WO2022206861A1 - T cell receptor for identifying afp - Google Patents
T cell receptor for identifying afp Download PDFInfo
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- WO2022206861A1 WO2022206861A1 PCT/CN2022/084190 CN2022084190W WO2022206861A1 WO 2022206861 A1 WO2022206861 A1 WO 2022206861A1 CN 2022084190 W CN2022084190 W CN 2022084190W WO 2022206861 A1 WO2022206861 A1 WO 2022206861A1
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Definitions
- the present invention relates to TCRs capable of recognizing short peptides derived from AFP antigens and their coding sequences.
- the present invention also relates to AFP-specific T cells obtained by transducing the above-mentioned TCRs, and their use in the prevention and treatment of AFP-related diseases.
- AFP ( ⁇ Fetoprotein), also known as ⁇ -fetoprotein, is a protein expressed during embryonic development and is the main component of embryonic serum. During development, AFP is expressed at relatively high levels in the yolk sac and liver, and is subsequently suppressed. In hepatocellular carcinoma, the expression of AFP is activated (Butterfield et al. J Immunol., 2001, Apr 15;166(8):5300-8). After AFP is generated in cells, it is degraded into small molecular polypeptides, and combined with MHC (major histocompatibility complex) molecules to form complexes, which are presented to the cell surface.
- KWVESIFLIF (SEQ ID NO: 9) is a short peptide derived from the AFP antigen and is a target for the treatment of AFP-related diseases.
- T cell adoptive immunotherapy is the transfer of T cells specific for target cell antigens into the patient's body, so that they can play a role against the target cells.
- T cell receptor TCR
- T cell receptor is a membrane protein on the surface of T cells that can recognize short antigenic peptides on the surface of the corresponding target cells.
- APCs antigen presenting cells
- T cells and The other cell membrane surface molecules of the two APCs interact, causing a series of subsequent cell signaling and other physiological responses, so that T cells with different antigen specificities can exert immune effects on their target cells.
- the object of the present invention is to provide a T cell receptor that recognizes AFP antigenic short peptides.
- the first aspect of the present invention provides a T cell receptor (TCR), the TCR can bind to the KWVESIFLIF-HLA A2402 complex.
- TCR T cell receptor
- the TCR comprises a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain
- the amino acid sequence of CDR3 of the TCR ⁇ chain variable domain is AVRDAGGTSYGKLT (SEQ ID NO: 12); and/or the The amino acid sequence of CDR3 of the variable domain of the TCR beta chain is ASSYPGSYGYT (SEQ ID NO: 15).
- the three complementarity determining regions (CDRs) of the variable domain of the TCR ⁇ chain are:
- the three complementarity determining regions of the TCR ⁇ chain variable domain are:
- the TCR comprises a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain
- the TCR ⁇ chain variable domain is an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1
- the TCR beta chain variable domain is an amino acid sequence with at least 90% sequence identity to SEQ ID NO:5.
- the TCR comprises ⁇ chain variable domain amino acid sequence SEQ ID NO: 1.
- the TCR comprises ⁇ chain variable domain amino acid sequence SEQ ID NO:5.
- the TCR is an ⁇ heterodimer, which comprises a TCR ⁇ chain constant region TRAC*01 and a TCR ⁇ chain constant region TRBC1*01 or TRBC2*01.
- amino acid sequence of the ⁇ chain of the TCR is SEQ ID NO: 3 and/or the amino acid sequence of the ⁇ chain of the TCR is SEQ ID NO: 7.
- the TCR is of human origin.
- the TCR is isolated and purified.
- the TCR is soluble.
- the TCR is a single chain.
- the TCR is formed by linking the ⁇ chain variable domain and the ⁇ chain variable domain through a peptide linker sequence.
- the TCR comprises an ⁇ chain constant region and a ⁇ chain constant region, the ⁇ chain constant region is a murine constant region and/or the ⁇ chain constant region is a murine constant region.
- the TCR is at the 11th, 13th, 19th, 21st, 53rd, 76th, 89th, 91st, or 94th amino acid position of the ⁇ chain variable region, and/or the reciprocal amino acid of the ⁇ chain J gene short peptide has one or more mutations in position 3, 5 from the bottom, or 7 from the bottom; and/or the TCR is at amino acids 11, 13, 19, 21, 53, 76, 89, 91 of the beta chain variable region , or position 94, and/or one or more mutations in the penultimate amino acid position 2, 4 or 6 of the ⁇ -chain J gene short peptide, wherein the amino acid positions are numbered according to IMGT (International Information on Immunogenetics) system) listed in the position number.
- IMGT International Information on Immunogenetics
- the amino acid sequence of the ⁇ -chain variable domain of the TCR comprises SEQ ID NO:32 and/or the amino acid sequence of the ⁇ -chain variable domain of the TCR comprises SEQ ID NO:34.
- amino acid sequence of the TCR is SEQ ID NO:30.
- the TCR comprises (a) all or part of the TCR ⁇ chain excluding the transmembrane domain; and (b) all or part of the TCR ⁇ chain excluding the transmembrane domain;
- cysteine residues form an artificial disulfide bond between the constant domains of the ⁇ and ⁇ chains of the TCR.
- cysteine residues forming artificial disulfide bonds in the TCR are substituted with one or more sites selected from the following groups:
- amino acid sequence of the ⁇ chain of the TCR is SEQ ID NO: 26 and/or the amino acid sequence of the ⁇ chain of the TCR is SEQ ID NO: 28.
- an artificial interchain disulfide bond is contained between the variable region of the ⁇ chain and the constant region of the ⁇ chain of the TCR.
- cysteine residues that form artificial interchain disulfide bonds in the TCR are substituted with one or more sites selected from the following groups:
- the TCR comprises 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 comprise an ⁇ -chain constant domain, and the TCR
- the ⁇ chain variable domain forms a heterodimer with the ⁇ chain.
- a conjugate is bound to the C- or N-terminus of the ⁇ chain and/or the ⁇ chain of the TCR.
- the conjugate bound to the T cell receptor is a detectable label, a therapeutic agent, a PK modification moiety or a combination of any of these substances.
- the therapeutic agent is an anti-CD3 antibody.
- 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 a complementary sequence thereof.
- the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 2 or SEQ ID NO: 33 encoding the variable domain of the TCR ⁇ chain.
- the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 6 or SEQ ID NO: 35 encoding the variable domain of the TCR beta chain.
- the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO:4 encoding the TCR ⁇ chain and/or the nucleotide sequence SEQ ID NO:8 encoding the TCR ⁇ chain.
- the fourth aspect of the present invention provides a vector, which contains the nucleic acid molecule described in the third aspect of the present invention; preferably, the vector is a viral vector; more preferably, the vector is a viral vector viral vector.
- the fifth aspect of the present invention provides an isolated host cell, wherein the host cell contains the vector described in the fourth aspect of the present invention or the exogenous nucleic acid molecule described in the third aspect of the present invention is integrated into the genome .
- the sixth aspect of the present invention provides a cell transduced with the nucleic acid molecule described in the third aspect of the present invention or the vector described in the fourth aspect of the present invention; preferably, the cell is a T cell, NK cells, NKT cells or stem cells.
- a seventh aspect of the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising a pharmaceutically acceptable carrier, the TCR described in the first aspect of the present invention, the TCR complex described in the second aspect of the present invention, the present
- the nucleic acid molecule of the third aspect of the present invention, the vector of the fourth aspect of the present invention, or the cell of the sixth aspect of the present invention is provided.
- the eighth aspect of the present invention provides the use of the T cell receptor described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, or the cell described in the sixth aspect of the present invention, for use in To prepare a drug for treating tumor or autoimmune disease, preferably, the tumor is liver cancer.
- the ninth aspect of the present invention provides the T cell receptor described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, or the cell described in the sixth aspect of the present invention for use in the treatment of tumors or Drugs for autoimmune diseases; preferably, the tumor is liver cancer.
- the tenth aspect of the present invention provides a method for treating a disease, comprising administering an appropriate amount of the T cell receptor described in the first aspect of the present invention or the TCR complex described in the second aspect of the present invention to a subject in need of treatment , or the cell according to the sixth aspect of the present invention, or the pharmaceutical composition according to the seventh aspect of the present invention; preferably, the disease is tumor, more preferably, the tumor is liver cancer.
- Fig. 1a, Fig. 1b, Fig. 1c, Fig. 1d, Fig. 1e and Fig. 1f are respectively the amino acid sequence of TCR ⁇ chain variable domain, the nucleotide sequence of TCR ⁇ chain variable domain, the amino acid sequence of TCR ⁇ chain, the nucleotide sequence of TCR ⁇ chain, with The amino acid sequence of the TCR ⁇ chain of the leader sequence and the nucleotide sequence of the TCR ⁇ chain with the leader sequence.
- Figure 2a, Figure 2b, Figure 2c, Figure 2d, Figure 2e and Figure 2f are the amino acid sequence of the variable domain of the TCR ⁇ chain, the nucleotide sequence of the variable domain of the TCR ⁇ chain, the amino acid sequence of the TCR ⁇ chain, and the nucleotide sequence of the TCR ⁇ chain, respectively.
- Figure 3 shows the results of CD8+ and tetramer-PE double-positive staining of monoclonal cells.
- Figure 4a and Figure 4b show the amino acid sequence and nucleotide sequence of the soluble TCR ⁇ chain, respectively.
- Figure 5a and Figure 5b are the amino acid sequence and nucleotide sequence of the soluble TCR beta chain, respectively.
- Figure 6a and Figure 6b are gel images of soluble TCR obtained after purification.
- the right lanes of Figure 6a and Figure 6b are reducing gel and non-reducing gel, respectively, and the left lanes are molecular weight markers.
- Figure 7a and Figure 7b are the amino acid sequence and nucleotide sequence of the single-chain TCR, respectively, and the amino acid sequence and nucleotide sequence of the linker are underlined.
- Figures 8a and 8b are the amino acid sequence and nucleotide sequence of the variable domain of the single-chain TCR alpha chain, respectively.
- Figures 9a and 9b are the amino acid sequence and nucleotide sequence of the variable domain of the single-chain TCR beta chain, respectively.
- Figure 10 is a gel image of the soluble single-chain TCR obtained after purification.
- the leftmost lane is the non-reducing gel
- the middle lane is the molecular weight marker
- the rightmost lane is the reducing gel.
- Figure 11 is a BIAcore kinetic map of the binding of soluble TCR of the present invention to KWVESIFLIF-HLA A2402 complex.
- Figure 12 is a BIAcore kinetic map of the binding of the soluble single-chain TCR of the present invention to the KWVESIFLIF-HLA A2402 complex.
- Fig. 13 shows the results of verification of the ELISPOT activation function of the obtained T cell clones.
- FIG. 14 shows the results of verification of the ELISPOT activation function of effector cells transduced with the TCR of the present invention.
- Fig. 15 shows the results of LDH killing function verification of effector cells transduced with the TCR of the present invention.
- the present invention provides a nucleic acid molecule encoding the TCR and a vector comprising the nucleic acid molecule.
- the present invention also provides cells transduced with the TCR of the present invention.
- MHC molecules are proteins of the immunoglobulin superfamily and can be class I or class II MHC molecules. Therefore, it is specific for antigen presentation, and different individuals have different MHCs, which can present different short peptides in a protein antigen to their respective APC cell surfaces.
- the human MHC is often referred to as the HLA gene or HLA complex.
- TCR The T cell receptor
- MHC major histocompatibility complex
- APCs antigen-presenting cells
- TCR is a glycoprotein on the surface of the cell membrane that exists in the form of heterodimers of ⁇ chain/ ⁇ chain or ⁇ chain/ ⁇ chain.
- TCR heterodimers consist of alpha and beta chains in 95% of T cells, whereas 5% of T cells have TCRs composed of gamma and delta chains.
- a native ⁇ heterodimeric TCR has an ⁇ chain and a ⁇ chain, and the ⁇ chain and the ⁇ chain constitute the subunits of the ⁇ heterodimeric TCR.
- the alpha and beta chains each contain a variable region, a linker region, and a constant region
- the beta chain usually also contains a short variable region between the variable region and the linker region, but the variable region is often regarded as the linker region a part of.
- Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, chimerically incorporated in framework regions.
- the CDR region determines the binding of TCR to the pMHC complex, and CDR3 is recombined from the variable region and the linker region, which is called the hypervariable region.
- the alpha and beta chains of a TCR are generally viewed as having two "domains” each, a variable domain and a constant domain, the variable domains being composed of linked variable and linking regions.
- the sequences of the TCR constant domains can be found in the public database of the International Immunogenetics Information System (IMGT). 01" or "TRBC2*01".
- IMGT International Immunogenetics Information System
- the ⁇ and ⁇ chains of TCR also contain a transmembrane region and a cytoplasmic region, and the cytoplasmic region is very short.
- polypeptide of the present invention TCR of the present invention
- T cell receptor of the present invention T cell receptor of the present invention
- the position numbers of the amino acid sequences of TRAC*01 and TRBC1*01 or TRBC2*01 in the present invention are numbered sequentially from the N-terminus to the C-terminus, such as TRBC1*01 or TRBC2*01 , the 60th amino acid in the sequence from the N-terminus to the C-terminus is P (proline), then in the present invention, it can be described as Pro60 of exon 1 of TRBC1*01 or TRBC2*01, or it can be described as Pro60 of exon 1 of TRBC1*01 It is expressed as the 60th amino acid of exon 1 of TRBC1*01 or TRBC2*01, and in TRBC1*01 or TRBC2*01, the 61st amino acid in the sequence from the N-terminus to the C-terminus is Q (glutamate).
- the position numbers of the amino acid sequences of the variable regions TRAV and TRBV are numbered according to the position numbers listed in IMGT. For example, for a certain amino acid in TRAV, the position number listed in IMGT is 46, then it is described as the 46th amino acid of TRAV in the present invention, and so on. In the present invention, if the sequence position numbering of other amino acids has special instructions, the special instructions are followed.
- a first aspect of the present invention provides a TCR molecule capable of binding the KWVESIFLIF-HLA A2402 complex.
- the TCR molecule is isolated or purified.
- the alpha and beta chains of this TCR each have three complementarity determining regions (CDRs).
- the alpha chain of the TCR comprises a CDR having the following amino acid sequence:
- the three complementarity determining regions of the TCR ⁇ chain variable domain are:
- Chimeric TCRs can be prepared by inserting the above-described amino acid sequences of the CDR regions of the present invention into any suitable framework structure.
- the framework structure is compatible with the CDR regions of the TCR of the present invention, those skilled in the art can design or synthesize TCR molecules with corresponding functions based on the CDR regions disclosed in the present invention.
- the TCR molecule of the present invention refers to a TCR molecule comprising the above-mentioned alpha and/or beta chain CDR region sequences and any suitable framework structure.
- the TCR ⁇ chain variable domain of the present invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity with SEQ ID NO: 1; and/or the TCR ⁇ chain variable domain of the present invention is an amino acid sequence with SEQ ID NO: 1 NO:5 has an amino acid sequence of at least 90%, preferably 95%, more preferably 98% sequence identity.
- the TCR molecule of the present invention is a heterodimer composed of ⁇ and ⁇ chains.
- the ⁇ chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain
- the amino acid sequence of the ⁇ chain variable domain comprises CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 10) and CDR2 (SEQ ID NO: 10) of the above-mentioned ⁇ chain. ID NO: 11) and CDR3 (SEQ ID NO: 12).
- the TCR molecule comprises the alpha chain variable domain amino acid sequence of SEQ ID NO: 1.
- the ⁇ chain variable domain amino acid sequence of the TCR molecule is SEQ ID NO: 1.
- the ⁇ chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the amino acid sequence of the ⁇ chain variable domain comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 13) of the above-mentioned ⁇ chain NO: 14) and CDR3 (SEQ ID NO: 15).
- the TCR molecule comprises the beta chain variable domain amino acid sequence of SEQ ID NO:5. More preferably, the ⁇ chain variable domain amino acid sequence of the TCR molecule is SEQ ID NO:5.
- the TCR molecule of the present invention is a single-chain TCR molecule composed of part or all of the ⁇ chain and/or part or all of the ⁇ chain.
- a description of single-chain TCR molecules can be found in Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-12658. Those skilled in the art can readily construct single-chain TCR molecules comprising the CDRs regions of the present invention as described in the literature.
- the single-chain TCR molecule comprises V ⁇ , V ⁇ and C ⁇ , preferably linked in order from the N-terminus to the C-terminus.
- the ⁇ chain variable domain amino acid sequence of the single-chain TCR molecule comprises CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the above-mentioned ⁇ chain.
- the single-chain TCR molecule comprises the alpha chain variable domain amino acid sequence of SEQ ID NO: 1. More preferably, the ⁇ -chain variable domain amino acid sequence of the single-chain TCR molecule is SEQ ID NO: 1.
- the beta chain variable domain amino acid sequence of the single-chain TCR molecule comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 14) and CDR3 (SEQ ID NO: 15) of the above beta chain.
- the single-chain TCR molecule comprises the beta chain variable domain amino acid sequence of SEQ ID NO:5. More preferably, the ⁇ -chain variable domain amino acid sequence of the single-chain TCR molecule is SEQ ID NO:5.
- the constant domain of the TCR molecule of the present invention is a human constant domain.
- the constant domain sequence of the alpha chain of the TCR molecule of the present invention can be "TRAC*01”
- the constant domain sequence of the beta chain of the TCR molecule can be "TRBC1*01” or "TRBC2*01”.
- the 53rd position of the amino acid sequence given in TRAC*01 of IMGT is Arg, which is represented here as: Arg53 of exon 1 of TRAC*01, and so on.
- the amino acid sequence of the ⁇ chain of the TCR molecule of the present invention is SEQ ID NO:3, and/or the amino acid sequence of the ⁇ chain is SEQ ID NO:7.
- TCR The naturally occurring TCR is a membrane protein that is stabilized by its transmembrane region. Like immunoglobulins (antibodies) as antigen recognition molecules, TCRs can also be developed for diagnostic and therapeutic applications, where soluble TCR molecules need to be obtained. Soluble TCR molecules do not include their transmembrane domains. Soluble TCR has a wide range of uses, not only to study the interaction of TCR with pMHC, but also as a diagnostic tool to detect infection or as a marker for autoimmune diseases.
- soluble TCRs can be used to deliver therapeutic agents (eg, cytotoxic or immunostimulatory compounds) to cells presenting specific antigens, and in addition, soluble TCRs can bind to other molecules (eg, anti-CD3 antibodies) to redirect T cells so that they target cells presenting specific antigens.
- therapeutic agents eg, cytotoxic or immunostimulatory compounds
- soluble TCRs can bind to other molecules (eg, anti-CD3 antibodies) to redirect T cells so that they target cells presenting specific antigens.
- the present invention also obtains a soluble TCR specific for AFP antigenic short peptide.
- the TCRs of the invention may be TCRs in which artificial disulfide bonds are introduced between residues of their alpha and beta chain constant domains.
- Cysteine residues form artificial interchain disulfide bonds between the constant domains of the alpha and beta chains of the TCR.
- Cysteine residues can be substituted for other amino acid residues at appropriate sites in the native TCR to form artificial interchain disulfide bonds. For example, substitution of Thr48 of exon 1 of TRAC*01 and substitution of cysteine residues of Ser57 of exon 1 of TRBC1*01 or TRBC2*01 to form a disulfide bond.
- cysteine residues are introduced to form disulfide bonds can also be: Thr45 in exon 1 of TRAC*01 and Ser77 in exon 1 of TRBC1*01 or TRBC2*01; exon 1 of TRAC*01 1 Tyr10 and TRBC1*01 or Ser17 of TRBC2*01 exon 1; Thr45 of TRAC*01 exon 1 and Asp59 of TRBC1*01 or TRBC2*01 exon 1; TRAC*01 exon 1 Ser15 and Glu15 in exon 1 of TRBC1*01 or TRBC2*01; Arg53 in exon 1 of TRAC*01 and Ser54 in exon 1 of TRBC1*01 or TRBC2*01; Pro89 and exon 1 of TRAC*01 Ala19 of exon 1 of TRBC1*01 or TRBC2*01; or Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01.
- cysteine residues are substituted for any set of sites in the constant domains of the alpha and beta chains described above.
- One or more C-termini of the TCR constant domains of the invention may be truncated by up to 50, or up to 30, or up to 15, or up to 10, or up to 8 or fewer amino acids so that they do not include
- a cysteine residue can be used to achieve the purpose of deleting the natural disulfide bond, and the above purpose can also be achieved by mutating the cysteine residue that forms the natural disulfide bond into another amino acid.
- the TCRs of the present invention may contain artificial disulfide bonds introduced between residues of their alpha and beta chain constant domains. It should be noted that the TCRs of the invention may contain both a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, with or without the artificial disulfide bonds introduced above between the constant domains.
- the TRAC constant domain sequence of the TCR and the TRBC1 or TRBC2 constant domain sequence may be linked by natural disulfide bonds present in the TCR.
- the TCR of the present invention also includes a TCR with mutation in its hydrophobic core region, and the mutation of these hydrophobic core regions is preferably a mutation that can improve the stability of the soluble TCR of the present invention, such as in Publication No. Described in the patent document of WO2014/206304.
- Such a TCR may be mutated at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable domain amino acids 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or Or the alpha chain J gene (TRAJ) short peptide amino acid position 3, 5, 7 from the bottom, and/or the beta chain J gene (TRBJ) short peptide amino acid position 2, 4, 6 from the bottom, where the position number of the amino acid sequence Numbered by position as listed in the International Information System on Immunogenetics (IMGT).
- IMGT International Information System on Immunogenetics
- the mutated TCR in the hydrophobic core region can be a stable soluble single-chain TCR composed of a flexible peptide chain linking the variable domains of the ⁇ and ⁇ chains of the TCR.
- the flexible peptide chain in the present invention can be any peptide chain suitable for linking the variable domains of TCR ⁇ and ⁇ chain.
- its ⁇ chain variable domain amino acid sequence is SEQ ID NO: 32, and the encoded nucleotide sequence is SEQ ID NO: 33; the ⁇ chain variable domain amino acid sequence is SEQ ID NO: 33; is SEQ ID NO:34, and the encoded nucleotide sequence is SEQ ID NO:35.
- Patent Document 201680003540.2 also discloses that the introduction of artificial interchain disulfide bonds between the ⁇ chain variable region and the ⁇ chain constant region of TCR can significantly improve the stability of TCR. Therefore, the TCR of the present invention may further contain an artificial interchain disulfide bond 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 for: amino acid 46 of TRAV and TRBC1*01 or TRBC2* 01 amino acid 60 of exon 1; TRAV amino acid 47 and TRBC1*01 or TRBC2*01 exon 1 amino acid 61; TRAV 46 amino acid and TRBC1*01 or TRBC2*01 exon Amino acid 61 of exon 1; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01.
- such a TCR may comprise (i) all or part of the TCR alpha chain excluding its transmembrane domain, and (ii) all or part of the TCR beta chain excluding its transmembrane domain, wherein (i) and (ii) ) both contain the variable domain and at least a part of the constant domain of the TCR chain, and the ⁇ chain and the ⁇ chain form a heterodimer.
- such a TCR may contain an alpha chain variable domain and a beta chain variable domain and all or part of the beta chain constant domain except the transmembrane domain, but it does not contain the alpha chain constant domain, the alpha chain of the TCR.
- the chain variable domains form heterodimers with beta chains.
- the TCRs of the present invention may also be provided in the form of multivalent complexes.
- the multivalent TCR complexes of the present invention comprise two, three, four or more multimers formed by combining the TCRs of the present invention, for example, the tetramerization domain of p53 can be used to generate tetramers, or multiple A complex formed by combining a TCR of the present invention with another molecule.
- the TCR complexes of the present invention can be used to track or target cells presenting specific antigens in vitro or in vivo, as well as to generate intermediates for other multivalent TCR complexes with such applications.
- the TCR of the present invention can be used alone, or can be combined with the conjugate in a covalent or other manner, preferably in a covalent manner.
- the conjugate includes a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the KWVESIFLIF-HLA A2402 complex), a therapeutic agent, a PK (protein kinase) modification moiety, or any of the above combination or conjugation.
- Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing detectable products enzyme.
- Therapeutic agents that can be bound or conjugated to the TCR of the present invention include, but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539); 2. Biotoxicity (Chaudhary et al., 1989) , Nature (Nature) 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy (Cancer Immunology and Immunotherapy) 51, 565); 3. Cytokines such as IL-2, etc.
- Viral particles (Peng et al, 2004, Gene Gene therapy 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research 65, 11631); 9. Nanomagnetic particles; 10. Prodrug-activating enzymes (eg, DT-myocardial flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. chemotherapeutic agents (eg, cisplatin) or nanoparticles in any form, etc.
- DTD DT-myocardial flavinase
- BPHL biphenyl hydrolase-like protein
- the TCRs of the present invention may also be hybrid TCRs comprising sequences derived from more than one species. For example, studies have shown that murine TCRs are more efficiently expressed in human T cells than human TCRs. Thus, the TCRs of the present invention may comprise human variable domains and murine constant domains. The downside of this approach is the potential to elicit an immune response. Therefore, there should be a regulatory regime for immunosuppression when it is used in adoptive T cell therapy to allow engraftment of murine expressing T cells.
- a second aspect of the invention provides a nucleic acid molecule encoding a TCR molecule of the first aspect of the invention, or a portion thereof, which portion may be one or more CDRs, variable domains of alpha and/or beta chains, and alpha chains and/or or beta chains.
- nucleotide sequence encoding the ⁇ chain CDR region of the TCR molecule according to the first aspect of the present invention is as follows:
- nucleotide sequence encoding the beta chain CDR region of the TCR molecule according to the first aspect of the present invention is as follows:
- nucleotide sequences of the nucleic acid molecules of the invention encoding the TCR alpha chains of the invention include SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and/or the nucleotide sequences of the nucleic acid molecules of the invention encoding the TCR beta chains of the invention
- Nucleotide sequences include SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.
- the nucleotide sequence of the nucleic acid molecule of the invention may be single-stranded or double-stranded, the nucleic acid molecule may be RNA or DNA, and may or may not contain introns.
- the nucleotide sequence of the nucleic acid molecule of the present invention does not contain introns but is capable of encoding the polypeptide of the present invention, for example, the nucleotide sequence of the nucleic acid molecule of the present invention encoding the variable domain of the TCR alpha chain of the present invention includes SEQ ID NO: 2 and /or the nucleotide sequence of the nucleic acid molecule of the present invention encoding the variable domain of the TCR beta chain of the present invention comprises SEQ ID NO:6.
- the nucleotide sequence of the nucleic acid molecule of the present invention encoding the variable domain of the TCR alpha chain of the present invention includes SEQ ID NO: 33 and/or the nucleotide sequence of the nucleic acid molecule of the present invention encoding the variable domain of the TCR beta chain of the present invention includes SEQ ID NO: 33 NO: 35. More preferably, the nucleotide sequence of the nucleic acid molecule of the invention comprises SEQ ID NO:4 and/or SEQ ID NO:8. Alternatively, the nucleotide sequence of the nucleic acid molecule of the present invention is SEQ ID NO:31.
- nucleic acid sequences encoding the TCRs of the present invention may be identical or degenerate variants of the nucleic acid sequences shown in the figures of the present invention.
- degenerate variant refers to a nucleic acid sequence that encodes the protein sequence of SEQ ID NO: 1, but differs from the sequence of SEQ ID NO: 2.
- the nucleotide sequence may be codon-optimized. Different cells differ in the use of specific codons. Depending on the type of cell, the codons in the sequence can be changed to increase the amount of expression. Codon usage tables for mammalian cells, as well as various other organisms, are well known to those skilled in the art.
- the full-length sequence of the nucleic acid molecule of the present invention or a fragment thereof can generally be obtained by, but not limited to, PCR amplification method, recombinant method or artificial synthesis method.
- the DNA sequences encoding the TCRs of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. DNA can be the coding or non-coding strand.
- the present invention also relates to vectors comprising the nucleic acid molecules of the present invention, including expression vectors, ie constructs capable of in vivo or in vitro expression.
- expression vectors include bacterial plasmids, bacteriophages, and animal and plant viruses.
- Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors, retroviral vectors, lentiviral vectors, baculovirus vectors.
- AAV adeno-associated virus
- the vector can transfer the nucleotides of the invention into cells, such as T cells, such that the cells express AFP antigen-specific TCRs.
- the vector should be able to express consistently high levels in T cells.
- the present invention also relates to host cells genetically engineered with the vectors or coding sequences of the present invention.
- the host cell contains the vector of the present invention or the nucleic acid molecule of the present invention is integrated into the chromosome.
- the host cell is selected from: prokaryotic cells and eukaryotic cells, such as E. coli, yeast cells, CHO cells, and the like.
- the present invention also includes isolated cells expressing the TCR of the present invention, which can be, but are not limited to, T cells, NK cells, NKT cells, especially T cells.
- the T cells can be derived from T cells isolated from the subject, or can be part of a mixed population of cells isolated from the subject, such as a peripheral blood lymphocyte (PBL) population.
- PBL peripheral blood lymphocyte
- the cells can be isolated from peripheral blood mononuclear cells (PBMCs) and can be CD4 + helper T cells or CD8 + cytotoxic T cells.
- PBMCs peripheral blood mononuclear cells
- the cells can be in a mixed population of CD4 + helper T cells/CD8 + cytotoxic T cells.
- the cells can be activated with antibodies (eg, anti-CD3 or anti-CD28 antibodies) to render them more receptive to transfection, eg, with a vector comprising a nucleotide sequence encoding a TCR molecule of the invention dye.
- antibodies eg, anti-CD3 or anti-CD28 antibodies
- the cells of the invention may also be or derived from stem cells, such as hematopoietic stem cells (HSCs).
- stem cells such as hematopoietic stem cells (HSCs).
- HSCs hematopoietic stem cells
- T cells expressing the TCR of the present invention can be used for adoptive immunotherapy.
- Those skilled in the art are aware of many suitable methods for adopting adoptive therapy (eg, Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).
- the present invention also relates to a method of treating and/or preventing an AFP-related disease in a subject comprising the step of adoptively transferring AFP-specific T cells to the subject.
- the AFP-specific T cells recognize the KWVESIFLIF-HLA A2402 complex.
- the AFP-specific T cells of the present invention can be used to treat any AFP-related diseases that present the AFP antigen short peptide KWVESIFLIF-HLA A2402 complex, including but not limited to tumors, such as liver cancer and the like.
- Treatment can be performed by isolating T cells from patients or volunteers suffering from AFP antigen-related diseases, introducing the TCR of the present invention into the above T cells, and then infusing these genetically engineered cells back into the patient. Therefore, the present invention provides a method for treating AFP-related diseases, comprising infusing into a patient isolated T cells expressing the TCR of the present invention, preferably, the T cells are derived from the patient itself. Typically, this involves (1) isolating T cells from a patient, (2) transducing T cells in vitro with a nucleic acid molecule of the invention or a nucleic acid molecule capable of encoding a TCR molecule of the invention, and (3) infusing genetically engineered T cells into a patient in vivo. The number of cells isolated, transfected, and reinfused can be determined by the physician.
- the TCR of the present invention can specifically bind to the AFP antigen short peptide complex KWVESIFLIF-HLA A2402, and the effector cells transduced with the TCR of the present invention can be specifically activated.
- the effector cells transduced with the TCR of the present invention can specifically kill the target cells.
- Peripheral blood lymphocytes from healthy volunteers with HLA A2402 genotype were stimulated with the synthetic short peptide KWVESIFLIF (SEQ ID NO: 9; Jiangsu GenScript Biotechnology Co., Ltd.).
- the KWVESIFLIF peptide was renatured with biotin-labeled HLA A2402 to prepare pHLA haploids.
- These haploids were combined with PE-labeled streptavidin (BD) to form PE-labeled tetramers, and the tetramers and anti-CD8-APC double positive cells were sorted. Sorted cells were expanded and subjected to secondary sorting as described above, followed by monocloning by limiting dilution. Monoclonal cells were stained with tetramers, and the screened double-positive clones were shown in Figure 3. The double-positive clones obtained through layer-by-layer screening still need to meet further functional tests.
- the function and specificity of the T cell clone were further tested by ELISPOT assay.
- ELISPOT assay Those skilled in the art are familiar with methods for detecting cell function using ELISPOT assays.
- the effector cells used in the IFN- ⁇ ELISPOT experiment in this example are the T cell clones obtained in the present invention, and the target cells are T2-A24 (T2 cells transfected with HLA-A2402), SNU398-AFP ( AFP overexpression), HepG2, and the control groups were T2-A24 and SNU398 loaded with other short peptides.
- ELISPOT plate First prepare the ELISPOT plate, add the components of the test to the ELISPOT plate in the following order: 20,000 target cells/well, 2000 effector cells/well, add 20 ⁇ l of the corresponding short peptides to the experimental group and control group, and add 20 ⁇ l to the blank group. culture medium (assay medium), and set up 2 replicate wells. It was then incubated overnight (37°C, 5% CO2 ). The plates were then washed and subjected to secondary detection and color development, the plates were dried for 1 hour, and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID Corporation). The experimental results are shown in Figure 13.
- T cell clones have obvious activation responses to the target cells loaded with KWVESIFLIF short peptide and SNU398-AFP and HepG2 overexpressing AFP antigen, while the cells loaded with other short peptides and SNU398 Basically no response.
- RNA of the antigen short peptide KWVESIFLIF-specific and HLA A2402-restricted T cell clone screened in Example 1 was extracted with Quick-RNA TM MiniPrep (ZYMO research).
- the cDNA was synthesized using the SMART RACE cDNA amplification kit from clontech, and the primers used were designed in the C-terminal conserved region of the human TCR gene.
- the sequence was cloned into T vector (TAKARA) for sequencing. It should be noted that this sequence is complementary and does not contain introns. After sequencing, the sequence structures of the ⁇ chain and ⁇ chain of the TCR expressed by the double-positive clone are shown in Figure 1 and Figure 2, respectively.
- the alpha chain was identified as comprising CDRs with the following amino acid sequence:
- the beta chain contains CDRs with the following amino acid sequence:
- the full-length genes of the TCR alpha chain and beta chain were cloned into the lentiviral expression vector pLenti (addgene), respectively, by overlapping PCR.
- the TCR ⁇ -2A-TCR ⁇ fragment was obtained by connecting the full-length genes of the TCR ⁇ chain and the TCR ⁇ chain by overlap PCR.
- the pLenti-TRA-2A-TRB-IRES-NGFR plasmid was obtained by ligating the lentiviral expression vector and TCR ⁇ -2A-TCR ⁇ restriction enzyme.
- a lentiviral vector pLenti-eGFP expressing eGFP was also constructed. Afterwards, 293T/17 was used to package the pseudovirus.
- the ⁇ and ⁇ chains of the TCR molecule of the present invention can respectively contain only their variable domains and part of their constant domains, and a cysteine residue is introduced into the constant domains of the ⁇ and ⁇ chains respectively.
- the amino acid sequence and nucleotide sequence of the ⁇ chain are shown in Figure 4a and Figure 4b, respectively, and the amino acid sequence and nucleotide sequence of the ⁇ chain are shown in Figure 5a and Figure 5b, respectively. .
- the target gene sequences of the above TCR ⁇ and ⁇ chains were synthesized and inserted into the expression vector pET28a+ (Novagene ), the upstream and downstream cloning sites are NcoI and NotI, respectively. The insert was confirmed by sequencing.
- 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). ) in 20 mM Tris (pH 8.1).
- the dissolved TCR ⁇ and ⁇ chains were rapidly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine, 6.6mM ⁇ -mercapoethylamine (4°C) at a mass ratio of 1:1 at a final concentration of 60 mg/mL. After mixing, the solution was dialyzed in 10 times the volume of deionized water (4°C). After 12 hours, the deionized water was replaced with a buffer (20 mM Tris, pH 8.0), and the dialysis was continued at 4°C for 12 hours.
- the solution after dialysis was filtered through a 0.45 ⁇ M filter membrane and purified by an anion exchange column (HiTrap Q HP, 5 ml, GE Healthcare).
- the eluted peaks of TCR containing successfully renatured ⁇ and ⁇ dimers were confirmed by SDS-PAGE gel.
- TCR was then further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare).
- the purity of the purified TCR was more than 90% determined by SDS-PAGE, and the concentration was determined by BCA method.
- the SDS-PAGE gel images of the soluble TCR obtained by the present invention are shown in Figure 6a and Figure 6b.
- variable domains of TCR ⁇ and ⁇ chains in Example 2 were constructed into a stable soluble single-chain TCR molecule linked by a flexible short peptide (linker) by site-directed mutagenesis.
- the amino acid sequence and nucleotide sequence of the single-chain TCR molecule are shown in Figure 7a and Figure 7b, respectively, wherein the amino acid sequence and nucleotide sequence of the linker sequence are underlined;
- the nucleotide sequences are shown in Figure 8a and Figure 8b, respectively; the amino acid sequence and nucleotide sequence of the ⁇ chain variable domain are shown in Figure 9a and Figure 9b, respectively.
- the target gene was double digested with Nco I and Not I, and then ligated with the pET28a vector that was double digested with Nco I and Not I.
- the ligation product was transformed into E.coli DH5 ⁇ , coated with LB plate containing kanamycin, and cultured overnight at 37°C by inversion. Positive clones were picked for PCR screening, and the positive recombinants were sequenced. After confirming that the sequences were correct, the recombinant plasmids were extracted and transformed. to E. coli BL21(DE3) for expression.
- the inclusion bodies were dissolved in buffer (20mM Tris-HCl pH 8.0, 8M urea), and insoluble matter was removed by high-speed centrifugation. The supernatant was quantified by BCA method, and then packed and stored at -80°C for later use.
- renaturation buffer 100mM Tris-HCl pH 8.1, 0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mM ⁇ -mercapthoethylamine, 1.87mM Cystamine
- renaturation buffer 100mM Tris-HCl pH 8.1, 0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mM ⁇ -mercapthoethylamine, 1.87mM Cystamine
- the dialysate was replaced with 1 L of pre-cooled buffer (20mM Tris-HCl pH 8.0), and the dialysis was continued for 8 h at 4°C, and then the dialysate was replaced with the same fresh buffer and continued dialysis overnight.
- the sample was filtered through a 0.45 ⁇ m filter membrane, degassed in vacuo and passed through an anion exchange column (HiTrap Q HP, GE Healthcare), and the protein was purified with a linear gradient of 0-1 M NaCl prepared in 20 mM Tris-HCl pH 8.0.
- the collected elution fractions were analyzed by SDS-PAGE, the fractions containing single-chain TCR were concentrated and further purified by gel filtration column (Superdex 75 10/300, GE Healthcare), and the target fractions were also analyzed by SDS-PAGE.
- the eluted fractions for BIAcore analysis were further tested for purity by gel filtration.
- the conditions are: Column Agilent Bio SEC-3 (300A, ), the mobile phase was 150 mM phosphate buffer, the flow rate was 0.5 mL/min, the column temperature was 25°C, and the UV detection wavelength was 214 nm.
- Figure 10 shows the SDS-PAGE gel image of the soluble single-chain TCR obtained by the present invention.
- the binding activity of the TCR molecules obtained in Example 3 and Example 5 to the KWVESIFLIF-HLA A2402 complex was detected using the BIAcore T200 real-time analysis system.
- Anti-streptavidin antibody (GenScript) was added to coupling buffer (10 mM sodium acetate buffer, pH 4.77), and the antibody was then flowed through a CM5 chip preactivated with EDC and NHS to immobilize the antibody on the chip surface , and finally blocked the unreacted activated surface with ethanolamine hydrochloric acid solution to complete the coupling process with a coupling level of about 15,000RU.
- Collect 100ml of E.coli bacteria that induces the expression of heavy or light chains centrifuge at 8000g at 4°C for 10min, wash the cells once with 10ml PBS, and then use 5ml BugBuster Master Mix Extraction Reagents (Merck) to vigorously shake the cells to resuspend the cells. Incubate with rotation at room temperature for 20 min, then centrifuge at 6000g for 15 min at 4°C, discard the supernatant, and collect the inclusion bodies.
- the synthetic short peptide KWVESIFLIF (Jiangsu GenScript Biotechnology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg/ml.
- the inclusion bodies of light chain and heavy chain were dissolved with 8M urea, 20mM Tris pH 8.0, 10mM DTT, and further denatured by adding 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA before renaturation.
- KWVESIFLIF peptide was added to renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4°C), then added 20mg/L light chain and 90mg/L heavy chain in sequence (final concentration, heavy chain was added in three times, 8h/time), and renatured at 4°C for at least 3 days To complete, SDS-PAGE test whether the renaturation is successful.
- renaturation buffer 0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4°C
- renaturation buffer by dialyzing against 10 volumes of 20 mM Tris pH 8.0, at least twice, to sufficiently reduce the ionic strength of the solution.
- the protein solution was filtered through a 0.45 ⁇ m cellulose acetate filter and loaded onto a HiTrap Q HP (GE) anion exchange column (5 ml bed volume).
- the protein was eluted with an Akta purifier (GE), a linear gradient of 0-400 mM NaCl prepared with 20 mM Tris pH 8.0, and pMHC was eluted at about 250 mM NaCl, and the peak fractions were collected, and the purity was checked by SDS-PAGE.
- GE HiTrap Q HP
- Purified pMHC molecules were concentrated with Millipore ultrafiltration tubes while buffer exchanged to 20mM Tris pH 8.0, followed by addition of biotinylation reagents 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50 ⁇ M D-Biotin, 100 ⁇ g/ml BirA Enzyme (GST-BirA), the mixture was incubated overnight at room temperature, and the complete biotinylation was checked by SDS-PAGE.
- the biotinylated pMHC molecules were concentrated to 1 ml with a Millipore ultrafiltration tube, and the biotinylated pMHC was purified by gel filtration chromatography. HiPrep was pre-equilibrated with filtered PBS using an Akta purifier (GE). A TM 16/60 S200 HR column (GE) was loaded with 1 ml of concentrated biotinylated pMHC molecules and eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules eluted as a single peak at about 55 ml.
- the fractions containing protein were combined, concentrated with Millipore ultrafiltration tube, the protein concentration was determined by BCA method (Thermo), and the protease inhibitor cocktail (Roche) was added to store the biotinylated pMHC molecules in aliquots at -80°C.
- IFN- ⁇ is a powerful immunoregulatory factor produced by activated T lymphocytes. Therefore, in this example, the number of IFN- ⁇ was detected by the ELISPOT experiment well-known to those skilled in the art to verify the activation function of the cells transfected with the TCR of the present invention. antigen specificity.
- the effector cells used in this experiment are CD3 + T cells expressing the TCR of the present invention, and the same volunteer transfected with other TCR (A6) or empty transfected (NC) CD3 + T cells was used as a control group.
- the target cells used were T2-A24 loaded with AFP antigen short peptide KWVESIFLIF, and T2-A24 loaded with other irrelevant peptides or empty were used as control.
- the components of the test were added to the ELISPOT well plate: 1 ⁇ 10 4 cells/well of target cells, 2 ⁇ 10 3 cells/well of effector cells (calculated according to the positive rate of transfection), and two duplicate wells were set up.
- KWVESIFLIF short peptide solution other short peptide solutions, and equal volume of medium were added to the corresponding wells respectively, so that the final concentration of KWVESIFLIF short peptide in the ELISPOT plate was 1 ⁇ 10 -6 M to 1 ⁇ 10 -12 M in turn, a total of 7 gradients; make the final concentration of irrelevant peptides in the ELISPOT plate from 1 x 10 -6 M to 1 x 10 -8 M, 3 gradients in total. It was then incubated overnight (37°C, 5% CO2 ). On the second day of the experiment, the plate was washed for secondary detection and color development, the plate was dried, and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID20 company).
- ELISPOT READER system an immunospot plate reader
- the T cells transfected with the TCR of the present invention have an obvious activation effect, while the T cells transfected with other TCRs or empty transfected have no response from the beginning; At the same time, T cells transfected with the TCR of the present invention did not activate cells loaded with other short peptides or empty.
- Example 8 LDH killing function experiment of effector cells transfected with TCR of the present invention
- non-radioactive cytotoxicity experiments well known to those skilled in the art are used to measure the release of LDH, thereby verifying the killing function of cells transfected with the TCR of the present invention.
- This assay is a colorimetric alternative to the 51Cr release cytotoxicity assay and quantifies lactate dehydrogenase (LDH) released after cell lysis.
- the isolated CD3+ T cells were transfected with the TCR of the present invention as effector cells, and the same volunteers were transfected with other TCR (A6) or empty-transfected (NC) CD3+ T cells as the control group.
- the target cells used were T2-A24 loaded with AFP antigen short peptide KWVESIFLIF, and T2-A24 loaded with other irrelevant peptides or empty were used as control.
- the LDH plate First prepare the LDH plate, firstly add 3*10 4 target cells/well and 3*10 4 effector cells/well into the corresponding medium, and then add KWVESIFLIF short peptide solution, other short peptide solutions, and equal volume of medium to the corresponding wells respectively.
- the final concentration of KWVESIFLIF peptide in the LDH well plate was 1 ⁇ 10 -6 M to 1 ⁇ 10 -13 M, a total of 8 gradients; the final concentration of the irrelevant peptide in the LDH well plate was 1 ⁇ 10 -6 M to 1 ⁇ 10 -8 M, 3 gradients in total. And set three duplicate holes.
- the effector cells transfected with the TCR of the present invention have a strong killing effect, and they react when the concentration of the above-mentioned short peptide is low, while other TCRs are transfected. At the same time, the effector cells transfected with the TCR of the present invention did not kill the target cells loaded with other short peptides or empty.
Abstract
The present invention provides a T cell receptor (TCR) capable of specifically binding to a short peptide KWVESIFLIF derived from an AFP antigen. The antigen short peptide KWVESIFLIF can form a complex together with HLA A2402 and be presented together with same to a cell surface. The present invention further provides a nucleic acid molecule encoding the TCR and a vector comprising the nucleic acid molecule. In addition, the present invention further provides a cell that transduces the TCR of the present invention.
Description
本发明涉及能够识别源自AFP抗原短肽的TCR及其编码序列，本发明还涉及转导上述TCR来获得的AFP特异性的T细胞，及他们在预防和治疗AFP相关疾病中的用途。The present invention relates to TCRs capable of recognizing short peptides derived from AFP antigens and their coding sequences. The present invention also relates to AFP-specific T cells obtained by transducing the above-mentioned TCRs, and their use in the prevention and treatment of AFP-related diseases.
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(Butterfield et al.J Immunol.,2001,Apr 15；166(8):5300-8)。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。KWVESIFLIF(SEQ ID NO:9)是衍生自AFP抗原的短肽，是AFP相关疾病治疗的一种靶标。AFP (αFetoprotein), also known as α-fetoprotein, is a protein expressed during embryonic development and is the main component of embryonic serum. During development, AFP is expressed at relatively high levels in the yolk sac and liver, and is subsequently suppressed. In hepatocellular carcinoma, the expression of AFP is activated (Butterfield et al. J Immunol., 2001, Apr 15;166(8):5300-8). After AFP is generated in cells, it is degraded into small molecular polypeptides, and combined with MHC (major histocompatibility complex) molecules to form complexes, which are presented to the cell surface. KWVESIFLIF (SEQ ID NO: 9) is a short peptide derived from the AFP antigen and is a target for the treatment of AFP-related diseases.
T细胞过继免疫治疗是将对靶细胞抗原具有特异性的反应性T细胞转入病人体内，使其针对靶细胞发挥作用。T细胞受体(TCR)是T细胞表面的一种膜蛋白，其能够识别相应的靶细胞表面的抗原短肽。在免疫系统中，通过抗原短肽特异性的TCR与短肽-主组织相容性复合体(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，引起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。因此，本领域技术人员致力于分离出对AFP抗原短肽具有特异性的TCR，以及将该TCR转导T细胞来获得对AFP抗原短肽具有特异性的T细胞，从而使他们在细胞免疫治疗中发挥作用。T cell adoptive immunotherapy is the transfer of T cells specific for target cell antigens into the patient's body, so that they can play a role against the target cells. T cell receptor (TCR) is a membrane protein on the surface of T cells that can recognize short antigenic peptides on the surface of the corresponding target cells. In the immune system, direct physical contact between T cells and antigen presenting cells (APCs) is triggered by the binding of antigen peptide-specific TCRs to the peptide-major histocompatibility complex (pMHC complex), and then T cells and The other cell membrane surface molecules of the two APCs interact, causing a series of subsequent cell signaling and other physiological responses, so that T cells with different antigen specificities can exert immune effects on their target cells. Therefore, those skilled in the art are devoted to isolating TCRs specific for AFP antigen short peptides, and transducing this TCR into T cells to obtain T cells specific for AFP antigen short peptides, so that they can be used in cellular immunotherapy. function in.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于提供一种识别AFP抗原短肽的T细胞受体。The object of the present invention is to provide a T cell receptor that recognizes AFP antigenic short peptides.
本发明的第一方面，提供了一种T细胞受体(TCR)，所述TCR能够与KWVESIFLIF-HLA A2402复合物结合。The first aspect of the present invention provides a T cell receptor (TCR), the TCR can bind to the KWVESIFLIF-HLA A2402 complex.
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链可变域的CDR3的氨基酸序列为AVRDAGGTSYGKLT(SEQ ID NO:12)；和/或所述TCRβ链可变域的CDR3的氨基酸序列为ASSYPGSYGYT(SEQ ID NO:15)。In another preferred example, the TCR comprises a TCRα chain variable domain and a TCRβ chain variable domain, and the amino acid sequence of CDR3 of the TCRα chain variable domain is AVRDAGGTSYGKLT (SEQ ID NO: 12); and/or the The amino acid sequence of CDR3 of the variable domain of the TCR beta chain is ASSYPGSYGYT (SEQ ID NO: 15).
在另一优选例中，所述TCRα链可变域的3个互补决定区(CDR)为：In another preferred embodiment, the three complementarity determining regions (CDRs) of the variable domain of the TCRα chain are:
αCDR1-VGISA(SEQ ID NO:10)αCDR1-VGISA (SEQ ID NO: 10)
αCDR2-LSSGK(SEQ ID NO:11)αCDR2-LSSGK (SEQ ID NO: 11)
αCDR3-AVRDAGGTSYGKLT(SEQ ID NO:12)；和/或αCDR3-AVRDAGGTSYGKLT (SEQ ID NO: 12); and/or
所述TCRβ链可变域的3个互补决定区为：The three complementarity determining regions of the TCRβ chain variable domain are:
βCDR1-MNHEY(SEQ ID NO:13)βCDR1-MNHEY (SEQ ID NO: 13)
βCDR2-SVGAGI(SEQ ID NO:14)βCDR2-SVGAGI (SEQ ID NO: 14)
βCDR3-ASSYPGSYGYT(SEQ ID NO:15)。βCDR3-ASSYPGSYGYT (SEQ ID NO: 15).
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链可变域为与SEQ ID NO:1具有至少90％序列相同性的氨基酸序列；和/或所述TCRβ链可变域为与SEQ ID NO:5具有至少90％序列相同性的氨基酸序列。In another preferred embodiment, the TCR comprises a TCRα chain variable domain and a TCRβ chain variable domain, and the TCRα chain variable domain is an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1; and/ Or the TCR beta chain variable domain is an amino acid sequence with at least 90% sequence identity to SEQ ID NO:5.
在另一优选例中，所述TCR包含α链可变域氨基酸序列SEQ ID NO:1。In another preferred embodiment, the TCR comprises α chain variable domain amino acid sequence SEQ ID NO: 1.
在另一优选例中，所述TCR包含β链可变域氨基酸序列SEQ ID NO:5。In another preferred embodiment, the TCR comprises β chain variable domain amino acid sequence SEQ ID NO:5.
在另一优选例中，所述TCR为αβ异质二聚体，其包含TCRα链恒定区TRAC*01和TCRβ链恒定区TRBC1*01或TRBC2*01。In another preferred embodiment, the TCR is an αβ heterodimer, which comprises a TCRα chain constant region TRAC*01 and a TCRβ chain constant region TRBC1*01 or TRBC2*01.
在另一优选例中，所述TCR的α链氨基酸序列为SEQ ID NO:3和/或所述TCR的β链氨基酸序列为SEQ ID NO:7。In another preferred embodiment, the amino acid sequence of the α chain of the TCR is SEQ ID NO: 3 and/or the amino acid sequence of the β chain of the TCR is SEQ ID NO: 7.
在另一优选例中，所述TCR是人源的。In another preferred embodiment, the TCR is of human origin.
在另一优选例中，所述TCR是分离与纯化的。In another preferred embodiment, the TCR is isolated and purified.
在另一优选例中，所述TCR是可溶的。In another preferred embodiment, the TCR is soluble.
在另一优选例中，所述TCR为单链。In another preferred embodiment, the TCR is a single chain.
在另一优选例中，所述TCR是由α链可变域与β链可变域通过肽连接序列连接而成。In another preferred embodiment, the TCR is formed by linking the α chain variable domain and the β chain variable domain through a peptide linker sequence.
在另一优选例中，所述TCR包含α链恒定区与β链恒定区，所述α链恒定区为鼠的恒定区和/或所述β链恒定区为鼠的恒定区。In another preferred embodiment, the TCR comprises an α chain constant region and a β chain constant region, the α chain constant region is a murine constant region and/or the β chain constant region is a murine constant region.
在另一优选例中，所述TCR在α链可变区氨基酸第11、13、19、21、53、76、89、91、或第94位，和/或α链J基因短肽氨基酸倒数第3位、倒数第5位或倒数第7位中具有一个或多个突变；和/或所述TCR在β链可变区氨基酸第11、13、19、21、53、76、89、91、或第94位，和/或β链J基因短肽氨基酸倒数第2位、倒数第4位或倒数第6位中具有一个或多个突变，其中氨基酸位置编号按IMGT(国际免疫遗传学信息系统)中列出的位置编号。In another preferred embodiment, the TCR is at the 11th, 13th, 19th, 21st, 53rd, 76th, 89th, 91st, or 94th amino acid position of the α chain variable region, and/or the reciprocal amino acid of the α chain J gene short peptide has one or more mutations in position 3, 5 from the bottom, or 7 from the bottom; and/or the TCR is at amino acids 11, 13, 19, 21, 53, 76, 89, 91 of the beta chain variable region , or position 94, and/or one or more mutations in the penultimate amino acid position 2, 4 or 6 of the β-chain J gene short peptide, wherein the amino acid positions are numbered according to IMGT (International Information on Immunogenetics) system) listed in the position number.
在另一优选例中，所述TCR的α链可变域氨基酸序列包含SEQ ID NO:32和/或所述TCR的β链可变域氨基酸序列包含SEQ ID NO:34。In another preferred embodiment, the amino acid sequence of the α-chain variable domain of the TCR comprises SEQ ID NO:32 and/or the amino acid sequence of the β-chain variable domain of the TCR comprises SEQ ID NO:34.
在另一优选例中，所述TCR的氨基酸序列为SEQ ID NO:30。In another preferred embodiment, the amino acid sequence of the TCR is SEQ ID NO:30.
在另一优选例中，所述TCR包括(a)除跨膜结构域以外的全部或部分TCRα链；以及(b)除跨膜结构域以外的全部或部分TCRβ链；In another preferred embodiment, the TCR comprises (a) all or part of the TCRα chain excluding the transmembrane domain; and (b) all or part of the TCRβ chain excluding the transmembrane domain;
并且(a)和(b)各自包含功能性可变结构域，或包含功能性可变结构域和所述TCR链恒定结构域的至少一部分。And (a) and (b) each comprise a functional variable domain, or comprise a functional variable domain and at least a portion of said TCR chain constant domain.
在另一优选例中，半胱氨酸残基在所述TCR的α和β链恒定域之间形成人工二硫键。In another preferred embodiment, cysteine residues form an artificial disulfide bond between the constant domains of the α and β chains of the TCR.
在另一优选例中，在所述TCR中形成人工二硫键的半胱氨酸残基取代了选自下列的一组或多组位点：In another preferred embodiment, the cysteine residues forming artificial disulfide bonds in the TCR are substituted with one or more sites selected from the following groups:
TRAC*01外显子1的Thr48和TRBC1*01或TRBC2*01外显子1的Ser57；Thr48 in exon 1 of TRAC*01 and Ser57 in exon 1 of TRBC1*01 or TRBC2*01;
TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；Thr45 in exon 1 of TRAC*01 and Ser77 in exon 1 of TRBC1*01 or TRBC2*01;
TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；Tyr10 of exon 1 of TRAC*01 and Ser17 of exon 1 of TRBC1*01 or TRBC2*01;
TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；Thr45 of exon 1 of TRAC*01 and Asp59 of exon 1 of TRBC1*01 or TRBC2*01;
TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；Ser15 of exon 1 of TRAC*01 and Glu15 of exon 1 of TRBC1*01 or TRBC2*01;
TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；Arg53 in exon 1 of TRAC*01 and Ser54 in exon 1 of TRBC1*01 or TRBC2*01;
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。Pro89 of exon 1 of TRAC*01 and Ala19 of exon 1 of TRBC1*01 or TRBC2*01; and Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01.
在另一优选例中，所述TCR的α链氨基酸序列为SEQ ID NO:26和/或所述TCR的β链氨基酸序列为SEQ ID NO:28。In another preferred embodiment, the amino acid sequence of the α chain of the TCR is SEQ ID NO: 26 and/or the amino acid sequence of the β chain of the TCR is SEQ ID NO: 28.
在另一优选例中，所述TCR的α链可变区与β链恒定区之间含有人工链间二硫键。In another preferred embodiment, an artificial interchain disulfide bond is contained between the variable region of the α chain and the constant region of the β chain of the TCR.
在另一优选例中，其特征在于，在所述TCR中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：In another preferred embodiment, it is characterized in that the cysteine residues that form artificial interchain disulfide bonds in the TCR are substituted with one or more sites selected from the following groups:
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；Amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01;
TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的第61位氨基酸；Amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01;
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Amino acid 46 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01; or
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。Amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01.
在另一优选例中，所述TCR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。In another preferred embodiment, the TCR comprises an α-chain variable domain and a β-chain variable domain and all or part of the β-chain constant domain except the transmembrane domain, but it does not comprise an α-chain constant domain, and the TCR The α chain variable domain forms a heterodimer with the β chain.
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。In another preferred embodiment, a conjugate is bound to the C- or N-terminus of the α chain and/or the β chain of the TCR.
在另一优选例中，与所述T细胞受体结合的偶联物为可检测标记物、治疗剂、PK修饰部分或任何这些物质的组合。优选地，所述治疗剂为抗-CD3抗体。In another preferred embodiment, the conjugate bound to the T cell receptor is a detectable label, a therapeutic agent, a PK modification moiety or a combination of any of these substances. Preferably, the therapeutic agent is an anti-CD3 antibody.
本发明的第二方面，提供了一种多价TCR复合物，其包含至少两个TCR分子，并且其中的至少一个TCR分子为本发明第一方面所述的TCR。The second aspect of the present invention provides a multivalent TCR complex comprising at least two TCR molecules, and at least one of the TCR molecules is the TCR described in the first aspect of the present invention.
本发明的第三方面，提供了一种核酸分子，所述核酸分子包含编码本发明第一方面所述的TCR分子的核酸序列或其互补序列。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 a complementary sequence thereof.
在另一优选例中，所述核酸分子包含编码TCRα链可变域的核苷酸序列SEQ ID NO:2或SEQ ID NO:33。In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 2 or SEQ ID NO: 33 encoding the variable domain of the TCRα chain.
在另一优选例中，所述的核酸分子包含编码TCRβ链可变域的核苷酸序列SEQ ID NO:6或SEQ ID NO:35。In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 6 or SEQ ID NO: 35 encoding the variable domain of the TCR beta chain.
在另一优选例中，所述核酸分子包含编码TCRα链的核苷酸序列SEQ ID NO:4和/或包含编码TCRβ链的核苷酸序列SEQ ID NO:8。In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO:4 encoding the TCRα chain and/or the nucleotide sequence SEQ ID NO:8 encoding the TCRβ chain.
本发明的第四方面，提供了一种载体，所述的载体含有本发明第三方面所述的核酸分子；优选地，所述的载体为病毒载体；更优选地，所述的载体为慢病毒载体。The fourth aspect of the present invention provides a vector, which contains the nucleic acid molecule described in the third aspect of the present invention; preferably, the vector is a viral vector; more preferably, the vector is a viral vector viral vector.
本发明的第五方面，提供了一种分离的宿主细胞，所述的宿主细胞中含有本发明第四方面所述的载体或基因组中整合有外源的本发明第三方面所述的核酸分子。The fifth aspect of the present invention provides an isolated host cell, wherein the host cell contains the vector described in the fourth aspect of the present invention or the exogenous nucleic acid molecule described in the third aspect of the present invention is integrated into the genome .
本发明的第六方面，提供了一种细胞，所述细胞转导有本发明第三方面所述的核酸分子或本发明第四方面所述的载体；优选地，所述细胞为T细胞、NK细胞、NKT细胞或干细胞。The sixth aspect of the present invention provides a cell transduced with the nucleic acid molecule described in the third aspect of the present invention or the vector described in the fourth aspect of the present invention; preferably, the cell is a T cell, NK cells, NKT cells or stem cells.
本发明的第七方面，提供了一种药物组合物，所述组合物含有药学上可接受的载体以及本发明第一方面所述的TCR、本发明第二方面所述的TCR复合物、本发明第三方面所述的核酸分子、本发明第四方面所述的载体、或本发明第六方面所述的细胞。A seventh aspect of the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, the TCR described in the first aspect of the present invention, the TCR complex described in the second aspect of the present invention, the present The nucleic acid molecule of the third aspect of the present invention, the vector of the fourth aspect of the present invention, or the cell of the sixth aspect of the present invention.
本发明的第八方面，提供了本发明第一方面所述的T细胞受体、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，用于制备治疗肿瘤或自身免疫疾病的药物，优选地，所述肿瘤为肝癌。The eighth aspect of the present invention provides the use of the T cell receptor described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, or the cell described in the sixth aspect of the present invention, for use in To prepare a drug for treating tumor or autoimmune disease, preferably, the tumor is liver cancer.
本发明的第九方面，提供了本发明第一方面所述的T细胞受体、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用作治疗肿瘤或自身免疫疾病的药物；优选地，所述肿瘤为肝癌。The ninth aspect of the present invention provides the T cell receptor described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, or the cell described in the sixth aspect of the present invention for use in the treatment of tumors or Drugs for autoimmune diseases; preferably, the tumor is liver cancer.
本发明的第十方面，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T细胞受体、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，所述的疾病为肿瘤，更优选地，所述肿瘤为肝癌。The tenth aspect of the present invention provides a method for treating a disease, comprising administering an appropriate amount of the T cell receptor described in the first aspect of the present invention or the TCR complex described in the second aspect of the present invention to a subject in need of treatment , or the cell according to the sixth aspect of the present invention, or the pharmaceutical composition according to the seventh aspect of the present invention; preferably, the disease is tumor, more preferably, the tumor is liver cancer.
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。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.
图1a、图1b、图1c、图1d、图1e和图1f分别为TCRα链可变域氨基酸序列、TCRα链可变域核苷酸序列、TCRα链氨基酸序列、TCRα链核苷酸序列、具有前导序列的TCRα链氨基酸序列以及具有前导序列的TCRα链核苷酸序列。Fig. 1a, Fig. 1b, Fig. 1c, Fig. 1d, Fig. 1e and Fig. 1f are respectively the amino acid sequence of TCRα chain variable domain, the nucleotide sequence of TCRα chain variable domain, the amino acid sequence of TCRα chain, the nucleotide sequence of TCRα chain, with The amino acid sequence of the TCRα chain of the leader sequence and the nucleotide sequence of the TCRα chain with the leader sequence.
图2a、图2b、图2c、图2d、图2e和图2f分别为TCRβ链可变域氨基酸序列、TCRβ链可变域核苷酸序列、TCRβ链氨基酸序列、TCRβ链核苷酸序列、具有前导序列的TCRβ链氨基酸序列以及具有前导序列的TCRβ链核苷酸序列。Figure 2a, Figure 2b, Figure 2c, Figure 2d, Figure 2e and Figure 2f are the amino acid sequence of the variable domain of the TCRβ chain, the nucleotide sequence of the variable domain of the TCRβ chain, the amino acid sequence of the TCRβ chain, and the nucleotide sequence of the TCRβ chain, respectively. The amino acid sequence of the TCR beta chain of the leader sequence and the nucleotide sequence of the TCR beta chain with the leader sequence.
图3为单克隆细胞的CD8+及四聚体-PE双阳性染色结果。Figure 3 shows the results of CD8+ and tetramer-PE double-positive staining of monoclonal cells.
图4a和图4b分别为可溶性TCRα链的氨基酸序列和核苷酸序列。Figure 4a and Figure 4b show the amino acid sequence and nucleotide sequence of the soluble TCRα chain, respectively.
图5a和图5b分别为可溶性TCRβ链的氨基酸序列和核苷酸序列。Figure 5a and Figure 5b are the amino acid sequence and nucleotide sequence of the soluble TCR beta chain, respectively.
图6a和图6b为纯化后得到的可溶性TCR的胶图。其中，图6a和图6b的右侧泳道分别为还原胶和非还原胶，左侧泳道都为分子量标记(marker)。Figure 6a and Figure 6b are gel images of soluble TCR obtained after purification. Among them, the right lanes of Figure 6a and Figure 6b are reducing gel and non-reducing gel, respectively, and the left lanes are molecular weight markers.
图7a和图7b分别为单链TCR的氨基酸序列和核苷酸序列，其连接序列(linker)的氨基酸序列和核苷酸序列用下划线标出。Figure 7a and Figure 7b are the amino acid sequence and nucleotide sequence of the single-chain TCR, respectively, and the amino acid sequence and nucleotide sequence of the linker are underlined.
图8a和图8b分别为单链TCRα链可变域的氨基酸序列和核苷酸序列。Figures 8a and 8b are the amino acid sequence and nucleotide sequence of the variable domain of the single-chain TCR alpha chain, respectively.
图9a和图9b分别为单链TCRβ链可变域的氨基酸序列和核苷酸序列。Figures 9a and 9b are the amino acid sequence and nucleotide sequence of the variable domain of the single-chain TCR beta chain, respectively.
图10为纯化后得到的可溶性单链TCR的胶图。最左侧泳道为非还原胶，中间泳道为分子量标记(marker)，最右侧泳道为还原胶。Figure 10 is a gel image of the soluble single-chain TCR obtained after purification. The leftmost lane is the non-reducing gel, the middle lane is the molecular weight marker, and the rightmost lane is the reducing gel.
图11为本发明可溶性TCR与KWVESIFLIF-HLA A2402复合物结合的BIAcore动力学图谱。Figure 11 is a BIAcore kinetic map of the binding of soluble TCR of the present invention to KWVESIFLIF-HLA A2402 complex.
图12为本发明可溶性单链TCR与KWVESIFLIF-HLA A2402复合物结合的BIAcore动力学图谱。Figure 12 is a BIAcore kinetic map of the binding of the soluble single-chain TCR of the present invention to the KWVESIFLIF-HLA A2402 complex.
图13为得到的T细胞克隆的ELISPOT激活功能验证结果。Fig. 13 shows the results of verification of the ELISPOT activation function of the obtained T cell clones.
图14为转导本发明的TCR的效应细胞的ELISPOT激活功能验证结果。FIG. 14 shows the results of verification of the ELISPOT activation function of effector cells transduced with the TCR of the present invention.
图15为转导本发明的TCR的效应细胞的LDH杀伤功能验证结果。Fig. 15 shows the results of LDH killing function verification of effector cells transduced with the TCR of the present invention.
本发明人经过广泛而深入的研究，找到了与AFP抗原短肽KWVESIFLIF(SEQ ID NO:9)能够特异性结合的TCR，所述抗原短肽KWVESIFLIF可与HLA A2402形成复合物并一起被呈递到细胞表面。本发明还提供了编码所述TCR的核酸分子以及包含所述核酸分子的载体。另外，本发明还提供了转导本发明TCR的细胞。After extensive and in-depth research, the inventors found a TCR that can specifically bind to the AFP antigenic short peptide KWVESIFLIF (SEQ ID NO: 9), which can form a complex with HLA A2402 and be presented to the cell surface. The present invention also provides a nucleic acid molecule encoding the TCR and a vector comprising the nucleic acid molecule. In addition, the present invention also provides cells transduced with the TCR of the present invention.
术语the term
MHC分子是免疫球蛋白超家族的蛋白质，可以是Ⅰ类或Ⅱ类MHC分子。因此，其对于抗原的呈递具有特异性，不同的个体有不同的MHC，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。MHC molecules are proteins of the immunoglobulin superfamily and can be class I or class II MHC molecules. Therefore, it is specific for antigen presentation, and different individuals have different MHCs, which can present different short peptides in a protein antigen to their respective APC cell surfaces. The human MHC is often referred to as the HLA gene or HLA complex.
T细胞受体(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。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。The T cell receptor (TCR) is the only receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC). In the immune system, direct physical contact between T cells and antigen-presenting cells (APCs) is triggered by the binding of antigen-specific TCRs to pMHC complexes, and then other cell membrane surface molecules of both T cells and APCs interact. It causes a series of subsequent cell signaling and other physiological responses, so that T cells with different antigen specificities exert immune effects on their target cells.
TCR是由α链/β链或者γ链/δ链以异质二聚体形式存在的细胞膜表面的糖蛋白。在95％的T细胞中TCR异质二聚体由α和β链组成，而5％的T细胞具有由γ和δ链组成的TCR。天然αβ异质二聚TCR具有α链和β链，α链和β链构成αβ异源二聚TCR的亚单位。广义上讲，α和β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。各可变区包含嵌合在框架结构(framework regions)中的3个CDR(互补决定区)，CDR1、CDR2和CDR3。CDR区决定了TCR与pMHC复合物的结合，其中CDR3由可变区和连接区重组而成，被称为超变区。TCR的α和β链一般看作各有两个“结构域”即可变域和恒定域，可变域由连接的可变区和连接区构成。TCR恒定域的序列可以在国际免疫遗传学信息系统(IMGT)的公开数据库中找到，如TCR分子α链的恒定域序列为“TRAC*01”，TCR分子β链的恒定域序列为“TRBC1*01”或“TRBC2*01”。此外，TCR的α和β链还包含跨膜区和胞质区，胞质区很短。TCR is a glycoprotein on the surface of the cell membrane that exists in the form of heterodimers of α chain/β chain or γ chain/δ chain. TCR heterodimers consist of alpha and beta chains in 95% of T cells, whereas 5% of T cells have TCRs composed of gamma and delta chains. A native αβ heterodimeric TCR has an α chain and a β chain, and the α chain and the β chain constitute the subunits of the αβ heterodimeric TCR. Broadly speaking, the alpha and beta chains each contain a variable region, a linker region, and a constant region, and the beta chain usually also contains a short variable region between the variable region and the linker region, but the variable region is often regarded as the linker region a part of. Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, chimerically incorporated in framework regions. The CDR region determines the binding of TCR to the pMHC complex, and CDR3 is recombined from the variable region and the linker region, which is called the hypervariable region. The alpha and beta chains of a TCR are generally viewed as having two "domains" each, a variable domain and a constant domain, the variable domains being composed of linked variable and linking regions. The sequences of the TCR constant domains can be found in the public database of the International Immunogenetics Information System (IMGT). 01" or "TRBC2*01". In addition, the α and β chains of TCR also contain a transmembrane region and a cytoplasmic region, and the cytoplasmic region is very short.
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。In the present invention, the terms "polypeptide of the present invention", "TCR of the present invention", "T cell receptor of the present invention" are used interchangeably.
天然链间二硫键与人工链间二硫键Natural interchain disulfide bonds and artificial interchain disulfide bonds
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”。There is a set of disulfide bonds between the Cα and Cβ chains in the near-membrane region of the native TCR, which are referred to as "native interchain disulfide bonds" in the present invention. In the present invention, the artificially introduced interchain covalent disulfide bond whose position is different from that of the natural interchain disulfide bond is referred to as "artificial interchain disulfide bond".
为方便描述二硫键的位置，本发明中TRAC*01与TRBC1*01或TRBC2*01氨基酸序列的位置编号按从N端到C端依次的顺序进行位置编号，如TRBC1*01或TRBC2*01中，按从N端到C端依次的顺序第60个氨基酸为P(脯氨酸)，则本发明中可将其描述为TRBC1*01或TRBC2*01外显子1的Pro60，也可将其表述为TRBC1*01或TRBC2*01外显子1的第60位氨基酸，又如TRBC1*01或TRBC2*01中，按从N端到C端依次的顺序第61个氨基酸为Q(谷氨酰胺)，则本发明中可将其描述为TRBC1*01或TRBC2*01外显子1的Gln61，也可将其表述为TRBC1*01或TRBC2*01外显子1的第61位氨基酸，其他以此类推。本发明中，可变区TRAV与TRBV的氨基酸序列的位置编号，按照IMGT中列出的位置编号。如TRAV中的某个氨基酸，IMGT中列出的位置编号为46，则本发明中将其描述为TRAV第46位氨基酸，其他以此类推。本发明中，其他氨基酸的序列位置编号有特殊说明的，则按特殊说明。For the convenience of describing the position of the disulfide bond, the position numbers of the amino acid sequences of TRAC*01 and TRBC1*01 or TRBC2*01 in the present invention are numbered sequentially from the N-terminus to the C-terminus, such as TRBC1*01 or TRBC2*01 , the 60th amino acid in the sequence from the N-terminus to the C-terminus is P (proline), then in the present invention, it can be described as Pro60 of exon 1 of TRBC1*01 or TRBC2*01, or it can be described as Pro60 of exon 1 of TRBC1*01 It is expressed as the 60th amino acid of exon 1 of TRBC1*01 or TRBC2*01, and in TRBC1*01 or TRBC2*01, the 61st amino acid in the sequence from the N-terminus to the C-terminus is Q (glutamate). amide), in the present invention, it can be described as Gln61 of TRBC1*01 or TRBC2*01 exon 1, or it can be described as the 61st amino acid of TRBC1*01 or TRBC2*01 exon 1, other And so on. In the present invention, the position numbers of the amino acid sequences of the variable regions TRAV and TRBV are numbered according to the position numbers listed in IMGT. For example, for a certain amino acid in TRAV, the position number listed in IMGT is 46, then it is described as the 46th amino acid of TRAV in the present invention, and so on. In the present invention, if the sequence position numbering of other amino acids has special instructions, the special instructions are followed.
发明详述Detailed description of the invention
TCR分子TCR molecule
在抗原加工过程中，抗原在细胞内被降解，然后通过MHC分子携带至细胞表面。T细胞受体能够识别抗原呈递细胞表面的肽-MHC复合物。因此，本发明的第一方面提供了一种能够结合KWVESIFLIF-HLA A2402复合物的TCR分子。优选地，所述TCR分子是分离的或纯化的。该TCR的α和β链各具有3个互补决定区(CDR)。During antigen processing, the antigen is degraded within the cell and then carried to the cell surface by MHC molecules. T cell receptors recognize peptide-MHC complexes on the surface of antigen-presenting cells. Accordingly, a first aspect of the present invention provides a TCR molecule capable of binding the KWVESIFLIF-HLA A2402 complex. Preferably, the TCR molecule is isolated or purified. The alpha and beta chains of this TCR each have three complementarity determining regions (CDRs).
在本发明的一个优选地实施方式中，所述TCR的α链包含具有以下氨基酸序列的CDR：In a preferred embodiment of the present invention, the alpha chain of the TCR comprises a CDR having the following amino acid sequence:
αCDR1-VGISA(SEQ ID NO:10)αCDR1-VGISA (SEQ ID NO: 10)
αCDR2-LSSGK(SEQ ID NO:11)αCDR2-LSSGK (SEQ ID NO: 11)
αCDR3-AVRDAGGTSYGKLT(SEQ ID NO:12)；和/或αCDR3-AVRDAGGTSYGKLT (SEQ ID NO: 12); and/or
所述TCRβ链可变域的3个互补决定区为：The three complementarity determining regions of the TCRβ chain variable domain are:
βCDR1-MNHEY(SEQ ID NO:13)βCDR1-MNHEY (SEQ ID NO: 13)
βCDR2-SVGAGI(SEQ ID NO:14)βCDR2-SVGAGI (SEQ ID NO: 14)
βCDR3-ASSYPGSYGYT(SEQ ID NO:15)。βCDR3-ASSYPGSYGYT (SEQ ID NO: 15).
可以将上述本发明的CDR区氨基酸序列嵌入到任何适合的框架结构中来制备嵌合TCR。只要框架结构与本发明的TCR的CDR区兼容，本领域技术人员根据本发明公开的CDR区就能够设计或合成出具有相应功能的TCR分子。因此，本发明TCR分子是指包含上述α和/或β链CDR区序列及任何适合的框架结构的TCR分子。本发明TCRα链可变域为与SEQ ID NO:1具有至少90％，优选地95％，更优选地98％序列相同性的氨基酸序列；和/或本发明TCRβ链可变域为与SEQ ID NO:5具有至少90％，优选地95％，更优选地98％序列相同性的氨基酸序列。Chimeric TCRs can be prepared by inserting the above-described amino acid sequences of the CDR regions of the present invention into any suitable framework structure. As long as the framework structure is compatible with the CDR regions of the TCR of the present invention, those skilled in the art can design or synthesize TCR molecules with corresponding functions based on the CDR regions disclosed in the present invention. Accordingly, the TCR molecule of the present invention refers to a TCR molecule comprising the above-mentioned alpha and/or beta chain CDR region sequences and any suitable framework structure. The TCRα chain variable domain of the present invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity with SEQ ID NO: 1; and/or the TCRβ chain variable domain of the present invention is an amino acid sequence with SEQ ID NO: 1 NO:5 has an amino acid sequence of at least 90%, preferably 95%, more preferably 98% sequence identity.
在本发明的一个优选例中，本发明的TCR分子是由α与β链构成的异质二聚体。具体地， 一方面所述异质二聚TCR分子的α链包含可变域和恒定域，所述α链可变域氨基酸序列包含上述α链的CDR1(SEQ ID NO:10)、CDR2(SEQ ID NO:11)和CDR3(SEQ ID NO:12)。优选地，所述TCR分子包含α链可变域氨基酸序列SEQ ID NO:1。更优选地，所述TCR分子的α链可变域氨基酸序列为SEQ ID NO:1。另一方面，所述异质二聚TCR分子的β链包含可变域和恒定域，所述β链可变域氨基酸序列包含上述β链的CDR1(SEQ ID NO:13)、CDR2(SEQ ID NO:14)和CDR3(SEQ ID NO:15)。优选地，所述TCR分子包含β链可变域氨基酸序列SEQ ID NO:5。更优选地，所述TCR分子的β链可变域氨基酸序列为SEQ ID NO:5。In a preferred embodiment of the present invention, the TCR molecule of the present invention is a heterodimer composed of α and β chains. Specifically, in one aspect, the α chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the amino acid sequence of the α chain variable domain comprises CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 10) and CDR2 (SEQ ID NO: 10) of the above-mentioned α chain. ID NO: 11) and CDR3 (SEQ ID NO: 12). Preferably, the TCR molecule comprises the alpha chain variable domain amino acid sequence of SEQ ID NO: 1. More preferably, the α chain variable domain amino acid sequence of the TCR molecule is SEQ ID NO: 1. On the other hand, the β chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the amino acid sequence of the β chain variable domain comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 13) of the above-mentioned β chain NO: 14) and CDR3 (SEQ ID NO: 15). Preferably, the TCR molecule comprises the beta chain variable domain amino acid sequence of SEQ ID NO:5. More preferably, the β chain variable domain amino acid sequence of the TCR molecule is SEQ ID NO:5.
在本发明的一个优选例中，本发明的TCR分子是由α链的部分或全部和/或β链的部分或全部组成的单链TCR分子。有关单链TCR分子的描述可以参考文献Chung et al(1994)Proc.Natl.Acad.Sci.USA 91,12654-12658。根据文献中所述，本领域技术人员能够容易地构建包含本发明CDRs区的单链TCR分子。具体地，所述单链TCR分子包含Vα、Vβ和Cβ，优选地按照从N端到C端的顺序连接。In a preferred embodiment of the present invention, the TCR molecule of the present invention is a single-chain TCR molecule composed of part or all of the α chain and/or part or all of the β chain. A description of single-chain TCR molecules can be found in Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-12658. Those skilled in the art can readily construct single-chain TCR molecules comprising the CDRs regions of the present invention as described in the literature. Specifically, the single-chain TCR molecule comprises Vα, Vβ and Cβ, preferably linked in order from the N-terminus to the C-terminus.
所述单链TCR分子的α链可变域氨基酸序列包含上述α链的CDR1(SEQ ID NO:10)、CDR2(SEQ ID NO:11)和CDR3(SEQ ID NO:12)。优选地，所述单链TCR分子包含α链可变域氨基酸序列SEQ ID NO:1。更优选地，所述单链TCR分子的α链可变域氨基酸序列为SEQ ID NO:1。所述单链TCR分子的β链可变域氨基酸序列包含上述β链的CDR1(SEQ ID NO:13)、CDR2(SEQ ID NO:14)和CDR3(SEQ ID NO:15)。优选地，所述单链TCR分子包含β链可变域氨基酸序列SEQ ID NO:5。更优选地，所述单链TCR分子的β链可变域氨基酸序列为SEQ ID NO:5。The α chain variable domain amino acid sequence of the single-chain TCR molecule comprises CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the above-mentioned α chain. Preferably, the single-chain TCR molecule comprises the alpha chain variable domain amino acid sequence of SEQ ID NO: 1. More preferably, the α-chain variable domain amino acid sequence of the single-chain TCR molecule is SEQ ID NO: 1. The beta chain variable domain amino acid sequence of the single-chain TCR molecule comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 14) and CDR3 (SEQ ID NO: 15) of the above beta chain. Preferably, the single-chain TCR molecule comprises the beta chain variable domain amino acid sequence of SEQ ID NO:5. More preferably, the β-chain variable domain amino acid sequence of the single-chain TCR molecule is SEQ ID NO:5.
在本发明的一个优选例中，本发明的TCR分子的恒定域是人的恒定域。本领域技术人员知晓或可以通过查阅相关书籍或IMGT(国际免疫遗传学信息系统)的公开数据库来获得人的恒定域氨基酸序列。例如，本发明TCR分子α链的恒定域序列可以为“TRAC*01”，TCR分子β链的恒定域序列可以为“TRBC1*01”或“TRBC2*01”。IMGT的TRAC*01中给出的氨基酸序列的第53位为Arg，在此表示为：TRAC*01外显子1的Arg53，其他以此类推。优选地，本发明TCR分子α链的氨基酸序列为SEQ ID NO:3，和/或β链的氨基酸序列为SEQ ID NO:7。In a preferred embodiment of the present invention, the constant domain of the TCR molecule of the present invention is a human constant domain. Those skilled in the art know or can obtain the human constant domain amino acid sequence by consulting relevant books or the public database of IMGT (International Immunogenetics Information System). For example, the constant domain sequence of the alpha chain of the TCR molecule of the present invention can be "TRAC*01", and the constant domain sequence of the beta chain of the TCR molecule can be "TRBC1*01" or "TRBC2*01". The 53rd position of the amino acid sequence given in TRAC*01 of IMGT is Arg, which is represented here as: Arg53 of exon 1 of TRAC*01, and so on. Preferably, the amino acid sequence of the α chain of the TCR molecule of the present invention is SEQ ID NO:3, and/or the amino acid sequence of the β chain is SEQ ID NO:7.
天然存在的TCR是一种膜蛋白，通过其跨膜区得以稳定。如同免疫球蛋白(抗体)作为抗原识别分子一样，TCR也可以被开发应用于诊断和治疗，这时需要获得可溶性的TCR分子。可溶性的TCR分子不包括其跨膜区。可溶性TCR有很广泛的用途，它不仅可用于研究TCR与pMHC的相互作用，也可用作检测感染的诊断工具或作为自身免疫病的标志物。类似地，可溶性TCR可以被用来将治疗剂(如细胞毒素化合物或免疫刺激性化合物)输送到呈递特异性抗原的细胞，另外，可溶性TCR还可与其他分子(如，抗-CD3抗体)结合来重新定向T细胞，从而使其靶向呈递特定抗原的细胞。本发明也获得了对AFP抗原短肽具有特异性的可溶性TCR。The naturally occurring TCR is a membrane protein that is stabilized by its transmembrane region. Like immunoglobulins (antibodies) as antigen recognition molecules, TCRs can also be developed for diagnostic and therapeutic applications, where soluble TCR molecules need to be obtained. Soluble TCR molecules do not include their transmembrane domains. Soluble TCR has a wide range of uses, not only to study the interaction of TCR with pMHC, but also as a diagnostic tool to detect infection or as a marker for autoimmune diseases. Similarly, soluble TCRs can be used to deliver therapeutic agents (eg, cytotoxic or immunostimulatory compounds) to cells presenting specific antigens, and in addition, soluble TCRs can bind to other molecules (eg, anti-CD3 antibodies) to redirect T cells so that they target cells presenting specific antigens. The present invention also obtains a soluble TCR specific for AFP antigenic short peptide.
为获得可溶性TCR，一方面，本发明TCR可以是在其α和β链恒定域的残基之间引入人工二硫键的TCR。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 的半胱氨酸残基来形成二硫键。引入半胱氨酸残基以形成二硫键的其他位点还可以是：TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；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50个、或最多30个、或最多15个、或最多10个、或最多8个或更少的氨基酸，以使其不包括半胱氨酸残基来达到缺失天然二硫键的目的，也可通过将形成天然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。To obtain soluble TCRs, in one aspect, the TCRs of the invention may be TCRs in which artificial disulfide bonds are introduced between residues of their alpha and beta chain constant domains. Cysteine residues form artificial interchain disulfide bonds between the constant domains of the alpha and beta chains of the TCR. Cysteine residues can be substituted for other amino acid residues at appropriate sites in the native TCR to form artificial interchain disulfide bonds. For example, substitution of Thr48 of exon 1 of TRAC*01 and substitution of cysteine residues of Ser57 of exon 1 of TRBC1*01 or TRBC2*01 to form a disulfide bond. Other sites where cysteine residues are introduced to form disulfide bonds can also be: Thr45 in exon 1 of TRAC*01 and Ser77 in exon 1 of TRBC1*01 or TRBC2*01; exon 1 of TRAC*01 1 Tyr10 and TRBC1*01 or Ser17 of TRBC2*01 exon 1; Thr45 of TRAC*01 exon 1 and Asp59 of TRBC1*01 or TRBC2*01 exon 1; TRAC*01 exon 1 Ser15 and Glu15 in exon 1 of TRBC1*01 or TRBC2*01; Arg53 in exon 1 of TRAC*01 and Ser54 in exon 1 of TRBC1*01 or TRBC2*01; Pro89 and exon 1 of TRAC*01 Ala19 of exon 1 of TRBC1*01 or TRBC2*01; or Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01. That is, cysteine residues are substituted for any set of sites in the constant domains of the alpha and beta chains described above. One or more C-termini of the TCR constant domains of the invention may be truncated by up to 50, or up to 30, or up to 15, or up to 10, or up to 8 or fewer amino acids so that they do not include A cysteine residue can be used to achieve the purpose of deleting the natural disulfide bond, and the above purpose can also be achieved by mutating the cysteine residue that forms the natural disulfide bond into another amino acid.
如上所述，本发明的TCR可以包含在其α和β链恒定域的残基间引入的人工二硫键。应注意，恒定域间含或不含上文所述的引入的人工二硫键，本发明的TCR均可含有TRAC恒定域序列和TRBC1或TRBC2恒定域序列。TCR的TRAC恒定域序列和TRBC1或TRBC2恒定域序列可通过存在于TCR中的天然二硫键连接。As mentioned above, the TCRs of the present invention may contain artificial disulfide bonds introduced between residues of their alpha and beta chain constant domains. It should be noted that the TCRs of the invention may contain both a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, with or without the artificial disulfide bonds introduced above between the constant domains. The TRAC constant domain sequence of the TCR and the TRBC1 or TRBC2 constant domain sequence may be linked by natural disulfide bonds present in the TCR.
为获得可溶性TCR，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可溶性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。In order to obtain a soluble TCR, on the other hand, the TCR of the present invention also includes a TCR with mutation in its hydrophobic core region, and the mutation of these hydrophobic core regions is preferably a mutation that can improve the stability of the soluble TCR of the present invention, such as in Publication No. Described in the patent document of WO2014/206304. Such a TCR may be mutated at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable domain amino acids 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or Or the alpha chain J gene (TRAJ) short peptide amino acid position 3, 5, 7 from the bottom, and/or the beta chain J gene (TRBJ) short peptide amino acid position 2, 4, 6 from the bottom, where the position number of the amino acid sequence Numbered by position as listed in the International Information System on Immunogenetics (IMGT). Those skilled in the art are aware of the above-mentioned International Immunogenetics Information System, and can obtain the position numbers of amino acid residues of different TCRs in IMGT according to the database.
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稳定性可溶单链TCR。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。如在本发明实施例4中构建的单链可溶性TCR，其α链可变域氨基酸序列为SEQ ID NO:32，编码的核苷酸序列为SEQ ID NO:33；β链可变域氨基酸序列为SEQ ID NO:34，编码的核苷酸序列为SEQ ID NO:35。In the present invention, the mutated TCR in the hydrophobic core region can be a stable soluble single-chain TCR composed of a flexible peptide chain linking the variable domains of the α and β chains of the TCR. It should be noted that the flexible peptide chain in the present invention can be any peptide chain suitable for linking the variable domains of TCRα and β chain. For the single-chain soluble TCR constructed in Example 4 of the present invention, its α chain variable domain amino acid sequence is SEQ ID NO: 32, and the encoded nucleotide sequence is SEQ ID NO: 33; the β chain variable domain amino acid sequence is SEQ ID NO: 33; is SEQ ID NO:34, and the encoded nucleotide sequence is SEQ ID NO:35.
另外，对于稳定性而言，专利文献201680003540.2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。In addition, in terms of stability, Patent Document 201680003540.2 also discloses that the introduction of artificial interchain disulfide bonds between the α chain variable region and the β chain constant region of TCR can significantly improve the stability of TCR. Therefore, the TCR of the present invention may further contain an artificial interchain disulfide bond between the α chain variable region and the β chain constant region. Specifically, the cysteine residue that forms an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR is substituted for: amino acid 46 of TRAV and TRBC1*01 or TRBC2* 01 amino acid 60 of exon 1; TRAV amino acid 47 and TRBC1*01 or TRBC2*01 exon 1 amino acid 61; TRAV 46 amino acid and TRBC1*01 or TRBC2*01 exon Amino acid 61 of exon 1; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01. Preferably, such a TCR may comprise (i) all or part of the TCR alpha chain excluding its transmembrane domain, and (ii) all or part of the TCR beta chain excluding its transmembrane domain, wherein (i) and (ii) ) both contain the variable domain and at least a part of the constant domain of the TCR chain, and the α chain and the β chain form a heterodimer. More preferably, such a TCR may contain an alpha chain variable domain and a beta chain variable domain and all or part of the beta chain constant domain except the transmembrane domain, but it does not contain the alpha chain constant domain, the alpha chain of the TCR. The chain variable domains form heterodimers with beta chains.
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。The TCRs of the present invention may also be provided in the form of multivalent complexes. The multivalent TCR complexes of the present invention comprise two, three, four or more multimers formed by combining the TCRs of the present invention, for example, the tetramerization domain of p53 can be used to generate tetramers, or multiple A complex formed by combining a TCR of the present invention with another molecule. The TCR complexes of the present invention can be used to track or target cells presenting specific antigens in vitro or in vivo, as well as to generate intermediates for other multivalent TCR complexes with such applications.
本发明的TCR可以单独使用，也可与偶联物以共价或其他方式结合，优选以共价方式结合。所述偶联物包括可检测标记物(为诊断目的，其中所述TCR用于检测呈递KWVESIFLIF-HLA A2402复合物的细胞的存在)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。The TCR of the present invention can be used alone, or can be combined with the conjugate in a covalent or other manner, preferably in a covalent manner. The conjugate includes a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the KWVESIFLIF-HLA A2402 complex), a therapeutic agent, a PK (protein kinase) modification moiety, or any of the above combination or conjugation.
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。Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing detectable products enzyme.
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。Therapeutic agents that can be bound or conjugated to the TCR of the present invention include, but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539); 2. Biotoxicity (Chaudhary et al., 1989) , Nature (Nature) 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy (Cancer Immunology and Immunotherapy) 51, 565); 3. Cytokines such as IL-2, etc. (Gillies et al., 1992, National Academy of Sciences Journal (PNAS) 89, 1428; Card et al, 2004, Cancer Immunology and Immunotherapy 53, 345; Halin et al, 2003, Cancer Research 63, 3202); 4. Antibody Fc Fragments (Mosquera et al., 2005, The Journal of Immunology 174, 4381); 5. Antibody scFv fragments (Zhu et al., 1995, International Journal of Cancer 62, 319); 6. Gold nanoparticles/nanoparticles Rod (Lapotko et al, 2005, Cancer letters 239, 36; Huang et al, 2006, Journal of the American Chemical Society 128, 2115); 7. Viral particles (Peng et al, 2004, Gene Gene therapy 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research 65, 11631); 9. Nanomagnetic particles; 10. Prodrug-activating enzymes (eg, DT-myocardial flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. chemotherapeutic agents (eg, cisplatin) or nanoparticles in any form, etc.
另外，本发明的TCR还可以是包含衍生自超过一种物种序列的杂合TCR。例如，有研究显示鼠科TCR在人T细胞中比人TCR能够更有效地表达。因此，本发明TCR可包含人可变域和鼠的恒定域。这一方法的缺陷是可能引发免疫应答。因此，在其用于过继性T细胞治疗时应当有调节方案来进行免疫抑制，以允许表达鼠科的T细胞的植入。Additionally, the TCRs of the present invention may also be hybrid TCRs comprising sequences derived from more than one species. For example, studies have shown that murine TCRs are more efficiently expressed in human T cells than human TCRs. Thus, the TCRs of the present invention may comprise human variable domains and murine constant domains. The downside of this approach is the potential to elicit an immune response. Therefore, there should be a regulatory regime for immunosuppression when it is used in adoptive T cell therapy to allow engraftment of murine expressing T cells.
应理解，本文中氨基酸名称采用国际通用的单英文字母或三英文字母表示，氨基酸名称的单英文字母与三英文字母的对应关系如下：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)。It should be understood that the names of amino acids in this paper are represented by the international single English letter or three English letters, and the correspondence between the single English letter and the three English letters of the amino acid name is as follows: 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).
核酸分子nucleic acid molecule
本发明的第二方面提供了编码本发明第一方面TCR分子或其部分的核酸分子，所述部分可以是一个或多个CDR，α和/或β链的可变域，以及α链和/或β链。A second aspect of the invention provides a nucleic acid molecule encoding a TCR molecule of the first aspect of the invention, or a portion thereof, which portion may be one or more CDRs, variable domains of alpha and/or beta chains, and alpha chains and/or or beta chains.
编码本发明第一方面TCR分子α链CDR区的核苷酸序列如下：The nucleotide sequence encoding the α chain CDR region of the TCR molecule according to the first aspect of the present invention is as follows:
CDR1α-gtaggaataagtgcc(SEQ ID NO:16)CDR1α-gtaggaataagtgcc (SEQ ID NO: 16)
CDR2α-ctgagctcagggaag(SEQ ID NO:17)CDR2α-ctgagctcagggaag (SEQ ID NO: 17)
CDR3α-gctgtcagggatgctggtggtactagctatggaaagctgaca(SEQ ID NO:18)CDR3α-gctgtcagggatgctggtggtactagctatggaaagctgaca (SEQ ID NO: 18)
编码本发明第一方面TCR分子β链CDR区的核苷酸序列如下：The nucleotide sequence encoding the beta chain CDR region of the TCR molecule according to the first aspect of the present invention is as follows:
CDR1β-atgaaccatgaatac(SEQ ID NO:19)CDR1β-atgaaccatgaatac (SEQ ID NO: 19)
CDR2β-tcagttggtgctggtatc(SEQ ID NO:20)CDR2β-tcagttggtgctggtatc (SEQ ID NO:20)
CDR3β-gccagcagttacccaggttcttatggctacacc(SEQ ID NO:21)CDR3β-gccagcagttacccaggttcttatggctacacc (SEQ ID NO:21)
因此，编码本发明TCRα链的本发明核酸分子的核苷酸序列包括SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18，和/或编码本发明TCRβ链的本发明核酸分子的核苷酸序列包括SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21。Accordingly, the nucleotide sequences of the nucleic acid molecules of the invention encoding the TCR alpha chains of the invention include SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and/or the nucleotide sequences of the nucleic acid molecules of the invention encoding the TCR beta chains of the invention Nucleotide sequences include SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.
本发明核酸分子的核苷酸序列可以是单链或双链的，该核酸分子可以是RNA或DNA，并且可以包含或不包含内含子。优选地，本发明核酸分子的核苷酸序列不包含内含子但能够编码本发明多肽，例如编码本发明TCRα链可变域的本发明核酸分子的核苷酸序列包括SEQ ID NO:2和/或编码本发明TCRβ链可变域的本发明核酸分子的核苷酸序列包括SEQ ID NO:6。或者，编码本发明TCRα链可变域的本发明核酸分子的核苷酸序列包括SEQ ID NO:33和/或编码本发明TCRβ链可变域的本发明核酸分子的核苷酸序列包括SEQ ID NO:35。更优选地，本发明核酸分子的核苷酸序列包含SEQ ID NO:4和/或SEQ ID NO:8。或者，本发明核酸分子的核苷酸序列为SEQ ID NO:31。The nucleotide sequence of the nucleic acid molecule of the invention may be single-stranded or double-stranded, the nucleic acid molecule may be RNA or DNA, and may or may not contain introns. Preferably, the nucleotide sequence of the nucleic acid molecule of the present invention does not contain introns but is capable of encoding the polypeptide of the present invention, for example, the nucleotide sequence of the nucleic acid molecule of the present invention encoding the variable domain of the TCR alpha chain of the present invention includes SEQ ID NO: 2 and /or the nucleotide sequence of the nucleic acid molecule of the present invention encoding the variable domain of the TCR beta chain of the present invention comprises SEQ ID NO:6. Alternatively, the nucleotide sequence of the nucleic acid molecule of the present invention encoding the variable domain of the TCR alpha chain of the present invention includes SEQ ID NO: 33 and/or the nucleotide sequence of the nucleic acid molecule of the present invention encoding the variable domain of the TCR beta chain of the present invention includes SEQ ID NO: 33 NO: 35. More preferably, the nucleotide sequence of the nucleic acid molecule of the invention comprises SEQ ID NO:4 and/or SEQ ID NO:8. Alternatively, the nucleotide sequence of the nucleic acid molecule of the present invention is SEQ ID NO:31.
应理解，由于遗传密码的简并，不同的核苷酸序列可以编码相同的多肽。因此，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。以本发明中的其中一个例子来说明，“简并的变异体”是指编码具有SEQ ID NO:1的蛋白序列，但与SEQ ID NO:2的序列有差别的核酸序列。It will be appreciated that due to the degeneracy of the genetic code, different nucleotide sequences can encode the same polypeptide. Accordingly, the nucleic acid sequences encoding the TCRs of the present invention may be identical or degenerate variants of the nucleic acid sequences shown in the figures of the present invention. To illustrate with one of the examples in the present invention, "degenerate variant" refers to a nucleic acid sequence that encodes the protein sequence of SEQ ID NO: 1, but differs from the sequence of SEQ ID NO: 2.
核苷酸序列可以是经密码子优化的。不同的细胞在具体密码子的利用上是不同的，可以根据细胞的类型，改变序列中的密码子来增加表达量。哺乳动物细胞以及多种其他生物的密码子选择表是本领域技术人员公知的。The nucleotide sequence may be codon-optimized. Different cells differ in the use of specific codons. Depending on the type of cell, the codons in the sequence can be changed to increase the amount of expression. Codon usage tables for mammalian cells, as well as various other organisms, are well known to those skilled in the art.
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。DNA可以是编码链或非编码链。The full-length sequence of the nucleic acid molecule of the present invention or a fragment thereof can generally be obtained by, but not limited to, PCR amplification method, recombinant method or artificial synthesis method. At present, the DNA sequences encoding the TCRs of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. DNA can be the coding or non-coding strand.
载体carrier
本发明还涉及包含本发明的核酸分子的载体，包括表达载体，即能够在体内或体外表达的构建体。常用的载体包括细菌质粒、噬菌体和动植物病毒。The present invention also relates to vectors comprising the nucleic acid molecules of the present invention, including expression vectors, ie constructs capable of in vivo or in vitro expression. Commonly used vectors include bacterial plasmids, bacteriophages, and animal and plant viruses.
病毒递送系统包括但不限于腺病毒载体、腺相关病毒(AAV)载体、疱疹病毒载体、逆转录病毒载体、慢病毒载体、杆状病毒载体。Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors, retroviral vectors, lentiviral vectors, baculovirus vectors.
优选地，载体可以将本发明的核苷酸转移至细胞中，例如T细胞中，使得该细胞表达AFP抗原特异性的TCR。理想的情况下，该载体应当能够在T细胞中持续高水平地表达。Preferably, the vector can transfer the nucleotides of the invention into cells, such as T cells, such that the cells express AFP antigen-specific TCRs. Ideally, the vector should be able to express consistently high levels in T cells.
细胞cell
本发明还涉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。所述宿主细胞中含有本发明的载体或染色体中整合有本发明的核酸分子。宿主细胞选自：原核细胞和真核细胞，例如大肠杆菌、酵母细胞、CHO细胞等。The present invention also relates to host cells genetically engineered with the vectors or coding sequences of the present invention. The host cell contains the vector of the present invention or the nucleic acid molecule of the present invention is integrated into the chromosome. The host cell is selected from: prokaryotic cells and eukaryotic cells, such as E. coli, yeast cells, CHO cells, and the like.
另外，本发明还包括表达本发明的TCR的分离的细胞，可以但不仅限为T细胞、NK细胞、NKT细胞，特别是T细胞。该T细胞可衍生自从受试者分离的T细胞，或者可以是从受试者中分离的混合细胞群，诸如外周血淋巴细胞(PBL)群的一部分。如，该细胞可以分离自外周血单核细胞(PBMC)，可以是CD4
+辅助T细胞或CD8
+细胞毒性T细胞。该细胞可在CD4
+辅助T细胞/CD8
+细胞毒性T细胞的混合群中。一般地，该细胞可以用抗体(如，抗-CD3或抗-CD28的抗体)活化，以便使它们能够更容易接受转染，例如用包含编码本发明TCR分子的核苷酸序列的载体进行转染。
In addition, the present invention also includes isolated cells expressing the TCR of the present invention, which can be, but are not limited to, T cells, NK cells, NKT cells, especially T cells. The T cells can be derived from T cells isolated from the subject, or can be part of a mixed population of cells isolated from the subject, such as a peripheral blood lymphocyte (PBL) population. For example, the cells can be isolated from peripheral blood mononuclear cells (PBMCs) and can be CD4 + helper T cells or CD8 + cytotoxic T cells. The cells can be in a mixed population of CD4 + helper T cells/CD8 + cytotoxic T cells. Typically, the cells can be activated with antibodies (eg, anti-CD3 or anti-CD28 antibodies) to render them more receptive to transfection, eg, with a vector comprising a nucleotide sequence encoding a TCR molecule of the invention dye.
备选地，本发明的细胞还可以是或衍生自干细胞，如造血干细胞(HSC)。将基因转移至HSC不会导致在细胞表面表达TCR，因为干细胞表面不表达CD3分子。然而，当干细胞分化为迁移至胸腺的淋巴前体(lymphoid precursor)时，CD3分子的表达将启动在胸腺细胞的表面表达该引入的TCR分子。Alternatively, the cells of the invention may also be or derived from stem cells, such as hematopoietic stem cells (HSCs). Gene transfer to HSCs does not result in TCR expression on the cell surface because the CD3 molecule is not expressed on the surface of stem cells. However, when stem cells differentiate into lymphoid precursors that migrate to the thymus, expression of the CD3 molecule will initiate expression of the introduced TCR molecule on the surface of thymocytes.
有许多方法适合于用编码本发明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TCR的T细胞可以用于过继免疫治疗。本领域技术人员能够知晓进行过继性治疗的许多合适方法(如，Rosenberg等.，(2008)Nat Rev Cancer 8(4)：299-308)。There are a number of methods suitable for transfection of T cells with DNA or RNA encoding the TCRs of the invention (eg, Robbins et al., (2008) J. Immunol. 180:6116-6131). T cells expressing the TCR of the present invention can be used for adoptive immunotherapy. Those skilled in the art are aware of many suitable methods for adopting adoptive therapy (eg, Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).
AFP抗原相关疾病AFP antigen-related diseases
本发明还涉及在受试者中治疗和/或预防与AFP相关疾病的方法，其包括过继性转移AFP特异性T细胞至该受试者的步骤。该AFP特异性T细胞可识别KWVESIFLIF-HLA A2402复合物。The present invention also relates to a method of treating and/or preventing an AFP-related disease in a subject comprising the step of adoptively transferring AFP-specific T cells to the subject. The AFP-specific T cells recognize the KWVESIFLIF-HLA A2402 complex.
本发明的AFP特异性的T细胞可用于治疗任何呈递AFP抗原短肽KWVESIFLIF-HLA A2402复合物的AFP相关疾病，包括但不限于肿瘤，如肝癌等。The AFP-specific T cells of the present invention can be used to treat any AFP-related diseases that present the AFP antigen short peptide KWVESIFLIF-HLA A2402 complex, including but not limited to tumors, such as liver cancer and the like.
治疗方法treatment method
可以通过分离患有与AFP抗原相关疾病的病人或志愿者的T细胞，并将本发明的TCR导入上述T细胞中，随后将这些基因工程修饰的细胞回输到病人体内来进行治疗。因此，本发明提供了一种治疗AFP相关疾病的方法，包括将分离的表达本发明TCR的T细胞，优选地，该T细胞来源于病人本身，输入到病人体内。一般地，包括(1)分离病人的T细胞，(2)用本发明核酸分子或能够编码本发明TCR分子的核酸分子体外转导T细胞，(3)将基因工程修饰的T细胞输入到病人体内。分离、转染及回输的细胞的数量可以由医师决定。Treatment can be performed by isolating T cells from patients or volunteers suffering from AFP antigen-related diseases, introducing the TCR of the present invention into the above T cells, and then infusing these genetically engineered cells back into the patient. Therefore, the present invention provides a method for treating AFP-related diseases, comprising infusing into a patient isolated T cells expressing the TCR of the present invention, preferably, the T cells are derived from the patient itself. Typically, this involves (1) isolating T cells from a patient, (2) transducing T cells in vitro with a nucleic acid molecule of the invention or a nucleic acid molecule capable of encoding a TCR molecule of the invention, and (3) infusing genetically engineered T cells into a patient in vivo. The number of cells isolated, transfected, and reinfused can be determined by the physician.
本发明的主要优点在于：The main advantages of the present invention are:
(1)本发明的TCR能够与AFP抗原短肽复合物KWVESIFLIF-HLA A2402特异性结合，同时转导了本发明TCR的效应细胞能够被特异性激活。(1) The TCR of the present invention can specifically bind to the AFP antigen short peptide complex KWVESIFLIF-HLA A2402, and the effector cells transduced with the TCR of the present invention can be specifically activated.
(2)转导了本发明TCR的效应细胞能够特异性杀伤靶细胞。(2) The effector cells transduced with the TCR of the present invention can specifically kill the target cells.
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。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。The following specific examples further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted specific conditions in the following examples, usually according to conventional conditions, such as (Sambrook and Russell et al., Molecular Cloning: Laboratory Manual (Molecular Cloning-A Laboratory Manual) (Third Edition) (2001) CSHL Publishing company), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.
实施例1 克隆AFP抗原短肽特异性T细胞Example 1 Cloning of AFP antigen peptide-specific T cells
利用合成短肽KWVESIFLIF(SEQ ID NO:9；江苏金斯瑞生物科技有限公司)刺激来自于基因型为HLA A2402的健康志愿者的外周血淋巴细胞(PBL)。将KWVESIFLIF短肽与带有生物素标记的HLA A2402复性,制备pHLA单倍体。这些单倍体与用PE标记的链霉亲和素(BD公司)组合成PE标记的四聚体，分选该四聚体及抗-CD8-APC双阳性细胞。扩增分选的细胞，并按上述方法进行二次分选，随后用有限稀释法进行单克隆。单克隆细胞用四聚体染色，筛选到的双阳性克隆如图3所示。经过层层筛选得到的双阳性克隆，还需要满足进一步的功能测试。Peripheral blood lymphocytes (PBL) from healthy volunteers with HLA A2402 genotype were stimulated with the synthetic short peptide KWVESIFLIF (SEQ ID NO: 9; Jiangsu GenScript Biotechnology Co., Ltd.). The KWVESIFLIF peptide was renatured with biotin-labeled HLA A2402 to prepare pHLA haploids. These haploids were combined with PE-labeled streptavidin (BD) to form PE-labeled tetramers, and the tetramers and anti-CD8-APC double positive cells were sorted. Sorted cells were expanded and subjected to secondary sorting as described above, followed by monocloning by limiting dilution. Monoclonal cells were stained with tetramers, and the screened double-positive clones were shown in Figure 3. The double-positive clones obtained through layer-by-layer screening still need to meet further functional tests.
通过ELISPOT实验进一步检测该T细胞克隆的功能及特异性。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实验中所用的效应细胞为本发明中获得的T细胞克隆，靶细胞为负载了KWVESIFLIF短肽的T2-A24(转染了HLA-A2402的T2细胞)、SNU398-AFP(AFP过表达)、HepG2，对照组为负载了其他短肽的T2-A24和SNU398。The function and specificity of the T cell clone were further tested by ELISPOT assay. Those skilled in the art are familiar with methods for detecting cell function using ELISPOT assays. The effector cells used in the IFN-γ ELISPOT experiment in this example are the T cell clones obtained in the present invention, and the target cells are T2-A24 (T2 cells transfected with HLA-A2402), SNU398-AFP ( AFP overexpression), HepG2, and the control groups were T2-A24 and SNU398 loaded with other short peptides.
首先准备ELISPOT平板，按以下顺序将试验的各个组分加入ELISPOT平板：靶细胞20,000个/孔、效应细胞2000个/孔后，在实验组和对照组加入20μl相应的短肽，空白组加入20μl培养基(试验培养基)，并设置2复孔。然后温育过夜(37℃，5％CO
2)。随后洗涤平板并进行二级检测和显色，干燥平板1小时，再利用免疫斑点平板读数计(ELISPOT READER system；AID公司)计数膜上形成的斑点。实验结果如图13所示，得到的T细胞克隆对负载了KWVESIFLIF短肽的靶细胞和过表达AFP抗原的SNU398-AFP、HepG2起明显的激活反应，而对负载了其他短肽的细胞及SNU398基本没有反应。
First prepare the ELISPOT plate, add the components of the test to the ELISPOT plate in the following order: 20,000 target cells/well, 2000 effector cells/well, add 20 μl of the corresponding short peptides to the experimental group and control group, and add 20 μl to the blank group. culture medium (assay medium), and set up 2 replicate wells. It was then incubated overnight (37°C, 5% CO2 ). The plates were then washed and subjected to secondary detection and color development, the plates were dried for 1 hour, and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID Corporation). The experimental results are shown in Figure 13. The obtained T cell clones have obvious activation responses to the target cells loaded with KWVESIFLIF short peptide and SNU398-AFP and HepG2 overexpressing AFP antigen, while the cells loaded with other short peptides and SNU398 Basically no response.
实施例2 获取AFP抗原短肽特异性T细胞克隆的TCR基因与载体的构建Example 2 Construction of TCR gene and vector for obtaining AFP antigen short peptide-specific T cell clones
用Quick-RNA
TM MiniPrep(ZYMO research)抽提实施例1中筛选到的抗原短肽 KWVESIFLIF特异性、HLA A2402限制性的T细胞克隆的总RNA。cDNA的合成采用clontech的SMART RACE cDNA扩增试剂盒，采用的引物是设计在人类TCR基因的C端保守区。将序列克隆至T载体(TAKARA)上进行测序。应注意，该序列为互补序列，不包含内含子。经测序，该双阳性克隆表达的TCR的α链和β链序列结构分别如图1和图2所示。
The total RNA of the antigen short peptide KWVESIFLIF-specific and HLA A2402-restricted T cell clone screened in Example 1 was extracted with Quick-RNA ™ MiniPrep (ZYMO research). The cDNA was synthesized using the SMART RACE cDNA amplification kit from clontech, and the primers used were designed in the C-terminal conserved region of the human TCR gene. The sequence was cloned into T vector (TAKARA) for sequencing. It should be noted that this sequence is complementary and does not contain introns. After sequencing, the sequence structures of the α chain and β chain of the TCR expressed by the double-positive clone are shown in Figure 1 and Figure 2, respectively.
经鉴定，α链包含具有以下氨基酸序列的CDR：The alpha chain was identified as comprising CDRs with the following amino acid sequence:
αCDR1-VGISA(SEQ ID NO:10)αCDR1-VGISA (SEQ ID NO: 10)
αCDR2-LSSGK(SEQ ID NO:11)αCDR2-LSSGK (SEQ ID NO: 11)
αCDR3-AVRDAGGTSYGKLT(SEQ ID NO:12)αCDR3-AVRDAGGTSYGKLT (SEQ ID NO: 12)
β链包含具有以下氨基酸序列的CDR：The beta chain contains CDRs with the following amino acid sequence:
βCDR1-MNHEY(SEQ ID NO:13)βCDR1-MNHEY (SEQ ID NO: 13)
βCDR2-SVGAGI(SEQ ID NO:14)βCDR2-SVGAGI (SEQ ID NO: 14)
βCDR3-ASSYPGSYGYT(SEQ ID NO:15)。βCDR3-ASSYPGSYGYT (SEQ ID NO: 15).
通过重叠(overlap)PCR分别将TCRα链和β链的全长基因克隆至慢病毒表达载体pLenti(addgene)。具体为：用overlap PCR将TCRα链和TCRβ链的全长基因进行连接得到TCRα-2A-TCRβ片段。将慢病毒表达载体及TCRα-2A-TCRβ酶切连接得到pLenti-TRA-2A-TRB-IRES-NGFR质粒。作为对照用,同时也构建表达eGFP的慢病毒载体pLenti-eGFP。之后再用293T/17包装假病毒。The full-length genes of the TCR alpha chain and beta chain were cloned into the lentiviral expression vector pLenti (addgene), respectively, by overlapping PCR. Specifically, the TCRα-2A-TCRβ fragment was obtained by connecting the full-length genes of the TCRα chain and the TCRβ chain by overlap PCR. The pLenti-TRA-2A-TRB-IRES-NGFR plasmid was obtained by ligating the lentiviral expression vector and TCRα-2A-TCRβ restriction enzyme. As a control, a lentiviral vector pLenti-eGFP expressing eGFP was also constructed. Afterwards, 293T/17 was used to package the pseudovirus.
实施例3 AFP抗原短肽特异性可溶TCR的表达、重折叠和纯化Example 3 Expression, refolding and purification of AFP antigen short peptide-specific soluble TCR
为获得可溶的TCR分子，本发明的TCR分子的α和β链可以分别只包含其可变域及部分恒定域，并且α和β链的恒定域中分别引入了一个半胱氨酸残基以形成人工链间二硫键，其α链的氨基酸序列与核苷酸序列分别如图4a和图4b所示，其β链的氨基酸序列与核苷酸序列分别如图5a和图5b所示。通过《分子克隆实验室手册》(Molecular Cloning a Laboratory Manual)(第三版，Sambrook和Russell)中描述的标准方法将上述TCRα和β链的目的基因序列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。插入片段经过测序确认无误。In order to obtain a soluble TCR molecule, the α and β chains of the TCR molecule of the present invention can respectively contain only their variable domains and part of their constant domains, and a cysteine residue is introduced into the constant domains of the α and β chains respectively. To form artificial interchain disulfide bonds, the amino acid sequence and nucleotide sequence of the α chain are shown in Figure 4a and Figure 4b, respectively, and the amino acid sequence and nucleotide sequence of the β chain are shown in Figure 5a and Figure 5b, respectively. . The target gene sequences of the above TCR α and β chains were synthesized and inserted into the expression vector pET28a+ (Novagene ), the upstream and downstream cloning sites are NcoI and NotI, respectively. The insert was confirmed by sequencing.
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。
The expression vectors of TCR α and β chains were transformed into expressing bacteria BL21 (DE3) by chemical transformation, the bacteria were grown in LB medium, and induced with a final concentration of 0.5 mM IPTG at OD 600 = 0.6, the α and β 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). ) in 20 mM Tris (pH 8.1).
溶解后的TCRα和β链以1：1的质量比快速混合于5M尿素，0.4M精氨酸，20mM Tris(pH 8.1)，3.7mM cystamine，6.6mMβ-mercapoethylamine(4℃)中，终浓度为60mg/mL。混合后将溶液置于10倍体积的去离子水中透析(4℃)，12小时后将去离子水换成缓冲液(20mM Tris,pH 8.0)继续于4℃透析12小时。透析完成后的溶液经0.45μM的滤膜过滤后，通过阴离子交换柱(HiTrap Q HP，5ml，GE Healthcare)纯化。洗脱峰含有复性成功的α和β二 聚体的TCR通过SDS-PAGE胶确认。TCR随后通过凝胶过滤层析(HiPrep 16/60，Sephacryl S-100HR，GE Healthcare)进一步纯化。纯化后的TCR纯度经过SDS-PAGE测定大于90％，浓度由BCA法确定。本发明得到的可溶性TCR的SDS-PAGE胶图如图6a和图6b所示。The dissolved TCRα and β chains were rapidly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine, 6.6mM β-mercapoethylamine (4°C) at a mass ratio of 1:1 at a final concentration of 60 mg/mL. After mixing, the solution was dialyzed in 10 times the volume of deionized water (4°C). After 12 hours, the deionized water was replaced with a buffer (20 mM Tris, pH 8.0), and the dialysis was continued at 4°C for 12 hours. The solution after dialysis was filtered through a 0.45 μM filter membrane and purified by an anion exchange column (HiTrap Q HP, 5 ml, GE Healthcare). The eluted peaks of TCR containing successfully renatured α and β dimers were confirmed by SDS-PAGE gel. TCR was then further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was more than 90% determined by SDS-PAGE, and the concentration was determined by BCA method. The SDS-PAGE gel images of the soluble TCR obtained by the present invention are shown in Figure 6a and Figure 6b.
实施例4 AFP抗原短肽特异性的可溶性单链TCR的产生Example 4 Production of soluble single-chain TCR specific for AFP antigen short peptides
根据专利文献WO2014/206304中所述，利用定点突变的方法将实施例2中TCRα与β链的可变域构建成了一个以柔性短肽(linker)连接的稳定的可溶性单链TCR分子。该单链TCR分子的氨基酸序列及核苷酸序列分别如图7a和图7b所示，其中linker序列的氨基酸序列及核苷酸序列用下划线标出；其α链可变域的氨基酸序列及核苷酸序列分别如图8a和图8b所示；其β链可变域的氨基酸序列及核苷酸序列分别如图9a和图9b所示。According to the patent document WO2014/206304, the variable domains of TCRα and β chains in Example 2 were constructed into a stable soluble single-chain TCR molecule linked by a flexible short peptide (linker) by site-directed mutagenesis. The amino acid sequence and nucleotide sequence of the single-chain TCR molecule are shown in Figure 7a and Figure 7b, respectively, wherein the amino acid sequence and nucleotide sequence of the linker sequence are underlined; The nucleotide sequences are shown in Figure 8a and Figure 8b, respectively; the amino acid sequence and nucleotide sequence of the β chain variable domain are shown in Figure 9a and Figure 9b, respectively.
将目的基因经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。The target gene was double digested with Nco I and Not I, and then ligated with the pET28a vector that was double digested with Nco I and Not I. The ligation product was transformed into E.coli DH5α, coated with LB plate containing kanamycin, and cultured overnight at 37°C by inversion. Positive clones were picked for PCR screening, and the positive recombinants were sequenced. After confirming that the sequences were correct, the recombinant plasmids were extracted and transformed. to E. coli BL21(DE3) for expression.
实施例5 AFP抗原短肽特异性的可溶性单链TCR的表达、复性和纯化Example 5 Expression, renaturation and purification of soluble single-chain TCR specific for AFP antigen short peptides
将实施例4中制备的含有重组质粒pET28a-模板链的BL21(DE 3)菌落全部接种于含有卡那霉素的LB培养基中，37℃培养至OD600为0.6-0.8，加入IPTG至终浓度为0.5mM，37℃继续培养4h。5000rpm离心15min收获细胞沉淀物，用Bugbuster Master Mix(Merck)裂解细胞沉淀物，6000rpm离心15min回收包涵体，再用Bugbuster(Merck)进行洗涤以除去细胞碎片和膜组分，6000rpm离心15min，收集包涵体。将包涵体溶解在缓冲液(20mM Tris-HCl pH 8.0，8M尿素)中，高速离心去除不溶物，上清液用BCA法定量后进行分装，于-80℃保存备用。All the BL21 (DE 3) colonies containing the recombinant plasmid pET28a-template chain prepared in Example 4 were inoculated into LB medium containing kanamycin, cultivated at 37°C to OD600 of 0.6-0.8, and IPTG was added to the final concentration 0.5mM, and continue to culture at 37°C for 4h. Cell pellets were harvested by centrifugation at 5000 rpm for 15 min, cell pellets were lysed with Bugbuster Master Mix (Merck), inclusion bodies were recovered by centrifugation at 6000 rpm for 15 min, and then washed with Bugbuster (Merck) to remove cell debris and membrane components, and the inclusion bodies were collected by centrifugation at 6000 rpm for 15 min. body. The inclusion bodies were dissolved in buffer (20mM Tris-HCl pH 8.0, 8M urea), and insoluble matter was removed by high-speed centrifugation. The supernatant was quantified by BCA method, and then packed and stored at -80°C for later use.
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。To 5mg of dissolved single-chain TCR inclusion body protein, add 2.5mL buffer (6M Gua-HCl, 50mM Tris-HCl pH 8.1, 100mM NaCl, 10mM EDTA), then add DTT to a final concentration of 10mM, and treat at 37°C for 30min . 125mL of renaturation buffer (100mM Tris-HCl pH 8.1, 0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mM β-mercapthoethylamine, 1.87mM Cystamine) was added dropwise with the single-chain TCR after the above treatment with a syringe, Stir at 4 °C for 10 min, then put the renaturation solution into a cellulose membrane dialysis bag with a cut-off of 4 kDa, put the dialysis bag in 1 L of pre-cooled water, and slowly stir at 4 °C overnight. After 17 hours, the dialysate was replaced with 1 L of pre-cooled buffer (20mM Tris-HCl pH 8.0), and the dialysis was continued for 8 h at 4°C, and then the dialysate was replaced with the same fresh buffer and continued dialysis overnight. After 17 hours, the sample was filtered through a 0.45 μm filter membrane, degassed in vacuo and passed through an anion exchange column (HiTrap Q HP, GE Healthcare), and the protein was purified with a linear gradient of 0-1 M NaCl prepared in 20 mM Tris-HCl pH 8.0. The collected elution fractions were analyzed by SDS-PAGE, the fractions containing single-chain TCR were concentrated and further purified by gel filtration column (Superdex 75 10/300, GE Healthcare), and the target fractions were also analyzed by SDS-PAGE.
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，
)，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。
The eluted fractions for BIAcore analysis were further tested for purity by gel filtration. The conditions are: Column Agilent Bio SEC-3 (300A, ), the mobile phase was 150 mM phosphate buffer, the flow rate was 0.5 mL/min, the column temperature was 25°C, and the UV detection wavelength was 214 nm.
本发明获得的可溶性单链TCR的SDS-PAGE胶图如图10所示。Figure 10 shows the SDS-PAGE gel image of the soluble single-chain TCR obtained by the present invention.
实施例6 结合表征Example 6 Binding Characterization
本实施例证明了可溶性的本发明TCR分子能够与KWVESIFLIF-HLA A2402复合物特异性结合。This example demonstrates that soluble TCR molecules of the invention are capable of specifically binding to the KWVESIFLIF-HLA A2402 complex.
使用BIAcore T200实时分析系统检测实施例3和实施例5中得到的TCR分子与KWVESIFLIF-HLA A2402复合物的结合活性。将抗链霉亲和素的抗体(GenScript)加入偶联缓冲液(10mM醋酸钠缓冲液，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KWVESIFLIF-HLA A2402复合物流过检测通道，另一通道作为参比通道，再将0.05mM的生物素以10μL/min的流速流过芯片2min，封闭链霉亲和素剩余的结合位点。The binding activity of the TCR molecules obtained in Example 3 and Example 5 to the KWVESIFLIF-HLA A2402 complex was detected using the BIAcore T200 real-time analysis system. Anti-streptavidin antibody (GenScript) was added to coupling buffer (10 mM sodium acetate buffer, pH 4.77), and the antibody was then flowed through a CM5 chip preactivated with EDC and NHS to immobilize the antibody on the chip surface , and finally blocked the unreacted activated surface with ethanolamine hydrochloric acid solution to complete the coupling process with a coupling level of about 15,000RU. Make a low concentration of streptavidin flow over the surface of the chip that has been coated with the antibody, then flow KWVESIFLIF-HLA A2402 complex through the detection channel, another channel is used as a reference channel, and then flow 0.05mM biotin at 10μL/min. flow through the chip for 2 min to block the remaining binding sites of streptavidin.
上述KWVESIFLIF-HLA A2402复合物的制备过程如下：The preparation process of above-mentioned KWVESIFLIF-HLA A2402 complex is as follows:
a.纯化a. Purification
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。Collect 100ml of E.coli bacteria that induces the expression of heavy or light chains, centrifuge at 8000g at 4°C for 10min, wash the cells once with 10ml PBS, and then use 5ml BugBuster Master Mix Extraction Reagents (Merck) to vigorously shake the cells to resuspend the cells. Incubate with rotation at room temperature for 20 min, then centrifuge at 6000g for 15 min at 4°C, discard the supernatant, and collect the inclusion bodies.
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。Resuspend the above inclusions in 5ml of BugBuster Master Mix, rotate and incubate at room temperature for 5min; add 30ml of BugBuster diluted 10 times, mix well, and centrifuge at 6000g at 4°C for 15min; discard the supernatant and add 30ml of BugBuster diluted 10 times to resuspend the inclusion bodies , mix well, centrifuge at 6000g at 4°C for 15min, repeat twice, add 30ml 20mM Tris-HCl pH 8.0 to resuspend the inclusion bodies, mix well, centrifuge at 6000g at 4°C for 15min, and finally dissolve the inclusion bodies with 20mM Tris-HCl 8M urea, SDS- The purity of inclusion bodies was detected by PAGE, and the concentration was determined by BCA kit.
b.复性b. Refolding
将合成的短肽KWVESIFLIF(江苏金斯瑞生物科技有限公司)溶解于DMSO至20mg/ml的浓度。轻链和重链的包涵体用8M尿素、20mM Tris pH 8.0、10mM DTT来溶解，复性前加入3M盐酸胍、10mM醋酸钠、10mM EDTA进一步变性。将KWVESIFLIF肽以25mg/L(终浓度)加入复性缓冲液(0.4M L-精氨酸、100mM Tris pH 8.3、2mM EDTA、0.5mM氧化性谷胱甘肽、5mM还原型谷胱甘肽、0.2mM PMSF，冷却至4℃)，然后依次加入20mg/L的轻链和90mg/L的重链(终浓度，重链分三次加入，8h/次)，复性在4℃进行至少3天至完成，SDS-PAGE检测能否复性成功。The synthetic short peptide KWVESIFLIF (Jiangsu GenScript Biotechnology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg/ml. The inclusion bodies of light chain and heavy chain were dissolved with 8M urea, 20mM Tris pH 8.0, 10mM DTT, and further denatured by adding 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA before renaturation. KWVESIFLIF peptide was added to renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4°C), then added 20mg/L light chain and 90mg/L heavy chain in sequence (final concentration, heavy chain was added in three times, 8h/time), and renatured at 4°C for at least 3 days To complete, SDS-PAGE test whether the renaturation is successful.
c.复性后纯化c. Purification after renaturation
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。Replace the renaturation buffer by dialyzing against 10 volumes of 20 mM Tris pH 8.0, at least twice, to sufficiently reduce the ionic strength of the solution. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and loaded onto a HiTrap Q HP (GE) anion exchange column (5 ml bed volume). The protein was eluted with an Akta purifier (GE), a linear gradient of 0-400 mM NaCl prepared with 20 mM Tris pH 8.0, and pMHC was eluted at about 250 mM NaCl, and the peak fractions were collected, and the purity was checked by SDS-PAGE.
d.生物素化d. Biotinylation
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。Purified pMHC molecules were concentrated with Millipore ultrafiltration tubes while buffer exchanged to 20mM Tris pH 8.0, followed by addition of biotinylation reagents 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50μM D-Biotin, 100μg/ml BirA Enzyme (GST-BirA), the mixture was incubated overnight at room temperature, and the complete biotinylation was checked by SDS-PAGE.
e.纯化生物素化后的复合物e. Purification of biotinylated complexes
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
TM16/60 S200 HR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℃。
The biotinylated pMHC molecules were concentrated to 1 ml with a Millipore ultrafiltration tube, and the biotinylated pMHC was purified by gel filtration chromatography. HiPrep was pre-equilibrated with filtered PBS using an Akta purifier (GE). A TM 16/60 S200 HR column (GE) was loaded with 1 ml of concentrated biotinylated pMHC molecules and eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules eluted as a single peak at about 55 ml. The fractions containing protein were combined, concentrated with Millipore ultrafiltration tube, the protein concentration was determined by BCA method (Thermo), and the protease inhibitor cocktail (Roche) was added to store the biotinylated pMHC molecules in aliquots at -80°C.
利用BIAcore Evaluation软件计算动力学参数，得到本发明可溶性的TCR分子以及本发明构建的可溶性单链TCR分子与KWVESIFLIF-HLA A2402复合物结合的动力学图谱分别如图11和图12所示。图谱显示，本发明得到的可溶性TCR分子以及可溶性单链TCR分子都能够与KWVESIFLIF-HLA A2402复合物结合。同时，还利用上述方法检测了本发明可溶性的TCR分子与其他几种无关抗原短肽与HLA复合物的结合活性，结果显示本发明TCR分子与其他无关抗原均无结合。Utilize BIAcore Evaluation software to calculate kinetic parameters, and obtain the soluble TCR molecule of the present invention and the kinetic spectrum of the soluble single-chain TCR molecule constructed by the present invention and KWVESIFLIF-HLA A2402 complex, as shown in Figure 11 and Figure 12, respectively. The map shows that both the soluble TCR molecule and the soluble single-chain TCR molecule obtained by the present invention can bind to the KWVESIFLIF-HLA A2402 complex. At the same time, the above method was used to detect the binding activity of the soluble TCR molecule of the present invention to several other irrelevant antigen short peptides and HLA complexes, and the results showed that the TCR molecule of the present invention did not bind to other irrelevant antigens.
实施例7 转染本发明TCR的效应细胞的ELISPOT激活效应实验Example 7 ELISPOT activation effect experiment of effector cells transfected with TCR of the present invention
IFN-γ是活化T淋巴细胞产生的一种强有力的免疫调节因子，因此本实施例通过本领域技术人员熟知的ELISPOT实验检测IFN-γ数以验证转染本发明TCR的细胞的激活功能及抗原特异性。IFN-γ is a powerful immunoregulatory factor produced by activated T lymphocytes. Therefore, in this example, the number of IFN-γ was detected by the ELISPOT experiment well-known to those skilled in the art to verify the activation function of the cells transfected with the TCR of the present invention. antigen specificity.
本实验中所用的效应细胞是表达本发明TCR的CD3
+T细胞，并以同一志愿者转染其他TCR(A6)的或空转染(NC)的CD3
+T细胞作为对照组。所用的靶细胞为负载了AFP抗原短肽KWVESIFLIF的T2-A24，并以负载其他无关肽的或空载的T2-A24作为对照。将试验的各组分加入ELISPOT孔板：靶细胞1×10
4个/孔、效应细胞2×10
3个/孔(按转染阳性率计算)，并设置两个复孔。然后在相应孔分别加入KWVESIFLIF短肽溶液、其他短肽溶液、等体积培养基，使KWVESIFLIF短肽在ELISPOT孔板中的终浓度依次为1×10
-6M到1×10
-12M，共7个梯度；使无关肽在ELISPOT孔板中的终浓度依次为1×10
-6M到1×10
-8M，共3个梯度。然后温育过夜(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。
The effector cells used in this experiment are CD3 + T cells expressing the TCR of the present invention, and the same volunteer transfected with other TCR (A6) or empty transfected (NC) CD3 + T cells was used as a control group. The target cells used were T2-A24 loaded with AFP antigen short peptide KWVESIFLIF, and T2-A24 loaded with other irrelevant peptides or empty were used as control. The components of the test were added to the ELISPOT well plate: 1×10 4 cells/well of target cells, 2×10 3 cells/well of effector cells (calculated according to the positive rate of transfection), and two duplicate wells were set up. Then, KWVESIFLIF short peptide solution, other short peptide solutions, and equal volume of medium were added to the corresponding wells respectively, so that the final concentration of KWVESIFLIF short peptide in the ELISPOT plate was 1×10 -6 M to 1×10 -12 M in turn, a total of 7 gradients; make the final concentration of irrelevant peptides in the ELISPOT plate from 1 x 10 -6 M to 1 x 10 -8 M, 3 gradients in total. It was then incubated overnight (37°C, 5% CO2 ). On the second day of the experiment, the plate was washed for secondary detection and color development, the plate was dried, and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID20 company).
实验结果如图14所示，针对负载KWVESIFLIF短肽的靶细胞，转染本发明TCR的T细胞起到明显的激活效应，而转染其他TCR或空转染的的T细胞自始无反应；同时，转染 本发明TCR的T细胞对负载其他短肽的或空载的细胞无激活。The experimental results are shown in Figure 14. For the target cells loaded with the KWVESIFLIF short peptide, the T cells transfected with the TCR of the present invention have an obvious activation effect, while the T cells transfected with other TCRs or empty transfected have no response from the beginning; At the same time, T cells transfected with the TCR of the present invention did not activate cells loaded with other short peptides or empty.
实施例8.转染本发明TCR的效应细胞的LDH杀伤功能实验Example 8. LDH killing function experiment of effector cells transfected with TCR of the present invention
本实施例通过本领域技术人员熟知的非放射性细胞毒性实验，测定LDH的释放，从而验证转染本发明TCR的细胞的杀伤功能。该试验是51Cr释放细胞毒性试验的比色替代试验，定量测定细胞裂解后释放的乳酸脱氢酶(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。计算公式:％细胞毒性＝100×(实验-效应细胞自发-靶细胞自发)/(靶细胞最大-靶细胞自发)In this example, non-radioactive cytotoxicity experiments well known to those skilled in the art are used to measure the release of LDH, thereby verifying the killing function of cells transfected with the TCR of the present invention. This assay is a colorimetric alternative to the 51Cr release cytotoxicity assay and quantifies lactate dehydrogenase (LDH) released after cell lysis. LDH released in the medium was detected using a 30 min coupled enzymatic reaction in which LDH converts a tetrazolium salt (INT) to red formazan. The amount of red product produced is proportional to the number of cells lysed. Visible absorbance data at 490 nm can be collected using a standard 96-well plate reader. Calculation formula: % cytotoxicity=100×(experimental-effector cell spontaneous-target cell spontaneous)/(target cell maximum-target cell spontaneous)
本实施例LDH实验用分离的CD3+T细胞转染本发明TCR作为效应细胞，并以同一志愿者转染其他TCR(A6)的或空转染(NC)的CD3+T细胞作为对照组。所用的靶细胞为负载了AFP抗原短肽KWVESIFLIF的T2-A24，并以负载其他无关肽的或空载的T2-A24作为对照。In the LDH experiment in this example, the isolated CD3+ T cells were transfected with the TCR of the present invention as effector cells, and the same volunteers were transfected with other TCR (A6) or empty-transfected (NC) CD3+ T cells as the control group. The target cells used were T2-A24 loaded with AFP antigen short peptide KWVESIFLIF, and T2-A24 loaded with other irrelevant peptides or empty were used as control.
首先准备LDH平板，先按靶细胞3*10
4个/孔、效应细胞3*10
4个/孔加入对应中，然后在相应孔分别加入KWVESIFLIF短肽溶液、其他短肽溶液、等体积培养基，使KWVESIFLIF短肽在LDH孔板中的终浓度依次为1×10
-6M到1×10
-13M，共8个梯度；使无关肽在LDH孔板中的终浓度依次为1×10
-6M到1×10
-8M，共3个梯度。并设置三个复孔。同时设置效应细胞自发孔，靶细胞自发孔，靶细胞最大孔，体积校正对照孔及培养基背景对照孔。温育过夜(37℃，5％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。
First prepare the LDH plate, firstly add 3*10 4 target cells/well and 3*10 4 effector cells/well into the corresponding medium, and then add KWVESIFLIF short peptide solution, other short peptide solutions, and equal volume of medium to the corresponding wells respectively. , the final concentration of KWVESIFLIF peptide in the LDH well plate was 1×10 -6 M to 1×10 -13 M, a total of 8 gradients; the final concentration of the irrelevant peptide in the LDH well plate was 1×10 -6 M to 1×10 -8 M, 3 gradients in total. And set three duplicate holes. Simultaneously set effector cell spontaneous wells, target cell spontaneous wells, target cell maximum wells, volume correction control wells and medium background control wells. Incubate overnight (37°C, 5% CO2 ). On the second day of the experiment, color development was detected, and the absorbance value was recorded at 490 nm with a microplate reader (Bioteck) after the reaction was terminated.
实验结果如图15所示，针对负载AFP抗原短肽KWVESIFLIF的靶细胞，转染本发明TCR的效应细胞有强杀伤效应，且在上述短肽浓度较低时即起反应，而转染其他TCR的或空转染的效应细胞自始无杀伤；同时，转染本发明TCR的效应细胞对负载其他短肽的或空载的靶细胞无杀伤。The experimental results are shown in Figure 15. For the target cells loaded with the AFP antigen short peptide KWVESIFLIF, the effector cells transfected with the TCR of the present invention have a strong killing effect, and they react when the concentration of the above-mentioned short peptide is low, while other TCRs are transfected. At the same time, the effector cells transfected with the TCR of the present invention did not kill the target cells loaded with other short peptides or empty.
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。All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.
Claims (26)
- 一种T细胞受体(TCR)，其特征在于，所述TCR能够与KWVESIFLIF-HLA A2402复合物结合；并且，所述TCR包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域的3个互补决定区(CDR)为：A T cell receptor (TCR), characterized in that the TCR can be combined with KWVESIFLIF-HLA A2402 complex; and, the TCR comprises a TCRα chain variable domain and a TCRβ chain variable domain, and the TCRα chain can be The three complementarity determining regions (CDRs) of the variable domains are:αCDR1-VGISA (SEQ ID NO:10)αCDR1-VGISA (SEQ ID NO:10)αCDR2-LSSGK (SEQ ID NO:11)αCDR2-LSSGK (SEQ ID NO:11)αCDR3-AVRDAGGTSYGKLT(SEQ ID NO:12)；和/或αCDR3-AVRDAGGTSYGKLT (SEQ ID NO: 12); and/or所述TCRβ链可变域的3个互补决定区为：The three complementarity determining regions of the TCRβ chain variable domain are:βCDR1-MNHEY (SEQ ID NO:13)βCDR1-MNHEY (SEQ ID NO:13)βCDR2-SVGAGI (SEQ ID NO:14)βCDR2-SVGAGI (SEQ ID NO:14)βCDR3-ASSYPGSYGYT (SEQ ID NO:15)。βCDR3-ASSYPGSYGYT (SEQ ID NO: 15).
- 如权利要求1所述的TCR，其特征在于，其包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域为与SEQ ID NO:1具有至少90％序列相同性的氨基酸序列；和/或所述TCRβ链可变域为与SEQ ID NO：5具有至少90％序列相同性的氨基酸序列。The TCR of claim 1, comprising a TCRα chain variable domain and a TCRβ chain variable domain, the TCRα chain variable domain being an amino acid having at least 90% sequence identity with SEQ ID NO: 1 sequence; and/or the TCR beta chain variable domain is an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5.
- 如权利要求1所述的TCR，其特征在于，所述TCR包含α链可变域氨基酸序列SEQ ID NO:1；和/或所述TCR包含β链可变域氨基酸序列SEQ ID NO:5。The TCR of claim 1, wherein the TCR comprises an alpha chain variable domain amino acid sequence of SEQ ID NO: 1; and/or the TCR comprises a beta chain variable domain amino acid sequence of SEQ ID NO: 5.
- 如权利要求1所述的TCR，其特征在于，所述TCR为αβ异质二聚体，其包含TCRα链恒定区TRAC*01和TCRβ链恒定区TRBC1*01或TRBC2*01；优选地，所述TCR的α链氨基酸序列为SEQ ID NO：3和所述TCR的β链氨基酸序列为SEQ ID NO:7。The TCR of claim 1, wherein the TCR is an αβ heterodimer, which comprises a TCRα chain constant region TRAC*01 and a TCRβ chain constant region TRBC1*01 or TRBC2*01; preferably, the The α chain amino acid sequence of the TCR is SEQ ID NO: 3 and the β chain amino acid sequence of the TCR is SEQ ID NO: 7.
- 如权利要求1所述的TCR，其特征在于，所述TCR是可溶的。The TCR of claim 1, wherein the TCR is soluble.
- 如权利要求5所述的TCR，其特征在于，所述TCR为单链；优选地，所述TCR是由α链可变域与β链可变域通过肽连接序列连接而成。The TCR of claim 5, wherein the TCR is a single chain; preferably, the TCR is formed by connecting the variable domain of the α chain and the variable domain of the β chain through a peptide linker sequence.
- 如权利要求6所述的TCR，其特征在于，所述TCR在α链可变区氨基酸第11、13、19、21、53、76、89、91、或第94位，和/或α链J基因短肽氨基酸倒数第3位、倒数第5位或倒数第7位中具有一个或多个突变；和/或所述TCR在β链可变区氨基酸第11、13、19、21、53、76、89、91、或第94位，和/或β链J基因短肽氨基酸倒数第2位、倒数第4位或倒数第6位中具有一个或多个突变，其中氨基酸位置编号按IMGT(国际免疫遗传学信息系统)中列出的位置编号；优选地，所述TCR的α链可变域氨基酸序列包含SEQ ID NO:32和/或所述TCR的β链可变域氨基酸序列包含SEQ ID NO:34；更优选地，所述TCR的氨基酸序列为SEQ ID NO:30。The TCR of claim 6, wherein the TCR is at amino acid position 11, 13, 19, 21, 53, 76, 89, 91, or 94 of the α chain variable region, and/or the α chain J gene short peptide has one or more mutations in the penultimate 3rd, 5th or 7th amino acid of the penultimate; and/or the TCR is at amino acid 11, 13, 19, 21, 53 of the β chain variable region , 76, 89, 91, or 94, and/or one or more mutations in the penultimate amino acid position 2, 4 or 6 of the β-chain J gene short peptide, wherein the amino acid positions are numbered according to IMGT (International Immunogenetics Information System) as listed in the position numbers; preferably, the alpha chain variable domain amino acid sequence of the TCR comprises SEQ ID NO: 32 and/or the beta chain variable domain amino acid sequence of the TCR comprises SEQ ID NO:34; more preferably, the amino acid sequence of the TCR is SEQ ID NO:30.
- 如权利要求1所述的TCR，其特征在于，所述TCR包含(i)TCRα链可变域和除跨 膜结构域以外的全部或部分TCRα链恒定区；和(ii)TCRβ链可变域和除跨膜结构域以外的全部或部分TCRβ链恒定区。The TCR of claim 1, wherein the TCR comprises (i) a TCRα chain variable domain and all or part of a TCRα chain constant region excluding the transmembrane domain; and (ii) a TCRβ chain variable domain and all or part of the TCRβ chain constant region except the transmembrane domain.
- 如权利要求8所述的TCR，其特征在于，半胱氨酸残基在所述TCR的α和β链恒定域之间形成人工二硫键；优选地，在所述TCR中形成人工二硫键的半胱氨酸残基取代了选自下列的一组或多组位点：The TCR of claim 8, wherein cysteine residues form an artificial disulfide bond between the constant domains of the alpha and beta chains of the TCR; preferably, an artificial disulfide is formed in the TCR The cysteine residue of the bond is substituted at one or more groups of sites selected from the group consisting of:TRAC*01外显子1的Thr48和TRBC1*01或TRBC2*01外显子1的Ser57；Thr48 in exon 1 of TRAC*01 and Ser57 in exon 1 of TRBC1*01 or TRBC2*01;TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；Thr45 in exon 1 of TRAC*01 and Ser77 in exon 1 of TRBC1*01 or TRBC2*01;TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；Tyr10 of exon 1 of TRAC*01 and Ser17 of exon 1 of TRBC1*01 or TRBC2*01;TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；Thr45 of exon 1 of TRAC*01 and Asp59 of exon 1 of TRBC1*01 or TRBC2*01;TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；Ser15 of exon 1 of TRAC*01 and Glu15 of exon 1 of TRBC1*01 or TRBC2*01;TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；Arg53 in exon 1 of TRAC*01 and Ser54 in exon 1 of TRBC1*01 or TRBC2*01;TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；和Pro89 of exon 1 of TRAC*01 and Ala19 of exon 1 of TRBC1*01 or TRBC2*01; andTRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01.
- 如权利要求9所述的TCR，其特征在于，所述TCR的α链氨基酸序列为SEQ ID NO:26和/或所述TCR的β链氨基酸序列为SEQ ID NO:28。The TCR of claim 9, wherein the α-chain amino acid sequence of the TCR is SEQ ID NO:26 and/or the β-chain amino acid sequence of the TCR is SEQ ID NO:28.
- 如权利要求8所述的TCR，其特征在于，所述TCR的α链可变区与β链恒定区之间含有人工链间二硫键；优选地，在所述TCR中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：The TCR of claim 8, wherein an artificial interchain disulfide bond is contained between the α chain variable region and the β chain constant region of the TCR; preferably, an artificial interchain disulfide bond is formed in the TCR Sulfur-bonded cysteine residues are substituted at one or more groups of sites selected from the group consisting of: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；Amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01;TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的61位氨基酸；Amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01;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Amino acid 46 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01; orTRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸。Amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01.
- 如权利要求1所述的TCR，其特征在于，所述TCR包含α链恒定区和β链恒定区，所述α链恒定区为鼠源的和/或所述β链恒定区为鼠源的。The TCR of claim 1, wherein the TCR comprises an α chain constant region and a β chain constant region, the α chain constant region is murine and/or the β chain constant region is murine .
- 如权利要求1所述的TCR，其特征在于，所述TCR的α链和/或β链的C-或N-末端结合有偶联物；优选地，与所述T细胞受体结合的偶联物为可检测标记物或治疗剂；更优选地，所述治疗剂为抗-CD3抗体。The TCR of claim 1, wherein a conjugate is bound to the C- or N-terminus of the α chain and/or β chain of the TCR; preferably, a conjugate bound to the T cell receptor The conjugate is a detectable label or a therapeutic agent; more preferably, the therapeutic agent is an anti-CD3 antibody.
- 如权利要求1所述的TCR，其特征在于，所述TCR是分离的或纯化的。The TCR of claim 1, wherein the TCR is isolated or purified.
- 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。A multivalent TCR complex, characterized in that it comprises at least two TCR molecules, and at least one of the TCR molecules is the TCR described in any one of the preceding claims.
- 一种核酸分子，其特征在于，所述核酸分子包含编码上述任一权利要求所述的TCR分子的核酸序列或其互补序列。A nucleic acid molecule, characterized in that the nucleic acid molecule comprises a nucleic acid sequence encoding the TCR molecule according to any one of the preceding claims or a complementary sequence thereof.
- 如权利要求16所述的核酸分子，其特征在于，其包含编码TCRα链可变域的核苷酸序列SEQ ID NO:2或SEQ ID NO:33。The nucleic acid molecule of claim 16, which comprises the nucleotide sequence SEQ ID NO:2 or SEQ ID NO:33 encoding the variable domain of the TCR alpha chain.
- 如权利要求16或17所述的核酸分子，其特征在于，其包含编码TCRβ链可变域的核苷酸序列SEQ ID NO:6或SEQ ID NO:35。The nucleic acid molecule of claim 16 or 17, characterized in that it comprises the nucleotide sequence SEQ ID NO:6 or SEQ ID NO:35 encoding the variable domain of the TCR beta chain.
- 如权利要求16所述的核酸分子，其特征在于，其包含编码TCRα链的核苷酸序列SEQ ID NO:4和/或包含编码TCRβ链的核苷酸序列SEQ ID NO:8。The nucleic acid molecule of claim 16, characterized in that it comprises the nucleotide sequence of SEQ ID NO:4 encoding a TCRα chain and/or the nucleotide sequence of SEQ ID NO:8 encoding a TCRβ chain.
- 一种载体，其特征在于，所述的载体含有权利要求16-19中任一所述的核酸分子；优选地，所述的载体为病毒载体；更优选地，所述的载体为慢病毒载体。A vector, characterized in that the vector contains the nucleic acid molecule according to any one of claims 16-19; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector .
- 一种分离的宿主细胞，其特征在于，所述的宿主细胞中含有权利要求20中所述的载体或染色体中整合有外源的权利要求16-19中任一所述的核酸分子。An isolated host cell, characterized in that the host cell contains the vector described in claim 20 or the exogenous nucleic acid molecule described in any one of claims 16-19 integrated into a chromosome.
- 一种细胞，其特征在于，所述细胞中转导有权利要求16-19中任一所述的核酸分子或权利要求20中所述载体；优选地，所述细胞为T细胞或干细胞。A cell, characterized in that the cell is transduced with the nucleic acid molecule described in any one of claims 16-19 or the vector described in claim 20; preferably, the cell is a T cell or a stem cell.
- 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1-14中任一项所述的TCR、权利要求15中所述的TCR复合物、或权利要求22中所述的细胞。A pharmaceutical composition, characterized in that the composition contains a pharmaceutically acceptable carrier and the TCR described in any one of claims 1-14, the TCR complex described in claim 15, or the claim cells as described in 22.
- 权利要求1-14中任一项所述的T细胞受体、或权利要求15中所述的TCR复合物或权利要求22中所述的细胞的用途，其特征在于，用于制备治疗肿瘤或自身免疫疾病的药物；优选地，所述肿瘤为肝癌。Use of the T cell receptor described in any one of claims 1-14, or the TCR complex described in claim 15, or the cell described in claim 22, characterized in that it is used to prepare the treatment of tumors or Drugs for autoimmune diseases; preferably, the tumor is liver cancer.
- 权利要求1-14中任一项所述的T细胞受体、或权利要求15中所述的TCR复合物或权利要求22中所述的细胞，用作治疗肿瘤或自身免疫疾病的药物；优选地，所述肿瘤为肝癌。The T cell receptor described in any one of claims 1-14, or the TCR complex described in claim 15, or the cell described in claim 22, for use as a drug for the treatment of tumors or autoimmune diseases; preferably Ground, the tumor is liver cancer.
- 一种治疗疾病的方法，其特征在于，包括给需要治疗的对象施用适量的权利要求1-14中任一所述的TCR、权利要求15中所述TCR复合物、权利要求22中所述的细胞或权利要求23中所述的药物组合物；A method for treating a disease, characterized in that it comprises administering an appropriate amount of the TCR described in any one of claims 1-14, the TCR complex described in claim 15, the TCR complex described in claim 22 to an object in need of treatment cells or the pharmaceutical composition of claim 23;优选地，所述的疾病为肿瘤，更优选地所述肿瘤为肝癌。Preferably, the disease is a tumor, more preferably the tumor is liver cancer.
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| WO (1) | WO2022206861A1 (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105408353A (en) * | 2013-07-26 | 2016-03-16 | 艾达普特免疫有限公司 | T cell receptors |
| CN111171137A (en) * | 2018-11-13 | 2020-05-19 | 中国科学院广州生物医药与健康研究院 | T cell receptor for identifying AFP antigen short peptide and its coding sequence |
| CN112390875A (en) * | 2019-08-16 | 2021-02-23 | 广东香雪精准医疗技术有限公司 | High-affinity T cell receptor for identifying AFP |
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2021
- 2021-04-02 CN CN202110363579.8A patent/CN115160432A/en active Pending
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2022
- 2022-03-30 WO PCT/CN2022/084190 patent/WO2022206861A1/en unknown
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105408353A (en) * | 2013-07-26 | 2016-03-16 | 艾达普特免疫有限公司 | T cell receptors |
| CN111171137A (en) * | 2018-11-13 | 2020-05-19 | 中国科学院广州生物医药与健康研究院 | T cell receptor for identifying AFP antigen short peptide and its coding sequence |
| CN112390875A (en) * | 2019-08-16 | 2021-02-23 | 广东香雪精准医疗技术有限公司 | High-affinity T cell receptor for identifying AFP |
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| JOACHIMS, M. L. ET AL.: "Single-Cell Analysis of Glandular T Cell Receptors in Sjögren’s Syndrome", JCI INSIGHT, vol. 1, no. 8, 2 June 2016 (2016-06-02), XP055966921, DOI: 10.1172/jci.insight.85609 * |
| LUO XIAOBING, CUI HUIJUAN, CAI LUN, ZHU WEI, YANG WEI-CHIH, PATRICK MICHAEL, ZHU SHIGUI, HUANG JIAQI, YAO XIN, YAO YIHONG, HE YUKA: "Selection of a Clinical Lead TCR Targeting Alpha-Fetoprotein-Positive Liver Cancer Based on a Balance of Risk and Benefit", FRONTIERS IN IMMUNOLOGY, vol. 11, 1 January 2020 (2020-01-01), pages 623, XP055972992, DOI: 10.3389/fimmu.2020.00623 * |
| ZHENJUAN LI; HAIPING GONG; QIUPING LIU; WANLI WU; JIANTING CHENG; YINGYI MEI; YAOLONG CHEN; HONGJUN ZHENG; XIAOHONG YU; SHI ZHONG;: "Identification of an HLA‐A*24:02‐restricted α‐fetoprotein signal peptide‐derived antigen and its specific T‐cell receptor for T‐cell immunotherapy", CANCER RESEARCH, WILEY-BLACKWELL PUBLISHING LTD., GB, vol. 159, no. 4, 10 January 2020 (2020-01-10), GB , pages 384 - 392, XP071277592, ISSN: 0019-2805, DOI: 10.1111/imm.13168 * |
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| Publication number | Publication date |
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| CN115160432A (en) | 2022-10-11 |
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