 and SEQ ID NO: 27, respectively, as shown in Figures 7a and 7b.
  • the amino acid sequences of the alpha and beta chain variable domains of the wild-type TCR capable of binding to the FMNKFIYEI-HLA A0201 complex are SEQ ID NO:1 and SEQ ID NO: 2, respectively, as shown in Figures 1a and 1b.
  • the terms "polypeptide of the present invention", “TCR of the present invention", and “T cell receptor of the present invention” are used interchangeably.
  • the position numbers of the amino acid sequence of TRAC*01 and TRBC1*01 or TRBC2*01 in the present invention are numbered from N-terminal to C-terminal.
  • N The 60th amino acid in the sequence from end to C end is P (proline), then it can be described as Pro60 of TRBC1*01 or TRBC2*01 exon 1 in the present invention, or it can be expressed as TRBC1* 01 or TRBC2*01 exon 1’s 60th amino acid, as in TRBC1*01 or TRBC2*01, the 61st amino acid from N-terminal to C-terminal is Q (glutamine), then this In the present invention, it can be described as Gln61 of TRBC1*01 or TRBC2*01 exon 1, or it can be expressed as the 61st amino acid of TRBC1*01 or TRBC2*01 exon 1, and so on.
  • tumor is meant to include all types of cancer cell growth or carcinogenic processes, metastatic tissues or malignant transformed cells, tissues or organs, regardless of the pathological type or the stage of infection.
  • tumors include, without limitation, solid tumors, soft tissue tumors, and metastatic lesions.
  • solid tumors include: malignant tumors of different organ systems, such as sarcoma, lung squamous cell carcinoma and cancer.
  • sarcoma for example: infected prostate, lung, breast, lymph, gastrointestinal (for example: colon), and genitourinary tract (for example: kidney, epithelial cells), pharynx.
  • Lung squamous cell carcinoma includes malignant tumors, for example, most colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer and esophageal cancer.
  • malignant tumors for example, most colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer and esophageal cancer.
  • the above-mentioned metastatic lesions of cancer can also be treated and prevented by the method and composition of the present invention.
  • the ⁇ chain variable domain and ⁇ chain variable domain of TCR each contain 3 CDRs, which are similar to the complementarity determining regions of antibodies.
  • CDR3 interacts with short antigen peptides
  • CDR1 and CDR2 interact with HLA. Therefore, the CDR of the TCR molecule determines its interaction with the antigen short peptide-HLA complex.
  • the alpha chain variable domain amino acid sequence and the ⁇ chain variable domain amino acid sequence of the wild-type TCR that can bind the antigen short peptide FMNKFIYEI and HLA A0201 complex are SEQ ID NO:1 and SEQ, respectively ID NO: 2, this sequence is the first discovery by the inventor. It has the following CDR regions:
  • the present invention obtains a high-affinity TCR whose affinity with the FMNKFIYEI-HLA A0201 complex is at least 5 times the affinity of the wild-type TCR with the FMNKFIYEI-HLA A0201 complex through mutation screening of the above CDR regions.
  • the present invention provides a T cell receptor (TCR), which has the activity of binding FMNKFIYEI-HLA A0201 complex.
  • the T cell receptor comprises a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain.
  • the TCR ⁇ chain variable domain comprises 3 CDR regions.
  • the reference sequences of the 3 CDR regions of the TCR ⁇ chain variable domain are as follows:
  • CDR3 ⁇ AVNSGGSNYKLT, and contains at least one of the following mutations:
  • the TCR ⁇ chain variable domain comprises 3 CDR regions, and the reference sequence of the 3 CDR regions of the TCR ⁇ chain variable domain is as follows,
  • the number of mutations in the CDR region of the TCR ⁇ chain may be 1, 2, 3, 4, 5, or 6.
  • the TCR of the present invention is an ⁇ heterodimeric TCR
  • the ⁇ chain variable domain of the TCR contains at least 85% of the amino acid sequence shown in SEQ ID NO:1; preferably, at least 90%; more preferably Preferably, at least 92%; more preferably, at least 94% (eg, it can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% %, 99% sequence homology); and/or the ⁇ -chain variable domain of the TCR contains at least 90% of the amino acid sequence shown in SEQ ID NO: 2, preferably , At least 92%; more preferably, at least 94% (eg, it can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same sequence Source) amino acid sequence of sequence homology.
  • the TCR of the present invention is a single-chain TCR, and the alpha chain variable domain of the TCR contains at least 85%, preferably at least 90%, and more preferably at least the amino acid sequence shown in SEQ ID NO: 3; 92%; most preferably, at least 94% (eg, it can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% % Sequence homology); and/or the ⁇ chain variable domain of the TCR contains at least 85% of the amino acid sequence shown in SEQ ID NO: 4, preferably at least 90% %; more preferably, at least 92%; most preferably, at least 94%; (eg, it can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% The sequence homology) of the amino acid sequence.
  • the three CDRs of the wild-type TCR ⁇ chain variable domain SEQ ID NO:1, namely CDR1, CDR2 and CDR3 are located at positions 27-32, 50-56 and 91-102 of SEQ ID NO:1, respectively . Accordingly, the numbering of amino acid residues adopts the numbering shown in SEQ ID NO:1, 93N is the third N of CDR3 ⁇ , 94S is the fourth S of CDR3 ⁇ , and 95G is the fifth G and 96G of CDR3 ⁇ .
  • the 6th G and 97S of CDR3 ⁇ are the 7th S of CDR3 ⁇ , and the 98N is the 8th N of CDR3 ⁇ .
  • the present invention provides a TCR that has the property of binding to the FMNKFIYEI-HLA A0201 complex, and includes an ⁇ chain variable domain and a ⁇ chain variable domain, characterized in that the TCR is in the ⁇ chain variable shown in SEQ ID NO:1 A mutation occurs in the domain, and the mutated amino acid residue positions include one or more of 93N, 94S, 95G, 96G, 97S, and 98N, wherein the numbering of the amino acid residues adopts the numbering shown in SEQ ID NO:1.
  • the TCR ⁇ chain variable domain after mutation includes one or more amino acid residues selected from the following group: 93D or 93E; 94D or 94G or 94A or 94W or 94T or 94H; 95Q or 95A or 95V or 95H Or 95W or 95Y or 95M or 95I; 96D or 96R or 96P or 96Q or 96T or 96Y; 97G or 97D; and 98G or 98D, wherein the amino acid residue numbering adopts the numbering shown in SEQ ID NO:1.
  • the specific forms of the mutation in the variable domain of the ⁇ chain include N93/D/E, S94/D/G/A/W/T/H; G95/Q/A/V/H/W/Y /M/I; G96/D/R/P/Q/T/Y; S97/G/D; and N98/G/D in one or several groups.
  • the Thr48 of the wild-type TCR ⁇ chain constant region TRAC*01 exon 1 was mutated to cysteine, and the ⁇ chain constant region TRBC1*01 or TRBC2*01 exon 1 Ser57 is mutated to cysteine to obtain a reference TCR.
  • the amino acid sequence is shown in Figures 7a and 7b, and the cysteine residues after mutation are shown in bold letters.
  • the above cysteine substitution can form artificial interchain disulfide bonds between the constant regions of the ⁇ and ⁇ chains of the reference TCR to form a more stable soluble TCR, which makes it easier to evaluate the complex of TCR and FMNKFIYEI-HLA A2
  • the binding affinity and/or binding half-life between substances It should be understood that the CDR region of the TCR variable region determines its affinity with the pMHC complex. Therefore, the above-mentioned cysteine substitution in the TCR constant region will not affect the binding affinity and/or binding half-life of the TCR.
  • the measured binding affinity between the reference TCR and the FMNKFIYEI-HLA A0201 complex is considered to be the binding affinity between the wild-type TCR and the FMNKFIYEI-HLA A0201 complex.
  • the binding affinity between the TCR of the present invention and the FMNKFIYEI-HLA A0201 complex is at least 10 times that between the reference TCR and the FMNKFIYEI-HLA A0201 complex, it is equivalent to the TCR of the present invention and FMNKFIYEI -The binding affinity between the HLA A0201 complex is at least 10 times that between the wild-type TCR and the FMNKFIYEI-HLA A0201 complex.
  • the binding affinity (inversely proportional to the dissociation equilibrium constant K D ) and the binding half-life (denoted as T 1/2 ) can be determined by any suitable method. It should be understood that doubling the affinity of TCR will cause K D to be halved. T 1/2 is calculated as In2 divided by the dissociation rate (K off ). Therefore, doubling T 1/2 will cause K off to be halved.
  • the same test protocol is used to detect the binding affinity or binding half-life of a given TCR several times, for example, 3 times or more, and the results are averaged.
  • the surface plasmon resonance (BIAcore) method in the examples herein is used for these detections.
  • This method detects that the dissociation equilibrium constant K D of the reference TCR to the FMNKFIYEI-HLAA2 complex is 2.08E-04M, that is, 208 ⁇ M, and in the present invention, the dissociation equilibrium constant K of the wild-type TCR to the FMNKFIYEI-HLA A2 complex is considered D is also 208 ⁇ M.
  • the affinity of the TCR and FMNKFIYEI-HLA A0201 complex is at least 5 times that of the wild-type TCR; preferably, at least 10 times; more preferably, at least 50 times.
  • the affinity of the TCR and FMNKFIYEI-HLA A0201 complex is at least 100 times that of the wild-type TCR; preferably, at least 500 times; more preferably, at least 1000 times.
  • the dissociation equilibrium constant K D of the TCR to the FMNKFIYEI-HLA A0201 complex is ⁇ 20 ⁇ M;
  • the dissociation equilibrium constant of the TCR to the FMNKFIYEI-HLA A0201 complex is 5 ⁇ M ⁇ K D ⁇ 10 ⁇ M; preferably, 0.1 ⁇ M ⁇ K D ⁇ 1 ⁇ M; more preferably, 1 nM ⁇ K D ⁇ 100 nM .
  • PCR polymerase chain reaction
  • cloning based on restriction enzymes or ligation-independent cloning (LIC) methods.
  • LIC ligation-independent cloning
  • the method for producing the TCR of the present invention can be, but is not limited to, screening a TCR with high affinity for the FMNKFIYEI-HLA-A2 complex from a diverse library of phage particles displaying such TCR, as shown in the literature (Li, et al. (2005) Nature Biotech 23(3):349-354).
  • genes expressing wild-type TCR alpha and beta chain variable domain amino acids or genes expressing slightly modified wild-type TCR alpha and beta chain variable domain amino acids can be used to prepare template TCRs.
  • the DNA encoding the variable domain of the template TCR then introduces the changes required to produce the high-affinity TCR of the present invention.
  • the high-affinity TCR of the present invention comprises one of the alpha chain variable domain amino acid sequence SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 and / Or ⁇ chain variable domain amino acid sequence SEQ ID NO: 2.
  • the amino acid sequences of the ⁇ -chain variable domain and ⁇ -chain variable domain forming the heterodimeric TCR molecule are preferably from Table 1 below:
  • SEQ ID NO: 1 11 2 2 12 2 3 13 2 4 14 2 5 15 2 6 16 2 7 17 2 8 18 2 9 19 2 10 20 2 11 twenty one 2 12 twenty two 2 13 twenty three 2 14 twenty four 2
  • the TCR of the present invention is a part having at least one TCR ⁇ and/or TCR ⁇ chain variable domain. They usually contain both the TCR ⁇ chain variable domain and the TCR ⁇ chain variable domain. They can be ⁇ heterodimers or single-stranded forms or any other forms that can exist stably. In adoptive immunotherapy, the full-length chain of ⁇ heterodimeric TCR (including cytoplasm and transmembrane domain) can be transfected.
  • the TCR of the present invention can be used as a targeting agent for delivering therapeutic agents to antigen-presenting cells or combined with other molecules to prepare bifunctional polypeptides to target effector cells. In this case, the TCR is preferably in a soluble form.
  • the prior art discloses that the introduction of artificial interchain disulfide bonds between the ⁇ and ⁇ chain constant domains of TCR can obtain soluble and stable TCR molecules, as described in patent document PCT/CN2015/093806 Narrated. Therefore, the TCR of the present invention may be a TCR in which an artificial interchain disulfide bond is introduced between the residues of the constant domain of its ⁇ and ⁇ chains. Cysteine residues form artificial interchain disulfide bonds between the alpha and beta chain constant domains of the TCR. Cysteine residues can be substituted for other amino acid residues at appropriate positions in the natural TCR to form artificial interchain disulfide bonds.
  • Thr48 in TRAC*01 exon 1 and replacing Ser57 in TRBC1*01 or TRBC2*01 exon 1 to form a disulfide bond can also be: Thr45 of TRAC*01 exon 1 and TRBC1*01 or Ser77 of TRBC2*01 exon 1; TRAC*01 exon Tyr10 of 1 and Ser17 of TRBC1*01 or TRBC2*01 exon 1; Thr45 of TRAC*01 exon 1 and Asp59 of TRBC1*01 or TRBC2*01 exon 1; TRAC*01 exon 1 Ser15 and TRBC1*01 or TRBC2*01 exon 1 Glu15; TRAC*01 exon 1 Arg53 and TRBC1*01 or TRBC2*01 exon 1 Ser54; TRAC*01 exon 1 Pro89 and Ala19 of TRBC1*01 or TRBC2*01 exon 1; or Tyr10 of TRAC*01 exon 1 and Glu20
  • cysteine residues replace any set of positions in the constant domains of the ⁇ and ⁇ chains.
  • One or more C-terminals of the TCR constant domain of the present invention can be truncated up to 15, or up to 10, or up to 8 or less amino acids so that it does not include cysteine residues to achieve the deletion of natural
  • the purpose of interchain disulfide bonds can also be achieved by mutating the cysteine residues that form natural interchain disulfide bonds to another amino acid.
  • the TCR of the present invention may contain artificial interchain disulfide bonds introduced between the residues of the constant domains of its ⁇ and ⁇ chains. It should be noted that, with or without the introduced artificial disulfide bonds between the constant domains, the TCR of the present invention can contain the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence.
  • the TRAC constant domain sequence of TCR and the TRBC1 or TRBC2 constant domain sequence can be linked by natural interchain disulfide bonds present in the TCR.
  • patent document PCT/CN2016/077680 also discloses that the introduction of artificial interchain disulfide bonds between the ⁇ chain variable region and the ⁇ chain constant region of the TCR can significantly improve the stability of the TCR. Therefore, the high-affinity TCR of the present invention may also contain artificial interchain disulfide bonds between the ⁇ chain variable region and the ⁇ chain constant region.
  • cysteine residue that forms an artificial interchain disulfide bond between the ⁇ chain variable region and the ⁇ chain constant region of the TCR is substituted: the 46th amino acid of TRAV and TRBC1*01 or TRBC2* The 60th amino acid of 01 exon 1; the 47th amino acid of TRAV and the 61st amino acid of TRBC1*01 or TRBC2*01 exon 1; the 46th amino acid of TRAV and the TRBC1*01 or TRBC2*01 exon The 61st amino acid of sub 1; or the 47th amino acid of TRAV and the 60th amino acid of TRBC1*01 or TRBC2*01 exon 1.
  • such a TCR may comprise (i) all or part of the TCR ⁇ chain excluding its transmembrane domain, and (ii) all or part of the TCR ⁇ chain excluding its transmembrane domain, wherein (i) and (ii) ) Contains the variable domain and at least a part of the constant domain of the TCR chain, and the ⁇ chain and the ⁇ chain form a heterodimer. More preferably, such a TCR may include an ⁇ chain variable domain and a ⁇ chain variable domain and all or part of the ⁇ chain constant domain except the transmembrane domain, but it does not include the ⁇ chain constant domain. The chain variable domain and the ⁇ chain form a heterodimer.
  • the TCR of the present invention also includes TCRs with mutations in the hydrophobic core region.
  • the mutations in these hydrophobic core regions are preferably mutations that can improve the stability of the TCR of the present invention, as described in Publication No. It is described in the patent document of WO2014/206304.
  • TCR can be mutated at the following variable domain hydrophobic core positions: ( ⁇ and/or ⁇ chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/ Or alpha chain J gene (TRAJ) short peptide amino acid position is the 3rd, 5th, and 7th position from the bottom, and/or ⁇ chain J gene (TRBJ) short peptide amino acid position is the 2nd, 4th, 6th position from the bottom, the position number of the amino acid sequence According to the position number listed in the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • the TCR with mutation in the hydrophobic core region of the present invention may be a highly stable single-chain TCR composed of a flexible peptide chain connecting the variable domains of the ⁇ and ⁇ chains of the TCR.
  • the CDR region of the TCR variable region determines its affinity with the short peptide-HLA complex. Mutations in the hydrophobic core can make the TCR more stable, but it will not affect its affinity with the short peptide-HLA complex.
  • the flexible peptide chain in the present invention can be any peptide chain suitable for connecting the variable domains of the TCR ⁇ and ⁇ chains.
  • the template chain constructed in Example 1 of the present invention for screening high-affinity TCRs is the high-stability single-chain TCR containing the hydrophobic core mutation. Using TCR with higher stability can more conveniently evaluate the affinity between TCR and FMNKFIYEI-HLA-A0201 complex.
  • the CDR regions of the ⁇ -chain variable domain and ⁇ -chain variable domain of the single-chain template TCR are exactly the same as those of the wild-type TCR. That is, the three CDRs of the ⁇ chain variable domain are CDR1 ⁇ : DSAIYN, CDR2 ⁇ : IQSSQRE,
  • CDR3 ⁇ AVNSGGSNYKLT and the 3 CDRs of the ⁇ chain variable domain are CDR1 ⁇ : SGHVS, CDR2 ⁇ : FQNEAQ, and CDR3 ⁇ : ASSLFGQGREKLF.
  • the amino acid sequence (SEQ ID NO: 9) and nucleotide sequence (SEQ ID NO: 10) of the single-stranded template TCR are shown in Figures 5a and 5b, respectively. Based on this, a single-chain TCR composed of ⁇ -chain variable domain and ⁇ -chain variable domain with high affinity to FMNKFIYEI-HLA A0201 complex was screened out.
  • the three CDRs of the single-chain template TCR ⁇ chain variable domain SEQ ID NO: 3, namely CDR1, CDR2, and CDR3 are located at positions 27-32, 50-56 and 91-102 of SEQ ID NO: 3, respectively Bit. Accordingly, the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 3.
  • 93N is the third N of CDR3 ⁇
  • 94S is the fourth S of CDR3 ⁇
  • 95G is the fifth G and 96G of CDR3 ⁇ .
  • the 6th G and 97S of CDR3 ⁇ are the 7th S of CDR3 ⁇
  • the 98N is the 8th N of CDR3 ⁇ .
  • the ⁇ heterodimer with high affinity to the FMNKFIYEI-HLA-A0201 complex of the present invention is obtained by transferring the CDR regions of the ⁇ and ⁇ chain variable domains of the selected high-affinity single-chain TCR To the corresponding positions of the wild-type TCR ⁇ chain variable domain (SEQ ID NO: 1) and ⁇ chain variable domain (SEQ ID NO: 2).
  • the TCR of the present invention can also be provided in the form of a multivalent complex.
  • the multivalent TCR complex of the present invention contains two, three, four or more TCRs of the present invention combined to form a polymer.
  • the tetramerization domain of p53 can be used to generate a tetramer, or more A complex formed by combining the TCR of the present invention with another molecule.
  • the TCR complex of the present invention can be used to track or target cells presenting a specific antigen in vitro or in vivo, and can also be used to produce intermediates of other multivalent TCR complexes with such applications.
  • the TCR of the present invention can be used alone or combined with the conjugate in a covalent or other manner, preferably in a covalent manner.
  • the conjugate includes a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the FMNKFIYEI-HLA-A0201 complex), a therapeutic agent, a PK (protein kinase) modified portion or any of the above Combination of substances combined or coupled.
  • Detectable markers used for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or capable of producing detectable products Of enzymes.
  • Therapeutic agents that can be combined or coupled with the TCR of the present invention include but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539); 2. Biotoxicity (Chaudhary et al., 1989) , Nature 339,394; Epel et al., 2002, Cancer Immunology and Immunotherapy (Cancer Immunology and Immunotherapy 51,565); 3. Cytokines such as IL-2, etc.
  • Gold nanoparticles/nano Stick (Lapotko et al., 2005, Cancer letters 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115); 7. Virus particles (Peng et al., 2004, Gene Treatment (Genetherapy) 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research (Cancer research) 65, 11631); 9. Nano magnetic particles; 10. Prodrug activating enzymes (for example, DT-cardiac Diazyme (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. Chemotherapeutics (for example, cisplatin) or any form of nanoparticles, etc.
  • DTD DT-cardiac Diazyme
  • BPHL biphenyl hydrolase-like protein
  • the antibodies or fragments thereof that bind to the TCR of the present invention include anti-T cell or NK-cell determining antibodies, such as anti-CD3 or anti-CD28 or anti-CD16 antibodies.
  • the combination of the aforementioned antibodies or fragments with TCR can affect effector cells. Orientation to better target target cells.
  • a preferred embodiment is that the TCR of the present invention is combined with an anti-CD3 antibody or a functional fragment or variant of the anti-CD3 antibody.
  • the fusion molecule of the TCR and the anti-CD3 single chain antibody of the present invention includes the amino acid sequence of the variable domain of the TCR ⁇ chain selected from the group consisting of SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23 and 24, and the amino acid sequence of the variable domain of the TCR ⁇ chain SEQ ID NO: 2.
  • the invention also relates to a nucleic acid molecule encoding the TCR of the invention.
  • the nucleic acid molecule of the present invention may be in the form of DNA or RNA.
  • DNA can be a coding strand or a non-coding strand.
  • the nucleic acid sequence encoding the TCR of the present invention may be the same as the nucleic acid sequence shown in the drawings of the present invention or a degenerate variant.
  • degenerate variant refers to a protein sequence that encodes SEQ ID NO: 3, but has the same sequence as SEQ ID NO: 5 Different nucleic acid sequences.
  • the full-length sequence of the nucleic acid molecule of the present invention or its fragments can usually be obtained by but not limited to PCR amplification method, recombination method or artificial synthesis method.
  • the DNA sequence encoding the TCR (or a fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • the present invention also relates to a vector containing the nucleic acid molecule of the present invention, and a host cell produced by genetic engineering using the vector or coding sequence of the present invention.
  • the invention also includes isolated cells expressing the TCR of the invention, particularly T cells.
  • T cells There are many methods suitable for T cell transfection with DNA or RNA encoding the high-affinity TCR of the present invention (eg, Robbins et al., (2008) J. Immunol. 180: 6116-6131).
  • T cells expressing the high-affinity TCR of the present invention can be used for adoptive immunotherapy.
  • Those skilled in the art can know many suitable methods for adoptive therapy (eg, Rosenberg et al., (2008) Nat Rev Cancer 8(4): 299-308).
  • the present invention also provides a pharmaceutical composition containing a pharmaceutically acceptable carrier and the TCR of the present invention, or the TCR complex of the present invention, or a cell presenting the TCR of the present invention.
  • the present invention also provides a method for treating diseases, which comprises administering an appropriate amount of the TCR of the present invention, or the TCR complex of the present invention, or cells presenting the TCR of the present invention, or the pharmaceutical composition of the present invention to a subject in need of treatment.
  • amino acids in this article are identified by internationally accepted single English letters, and the corresponding three-letter abbreviations of amino acid names are: Ala(A), Arg(R), Asn(N), Asp(D), Cys (C), Gln(Q), Glu(E), Gly(G), His(H), Ile(I), Leu(L), Lys(K), Met(M), Phe(F), Pro (P), Ser(S), Thr(T), Trp(W), Tyr(Y), Val(V);
  • Pro60 or 60P represents proline at position 60.
  • the specific form of the mutation in the present invention is expressed as "N93D” represents that the N at position 93 is replaced by D.
  • N93D/E represents that the N at position 93 is replaced by D or E. The other analogy is similar.
  • the TCR of the present invention also includes at most 5, preferably at most 3, more preferably at most 2, and most preferably 1 amino acid (especially the amino acid located outside the CDR region) of the TCR of the present invention. Or similar amino acids are replaced, and still maintain its functional TCR.
  • the present invention also includes a TCR slightly modified from the TCR of the present invention.
  • Modified (usually not changing the primary structure) forms include: chemically derived forms of the TCR of the present invention such as acetylation or carboxylation.
  • Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of the TCR of the present invention or further processing steps. This modification can be accomplished by exposing the TCR to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase).
  • Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes TCR that has been modified to improve its resistance to proteolysis or optimize its solubility.
  • the TCR, TCR complex of the present invention or T cells transfected with the TCR of the present invention can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the TCR, multivalent TCR complex or cell of the present invention is usually provided as part of a sterile pharmaceutical composition, which usually includes a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be in any suitable form (depending on the desired method of administration to the patient). It can be provided in a unit dosage form, usually in a sealed container, and can be provided as part of a kit. Such kits (but not required) include instructions for use. It may include a plurality of such unit dosage forms.
  • the TCR of the present invention can be used alone, or can be combined or coupled with other therapeutic agents (for example, formulated in the same pharmaceutical composition).
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent.
  • medicament carriers they themselves do not induce the production of antibodies that are harmful to the individual receiving the composition and do not have excessive toxicity after administration.
  • Such vectors are well known to those of ordinary skill in the art.
  • Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • the pharmaceutically acceptable carrier in the therapeutic composition may contain liquids such as water, saline, glycerol and ethanol.
  • these carriers may also contain auxiliary substances, such as wetting or emulsifying agents, and pH buffering substances.
  • the therapeutic composition can be made into an injectable, such as a liquid solution or suspension; it can also be made into a solid form suitable for being formulated into a solution or suspension in a liquid carrier before injection.
  • an injectable such as a liquid solution or suspension
  • it can also be made into a solid form suitable for being formulated into a solution or suspension in a liquid carrier before injection.
  • composition of the invention can be administered by conventional routes, including (but not limited to): intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably gastrointestinal
  • the outside includes subcutaneous, intramuscular or intravenous.
  • the objects to be prevented or treated can be animals; especially humans.
  • composition of the present invention When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition can be used according to the use situation. Preferably, injections and oral preparations can be exemplified.
  • compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic (Isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a usual manner according to the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic (Isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a usual manner according to the dosage form.
  • the pharmaceutical composition of the present invention can also be administered in the form of a sustained-release formulation.
  • the TCR of the present invention can be incorporated into a pill or microcapsule with a sustained-release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated.
  • sustained-release polymers ethylene-vinyl acetate copolymers, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, Lactic acid-glycolic acid copolymers and the like, preferably exemplified are biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.
  • the TCR or TCR complex of the present invention as the active ingredient or the cells presenting the TCR of the present invention can be based on the weight, age, sex, and degree of symptoms of each patient to be treated. The reasonable dosage is determined by the doctor.
  • the affinity and/or binding half-life of the TCR of the present invention for the FMNKFIYEI-HLA-A2 complex is at least 5 times, preferably at least 10 times, that of the wild-type TCR.
  • the affinity and/or binding half-life of the TCR of the present invention for the FMNKFIYEI-HLA-A2 complex is at least 100 times, preferably at least 1000 times, that of the wild-type TCR.
  • the effector cells transduced with the high-affinity TCR of the present invention have a strong killing effect on target cells.
  • E. coli DH5 ⁇ was purchased from Tiangen
  • E. coli BL21 (DE3) was purchased from Tiangen
  • E. coli Tuner (DE3) was purchased.
  • plasmid pET28a was purchased from Novagen.
  • Example 1 Stability of hydrophobic core mutations. Generation of single-stranded TCR template chains
  • the present invention uses the method of site-directed mutagenesis, according to the patent document WO2014/206304, to construct a stable single-stranded TCR molecule composed of a flexible short peptide (linker) connecting TCR ⁇ and ⁇ chain variable domains, its amino acids and DNA
  • the sequences are SEQ ID NO: 9 and SEQ ID NO: 10, respectively, as shown in Figure 5a and Figure 5b.
  • the amino acid sequences of the ⁇ variable domain (SEQ ID NO: 3) and ⁇ variable domain (SEQ ID NO: 4) of the template chain are shown in Figure 2a and 2b; the corresponding DNA sequences are respectively SEQ ID NO: 5 And 6, as shown in Figures 3a and 3b; the amino acid sequence and DNA sequence of the flexible short peptide (linker) are SEQ ID NO: 7 and 8, respectively, as shown in Figures 4a and 4b.
  • the target gene carrying the template chain was digested with Nco I and Not I, and ligated with the pET28a vector that was digested with Nco I and Not I.
  • the ligation product was transformed into E.coli DH5 ⁇ , spread on an LB plate containing kanamycin, and incubated overnight at 37°C. Positive clones were selected for PCR screening, the positive recombinants were sequenced, and the recombinant plasmids were extracted after confirming the correct sequence.
  • E.coli BL21(DE3) for expression.
  • Example 2 Expression, renaturation and purification of the stable single-chain TCR constructed in Example 1
  • the inclusion bodies were dissolved in a buffer (20mM Tris-HCl pH 8.0, 8M urea), centrifuged at a high speed to remove insoluble materials, the supernatant was quantified by BCA method and then aliquoted and stored at -80°C for later use.
  • a syringe to drop the single-stranded TCR treated above into 125mL of refolding buffer (100mM Tris-HCl pH 8.1, 0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mM ⁇ -mercapthoethylamine, 1.87mM Cystamine), Stir at 4°C for 10 minutes, then put the refolding solution into a cellulose membrane dialysis bag with a cutoff of 4kDa, place the dialysis bag in 1L of pre-cooled water, and stir slowly at 4°C overnight.
  • refolding buffer 100mM Tris-HCl pH 8.1, 0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mM ⁇ -mercapthoethylamine, 1.87mM Cystamine
  • the collected elution fractions were analyzed by SDS-PAGE, and the fractions containing single-stranded TCR were concentrated and further purified with a gel filtration column (Superdex 75 10/300, GE Healthcare), and the target fractions were also analyzed by SDS-PAGE.
  • the eluted fractions used for BIAcore analysis were further tested for purity by gel filtration.
  • the conditions are: chromatographic column Agilent Bio SEC-3 (300A, ⁇ 7.8 ⁇ 300mm), mobile phase is 150mM phosphate buffer, flow rate 0.5mL/min, column temperature 25°C, UV detection wavelength 214nm.
  • the BIAcore T200 real-time analysis system was used to detect the binding activity of TCR molecules and the FMNKFIYEI-HLA-A0201 complex.
  • the TCR was diluted with HEPES-EP buffer (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.005% P20, pH 7.4) into several different concentrations at a flow rate of 30 ⁇ L/min , Flow through the chip surface in sequence, the binding time of each injection is 120s, and let it dissociate for 600s after the last injection. After each round of measurement, the chip was regenerated with 10mM Gly-HCl at pH 1.75. Use BIAcore Evaluation software to calculate kinetic parameters.
  • the synthetic short peptide FMNKFIYEI (Beijing Saibaisheng Gene Technology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg/ml.
  • the inclusion bodies of the light chain and the heavy chain were dissolved with 8M urea, 20mM Tris pH 8.0, 10mM DTT, and 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA were added before renaturation to further denature.
  • FMNKFIYEI peptide at 25mg/L (final concentration) to refolding buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4°C), then add 20mg/L light chain and 90mg/L heavy chain (final concentration, heavy chain is added in three times, 8h/time), and renaturate at 4°C for at least 3 days Upon completion, SDS-PAGE will test whether the refolding is successful.
  • refolding buffer 0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4°C
  • the protein-containing fractions were combined, concentrated with a Millipore ultrafiltration tube, the protein concentration was determined by BCA method (Thermo), and the protease inhibitor cocktail (Roche) was added to store the biotinylated pMHC molecules in aliquots at -80°C.
  • Phage display technology is a means of generating TCR high-affinity variant libraries to screen high-affinity variants.
  • the TCR phage display and screening method described by Li et al. ((2005) Nature Biotech 23(3):349-354) was applied to the single-stranded TCR template in Example 1.
  • a high-affinity TCR library was established and panned. After several rounds of panning, the phage library has specific binding to the corresponding antigen, and a single clone is selected from it and analyzed.
  • the BIAcore method in Example 3 was used to analyze the interaction between TCR molecules and the FMNKFIYEI-HLA-A0201 complex, and high-affinity TCRs with affinity and/or binding half-life at least 5 times that of wild-type TCR were screened, that is, the screened high affinity TCR binding solution FMNKFIYEI-HLA-A0201 complex dissociation equilibrium constant K D or less of wild-type TCR binding solution FMNKFIYEI-HLA-A0201 complex from one-fifth of the equilibrium constant K D, the following results in table 3 Shown.
  • the K D value of the interaction between the reference TCR and the FMNKFIYEI-HLA-A0201 complex was 208 ⁇ M, and the interaction curve is shown in Figure 9, that is, the interaction between the wild-type TCR and the FMNKFIYEI-HLA-A0201 complex
  • the KD value is also 208 ⁇ M, which is 2.08E-04M.
  • a single-chain TCR whose affinity with the FMNKFIYEI-HLA-A0201 complex is at least 5 times that of the wild-type TCR and the FMNKFIYEI-HLA-A0201 complex is selected.
  • the CDR region mutation of the high-affinity single-chain TCR screened in Example 4 was introduced into the corresponding position of the variable domain of ⁇ heterodimeric TCR, and the complex with FMNKFIYEI-HLA-A0201 was detected by BIAcore Affinity.
  • the introduction of the above-mentioned high-affinity mutation points in the CDR region adopts a site-directed mutation method well known to those skilled in the art.
  • the amino acid sequences of the alpha chain and beta chain variable domains of the wild-type TCR are shown in Figure 1a (SEQ ID NO: 1) and 1b (SEQ ID NO: 2), respectively.
  • the ⁇ heterodimeric TCR can be constant in the ⁇ and ⁇ chains.
  • a cysteine residue was introduced into the regions to form the TCR of the artificial inter-chain disulfide bond.
  • the amino acid sequences of the TCR ⁇ and ⁇ chains after the introduction of cysteine residues are shown in Figure 7a (SEQ ID NO : 26) and 7b (SEQ ID NO: 27), the introduced cysteine residues are indicated in bold letters.
  • the extracellular sequence genes of the TCR ⁇ and ⁇ chains to be expressed were synthesized and inserted into the expression vector by the standard method described in "Molecular Cloning a Laboratory Manual” (third edition, Sambrook and Russell) pET28a+ (Novagene), the upstream and downstream cloning sites are NcoI and NotI respectively. Mutations in the CDR region are introduced by overlapping PCR (overlap PCR) well known to those skilled in the art. The inserted fragment was confirmed by sequencing.
  • the ⁇ and ⁇ chains of TCR were expressed
  • the inclusion bodies formed later were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution.
  • the inclusion bodies were finally dissolved in 6M guanidine hydrochloride, 10mM dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA) ), 20mM Tris (pH 8.1).
  • the dissolved TCR ⁇ and ⁇ chains are quickly mixed with 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine, 6.6mM ⁇ -mercapoethylamine (4°C) at a mass ratio of 1:1, and the final concentration is 60mg/mL.
  • 5M urea 20mM Tris (pH 8.1)
  • 20mM Tris 20mM Tris (pH 8.1)
  • cystamine 6.6mM ⁇ -mercapoethylamine
  • 4°C a mass ratio of 1:1
  • the solution is filtered by a 0.45 ⁇ M filter membrane and purified by an anion exchange column (HiTrap Q HP, 5ml, GE Healthcare).
  • the eluted peak contains the TCR of the successfully renatured ⁇ and ⁇ dimers and confirmed by SDS-PAGE gel.
  • TCR was then further purified by gel filtration chromatography (HiPrep16/60, Sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was determined by SDS-PAGE to be greater than 90%, and the concentration was determined by the BCA method.
  • Example 3 The method described in Example 3 was used to detect the affinity of the ⁇ heterodimeric TCR introduced into the high-affinity CDR region and the FMNKFIYEI-HLA-A0201 complex.
  • the CDR regions selected from the high-affinity single-chain TCR ⁇ and ⁇ chains are transferred to the corresponding positions of the wild-type TCR ⁇ chain variable domain SEQ ID NO:1 and ⁇ chain variable domain SEQ ID NO: 2 to form ⁇ Quality dimerization TCR.
  • the obtained amino acid sequence of the new TCR ⁇ chain variable domain is shown in Figure 6(1)-(14). Since the CDR region of the TCR molecule determines its affinity with the corresponding pMHC complex, those skilled in the art can expect that the ⁇ heterodimeric TCR introduced with high affinity mutation points will also have high affinity for the FMNKFIYEI-HLA-A0201 complex .
  • the expression vector was constructed using the method described in Example 5, and the above-mentioned ⁇ heterodimeric TCR introduced with high affinity mutations was expressed, renatured and purified using the method described in Example 6, and then the BIAcore T200 was used to determine its relationship with FMNKFIYEI-
  • the affinity of the HLA-A0201 complex is shown in Table 2 below.
  • the ⁇ heterodimeric TCR introduced into the mutation point of the CDR region maintains a high affinity for the FMNKFIYEI-HLA-A0201 complex.
  • the affinity of the heterodimeric TCR is at least 5 times that of the wild-type TCR for the FMNKFIYEI-HLA-A0201 complex.
  • Example 8 Expression, renaturation and purification of the fusion of anti-CD3 antibody and high-affinity ⁇ heterodimeric TCR
  • the anti-CD3 single chain antibody (scFv) was fused with ⁇ heterodimeric TCR to prepare a fusion molecule.
  • the anti-CD3 scFv is fused with the ⁇ chain of the TCR.
  • the TCR ⁇ chain may include any of the above-mentioned high-affinity ⁇ heterodimeric TCR ⁇ -chain variable domains
  • the TCR ⁇ chain of the fusion molecule may include any of the above-mentioned high-affinity The alpha chain variable domain of a sexual alpha beta heterodimeric TCR.
  • the target gene carrying the ⁇ chain of ⁇ heterodimeric TCR was digested with Nco I and Not I, and then linked to the pET28a vector that was digested with Nco I and Not I.
  • the ligation product was transformed into E.coli DH5 ⁇ , spread on an LB plate containing kanamycin, and incubated overnight at 37°C. Positive clones were selected for PCR screening, positive recombinants were sequenced, and the recombinant plasmids were extracted after the sequence was correct. Transform to E.coli Tuner (DE3) for expression.
  • primers are designed to link the anti-CD3scFv and the high-affinity heterodimeric TCR ⁇ chain gene, the linker in the middle is GGGGS (SEQ ID NO: 30), and
  • the gene fragment of the fusion protein of the anti-CD3 scFv and the high-affinity heterodimeric TCR ⁇ chain has restriction endonuclease sites Nco I (CCATGG (SEQ ID NO: 31)) and Not I (GCGGCCGC (SEQ ID NO: 32)).
  • the PCR amplified product was digested with Nco I and Not I, and ligated with the pET28a vector digested with Nco I and Not I.
  • the ligation product was transformed into E.coli DH5 ⁇ competent cells, spread on LB plates containing kanamycin, and incubated overnight at 37°C. Positive clones were selected for PCR screening, and the positive recombinants were sequenced to determine the correct sequence and then extracted The recombinant plasmid is transformed into E. coli Tuner (DE3) competent cells for expression.
  • the expression plasmids were respectively transformed into E. coli Tuner (DE3) competent cells, spread on LB plates (kanamycin 50 ⁇ g/mL) and incubated overnight at 37°C. On the next day, pick clones and inoculate 10mL LB liquid medium (kanamycin 50 ⁇ g/mL) for 2-3 hours, inoculate into 1L LB medium at a volume ratio of 1:100, continue to cultivate until OD600 is 0.5-0.8, add The final concentration is 1mM IPTG induces the expression of the target protein. After 4 hours of induction, the cells were harvested by centrifugation at 6000 rpm for 10 min. Wash the cells once with PBS buffer, and divide them into cells.
  • the dissolved TCR ⁇ chain and anti-CD3(scFv)- ⁇ chain are quickly mixed with 5M urea (urea), 0.4M L-arginine (L-arginine), 20mM Tris pH 8.1, 3.7 at a mass ratio of 2:5 mM cystamine, 6.6mM ⁇ -mercapoethylamine (4°C), the final concentration of ⁇ chain and anti-CD3 (scFv)- ⁇ chain are 0.1mg/mL and 0.25mg/mL respectively.
  • the TCR fusion molecule is then further purified by size exclusion chromatography (S-100 16/60, GE healthcare), and again purified by anion exchange column (HiTrap Q HP 5ml, GE healthcare).
  • the purity of the purified TCR fusion molecule was determined by SDS-PAGE to be greater than 90%, and the concentration was determined by the BCA method.
  • the following experiments were performed to prove the specific activation response of T cells transduced by TCR of the present invention to target cells.
  • the IFN- ⁇ production detected by the ELISPOT test was used as the readout value of T cell activation.
  • Test medium 10% FBS (Gibco, catalog number 16000-044), RPMI 1640 (Gibco, catalog number C11875500bt)
  • PVDF ELISPOT 96-well plate (Merck Millipore, catalog number MSIPS4510)
  • Human IFN- ⁇ ELISPOT PVDF-Enzyme Kit contains all the other reagents needed (capture and detection antibody, streptavidin-alkaline phosphatase and BCIP/NBT solution)
  • the target cells used in this experiment were T2 cells loaded with the specific short peptide FMNKFIYEI. Prepare target cells in the experimental medium: adjust the target cell concentration to 1.0 ⁇ 10 5 cells/ml, and take 100 microliters per well to obtain 1.0 ⁇ 10 4 cells/well.
  • the effector cells (T cells) in this experiment are CD3+ T cells transfected with high-affinity TCR specific for the AFP antigen short peptide of the present invention.
  • the transfected high-affinity TCR molecules are as follows (specifically used in this example and the following examples) TCR names, such as TCR1, TCR2, etc.
  • TCR1 ( ⁇ chain variable domain SEQ ID NO: 11, ⁇ chain variable domain SEQ ID NO: 2)
  • TCR2 ( ⁇ chain variable domain SEQ ID NO: 13, ⁇ chain variable domain SEQ ID NO: 2)
  • TCR3 ( ⁇ chain variable domain SEQ ID NO: 14, ⁇ chain variable domain SEQ ID NO: 2)
  • TCR4 ( ⁇ chain variable domain SEQ ID NO: 15, ⁇ chain variable domain SEQ ID NO: 2)
  • TCR5 ( ⁇ chain variable domain SEQ ID NO: 17, ⁇ chain variable domain SEQ ID NO: 2)
  • TCR6 ( ⁇ chain variable domain SEQ ID NO: 18, ⁇ chain variable domain SEQ ID NO: 2).
  • the same volunteer was used to transfect the wild-type TCR corresponding to the high-affinity TCR of the present invention (named: A0B0, ⁇ chain SEQ ID NO: 28, ⁇ chain SEQ ID NO: 29), and transfected with other high-affinity TCRs (named A6) CD3+ T cells served as a control group.
  • the well plates as follows: Dilute the anti-human IFN- ⁇ capture antibody 1:200 with 10 ml sterile PBS per plate, and then add 50 ⁇ l aliquots of the diluted capture antibody to each well . Incubate the plate overnight at 4°C. After incubation, wash the well plate to remove excess capture antibody. Add 200 ⁇ l/well of PBS medium containing 5% FBS, and incubate the well plate at room temperature for 2 hours to seal the well. The medium is then washed away from the well plate, and any residual wash buffer is removed by flicking and tapping the ELISPOT well plate on the paper.
  • test components added to the ELISPOT plate in the following order:
  • target cells 1*10 5 cells/ml (to get a total of about 1*10 4 target cells/well).
  • effector cells 1*10 3 control effector cells/well and AFP TCR positive T cells/well.
  • Dilute streptavidin-alkaline phosphatase 1:100 with PBS containing 5% FBS add 50 ⁇ l of the diluted streptavidin-alkaline phosphatase to each well and incubate the plate at room temperature 1 hour. Then wash 4 times with washing buffer and 2 times with PBS, tap the well plate on a paper towel to remove excess washing buffer and PBS. After washing, add 50 ⁇ l/well of BCIP/NBT solution provided by the kit for development. During development, cover the orifice plate with tin foil to protect it from light, and let it stand for 2-5 minutes. During this period, routinely check the spots of the developing well plate to determine the best time to terminate the reaction.
  • the ELISPOT experiment (as described above) was used to test the IFN- ⁇ release of T cells transduced with the TCR of the present invention in response to target cells loaded with the AFP antigen short peptide FMNKFIYEI.
  • T cells (effector cells) transduced with the high-affinity TCR of the present invention have a good activation response against target cells loaded with specific short peptides.
  • the release of IFN- ⁇ is much higher than that of effector cells that transduce wild-type TCR, and T cells (effector cells) that transduce other TCRs (A6) have basically no activation response to the corresponding target cells.
  • Example 10 The activation function experiment of effector cells transfected with the high-affinity TCR of the present invention against tumor cell lines
  • the function and specificity of the high-affinity TCR of the present invention in cells are tested by ELISPOT experiment.
  • ELISPOT experiment Those skilled in the art are familiar with the method of using ELISPOT assay to detect cell function.
  • IFN- ⁇ ELISPOT experiment of this example CD3+ T cells isolated from the blood of healthy volunteers were used to transfect the high-affinity TCR of the present invention as effector cells.
  • the TCR transfected effector cells of the present invention are randomly selected, TCR1 ( ⁇ chain variable domain SEQ ID NO: 11, ⁇ chain variable domain SEQ ID NO: 2), TCR3 ( ⁇ chain variable domain SEQ ID NO: 14, ⁇ chain Variable domain SEQ ID NO: 2), TCR5 ( ⁇ chain variable domain SEQ ID NO: 17, ⁇ chain variable domain SEQ ID NO: 2) and TCR6 ( ⁇ chain variable domain SEQ ID NO: 18, ⁇ chain Variable domain SEQ ID NO: 2).
  • the effector cells of the control group were labeled as A0B0 (transfected with wild-type TCR, ⁇ chain SEQ ID NO: 28, ⁇ chain SEQ ID NO: 29) and A6 (transfected with other TCRs other than the present invention).
  • the target cell lines are HepG2, HUH-6, Hep3B, HCCC9810 and SNU-398 cells.
  • the target cell line HepG2 expresses related antigens and the genotype is also consistent with positive cell lines, and HUH-6, Hep3B, HCCC9810 and SNU-398 are negative cell lines as controls.
  • ELISPOT plate was activated and coated with ethanol at 4°C overnight. On the first day of the experiment, remove the coating solution, wash and block, incubate at room temperature for two hours, remove the blocking solution, and add the test components to the ELISPOT plate in the following order: adjust the medium to 1X 10 4 cells/ml , Adjust the medium to each target cell line to 2X 10 5 cells/ml. After mixing uniformly, take 100 ⁇ L of target cells (ie 20,000 cells/well) and 100 ⁇ L of effector cells (ie, 1000 cells/well) into the corresponding wells, and set up two multiple wells. Incubate overnight (37°C, 5% CO 2 ). On the second day of the experiment, the plate was washed and subjected to secondary detection and color development, the plate was dried, and then the spots formed on the membrane were counted with an immunospot plate reader (ELISPOT READER system; AID20 company).
  • ELISPOT READER system AID20 company
  • the effector cells transfected with the high-affinity TCR of the present invention have basically no activation effect, while for positive target cells, there is a very good specific activation effect, and its effect is far better than that of transfection. Effector cells stained with wild-type TCR.
  • a non-radioactive cytotoxicity experiment was used to measure the release of LDH to verify the killing function of the cells transduced with the TCR of the present invention.
  • This test is a colorimetric alternative to the 51Cr release cytotoxicity test, which quantitatively measures the lactate dehydrogenase (LDH) released after cell lysis.
  • LDH lactate dehydrogenase
  • a 30-minute coupled enzyme reaction is used to detect the LDH released in the medium.
  • LDH can convert a tetrazolium salt (INT) into red formazan (formazan).
  • the amount of red product produced is proportional to the number of cells lysed.
  • a standard 96-well plate reader can be used to collect 490nm visible light absorbance data.
  • HCCC9810 and SNU-398 are negative cell lines as controls.
  • the effector cells were transfected with TCR1 ( ⁇ chain variable domain SEQ ID NO: 11, ⁇ chain variable domain SEQ ID NO: 2), TCR3 ( ⁇ chain variable domain SEQ ID NO: 14, ⁇ chain variable domain SEQ ID NO: 2), TCR5 ( ⁇ chain variable domain SEQ ID NO: 17, ⁇ chain variable domain SEQ ID NO: 2) and TCR6 ( ⁇ chain variable domain SEQ ID NO: 18, ⁇ chain variable domain SEQ ID NO: 2), the control group effector cells are labeled A6 (transfected with other TCRs other than the present invention).
  • the effector cells transduced with the TCR of the present invention have a strong killing effect on target cells expressing the relevant antigen, but basically no killing effect on the target cells that do not express the relevant antigen.
  • Example 12 In vivo efficacy of high-affinity TCR molecules of the present invention
  • the T cells transfected with the high-affinity TCR of the present invention are injected into mice of human liver cancer cell xenotransplantation models, and their inhibitory effects on tumors in vivo are tested.
  • the experiment uses NSG mice (Beijing Biocytogene Biotechnology Co., Ltd.) (female, experimental age 6-8 weeks) as the experimental object.
  • the HEPG2 tumor cells (ATCC) were collected and suspended 20 days before the experiment.
  • Suspensions were injected unilaterally into the abdomen of mice with 1*10 ⁇ 7/mouse (injection volume 200ul) to establish a mouse model of human liver cancer xenotransplantation.
  • the prepared IL-2 solution (50000IU/100UL) was injected into the intraperitoneal cavity of each mouse with 100ul, and then the same amount of IL-2 solution was continuously injected every day for 4 days. Since the beginning of the experiment, the mouse tumor tumor diameter and calculated volume were measured every 3 days according to the above method, and continued until the mice were affected by the excessive tumor movement or the tumor subsided. The above data was sorted out and the tumor volume of each group of mice was analyzed. deal with.

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Abstract

本发明提供了一种T细胞受体(TCR)，其具有结合FMNKFIYEI-HLA A0201复合物的特性；并且所述TCR对所述FMNKFIYEI-HLA A0201复合物的结合亲和力是野生型TCR对FMNKFIYEI-HLA A0201复合物的结合亲和力的至少5倍。本发明还提供了此类TCR与治疗剂的融合分子。此类TCR可以单独使用，也可与治疗剂联用，以靶向呈递FMNKFIYEI-HLA A0201复合物肿瘤细胞。

Description

一种识别AFP抗原的高亲和力TCR 技术领域
本发明涉及生物技术领域，更具体地涉及能够识别衍生自AFP蛋白多肽的T细胞受体(T cell receptor,TCR)。本发明还涉及所述受体的制备和用途。
背景技术
仅仅有两种类型的分子能够以特异性的方式识别抗原。其中一种是免疫球蛋白或抗体；另一种是T细胞受体(TCR)，它是由α链/β链或者γ链/δ链以异二聚体形式存在的细胞膜表面的糖蛋白。免疫系统的TCR总谱的组成是在胸腺中通过V(D)J重组，然后进行阳性和阴性选择而产生的。在外周环境中，TCR介导了T细胞对主组织相容性复合体-肽复合物(pMHC)的特异性识别，因此其对免疫系统的细胞免疫功能是至关重要的。
TCR是呈递在主组织相容性复合体(MHC)上的特异性抗原肽的唯一受体，这种外源肽或内源肽可能会是细胞出现异常的唯一迹象。在免疫系统中，通过抗原特异性的TCR与pMHC复合物的结合引发T细胞与抗原呈递细胞(APC)直接的物理接触，然后T细胞及APC两者的其他细胞膜表面分子就发生相互作用，这就引起了一系列后续的细胞信号传递和其他生理反应，从而使得不同抗原特异性的T细胞对其靶细胞发挥免疫效应。
与TCR相对应的MHC I类和II类分子配体也是免疫球蛋白超家族的蛋白质但对于抗原的呈递具有特异性，不同的个体有不同的MHC，从而能呈递一种蛋白抗原中不同的短肽到各自的APC细胞表面。人类的MHC通常称为HLA基因或HLA复合体。
AFP(αFetoprotein)也称α胎蛋白，是胚胎发育过程中表达的一种蛋白，是胚胎血清的主要成分。在发育过程中，AFP在卵黄囊及肝脏中有比较高的表达水平，随后被抑制。在肝细胞癌中，AFP的表达被激活。AFP在细胞内生成后被降解成小分子多肽，并与MHC(主组织相容性复合体)分子结合形成复合物，被呈递到细胞表面。FMNKFIYEI(SEQ ID NO:25)是衍生自AFP抗原的短肽，是AFP相关疾病治疗的一种靶标。
因此，FMNKFIYEI-HLA A0201复合物提供了一种TCR可靶向肿瘤细胞的标记。能够结合FMNKFIYEI-HLA A0201复合物的TCR对肿瘤的治疗具有很高的应用价值。例如，能够靶向该肿瘤细胞标记的TCR可用于将细胞毒性剂或免疫刺激剂递送到靶细胞，或被转化入T细胞，使表达该TCR的T细胞能够破坏肿瘤细胞，以便在被称为过继免疫治疗的治疗过程中给予患者。对于前一目的，理想的TCR是具有较高的亲和力的，从而使该TCR能够长期驻留在所靶向的细胞上面。对于后一目的，则优选使用中等亲和力的TCR。因此，本领域技术人员致力于开发可用于满足不同目的的靶向肿瘤细胞标记的TCR。
发明内容
本发明的目的在于提供一种对FMNKFIYEI-HLA A0201复合物具有较高亲和力的TCR。
本发明的再一目的是提供一种上述类型TCR的制备方法及上述类型TCR的用途。
本发明的第一方面，提供了一种T细胞受体(TCR)，其具有结合FMNKFIYEI-HLA A0201复合物的活性。
在另一优选例中，所述T细胞受体(TCR)具有结合FMNKFIYEI-HLA A0201复合物的活性，并且所述T细胞受体包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域包含3个CDR区，所述TCRα链可变域的3个CDR区的基准序列如下，
CDR1α：DSAIYN
CDR2α：IQSSQRE
CDR3α：AVNSGGSNYKLT，并且CDR3α含有至少一个下列突变：
突变前的残基 突变后的残基
CDR3α的第3位N D或E
CDR3α的第4位S D或G或A或W或T或H
CDR3α的第5位G Q或A或V或H或W或Y或M或I
CDR3α的第6位G D或R或P或Q或T或Y
CDR3α的第7位S G或D
CDR3α的第8位N G或D
和/或所述TCR的β链可变域为与SEQ ID NO:2所示的氨基酸序列有至少90％的序列同源性的氨基酸序列。
在另一优选例中，所述TCR的β链可变域为与SEQ ID NO:2所示的氨基酸序列有至少90％，91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同源性的氨基酸序列。
在另一优选例中，所述TCRα链可变域中CDR3α的突变个数为1至4个。
在另一优选例中，所述TCR与FMNKFIYEI-HLA A0201复合物的亲和力是野生型TCR的至少5倍。
在另一优选例中，所述TCR的α链可变域包含与SEQ ID NO:1所示的氨基酸序列有至少90％，91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性的氨基酸序列。
在另一优选例中，所述TCRβ链可变域包含3个CDR区，所述TCRβ链可变域的3个CDR区的氨基酸序列如下：
CDR1β：SGHVS
CDR2β：FQNEAQ
CDR3β：ASSLFGQGREKLF。
在另一优选例中，所述TCRβ链可变域的氨基酸序列为SEQ ID NO：2。
在另一优选例中，所述TCR包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域包含CDR1α、CDR2α和CDR3α，其中CDR1α的氨基酸序列为DSAIYN，CDR2α的氨基酸序列为IQSSQRE；和所述TCRβ链可变域包含CDR1β、CDR2β和CDR3β，其中CDR1β的氨基酸序列为SGHVS，CDR2β的氨基酸序列为FQNEAQ，CDR3β的氨基酸序列为ASSLFGQGREKLF。
在另一优选例中，所述TCR包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变 域包含CDR1α、CDR2α和CDR3α，其中CDR1α的氨基酸序列为DSAIYN，CDR2α的氨基酸序列为IQSSQRE，并且CDR3α的氨基酸序列为：
AV[3αX1][3αX2][3αX3][3αX4][3αX5][3αX6]YKLT。
在另一优选例中，所述[3αX1]为N或D或E。
在另一优选例中，所述[3αX2]为S或D或G或A或W或T或H。
在另一优选例中，所述[3αX3]为G或Q或A或V或H或W或Y或M或I。
在另一优选例中，所述[3αX4]为G或D或R或P或Q或T或Y。
在另一优选例中，所述[3αX5]为S或G或D。
在另一优选例中，所述[3αX6]为N或G或D。
在另一优选例中，所述TCR具有选自下组的CDR:
CDR编号 CDR1α CDR2α CDR3α CDR1β CDR2β CDR3β
1 DSAIYN IQSSQRE AVDSGGSNYKLT SGHVS FQNEAQ ASSLFGQGREKLF
2 DSAIYN IQSSQRE AVEDQGSNYKLT SGHVS FQNEAQ ASSLFGQGREKLF
3 DSAIYN IQSSQRE AVDGADSNYKLT SGHVS FQNEAQ ASSLFGQGREKLF
4 DSAIYN IQSSQRE AVNSVRGGYKLT SGHVS FQNEAQ ASSLFGQGREKLF
5 DSAIYN IQSSQRE AVEGARSNYKLT SGHVS FQNEAQ ASSLFGQGREKLF
6 DSAIYN IQSSQRE AVDSHPSNYKLT SGHVS FQNEAQ ASSLFGQGREKLF
7 DSAIYN IQSSQRE AVDAAQSNYKLT SGHVS FQNEAQ ASSLFGQGREKLF
8 DSAIYN IQSSQRE AVNSWTGGYKLT SGHVS FQNEAQ ASSLFGQGREKLF
9 DSAIYN IQSSQRE AVDWHPSNYKLT SGHVS FQNEAQ ASSLFGQGREKLF
10 DSAIYN IQSSQRE AVDSQDSNYKLT SGHVS FQNEAQ ASSLFGQGREKLF
11 DSAIYN IQSSQRE AVNSYYDGYKLT SGHVS FQNEAQ ASSLFGQGREKLF
12 DSAIYN IQSSQRE AVDTMDSNYKLT SGHVS FQNEAQ ASSLFGQGREKLF
13 DSAIYN IQSSQRE AVDHHPSNYKLT SGHVS FQNEAQ ASSLFGQGREKLF
14 DSAIYN IQSSQRE AVNSIYGDYKLT SGHVS FQNEAQ ASSLFGQGREKLF
在另一优选例中，所述TCR是可溶的。
在另一优选例中，所述TCR为αβ异质二聚TCR，包含α链TRAC恒定区序列和β链TRBC1或TRBC2恒定区序列。
在另一优选例中，所述TCR包含(ⅰ)除其跨膜结构域以外的全部或部分TCRα链，和(ⅱ)除其跨膜结构域以外的全部或部分TCRβ链，其中(ⅰ)和(ⅱ)均包含TCR链的可变域和至少一部分恒定域。
在另一优选例中，所述TCR的α链恒定区与β链恒定区之间含有人工链间二硫键。
在另一优选例中，在所述TCRα与β链的恒定区之间形成人工链间二硫键的半胱氨酸残基取代了选自下列的一组或多组位点：
TRAC*01外显子1的Thr48和TRBC1*01或TRBC2*01外显子1的Ser57；
TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；
TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；
TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；
TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；
TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；
TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；
和
TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。
在另一优选例中，所述TCR的α链可变域氨基酸序列选自：SEQ ID NO:11-24；和/或所述TCR的β链可变域氨基酸序列为SEQ ID NO:2。
在另一优选例中，所述TCR选自下组：
Figure PCTCN2020078271-appb-000001
在另一优选例中，所述TCR为单链TCR。
在另一优选例中，所述TCR是由α链可变域和β链可变域组成的单链TCR，所述α链可变域和β链可变域由一柔性短肽序列(linker)连接。
在另一优选例中，所述TCR的α链和/或β链的C-或N-末端结合有偶联物。
在另一优选例中，与所述TCR结合的偶联物为可检测标记物、治疗剂、PK修饰部分或任何这些物质的组合。
在另一优选例中，与所述TCR结合的治疗剂为连接于所述TCR的α或β链的C-或N-末端的抗-CD3抗体。
在本发明的一个优选例中，所述TCR与FMNKFIYEI-HLA A0201复合物的亲和力是野生型TCR的至少5倍；优选地，至少10倍；更优选地，至少50倍。
在另一优选例中，所述TCR与FMNKFIYEI-HLA A0201复合物的亲和力是野生型TCR的至少100倍；优选地，至少500倍；更优选地，至少1000倍。
具体地，所述TCR对FMNKFIYEI-HLA A0201复合物的解离平衡常数K D≤20μM；优选地，5μM≤K D≤10μM。
在另一优选例中，所述TCR对FMNKFIYEI-HLA A0201复合物的解离平衡常数0.1μM≤K D≤1μM；优选地，1nM≤K D≤100nM。
在本发明的一个优选的实施方式中，所述T细胞受体(TCR)，其具有结合FMNKFIYEI-HLA A0201复合物的活性，并包含TCRα链可变域和TCRβ链可变域，所述TCR在SEQ ID NO：1所示的α链可变域中发生突变，所述突变的氨基酸残基位点包括93N、94S、95G、96G、97S和98N中的一个或多个，其中，氨基酸残基编号采用SEQ ID NO:1所示的编号；
优选地，突变后的所述TCRα链可变域包括选自下组的一个或多个氨基酸残基：93D或93E；94D或94G或94A或94W或94T或94H；95Q或95A或95V或95H或95W或95Y或95M或95I；96D或96R或96P或96Q或96T或96Y；97G或97D；和98G或98D，其中，氨基酸残基编号采用SEQ ID NO:1所示的编号。
本发明的第二方面，提供了一种多价TCR复合物，包含至少两个TCR分子，并且其中的至少一个TCR分子为本发明第一方面所述的TCR。
本发明的第三方面，提供了一种核酸分子，所述核酸分子包含编码本发明第一方面所述的TCR分子或者本发明第二方面所述的多价TCR复合物的核酸序列或其互补序列。
本发明的第四方面，提供了一种载体，所述的载体含有本发明第三方面所述的所述的核酸分子。
本发明的第五方面，提供了一种宿主细胞，所述的宿主细胞中含有本发明第四方面所述的载体或染色体中整合有外源的本发明第三方面所述的核酸分子。
本发明的第六方面，提供了一种分离的细胞，所述细胞表达本发明第一方面所述的TCR。
本发明的第七方面，提供了一种药物组合物，所述组合物含有药学上可接受的载体以及本发明第一方面所述的TCR、或本发明第二方面所述的TCR复合物、或本发明第六方面所述的细胞。
本发明的第八方面，提供了一种治疗疾病的方法，包括给需要治疗的对象施用适量的本发明第一方面所述的TCR、或本发明第二方面所述的TCR复合物、或本发明第六方面所述的细胞、或本发明第七方面所述的药物组合物。
优选地，所述疾病为AFP阳性肿瘤。
优选地，所述AFP阳性肿瘤为肝癌、乳腺癌或生殖细胞肿瘤；更优选地，所述AFP阳性肿瘤为肝细胞癌。
本发明的第九方面，提供了本发明第一方面所述的TCR、或本发明第二方面所述的TCR复合物、或本发明第六方面所述的细胞的用途，用于制备治疗肿瘤的药物。
优选地，所述肿瘤为AFP阳性肿瘤。
优选地，所述AFP阳性肿瘤为肝癌、乳腺癌或生殖细胞肿瘤；更优选地，所述AFP阳性肿瘤为肝细胞癌。
本发明的第十方面，提供了一种制备本发明第一方面所述的T细胞受体的方法，包括步骤：
(i)培养本发明第五方面所述的宿主细胞，从而表达本发明第一方面所述的T细胞受体；
(ii)分离或纯化出所述的T细胞受体。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1a和图1b分别显示了对FMNKFIYEI-HLA A0201复合物能够特异性结合的野生型TCRα与β链可变域氨基酸序列。
图2a和图2b分别为本发明构建的单链模板TCR的α可变域的氨基酸序列和β链可变域的氨基酸序列。
图3a和图3b分别为本发明构建的单链模板TCR的α可变域的DNA序列和β链可变域的DNA序列。
图4a和图4b分别为本发明构建的单链模板TCR的连接短肽(linker)的氨基酸序列和核苷酸序列。
图5a和图5b分别为本发明构建的单链模板TCR的氨基酸序列和DNA序列。
图6(1)-(14)分别显示了对FMNKFIYEI-HLA A0201复合物具有高亲和力的异质二聚TCR的α链可变域氨基酸序列，突变的残基以加下划线表示。
图7a和图7b分别显示了本发明中参比TCRα与β链的氨基酸序列。
图8a和图8b分别显示了对FMNKFIYEI-HLA A0201复合物能够特异性结合的野生型TCRα与β链氨基酸序列。
图9为参比TCR即野生型TCR与FMNKFIYEI-HLA A0201复合物的结合曲线。
图10a-f为针对负载特异短肽的T2细胞，转染本发明高亲和力TCR的效应细胞的激活功能实验结果图。
图11为针对肿瘤细胞系，转染本发明高亲和力TCR的效应细胞的激活功能实验结果图。
图12为转染本发明高亲和力TCR的效应细胞的杀伤功能实验结果。
图13为转染本发明高亲和力TCR的T细胞的体内效力实验结果。
具体实施方式
本发明通过广泛而深入的研究，获得一种识别FMNKFIYEI短肽(衍生自AFP蛋白)的高亲和性T细胞受体(TCR)，所述FMNKFIYEI短肽以肽-HLA A0201复合物的形式被呈递。所述高亲和性TCR在其α链可变域的3个CDR区：
CDR1α：DSAIYN
CDR2α：IQSSQRE
CDR3α：AVNSGGSNYKLT中发生突变；
并且，突变后本发明TCR对上述FMNKFIYEI-HLA A0201复合物的亲和力和/或结合半衰期是野生型TCR的至少5倍。
在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且其意图不是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。
虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。
术语
T细胞受体(T cell receptor,TCR)
可以采用国际免疫遗传学信息系统(IMGT)来描述TCR。天然αβ异源二聚TCR具有α链和β链。广义上讲，各链包含可变区、连接区和恒定区，β链通常还在可变区和连接区之间含有短的多变区，但该多变区常视作连接区的一部分。通过独特的IMGT的TRAJ和TRBJ确定TCR的连接区，通过IMGT的TRAC和TRBC确定TCR的恒定区。
各可变区包含嵌合在框架序列中的3个CDR(互补决定区)，CDR1、CDR2和CDR3。在IMGT命名法中，TRAV和TRBV的不同编号分别指代不同Vα类型和Vβ的类型。在IMGT系统中，α链恒定结构域具有以下的符号：TRAC*01，其中“TR”表示T细胞受体基因；“A”表示α链基因；C表示恒定区；“*01”表示等位基因1。β链恒定结构域具有以下的符号：TRBC1*01或TRBC2*01，其中“TR”表示T细胞受体基因；“B”表示β链基因；C表示恒定区；“*01”表示等位基因1。α链的恒定区是唯一确定的，在β链的形式中，存在两个可能的恒定区基因“C1”和“C2”。本领域技术人员通过公开的IMGT数据库可以获得TCRα与β链的恒定区基因序列。
TCR的α和β链一般看作各有两个“结构域”即可变域和恒定结构域。可变域由连接的可变区和连接区构成。因此，在本申请的说明书和权利要求书中，“TCRα链可变域”指连接的TRAV和TRAJ区，同样地，“TCRβ链可变域”指连接的TRBV和TRBD/TRBJ区。TCRα链可变域的3个CDR分别为CDR1α、CDR2α和CDR3α；TCRβ链可变域的3个CDR分别为CDR1β、CDR2β和CDR3β。本发明TCR可变域的框架序列可以为鼠源的或人源的，优选为人源的。 TCR的恒定结构域包含胞内部分、跨膜区和胞外部分。
本发明中所述“野生型TCR”的α链氨基酸序列及β链氨基酸序列分别为SEQ ID NO:28和SEQ ID NO：29，如图8a和8b所示。本发明中所述“参比TCR”的α链氨基酸序列及β链氨基酸序列分别为SEQ ID NO:26和SEQ ID NO：27，如图7a和7b所示。本发明中，能够结合FMNKFIYEI-HLA A0201复合物的野生型TCR的α与β链可变域氨基酸序列分别为SEQ ID NO:1和SEQ ID NO：2，如图1a和1b所示。在本发明中，术语“本发明多肽”、“本发明的TCR”、“本发明的T细胞受体”可互换使用。
天然链间二硫键与人工链间二硫键
在天然TCR的近膜区Cα与Cβ链间存在一组二硫键，本发明中称为“天然链间二硫键”。在本发明中，将人工引入的，位置与天然链间二硫键的位置不同的链间共价二硫键称为“人工链间二硫键”。
为方便描述，本发明中TRAC*01与TRBC1*01或TRBC2*01氨基酸序列的位置编号按从N端到C端依次的顺序进行位置编号，如TRBC1*01或TRBC2*01中，按从N端到C端依次的顺序第60个氨基酸为P(脯氨酸)，则本发明中可将其描述为TRBC1*01或TRBC2*01外显子1的Pro60，也可将其表述为TRBC1*01或TRBC2*01外显子1的第60位氨基酸，又如TRBC1*01或TRBC2*01中，按从N端到C端依次的顺序第61个氨基酸为Q(谷氨酰胺)，则本发明中可将其描述为TRBC1*01或TRBC2*01外显子1的Gln61，也可将其表述为TRBC1*01或TRBC2*01外显子1的第61位氨基酸，其他以此类推。本发明中，其他氨基酸的序列位置编号有特殊说明的，则按特殊说明。
肿瘤
术语“肿瘤”指包括所有类型的癌细胞生长或致癌过程，转移性组织或恶性转化细胞、组织或器官，不管病理类型或侵染的阶段。肿瘤的实施例非限制性地包括：实体瘤，软组织瘤，和转移性病灶。实体瘤的实施例包括：不同器官系统的恶性肿瘤，例如肉瘤，肺鳞癌和癌症。例如：感染的前列腺，肺，乳房，淋巴，肠胃(例如：结肠)，和生殖泌尿道(例如：肾脏，上皮细胞)，咽头。肺鳞癌包括恶性肿瘤，例如，多数的结肠癌，直肠癌，肾细胞癌，肝癌，肺部的非小细胞癌，小肠癌和食道癌。上述癌症的转移性病变可同样用本发明的方法和组合物来治疗和预防。
发明详述
众所周知，TCR的α链可变域与β链可变域各含有3个CDR，类似于抗体的互补决定区。CDR3与抗原短肽相互作用，CDR1和CDR2与HLA相互作用。因此，TCR分子的CDR决定了其与抗原短肽-HLA复合物的相互作用。能够结合抗原短肽FMNKFIYEI与HLA A0201复合物(即，FMNKFIYEI-HLA A0201复合物)的野生型TCR的α链可变域氨基酸序列与β链可变域氨基酸序列分别为SEQ ID NO:1和SEQ ID NO：2,该序列为本发明人首次发现。其具有下列CDR区：
α链可变域CDR CDR1α：DSAIYN
CDR2α：IQSSQRE
CDR3α：AVNSGGSNYKLT
和β链可变域CDR CDR1β：SGHVS
CDR2β：FQNEAQ
CDR3β：ASSLFGQGREKLF
本发明通过对上述CDR区进行突变筛选，获得了与FMNKFIYEI-HLA A0201复合物的亲和力是野生型TCR与FMNKFIYEI-HLA A0201复合物亲和力至少5倍的高亲和力TCR。
本发明提供了一种T细胞受体(TCR)，其具有结合FMNKFIYEI-HLA A0201复合物的活性。
所述T细胞受体包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域包含3个CDR区，所述TCRα链可变域的3个CDR区的基准序列如下，
CDR1α：DSAIYN
CDR2α：IQSSQRE
CDR3α：AVNSGGSNYKLT，并且含有至少一个下列突变：
Figure PCTCN2020078271-appb-000002
和/或，所述TCRβ链可变域包含3个CDR区，所述TCRβ链可变域的3个CDR区的基准序列如下，
CDR1β：SGHVS
CDR2β：FQNEAQ
CDR3β：ASSLFGQGREKLF。
更详细地，所述TCRα链CDR区的突变个数可以为1个、2个、3个、4个、5个、或6个。
进一步，本发明所述TCR是αβ异质二聚TCR，所述TCR的α链可变域包含与SEQ ID NO:1所示的氨基酸序列有至少85％；优选地，至少90％；更优选地，至少92％；更优选地，至少94％(如，可以是至少88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同源性)的序列同源性的氨基酸序列；和/或所述TCR的β链可变域包含与SEQ ID NO:2所示的氨基酸序列有至少90％，优选地，至少92％；更优选地，至少94％(如，可以是至少91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同源性)的序列同源性的氨基酸序列。
进一步，本发明所述TCR是单链TCR，所述TCR的α链可变域包含与SEQ ID NO:3所示 的氨基酸序列有至少85％，优选地，至少90％；更优选地，至少92％；最优选地，至少94％(如，可以是至少88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同源性)的序列同源性的氨基酸序列；和/或所述TCR的β链可变域包含与SEQ ID NO:4所示的氨基酸序列有至少85％，优选地，至少90％；更优选地，至少92％；最优选地，至少94％；(如，可以是至少91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同源性)的序列同源性的氨基酸序列。
本发明中野生型TCRα链可变域SEQ ID NO：1的3个CDR即CDR1、CDR2和CDR3分别位于SEQ ID NO：1的第27-32位、第50-56位和第91-102位。据此，氨基酸残基编号采用SEQ ID NO:1所示的编号，93N即为CDR3α的第3位N、94S即为CDR3α的第4位S、95G即为CDR3α的第5位G、96G即为CDR3α的第6位G、97S即为CDR3α的第7位S，98N即为CDR3α的第8位N。
本发明提供具有结合FMNKFIYEI-HLA A0201复合物的特性的TCR，并包含α链可变域和β链可变域，其特征在于，所述TCR在SEQ ID NO：1所示的α链可变域中发生突变，所述突变的氨基酸残基位点包括93N、94S、95G、96G、97S和98N中的一个或多个，其中，氨基酸残基编号采用SEQ ID NO:1所示的编号。
优选地，突变后的所述TCRα链可变域包括选自下组的一个或多个氨基酸残基：93D或93E；94D或94G或94A或94W或94T或94H；95Q或95A或95V或95H或95W或95Y或95M或95I；96D或96R或96P或96Q或96T或96Y；97G或97D；和98G或98D，其中，氨基酸残基编号采用SEQ ID NO:1所示的编号。
更具体地，α链可变域中所述突变的具体形式包括N93/D/E、S94/D/G/A/W/T/H；G95/Q/A/V/H/W/Y/M/I；G96/D/R/P/Q/T/Y；S97/G/D；和N98/G/D中的一组或几组。
根据本领域技术人员熟知的定点突变的方法，将野生型TCRα链恒定区TRAC*01外显子1的Thr48突变为半胱氨酸，β链恒定区TRBC1*01或TRBC2*01外显子1的Ser57突变为半胱氨酸，即得到参比TCR，其氨基酸序列分别如图7a和7b所示，突变后的半胱氨酸残基以加粗字母表示。上述半胱氨酸取代能使参比TCR的α与β链的恒定区之间形成人工链间二硫键，以形成更加稳定的可溶性TCR，从而能够更加方便地评估TCR与FMNKFIYEI-HLA A2复合物之间的结合亲和力和/或结合半衰期。应理解，TCR可变区的CDR区决定了其与pMHC复合物之间的亲和力，因此，上述TCR恒定区的半胱氨酸取代并不会对TCR的结合亲和力和/或结合半衰期产生影响。所以，在本发明中，测得的参比TCR与FMNKFIYEI-HLA A0201复合物之间的结合亲和力即认为是野生型TCR与FMNKFIYEI-HLA A0201复合物之间的结合亲和力。同样地，如果测得本发明TCR与FMNKFIYEI-HLA A0201复合物之间的结合亲和力是参比TCR与FMNKFIYEI-HLA A0201复合物之间的结合亲和力的至少10倍，即等同于本发明TCR与FMNKFIYEI-HLA A0201复合物之间的结合亲和力是野生型TCR与FMNKFIYEI-HLA A0201复合 物之间的结合亲和力的至少10倍。
可通过任何合适的方法测定结合亲和力(与解离平衡常数K D成反比)和结合半衰期(表示为T 1/2)。应了解，TCR的亲和力翻倍将导致K D减半。T 1/2计算为In2除以解离速率(K off)。因此，T 1/2翻倍会导致K off减半。优选采用相同的试验方案检测给定TCR的结合亲和力或结合半衰期数次，例如3次或更多，取结果的平均值。在优选的实施方式中，采用本文实施例中的表面等离振子共振(BIAcore)方法进行这些检测。该方法检测到参比TCR对FMNKFIYEI-HLAA2复合物的解离平衡常数K D为2.08E-04M，即208μM，本发明中即认为野生型TCR对FMNKFIYEI-HLA A2复合物的解离平衡常数K D也为208μM。由于TCR的亲和力翻倍将导致K D减半，所以若检测到高亲和力TCR对FMNKFIYEI-HLA A0201复合物的解离平衡常数K D为2.08E-05M，即20.8μM，则说明该高亲和力TCR对FMNKFIYEI-HLA A0201复合物的亲和力是野生型TCR对FMNKFIYEI-HLA A0201复合物的亲和力的10倍。本领域技术人员熟知K D值单位间的换算关系，即1M＝1000μM,1μM＝1000nM，1nM＝1000pM。
在本发明的一个优选例中，所述TCR与FMNKFIYEI-HLA A0201复合物的亲和力是野生型TCR的至少5倍；优选地，至少10倍；更优选地，至少50倍。
在另一优选例中，所述TCR与FMNKFIYEI-HLA A0201复合物的亲和力是野生型TCR的至少100倍；优选地，至少500倍；更优选地，至少1000倍。
具体地，所述TCR对FMNKFIYEI-HLA A0201复合物的解离平衡常数K D≤20μM；
在另一优选例中，所述TCR对FMNKFIYEI-HLA A0201复合物的解离平衡常数5μM≤K D≤10μM；优选地，0.1μM≤K D≤1μM；更优选地，1nM≤K D≤100nM。
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 Biotechnol6(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FMNKFIYEI-HLA-A2复合物具有高亲和性的TCR，如文献(Li,et al(2005)Nature Biotech 23(3):349-354)中所述。
应理解，表达野生型TCRα和β链可变域氨基酸的基因或者表达略作修饰的野生型TCR的α和β链可变域氨基酸的基因都可用来制备模板TCR。然后在编码该模板TCR的可变域的DNA中引入产生本发明的高亲和力TCR所需的改变。
本发明的高亲和性TCR包含α链可变域氨基酸序列SEQ ID NO:11、12、13、14、15、16、17、18、19、20、21、22、23或24之一和/或β链可变域氨基酸序列SEQ ID NO:2。本发明中，形成异质二聚TCR分子的α链可变域与β链可变域的氨基酸序列优选自下表1：
表1
TCR编号 α链可变域序列 β链可变域序列
  SEQ ID NO: SEQ ID NO:
1 11 2
2 12 2
3 13 2
4 14 2
5 15 2
6 16 2
7 17 2
8 18 2
9 19 2
10 20 2
11 21 2
12 22 2
13 23 2
14 24 2
基于本发明的目的，本发明TCR是具有至少一个TCRα和/或TCRβ链可变域的部分。它们通常同时包含TCRα链可变域和TCRβ链可变域。它们可以是αβ异源二聚体或是单链形式或是其他任何能够稳定存在的形式。在过继性免疫治疗中，可将αβ异源二聚TCR的全长链(包含胞质和跨膜结构域)进行转染。本发明TCR可用作将治疗剂递送至抗原呈递细胞的靶向剂或与其他分子结合制备双功能多肽来定向效应细胞，此时TCR优选为可溶形式。
对于稳定性而言，现有技术中公开了在TCR的α与β链恒定域之间引入人工链间二硫键能够获得可溶且稳定的TCR分子，如专利文献PCT/CN2015/093806中所述。因此，本发明TCR可以是在其α和β链恒定域的残基之间引入人工链间二硫键的TCR。半胱氨酸残基在所述TCR的α和β链恒定域间形成人工链间二硫键。半胱氨酸残基可以取代在天然TCR中合适位点的其他氨基酸残基以形成人工链间二硫键。例如，取代TRAC*01外显子1的Thr48和取代TRBC1*01或TRBC2*01外显子1的Ser57来形成二硫键。引入半胱氨酸残基以形成二硫键的其他位点还可以是：TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；或TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。即半胱氨酸残基取代了上述α与β链恒定域中任一组位点。可在本发明TCR恒定域的一个或多个C末端截短最多15个、或最多10个、或最多8个或更少的氨基酸，以使其不包括半胱氨酸残基来达到缺失天然链间二硫键的目的，也可通过将形成天然链间二硫键的半胱氨酸残基突变为另一氨基酸来达到上述目的。
如上所述，本发明的TCR可以包含在其α和β链恒定域的残基间引入的人工链间二硫键。应注意，恒定域间含或不含上文所述的引入的人工二硫键，本发明的TCR均可含有TRAC恒定域序列和TRBC1或TRBC2恒定域序列。TCR的TRAC恒定域序列和TRBC1或TRBC2恒定域 序列可通过存在于TCR中的天然链间二硫键连接。
另外，对于稳定性而言，专利文献PCT/CN2016/077680还公开了在TCR的α链可变区与β链恒定区之间引入人工链间二硫键能够使TCR的稳定性显著提高。因此，本发明的高亲和力TCR的α链可变区与β链恒定区之间还可以含有人工链间二硫键。具体地，在所述TCR的α链可变区与β链恒定区之间形成人工链间二硫键的半胱氨酸残基取代了：TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸；TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的61位氨基酸；TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第61位氨基酸；或TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸。优选地，这样的TCR可以包含(ⅰ)除其跨膜结构域以外的全部或部分TCRα链，和(ⅱ)除其跨膜结构域以外的全部或部分TCRβ链，其中(ⅰ)和(ⅱ)均包含TCR链的可变域和至少一部分恒定域，α链与β链形成异质二聚体。更优选地，这样的TCR可以包含α链可变域和β链可变域以及除跨膜结构域以外的全部或部分β链恒定域，但其不包含α链恒定域，所述TCR的α链可变域与β链形成异质二聚体。
对于稳定性而言，另一方面，本发明TCR还包括在其疏水芯区域发生突变的TCR，这些疏水芯区域的突变优选为能够使本发明TCR的稳定性提高的突变，如在公开号为WO2014/206304的专利文献中所述。这样的TCR可在其下列可变域疏水芯位置发生突变：(α和/或β链)可变区氨基酸第11，13，19，21，53，76，89，91，94位，和/或α链J基因(TRAJ)短肽氨基酸位置倒数第3,5,7位,和/或β链J基因(TRBJ)短肽氨基酸位置倒数第2,4,6位，其中氨基酸序列的位置编号按国际免疫遗传学信息系统(IMGT)中列出的位置编号。本领域技术人员知晓上述国际免疫遗传学信息系统，并可根据该数据库得到不同TCR的氨基酸残基在IMGT中的位置编号。
更具体地，本发明中疏水芯区域发生突变的TCR可以是由一柔性肽链连接TCR的α与β链的可变域而构成的高稳定性单链TCR。TCR可变区的CDR区决定了其与短肽-HLA复合物之间的亲和力，疏水芯的突变能够使TCR更加稳定，但并不会影响其与短肽-HLA复合物之间的亲和力。应注意，本发明中柔性肽链可以是任何适合连接TCRα与β链可变域的肽链。本发明实施例1中构建的用于筛选高亲和性TCR的模板链即为上述含有疏水芯突变的高稳定性单链TCR。采用稳定性较高的TCR，能够更方便的评估TCR与FMNKFIYEI-HLA-A0201复合物之间的亲和力。
该单链模板TCR的α链可变域及β链可变域的CDR区与野生型TCR的CDR区完全相同。即α链可变域的3个CDR分别为CDR1α：DSAIYN，CDR2α：IQSSQRE，
CDR3α：AVNSGGSNYKLT和β链可变域的3个CDR分别为CDR1β：SGHVS，CDR2β：FQNEAQ，CDR3β：ASSLFGQGREKLF。该单链模板TCR的氨基酸序列(SEQ ID NO:9)及核苷酸序列(SEQ ID NO:10)分别如图5a和5b所示。以此筛选出对FMNKFIYEI-HLA A0201复合物具有高亲和性的由α链可变域和β链可变域构成的单链TCR。
本发明中单链模板TCRα链可变域SEQ ID NO：3的3个CDR即CDR1、CDR2和CDR3分别位于SEQ ID NO：3的第27-32位、第50-56位和第91-102位。据此，氨基酸残基编号采 用SEQ ID NO:3所示的编号，93N即为CDR3α的第3位N、94S即为CDR3α的第4位S、95G即为CDR3α的第5位G、96G即为CDR3α的第6位G、97S即为CDR3α的第7位S，98N即为CDR3α的第8位N。
本发明的对FMNKFIYEI-HLA-A0201复合物具有高亲和性的αβ异质二聚体的获得是通过将筛选出的高亲和性单链TCR的α与β链可变域的CDR区转移到野生型TCRα链可变域(SEQ ID NO:1)与β链可变域(SEQ ID NO:2)的相应位置而得到。
本发明的TCR也可以多价复合体的形式提供。本发明的多价TCR复合体包含两个、三个、四个或更多个本发明TCR相结合而形成的多聚物，如可以用p53的四聚结构域来产生四聚体，或多个本发明TCR与另一分子结合而形成的复合物。本发明的TCR复合物可用于体外或体内追踪或靶向呈递特定抗原的细胞，也可用于产生具有此类应用的其他多价TCR复合物的中间体。
本发明的TCR可以单独使用，也可与偶联物以共价或其他方式结合，优选以共价方式结合。所述偶联物包括可检测标记物(为诊断目的，其中所述TCR用于检测呈递FMNKFIYEI-HLA-A0201复合物的细胞的存在)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明TCR结合或偶联的治疗剂包括但不限于：1.放射性核素(Koppe等，2005，癌转移评论(Cancer metastasis reviews)24，539)；2.生物毒(Chaudhary等，1989，自然(Nature)339，394；Epel等，2002，癌症免疫学和免疫治疗(Cancer Immunology and Immunotherapy)51，565)；3.细胞因子如IL-2等(Gillies等，1992，美国国家科学院院刊(PNAS)89，1428；Card等，2004，癌症免疫学和免疫治疗(Cancer Immunology and Immunotherapy)53，345；Halin等，2003，癌症研究(Cancer Research)63，3202)；4.抗体Fc片段(Mosquera等，2005，免疫学杂志(The Journal Of Immunology)174，4381)；5.抗体scFv片段(Zhu等，1995，癌症国际期刊(International Journal of Cancer)62,319)；6.金纳米颗粒/纳米棒(Lapotko等，2005，癌症通信(Cancer letters)239，36；Huang等，2006，美国化学学会杂志(Journal of the American Chemical Society)128，2115)；7.病毒颗粒(Peng等，2004，基因治疗(Gene therapy)11，1234)；8.脂质体(Mamot等，2005，癌症研究(Cancer research)65，11631)；9.纳米磁粒；10.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；11.化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
与本发明TCR结合的抗体或其片段包括抗-T细胞或NK-细胞决定抗体，如抗-CD3或抗-CD28或抗-CD16抗体，上述抗体或其片段与TCR的结合能够对效应细胞进行定向来更好地靶向靶细胞。一个优选的实施方式是本发明TCR与抗-CD3抗体或所述抗-CD3抗体的功能片段或变体结合。具体地，本发明的TCR与抗CD3单链抗体的融合分子包括选自下组的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、23和24，和TCRβ链可变域氨基酸序列SEQ ID NO:2。
本发明还涉及编码本发明TCR的核酸分子。本发明的核酸分子可以是DNA形式或RNA形式。DNA可以是编码链或非编码链。例如，编码本发明TCR的核酸序列可以与本发明附图中所示的核酸序列相同或是简并的变异体。举例说明“简并的变异体”的含义，如本文所用，“简并的变异体”在本发明中是指编码具有SEQ ID NO:3的蛋白序列，但与SEQ ID NO:5的序列有差别的核酸序列。
本发明的核酸分子全长序列或其片段通常可以用但不限于PCR扩增法、重组法或人工合成的方法获得。目前，已经可以完全通过化学合成来得到编码本发明TCR(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本发明也涉及包含本发明的核酸分子的载体，以及用本发明的载体或编码序列经基因工程产生的宿主细胞。
本发明还包括表达本发明TCR的分离细胞，特别是T细胞。有许多方法适合于用编码本发明的高亲和力TCR的DNA或RNA进行T细胞转染(如，Robbins等.，(2008)J.Immunol.180:6116-6131)。表达本发明高亲和性TCR的T细胞可以用于过继免疫治疗。本领域技术人员能够知晓进行过继性治疗的许多合适方法(如，Rosenberg等.，(2008)Nat Rev 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)；
本发明中，Pro60或者60P均表示第60位脯氨酸。另外，本发明中所述突变的具体形式的表述方式如“N93D”代表第93位的N被D取代，同理，“N93D/E”代表第93位的N被D取代或被E取代。其他以此类推。
在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的结构和功能。因此，本发明TCR还包括本发明TCR的至多5个，较佳地至多3个，更佳地至多2个，最佳地1个氨基酸(尤其是位于CDR区之外的氨基酸)，被性质相似或相近的氨基酸所替换，并仍能够保持其功能性的TCR。
本发明还包括对本发明TCR略作修饰后的TCR。修饰(通常不改变一级结构)形式包括：本发明TCR的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在本发明TCR的 合成和加工中或进一步加工步骤中进行糖基化修饰而产生的TCR。这种修饰可以通过将TCR暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的TCR。
本发明的TCR、TCR复合物或本发明TCR转染的T细胞可与药学上可接受的载体一起在药物组合物中提供。本发明的TCR、多价TCR复合物或细胞通常作为无菌药物组合物的一部分提供，所述组合物通常包括药学上可接受的载体。该药物组合物可以是任何合适的形式(取决于给予患者的所需方法)。其可采用单位剂型提供，通常在密封的容器中提供，可作为试剂盒的一部分提供。此类试剂盒(但非必需)包括使用说明书。其可包括多个所述单位剂型。
此外，本发明的TCR可以单用，也可与其他治疗剂结合或偶联在一起使用(如配制在同一药物组合物中)。
药物组合物还可含有药学上可接受的载体。术语“药学上可接受的载体”指用于治疗剂给药的载体。该术语指这样一些药剂载体：它们本身不诱导产生对接受该组合物的个体有害的抗体，且给药后没有过分的毒性。这些载体是本领域普通技术人员所熟知的。在雷明顿药物科学(Remington's Pharmaceutical Sciences(Mack Pub.Co.，N.J.1991))中可找到关于药学上可接受的赋形剂的充分讨论。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、佐剂、及其组合。
治疗性组合物中药学上可接受的载体可含有液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如润湿剂或乳化剂、pH缓冲物质等。
通常，可将治疗性组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液中、液体载体的固体形式。
一旦配成本发明的组合物，可将其通过常规途径进行给药，其中包括(但并不限于)：眼内、肌内、静脉内、皮下、皮内、或局部给药，优选为胃肠外包括皮下、肌肉内或静脉内。待预防或治疗的对象可以是动物；尤其是人。
当本发明的药物组合物被用于实际治疗时，可根据使用情况而采用各种不同剂型的药物组合物。较佳地，可以例举的有针剂、口服剂等。
这些药物组合物可根据常规方法通过混合、稀释或溶解而进行配制，并且偶尔添加合适的药物添加剂，如赋形剂、崩解剂、粘合剂、润滑剂、稀释剂、缓冲剂、等渗剂(isotonicities)、防腐剂、润湿剂、乳化剂、分散剂、稳定剂和助溶剂，而且该配制过程可根据剂型用惯常方式进行。
本发明的药物组合物还可以缓释剂形式给药。例如，本发明TCR可被掺入以缓释聚合物为载体的药丸或微囊中，然后将该药丸或微囊通过手术植入待治疗的组织。作为缓释聚合物的例子，可例举的有乙烯-乙烯基乙酸酯共聚物、聚羟基甲基丙烯酸酯(polyhydrometaacrylate)、聚丙烯酰胺、聚乙烯吡咯烷酮、甲基纤维素、乳酸聚合物、乳酸-乙醇酸共聚物等，较佳地可例举的是可生物降解的聚合物如乳酸聚合物和乳酸-乙醇酸共聚物。
当本发明的药物组合物被用于实际治疗时，作为活性成分的本发明TCR或TCR复合物或呈递本发明TCR的细胞，可根据待治疗的每个病人的体重、年龄、性别、症状程度而合理地加以确定，最终由医师决定合理的用量。
本发明的主要优点在于：
(1)本发明的TCR对所述FMNKFIYEI-HLA-A2复合物的亲和力和/或结合半衰期是野生型TCR的至少5倍，优选地，至少10倍。
(2)本发明的TCR对所述FMNKFIYEI-HLA-A2复合物的亲和力和/或结合半衰期是野生型TCR的至少100倍，优选地，至少1000倍。
(3)转导本发明的高亲和力TCR的效应细胞对靶细胞有很强的杀伤作用。
下面的具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如(Sambrook和Russell等人，分子克隆：实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)(2001)CSHL出版社)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
材料和方法
本发明实施例中所用的实验材料如无特殊说明均可从市售渠道获得，其中，E.coli DH5α购自Tiangen、E.coli BL21(DE3)购自Tiangen、E.coli Tuner(DE3)购自Novagen、质粒pET28a购自Novagen。
实施例1 疏水芯突变的稳定性单链TCR模板链的产生
本发明利用定点突变的方法，根据专利文献WO2014/206304中所述，构建了以一个柔性短肽(linker)连接TCRα与β链可变域而构成的稳定性单链TCR分子，其氨基酸及DNA序列分别为SEQ ID NO:9和SEQ ID NO:10，如图5a和图5b所示。并以该单链TCR分子为模板进行高亲和性TCR分子的筛选。该模板链的α可变域(SEQ ID NO:3)及β可变域(SEQ ID NO:4)的氨基酸序列如图2a和2b所示；其对应的DNA序列分别为SEQ ID NO:5和6，如图3a和3b所示；柔性短肽(linker)的氨基酸序列及DNA序列分别为SEQ ID NO:7和8，如图4a和4b所示。
将携带模板链的目的基因经Nco Ⅰ和Not Ⅰ双酶切，与经过Nco Ⅰ和Not Ⅰ双酶切的pET28a载体连接。连接产物转化至E.coli DH5α，涂布含卡那霉素的LB平板，37℃倒置培养过夜，挑取阳性克隆进行PCR筛选，对阳性重组子进行测序，确定序列正确后抽提重组质粒转化至E.coli BL21(DE3),用于表达。
实施例2 实施例1中构建的稳定性单链TCR的表达、复性和纯化
将实施例1中制备的含有重组质粒pET28a-模板链的BL21(DE 3)菌落全部接种于含有卡那霉素的LB培养基中,37℃培养至OD 600为0.6-0.8，加入IPTG至终浓度为0.5mM，37℃继续培养4h。5000rpm离心15min收获细胞沉淀物，用Bugbuster Master Mix(Merck)裂解细胞沉淀物，6000rpm离心15min回收包涵体，再用Bugbuster(Merck)进行洗涤以除去细胞碎片和膜组分，6000rpm离心15min,收集包涵体。将包涵体溶解在缓冲液(20mM Tris-HCl pH 8.0,8M尿素)中，高速离心去除不溶物，上清液用BCA法定量后进行分装，于-80℃保存备用。
向5mg溶解的单链TCR包涵体蛋白中，加入2.5mL缓冲液(6M Gua-HCl，50mM Tris-HCl pH 8.1，100mM NaCl，10mM EDTA)，再加入DTT至终浓度为10mM,37℃处理30min。用注射器向125mL复性缓冲液(100mM Tris-HCl pH 8.1,0.4M L-精氨酸,5M尿素,2mM EDTA,6.5mMβ-mercapthoethylamine,1.87mM Cystamine)中滴加上述处理后的单链TCR，4℃搅拌10min，然后将复性液装入截留量为4kDa的纤维素膜透析袋,透析袋置于1L预冷的水中，4℃缓慢搅拌过夜。17小时后，将透析液换成1L预冷的缓冲液(20mM Tris-HCl pH 8.0)，4℃继续透析8h，然后将透析液换成相同的新鲜缓冲液继续透析过夜。17小时后，样品经0.45μm滤膜过滤，真空脱气后通过阴离子交换柱(HiTrap Q HP,GE Healthcare)，用20mM Tris-HCl pH 8.0配制的0-1M NaCl线性梯度洗脱液纯化蛋白，收集的洗脱组分进行SDS-PAGE分析，包含单链TCR的组分浓缩后进一步用凝胶过滤柱(Superdex 75 10/300,GE Healthcare)进行纯化，目标组分也进行SDS-PAGE分析。
用于BIAcore分析的洗脱组分进一步采用凝胶过滤法测试其纯度。条件为：色谱柱Agilent Bio SEC-3(300A，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TCR分子与FMNKFIYEI-HLA-A0201复合物的结合活性。将抗链霉亲和素的抗体(GenScript)加入偶联缓冲液(10mM醋酸钠缓冲液，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FMNKFIYEI-HLA-A0201复合物流过检测通道，另一通道作为参比通道，再将0.05mM的生物素以10μL/min的流速流过芯片2min，封闭链霉亲和素剩余的结合位点。采用单循环动力学分析方法测定其亲和力，将TCR用HEPES-EP缓冲液(10mM HEPES,150mM NaCl，3mM EDTA,0.005％P20，pH 7.4)稀释成几个不同的浓度，以30μL/min的流速，依次流过芯片表面，每次进样的结合时间为120s，最后一次进样结束后让其解离600s。每一轮测定结束后用pH 1.75的10mM Gly-HCl再生芯片。利用BIAcore Evaluation软件计算动力学参数。
上述FMNKFIYEI-HLA-A0201复合物的制备过程如下：
a.纯化
收集100ml诱导表达重链或轻链的E.coli菌液，于4℃8000g离心10min后用10ml PBS洗涤菌体一次，之后用5ml BugBuster Master Mix Extraction Reagents(Merck)剧烈震荡重悬菌体，并于室温旋转孵育20min，之后于4℃，6000g离心15min，弃去上清，收集包涵体。
将上述包涵体重悬于5ml BugBuster Master Mix中，室温旋转孵育5min；加30ml稀释10倍的BugBuster，混匀，4℃6000g离心15min；弃去上清，加30ml稀释10倍的BugBuster重悬包涵体，混匀，4℃6000g离心15min，重复两次，加30ml 20mM Tris-HCl pH 8.0重悬包涵体，混匀，4℃6000g离心15min，最后用20mM Tris-HCl 8M尿素溶解包涵体，SDS-PAGE检测包涵体纯度，BCA试剂盒测浓度。
b.复性
将合成的短肽FMNKFIYEI(北京赛百盛基因技术有限公司)溶解于DMSO至20mg/ml的浓度。轻链和重链的包涵体用8M尿素、20mM Tris pH 8.0、10mM DTT来溶解，复性前加入3M盐酸胍、10mM醋酸钠、10mM EDTA进一步变性。将FMNKFIYEI肽以25mg/L(终浓度)加入复性缓冲液(0.4M L-精氨酸、100mM Tris pH 8.3、2mM EDTA、0.5mM氧化性谷胱甘肽、5mM还原型谷胱甘肽、0.2mM PMSF，冷却至4℃)，然后依次加入20mg/L的轻链和90mg/L的重链(终浓度，重链分三次加入，8h/次)，复性在4℃进行至少3天至完成，SDS-PAGE检测能否复性成功。
c.复性后纯化
用10体积的20mM Tris pH 8.0作透析来更换复性缓冲液，至少更换缓冲液两次来充分降低溶液的离子强度。透析后用0.45μm醋酸纤维素滤膜过滤蛋白质溶液，然后加载到HiTrap Q HP(GE通用电气公司)阴离子交换柱上(5ml床体积)。利用Akta纯化仪(GE通用电气公司)，20mM Tris pH 8.0配制的0-400mM NaCl线性梯度液洗脱蛋白，pMHC约在250mM NaCl处洗脱，收集诸峰组分，SDS-PAGE检测纯度。
d.生物素化
用Millipore超滤管将纯化的pMHC分子浓缩，同时将缓冲液置换为20mM Tris pH 8.0，然后加入生物素化试剂0.05M Bicine pH 8.3、10mM ATP、10mM MgOAc、50μM D-Biotin、100μg/ml BirA酶(GST-BirA)，室温孵育混合物过夜，SDS-PAGE检测生物素化是否完全。
e.纯化生物素化后的复合物
用Millipore超滤管将生物素化标记后的pMHC分子浓缩至1ml，采用凝胶过滤层析纯化生物素化的pMHC，利用Akta纯化仪(GE通用电气公司)，用过滤过的PBS预平衡HiPrep TM16/60S200HR柱(GE通用电气公司)，加载1ml浓缩过的生物素化pMHC分子，然后用PBS以1ml/min流速洗脱。生物素化的pMHC分子在约55ml时作为单峰洗脱出现。合并含有蛋白质的组分，用Millipore超滤管浓缩，BCA法(Thermo)测定蛋白质浓度，加入蛋白酶抑制剂cocktail(Roche)将生物素化的pMHC分子分装保存在-80℃。
实施例4 高亲和性单链TCR的产生
噬菌体展示技术是产生TCR高亲和力变体文库以筛选高亲和力变体的一种手段。将Li等((2005)Nature Biotech 23(3):349-354)描述的TCR噬菌体展示和筛选方法应用于实施例1中的单链TCR模板。通过突变该模板链的CDR区来建立高亲和性TCR的文库并进行淘选。经过几轮淘选后的噬菌体文库均和相应抗原有特异性结合，从中挑取单克隆，并进行分析。
采用实施例3中BIAcore方法分析TCR分子与FMNKFIYEI-HLA-A0201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TCR的至少5倍的高亲和性TCR，即筛选出的高亲和性TCR结合FMNKFIYEI-HLA-A0201复合物的解离平衡常数K D小于等于野生型TCR结合FMNKFIYEI-HLA-A0201复合物的解离平衡常数K D的五分之一，结果如下表3所示。利用上述方法检测到参比TCR与FMNKFIYEI-HLA-A0201复合物相互作用的K D值为208μM，其相互作用曲线如图9所示，即野生型TCR与FMNKFIYEI-HLA-A0201复合物相互作用的KD值也为208μM，即2.08E-04M。
经检测筛选出了与FMNKFIYEI-HLA-A0201复合物的亲和力是野生型TCR与FMNKFIYEI-HLA-A0201复合物的亲和力至少5倍的单链TCR。
实施例5 高亲和性αβ异质二聚TCR的产生
将实施例4中筛选到的高亲和力的单链TCR的CDR区突变引入到αβ异质二聚TCR的可变域的相应位点中，并通过BIAcore来检测其与FMNKFIYEI-HLA-A0201复合物的亲和力。上述CDR区高亲和力突变点的引入采用本领域技术人员熟知的定点突变的方法。上述野生型TCR的α链与β链可变域氨基酸序列分别如图1a(SEQ ID NO:1)和1b(SEQ ID NO:2)所示。
应注意，为获得更加稳定的可溶性TCR，以便更方便地评估TCR与FMNKFIYEI-HLA A0201复合物之间的结合亲和力和/或结合半衰期，αβ异质二聚TCR可以是在α和β链的恒定区中分别引入了一个半胱氨酸残基以形成人工链间二硫键的TCR，本实施例中引入半胱氨酸残基后TCRα与β链的氨基酸序列分别如图7a(SEQ ID NO:26)和7b所示(SEQ ID NO:27)，引入的半胱氨酸残基以加粗字母表示。
通过《分子克隆实验室手册》(Molecular Cloning a Laboratory Manual)(第三版，Sambrook和Russell)中描述的标准方法将待表达的TCRα和β链的胞外序列基因经合成后分别插入到表达载体pET28a+(Novagene)，上下游的克隆位点分别是NcoI和NotI。CDR区的突变通过本领域技术人员熟知的重叠PCR(overlap PCR)引入。插入片段经过测序确认无误。
实施例6 αβ异质二聚TCR的表达、复性和纯化
将TCRα和β链的表达载体分别通过化学转化法转化进入表达细菌BL21(DE3),细菌用LB培养液生长，于OD 600＝0.6时用终浓度0.5mM IPTG诱导，TCR的α和β链表达后形成的包涵体通过BugBuster Mix(Novagene)进行提取，并且经BugBuster溶液反复多次洗涤，包涵体最后溶解于6M盐酸胍,10mM二硫苏糖醇(DTT),10mM乙二胺四乙酸(EDTA),20mM Tris(pH 8.1)中。
溶解后的TCRα和β链以1：1的质量比快速混合于5M尿素,0.4M精氨酸,20mM Tris(pH 8.1),3.7mM cystamine,6.6mMβ-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(HiPrep16/60，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7 BIAcore分析结果
采用实施例3中所述方法检测引入高亲和力CDR区的αβ异质二聚TCR与FMNKFIYEI-HLA-A0201复合物的亲和力。
将高亲和性单链TCRα与β链中筛选出的CDR区分别转移到野生型TCRα链可变域SEQ ID NO:1和β链可变域SEQ ID NO:2的相应位置，形成αβ异质二聚TCR。得到的新的TCRα链可变域氨基酸序列，如图6(1)-(14)所示。由于TCR分子的CDR区决定了其与相应的pMHC复合物的亲和力，所以本领域技术人员能够预料引入高亲和力突变点的αβ异质二聚TCR也具有对FMNKFIYEI-HLA-A0201复合物的高亲和力。利用实施例5中所述方法构建表达载体，利用实施例6中所述方法对上述引入高亲和力突变的αβ异质二聚TCR进行表达、复性和纯化，然后利用BIAcore T200测定其与FMNKFIYEI-HLA-A0201复合物的亲和力，如下表2所示。
表2
Figure PCTCN2020078271-appb-000003
由上表2可知，引入CDR区突变点的αβ异质二聚TCR保持了对FMNKFIYEI-HLA-A0201复合物的高亲和力。所述异质二聚TCR的亲和力是野生型TCR对FMNKFIYEI-HLA-A0201复合物的亲和力的至少5倍。
实施例8 抗-CD3抗体与高亲和性αβ异质二聚TCR的融合体的表达、复性和纯化
将抗-CD3的单链抗体(scFv)与αβ异质二聚TCR融合，制备融合分子。抗-CD3的scFv与TCR的β链融合，该TCRβ链可以包含任一上述高亲和性αβ异质二聚TCR的β链可变域，融合分子的TCRα链可以包含任一上述高亲和性αβ异质二聚TCR的α链可变域。
融合分子表达载体的构建
1.α链表达载体的构建
将携带αβ异质二聚TCR的α链的目的基因经Nco Ⅰ和Not Ⅰ双酶切，与经过Nco Ⅰ和Not Ⅰ双酶切的pET28a载体连接。连接产物转化至E.coli DH5α，涂布于含卡那霉素的LB平板，37℃倒置培养过夜，挑取阳性克隆进行PCR筛选，对阳性重组子进行测序，确定序列正确后抽提重组质粒转化至E.coli Tuner(DE3),用于表达。
2.抗-CD3(scFv)-β链表达载体的构建
通过重叠(overlap)PCR的方法，设计引物将抗-CD3scFv和高亲和性异质二聚TCRβ链基因连接起来，中间的连接短肽(linker)为GGGGS(SEQ ID NO:30)，并且使抗-CD3的scFv与高亲和性异质二聚TCRβ链的融合蛋白的基因片段带上限制性内切酶位点Nco Ⅰ(CCATGG(SEQ ID NO:31))和Not Ⅰ(GCGGCCGC(SEQ ID NO:32))。将PCR扩增产物经Nco Ⅰ和Not Ⅰ双酶切，与经过Nco Ⅰ和Not Ⅰ双酶切的pET28a载体连接。连接产物转化至E.coli DH5α感受态细胞，涂布含卡那霉素的LB平板，37℃倒置培养过夜，挑取阳性克隆进行PCR筛选，对阳性重组子进行测序，确定序列正确后抽提重组质粒转化至E.coli Tuner(DE3)感受态细胞，用于表达。
融合蛋白的表达、复性及纯化
将表达质粒分别转化进入E.coli Tuner(DE3)感受态细胞，涂布LB平板(卡那霉素50μg/mL)置于37℃培养过夜。次日，挑克隆接种至10mL LB液体培养基(卡那霉素50μg/mL)培养2-3h，按体积比1:100接种至1L LB培养基中，继续培养至OD600为0.5-0.8，加入终浓度为1mM IPTG诱导目的蛋白的表达。诱导4小时以后，以6000rpm离心10min收获细胞。PBS缓冲液洗涤菌体一次，并且分装菌体，取相当于200mL的细菌培养物的菌体用5mL BugBuster Master Mix(Merck)裂解细菌，以6000g离心15min收集包涵体。然后进行4次洗涤剂洗涤以去除细胞碎片和膜组分。然后，用缓冲液如PBS洗涤包涵体以除去洗涤剂和盐。最终，将包涵体用含6M盐酸胍,10mM二硫苏糖醇(DTT),10mM乙二胺四乙酸(EDTA),20mM Tris,pH 8.1缓冲溶液溶解，并测定包涵体浓度，将其分装后置于-80℃冷冻保存。
溶解后的TCRα链和抗-CD3(scFv)-β链以2：5的质量比快速混合于5M尿素(urea),0.4M L-精氨酸(L-arginine),20mM Tris pH 8.1,3.7mM cystamine,6.6mMβ-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-100 16/60，GE healthcare)进一步纯化，以及阴离子交换柱(HiTrap Q HP 5ml,GE healthcare)再次纯化。纯化后的TCR融合分子纯度经过SDS-PAGE测定大于90％，浓度由BCA法测定。
实施例9 针对负载特异短肽的T2细胞，转染本发明高亲和力TCR的效应细胞的激活功能实验
ELISPOT方案
进行以下试验以证明本发明TCR转导的T细胞对靶细胞特异性的激活反应。利用ELISPOT试验检测的IFN-γ产量作为T细胞激活的读出值。
试剂
试验培养基：10％FBS(吉布可公司(Gibco)，目录号16000-044)，RPMI 1640(吉布可公司(Gibco)，目录号C11875500bt)
洗涤缓冲液(PBST)：0.01M PBS/0.05％吐温20
PBS(吉布可公司(Gibco)，目录号C10010500BT)
PVDF ELISPOT 96孔板(默克密理博(Merck Millipore)，目录号MSIPS4510)
人IFN-γELISPOT PVDF-酶试剂盒(BD)装有所需的所有其他试剂(捕捉和检测抗体，链霉亲和素-碱性磷酸酶和BCIP/NBT溶液)
方法
靶细胞
本实验中所用的靶细胞为负载了特异短肽FMNKFIYEI的T2细胞。在实验培养基中制备靶细胞：靶细胞浓度调至1.0×10 5个/毫升，每孔取100微升从而得1.0×10 4个细胞/孔。
效应细胞
本实验的效应细胞(T细胞)是转染本发明AFP抗原短肽特异性高亲和力TCR的CD3+T细胞，转染的高亲和力TCR分子如下(在本实施例以及以下实施例中利用的具体TCR名称，例如TCR1、TCR2等与上表1和表2中的TCR编号并不相同，具体以α链可变域和β链可变域的序列为准)：TCR1(α链可变域SEQ ID NO:11,β链可变域SEQ ID NO:2)、TCR2(α链可变域SEQ ID NO:13,β链可变域SEQ ID NO:2)、TCR3(α链可变域SEQ ID NO:14,β链可变域SEQ ID NO:2)、TCR4(α链可变域SEQ ID NO:15,β链可变域SEQ ID NO:2)、TCR5(α链可变域SEQ ID NO:17,β链可变域SEQ ID NO:2)和TCR6(α链可变域SEQ ID NO:18,β链可变域SEQ ID NO:2)。并以同一志愿者转染本发明高亲和力TCR对应的野生型TCR(命名为：A0B0，α链SEQ ID NO:28,β链SEQ ID NO:29)，以及转染其他高亲和力TCR(命名为A6)的CD3+T细胞作为对照组。
短肽溶液
在相应靶细胞(T2)实验组加入对应短肽，然后梯度稀释，使终浓度为10 -8M-10 -13M。
ELISPOT
按照生产商提供的说明书，如下所述准备孔板：以每块板10毫升无菌PBS按1：200稀释抗人IFN-γ捕捉抗体，然后将50微升的稀释捕捉抗体等分加入各孔。4℃下孵育孔板过夜。孵育后，洗涤孔板以除去多余的捕捉抗体。加入200微升/孔含有5％FBS的PBS培养基，并在室温下温育孔板2小时以封闭孔板。然后从孔板中洗去培养基，通过在纸上轻弹和轻拍ELISPOT孔板以除去任何残余的洗涤缓冲液。
然后采用以下顺序将试验的诸组分加入ELISPOT孔板：
100微升靶细胞1*10 5个细胞/毫升(得到总共约1*10 4个靶细胞/孔)。
100微升效应细胞(1*10 3个对照效应细胞/孔和AFP TCR阳性T细胞/孔)。
所有孔一式两份制备添加。
然后温育孔板过夜(37℃/5％CO 2)第二天，弃培养基，用双蒸水洗涤孔板2次，再用洗涤缓冲液洗涤3次，在纸巾上轻拍以除去残余的洗涤缓冲液。然后用含有5％FBS的PBS按1：200稀释检测抗体，按50微升/孔加入各孔。室温下温育孔板2小时，再用洗涤缓冲液洗涤3次，在纸巾上轻拍孔板以除去过量的洗涤缓冲液。
用含有5％FBS的PBS按1：100稀释链霉亲和素-碱性磷酸酶，将50微升稀释的链霉亲和素-碱性磷酸酶加入各孔并在室温下温育孔板1小时。然后用洗涤缓冲液洗涤4次PBS洗涤2次，在纸巾上轻拍孔板以除去过量的洗涤缓冲液和PBS。洗涤完毕后加入试剂盒提供的BCIP/NBT溶液50微升/孔进行显影。在显影期间用锡箔纸覆盖孔板避光，静置2-5分钟。在此期间常规检测显影孔板的斑点，确定终止反应的最佳时间。去除BCIP/NBT溶液并用双蒸水冲洗孔板以中止显影反应，甩干，然后将孔板底部去除，在室温下干燥孔板直至每个孔完全干燥，再利用免疫斑点平板计数计(CTL，细胞技术有限公司(Cellular Technology Limited))计数孔板内底膜形成的斑点。
结果
通过ELISPOT实验(如上所述)检验本发明TCR转导的T细胞对负载AFP抗原短肽FMNKFIYEI的靶细胞起反应的IFN-γ释放。利用graphpad prism6绘制各孔中观察到的ELSPOT斑点数量。
实验结果分别如图10a、10b、10c、10d、10e和10f所示，针对负载特异的短肽的靶细胞，转导本发明高亲和力TCR的T细胞(效应细胞)有很好的激活反应，IFN-γ的释放要远远高于转导野生型TCR的效应细胞，而转导其他TCR(A6)的T细胞(效应细胞)对相应的靶细胞基本没有激活反应。
实施例10 针对肿瘤细胞系，转染本发明高亲和力TCR的效应细胞的激活功能实验
本实施例验证了针对靶细胞,转染本发明高亲和力TCR的效应细胞有很好的特异性激活作用。
通过ELISPOT实验检测本发明高亲和力TCR在细胞中的功能及特异性。本领域技术人员熟知利用ELISPOT实验检测细胞功能的方法。本实施例IFN-γELISPOT实验用从健康志 愿者的血液中分离到的CD3+T细胞转染本发明高亲和力TCR作为效应细胞。
随机选择本发明TCR转染效应细胞，TCR1(α链可变域SEQ ID NO:11,β链可变域SEQ ID NO:2)、TCR3(α链可变域SEQ ID NO:14,β链可变域SEQ ID NO:2)、TCR5(α链可变域SEQ ID NO:17,β链可变域SEQ ID NO:2)和TCR6(α链可变域SEQ ID NO:18,β链可变域SEQ ID NO:2)。对照组效应细胞标记为A0B0(转染野生型TCR,α链SEQ ID NO:28,β链SEQ ID NO:29)和A6(转染非本发明其他TCR)。靶细胞系为HepG2、HUH-6、Hep3B、HCCC9810和SNU-398细胞。其中，靶细胞系HepG2表达相关抗原并且基因型也符合为阳性细胞系，HUH-6、Hep3B、HCCC9810和SNU-398为阴性细胞系作为对照。
首先准备ELISPOT平板。ELISPOT平板乙醇活化包被，4℃过夜。实验第1天，去掉包被液，洗涤封闭，室温下孵育两个小时，去除封闭液，按以下顺序将试验的各个组分加入ELISPOT平板：培养基调整效应细胞至1X 10 4个细胞/毫升，培养基调整各靶细胞系至2X 10 5个细胞/毫升。混合均匀后取100μL靶细胞(即20，000个细胞/孔)、100μL效应细胞(即1000个细胞/孔)加入对应孔中，并设置二个复孔。温育过夜(37℃，5％CO 2)。实验第2天，洗涤平板并进行二级检测和显色，干燥平板，再利用免疫斑点平板读数计(ELISPOT READER system；AID20公司)计数膜上形成的斑点。
实验结果如图11所示，针对阴性靶细胞，转染本发明高亲和力TCR的效应细胞基本没有激活作用，而针对阳性靶细胞，有很好的特异性激活作用，其效果远远好于转染野生型TCR的效应细胞。
实施例11 转染本发明高亲和力TCR的效应细胞的杀伤功能实验
本实施例通过非放射性细胞毒性实验，测定LDH的释放，从而验证转导本发明TCR的细胞的杀伤功能。该试验是51Cr释放细胞毒性试验的比色替代试验，定量测定细胞裂解后释放的乳酸脱氢酶(LDH)。采用30分钟偶联的酶反应来检测释放在培养基中的LDH，在酶反应中LDH可使一种四唑盐(INT)转化为红色的甲臜(formazan)。生成的红色产物的量与裂解的细胞数成正比。可以用标准的96孔读板计收集490nm可见光吸光值数据。
本领域技术人员熟知利用LDH的释放实验检测细胞功能的方法。本实施例LDH实验用从健康志愿者的血液中分离到的PBL细胞转染本发明高亲和力TCR作为效应细胞。靶细胞系为HepG2、HCCC9810和SNU-398，其中HepG2表达相关抗原并且基因型也符合为阳性细胞系，HCCC9810和SNU-398为阴性细胞系作为对照。
效应细胞分别转染了TCR1(α链可变域SEQ ID NO:11,β链可变域SEQ ID NO:2)、TCR3(α链可变域SEQ ID NO:14,β链可变域SEQ ID NO:2)、TCR5(α链可变域SEQ ID NO:17,β链可变域SEQ ID NO:2)和TCR6(α链可变域SEQ ID NO:18,β链可变域SEQ ID NO:2)，对照组效应细胞标号为A6(转染非本发明其他TCR)。
首先准备LDH平板。实验第1天，按以下顺序将试验的各个组分加入平板：培养基调整效应细胞至3X10 5个细胞/毫升，培养基调整各靶细胞系至3X105个细胞/毫升。混合均匀后取100μL靶细胞(即30，000个细胞/孔)、100μL效应细胞(即30，000个细胞/孔)加入对 应孔中，并设置三个复孔。同时设置效应细胞自发孔，靶细胞自发孔，靶细胞最大孔，体积校正对照孔及培养基背景对照孔，均为200μL。温育过夜(37℃，5％CO 2)。实验第2天，检测显色，终止反应后用酶标仪(Bioteck)在490nm记录吸光值。
实验结果如图12所示，转导本发明TCR的效应细胞对表达相关抗原的靶细胞具有很强的杀伤作用，而对不表达相关抗原的靶细胞基本没有杀伤作用。
实施例12 本发明高亲和力TCR分子的体内效力
将本发明高亲和力TCR转染的T细胞注射进人肝癌细胞异种移植模型的小鼠体内，检测其在体内对肿瘤的抑制效果。
实验采用NSG小鼠(北京百奥赛图基因生物技术有限公司)(雌性，实验周龄6-8周)作为实验对象，实验开始前20天以培养后收集并混悬好的HEPG2肿瘤细胞(ATCC)悬液按1*10^7个/只(注射体积200ul)的数量对小鼠进行腹部单侧皮下注射，建立人肝癌细胞异种移植小鼠模型。
实验开始当天用游标卡尺对每只小鼠已成型肿瘤的长径(a)、短径(b)分别进行测量，并按如下公式计算肿瘤体积：V＝a*b^2/2；随后根据分组设置：对照组(转染无关TCR的T细胞)6只标记为A6、转染TCR1(α链可变域SEQ ID NO:11,β链可变域SEQ ID NO:2)的T细胞组6只和转染TCR5(α链可变域SEQ ID NO:17,β链可变域SEQ ID NO:2)的T细胞组6只，按肿瘤体积对小鼠进行随机分组。分组完毕后取已制备好的T细胞按2.5*10^7个/只分别对上述分组小鼠进行尾部静脉注射。
细胞注射后，再取制备好的IL-2溶液(50000IU/100UL)对每只小鼠腹腔注射100ul，随后4天每天连续注射等量IL-2溶液。自实验开始，按上述方法每3天测量一次小鼠肿瘤瘤径和计算体积，持续至小鼠因肿瘤过大影响行动或者肿瘤消退为止，整理以上数据对每组小鼠肿瘤体积作数据统计分析处理。
所得实验结果如图13所示，注射了转染本发明高亲和力TCR的T细胞的小鼠组，肿瘤的生长明显受到抑制并且呈现缩小的趋势，而注射了转染无关TCR的T细胞的小鼠组的肿瘤体积增长非常快。
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 (39)

  1. 一种T细胞受体(TCR)，其特征在于，其具有结合FMNKFIYEI-HLA A0201复合物的活性，并且所述T细胞受体包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域包含3个CDR区，所述TCRα链可变域的3个CDR区的基准序列如下，
    CDR1α：DSAIYN
    CDR2α：IQSSQRE
    CDR3α：AVNSGGSNYKLT，并且CDR3α含有至少一个下列突变：
    突变前的残基 突变后的残基 CDR3α的第3位N D或E CDR3α的第4位S D或G或A或W或T或H CDR3α的第5位G Q或A或V或H或W或Y或M或I CDR3α的第6位G D或R或P或Q或T或Y CDR3α的第7位S G或D CDR3α的第8位N G或D
    和/或所述TCR的β链可变域为与SEQ ID NO:2所示的氨基酸序列有至少90％的序列同源性的氨基酸序列。
  2. 如权利要求1所述的TCR，其特征在于，所述TCR的β链可变域为与SEQ ID NO:2所示的氨基酸序列有至少90％，91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同源性的氨基酸序列。
  3. 如权利要求1所述的TCR，其特征在于，所述TCRα链可变域中CDR3α的突变个数为1至4个。
  4. 如权利要求1所述的TCR，其特征在于，所述TCR与FMNKFIYEI-HLA A0201复合物的亲和力是野生型TCR的至少5倍。
  5. 如权利要求1所述的TCR，其特征在于，所述TCR的α链可变域包含与SEQ ID NO:1所示的氨基酸序列有至少90％，91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性的氨基酸序列。
  6. 如权利要求1所述的TCR，其特征在于，所述TCRβ链可变域包含3个CDR区，所述TCRβ链可变域的3个CDR区的氨基酸序列如下：
    CDR1β：SGHVS
    CDR2β：FQNEAQ
    CDR3β：ASSLFGQGREKLF。
  7. 如权利要求1所述的TCR，其特征在于，所述TCRβ链可变域的氨基酸序列为SEQ ID NO：2。
  8. 如权利要求1所述的TCR，其特征在于，所述TCR包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域包含CDR1α、CDR2α和CDR3α，其中CDR1α的氨基酸序列为DSAIYN，CDR2α的氨基酸序列为IQSSQRE；和所述TCRβ链可变域包含CDR1β、CDR2β和CDR3β，其 中CDR1β的氨基酸序列为SGHVS，CDR2β的氨基酸序列为FQNEAQ，CDR3β的氨基酸序列为ASSLFGQGREKLF。
  9. 如权利要求1所述的TCR，其特征在于，所述TCR包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域包含CDR1α、CDR2α和CDR3α，其中CDR1α的氨基酸序列为DSAIYN，CDR2α的氨基酸序列为IQSSQRE，并且CDR3α的氨基酸序列为：
    AV[3αX1][3αX2][3αX3][3αX4][3αX5][3αX6]YKLT。
  10. 如权利要求9所述的TCR，其特征在于，所述[3αX1]为N或D或E。
  11. 如权利要求9所述的TCR，其特征在于，所述[3αX2]为S或D或G或A或W或T或H。
  12. 如权利要求9所述的TCR，其特征在于，所述[3αX3]为G或Q或A或V或H或W或Y或M或I。
  13. 如权利要求9所述的TCR，其特征在于，所述[3αX4]为G或D或R或P或Q或T或Y。
  14. 如权利要求9所述的TCR，其特征在于，所述[3αX5]为S或G或D。
  15. 如权利要求9所述的TCR，其特征在于，所述[3αX6]为N或G或D。
  16. 如权利要求1所述的TCR，其特征在于，所述TCR具有选自下组的CDR:
    CDR编号 CDR1α CDR2α CDR3α CDR1β CDR2β CDR3β 1 DSAIYN IQSSQRE AVDSGGSNYKLT SGHVS FQNEAQ ASSLFGQGREKLF 2 DSAIYN IQSSQRE AVEDQGSNYKLT SGHVS FQNEAQ ASSLFGQGREKLF 3 DSAIYN IQSSQRE AVDGADSNYKLT SGHVS FQNEAQ ASSLFGQGREKLF 4 DSAIYN IQSSQRE AVNSVRGGYKLT SGHVS FQNEAQ ASSLFGQGREKLF 5 DSAIYN IQSSQRE AVEGARSNYKLT SGHVS FQNEAQ ASSLFGQGREKLF 6 DSAIYN IQSSQRE AVDSHPSNYKLT SGHVS FQNEAQ ASSLFGQGREKLF 7 DSAIYN IQSSQRE AVDAAQSNYKLT SGHVS FQNEAQ ASSLFGQGREKLF 8 DSAIYN IQSSQRE AVNSWTGGYKLT SGHVS FQNEAQ ASSLFGQGREKLF 9 DSAIYN IQSSQRE AVDWHPSNYKLT SGHVS FQNEAQ ASSLFGQGREKLF 10 DSAIYN IQSSQRE AVDSQDSNYKLT SGHVS FQNEAQ ASSLFGQGREKLF 11 DSAIYN IQSSQRE AVNSYYDGYKLT SGHVS FQNEAQ ASSLFGQGREKLF 12 DSAIYN IQSSQRE AVDTMDSNYKLT SGHVS FQNEAQ ASSLFGQGREKLF 13 DSAIYN IQSSQRE AVDHHPSNYKLT SGHVS FQNEAQ ASSLFGQGREKLF 14 DSAIYN IQSSQRE AVNSIYGDYKLT SGHVS FQNEAQ ASSLFGQGREKLF。
  17. 如权利要求1所述的TCR，其特征在于，所述TCR是可溶的。
  18. 如权利要求1所述的TCR，其特征在于，所述TCR为αβ异质二聚TCR，包含α链TRAC恒定区序列和β链TRBC1或TRBC2恒定区序列。
  19. 如权利要求1所述的TCR，其特征在于，所述TCR包含(ⅰ)除其跨膜结构域以外的全部或部分TCRα链，和(ⅱ)除其跨膜结构域以外的全部或部分TCRβ链，其中(ⅰ)和(ⅱ) 均包含TCR链的可变域和至少一部分恒定域。
  20. 如权利要求18所述的TCR，其特征在于，所述TCR的α链恒定区与β链恒定区之间含有人工链间二硫键。
  21. 如权利要求20所述的TCR，其特征在于，在所述TCRα与β链的恒定区之间形成人工链间二硫键的半胱氨酸残基取代了选自下列的一组或多组位点：
    TRAC*01外显子1的Thr48和TRBC1*01或TRBC2*01外显子1的Ser57；
    TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；
    TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；
    TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；
    TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；
    TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；
    TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；
    和
    TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。
  22. 如权利要求1所述的TCR，其特征在于，所述TCR的α链可变域氨基酸序列选自：SEQ ID NO:11-24；和/或所述TCR的β链可变域氨基酸序列为SEQ ID NO:2。
  23. 如权利要求1所述的TCR，其特征在于，所述TCR选自下组：
    Figure PCTCN2020078271-appb-100001
  24. 如权利要求1所述的TCR，其特征在于，所述TCR为单链TCR。
  25. 如权利要求1所述的TCR，其特征在于，所述TCR是由α链可变域和β链可变域组成的单链TCR，所述α链可变域和β链可变域由一柔性短肽序列(linker)连接。
  26. 如以上任一权利要求所述的TCR，其特征在于，所述TCR的α链和/或β链的C-或N-末端结合有偶联物。
  27. 如权利要求26所述的TCR，其特征在于，与所述TCR结合的偶联物为可检测标记物、 治疗剂、PK修饰部分或任何这些物质的组合。
  28. 如权利要求27所述的TCR，其特征在于，与所述TCR结合的治疗剂为连接于所述TCR的α或β链的C-或N-末端的抗-CD3抗体。
  29. 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。
  30. 一种核酸分子，其特征在于，所述核酸分子包含编码权利要求1-28中任一项所述的TCR的核酸序列或其互补序列。
  31. 一种载体，其特征在于，所述的载体含有权利要求30中所述的核酸分子。
  32. 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31中所述的载体或染色体中整合有外源的权利要求30中所述的核酸分子。
  33. 一种分离的细胞，其特征在于，所述细胞表达权利要求1-28中任一项所述的TCR。
  34. 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1-28中任一项所述的TCR、或权利要求29中所述的TCR复合物、或权利要求33中所述的细胞。
  35. 一种治疗疾病的方法，其特征在于，包括给需要治疗的对象施用权利要求1-28中任一项所述的TCR、或权利要求29中所述的TCR复合物、或权利要求33中所述的细胞、或权利要求34中所述的药物组合物。
  36. 如权利要求35中所述的方法，其特征在于，所述疾病为AFP阳性肿瘤；优选地，所述AFP阳性肿瘤为肝癌、乳腺癌或生殖细胞肿瘤；更优选地，所述AFP阳性肿瘤为肝细胞癌。
  37. 权利要求1-28中任一项所述的T细胞受体、权利要29中所述的TCR复合物或权利要求33中所述细胞的用途，其特征在于，用于制备治疗肿瘤的药物。
  38. 如权利要求37中所述治疗肿瘤的药物，其特征在于，所述肿瘤为AFP阳性肿瘤；优选地，所述AFP阳性肿瘤为肝癌、乳腺癌或生殖细胞肿瘤；更优选地，所述AFP阳性肿瘤为肝细胞癌。
  39. 一种制备权利要求1-28中任一项所述的T细胞受体的方法，其特征在于，包括步骤：
    (i)培养权利要求32中所述的宿主细胞，从而表达权利要求1-28中任一项所述的T细胞受体；
    (ii)分离或纯化出所述的T细胞受体。
PCT/CN2020/078271 2019-03-08 2020-03-06 一种识别afp抗原的高亲和力tcr WO2020182082A1 (zh)

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KR1020217032584A KR20210142666A (ko) 2019-03-08 2020-03-06 Ａｆｐ 항원을 식별하기 위한 고친화력 ｔｃｒ
EP20771141.7A EP3936520A4 (en) 2019-03-08 2020-03-06 HIGH AFFINITY TCR FOR RECOGNIZING AFP ANTIGENS
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