WO2022002014A1 - T细胞抗原受体、其多聚体复合物及其制备方法和应用 - Google Patents
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- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
- G01N2333/04—Varicella-zoster virus
- G01N2333/045—Cytomegalovirus
Definitions
- the present invention relates to the technical field of T cell antigen receptors capable of recognizing CMV pp65, in particular to various TCRs and uses in the treatment and/or prevention of CMV-related diseases.
- T cell receptors are crucial to the cellular immune function of the immune system. TCRs are the only receptors for specific antigenic peptides presented on the major histocompatibility complex (MHC). Through the combination of antigen-specific TCR and pMHC complex, the direct physical contact between T cells and antigen-presenting cells is triggered, and the interaction leads to a series of subsequent cell signaling and other physiological responses, so that T cells with different antigen specificities can interact with their target cells. exert immune effect.
- MHC major histocompatibility complex
- CN107827959A discloses a method for isolating antigen-specific T cells and cloning antigen-specific TCR genes in antigen-specific T cells.
- the TCR can be combined with the HBVS183-91 antigen short peptide complex FLLTRILTI-HLA*A0201, and the cells transduced with the TCR of the present invention can be specifically activated and have a strong killing effect on the target cells.
- CN106279404A discloses a soluble and stable heterodimeric TCR containing artificial interchain disulfide bonds between the ⁇ chain variable region and the ⁇ chain constant region, the TCR is soluble and stable , can be well renatured, refolded, purified and can specifically bind to the original ligand.
- none of the above patents discloses specific TCR for cytomegalovirus.
- Cytomegalovirus (Cytomegalocirus, CMV) is a herpesvirus DNA virus, also known as cellular inclusion virus. Cytomegalovirus is widely distributed, and infection is very common in the population. The infection rate of Chinese adults is more than 95%, and it is usually negative for infection. Most infected people have no clinical symptoms, but under certain conditions, it can invade multiple organs and systems and cause inflammatory diseases.
- CN102656188A discloses a T cell receptor capable of recognizing antigens from cytomegalovirus, which specifically binds to a peptide from the cytomegalovirus (CMV) phosphoprotein pp65 when presented by the major histocompatibility complex (MHC), so The peptide has the amino acid sequence NLVPMVATV.
- CMV cytomegalovirus
- MHC major histocompatibility complex
- the present invention provides a T cell antigen receptor, which can be specifically activated by virus antigen peptide presenting cells, increase the release level of extracellular cytokines IFN ⁇ , IL2 and the release amount of lactate dehydrogenase, and significantly kill target cells.
- the present invention provides a variety of T cell antigen receptors (TCRs), which specifically bind to the cytomegalovirus (CMV) phosphoprotein pp65 antigen peptide presented by MHC molecules, and the cytomegalovirus (CMV) phosphoprotein pp65 comprises SEQ The amino acid sequence shown in ID NO:34.
- TCRs T cell antigen receptors
- CMV cytomegalovirus
- CMV cytomegalovirus
- phosphoprotein pp65 comprises SEQ The amino acid sequence shown in ID NO:34.
- the TCR of the present invention can specifically recognize the corresponding CMV pp65 antigen peptide-MHC molecular complex, activate TCR T cells, and then produce high levels of cytokines IFN ⁇ , IL2, TNF ⁇ , and both in vivo and in vitro tests significantly kill tumor cells. details as follows:
- the first aspect of the present invention provides a determinant complementary region (CDR) that binds to CMV pp65, wherein the CDR is selected from one or more combinations of SEQ ID NOs: 4-33.
- CDR determinant complementary region
- the CDRs include CDR1 ⁇ -CDR3 ⁇ and/or CDR1 ⁇ -CDR3 ⁇ .
- the amino acid sequence of CDR1 ⁇ comprises any one of SEQ ID NO: 4-7 or comprises at least 80% homology with the amino acid sequence shown in any one of SEQ ID NO: 4-7
- the amino acid sequence of CDR2 ⁇ comprises Any of SEQ ID NOs: 8-11 or comprising at least 80% homology with the amino acid sequence shown in any of SEQ ID NOs: 8-11
- the amino acid sequence of CDR3 ⁇ comprising SEQ ID NOs: 12-17
- the amino acid sequence of CDR1 ⁇ comprises any one of SEQ ID NO: 18-22 or comprises It has at least 80% homology with the amino acid sequence shown in any one of SEQ ID NOs: 18-22
- the amino acid sequence of CDR2 ⁇ comprises any one of SEQ ID NO: 23-27 or contains any one of SEQ
- the CDRs are selected from any of the following groups:
- the second aspect of the present invention provides an ⁇ -chain polypeptide that binds to CMV pp65, the ⁇ -chain polypeptide comprising CDR1 ⁇ , CDR2 ⁇ , and/or CDR3 ⁇ .
- the amino acid sequence of CDR1 ⁇ comprises any one of SEQ ID NO: 4-7 or comprises at least 80% homology with the amino acid sequence shown in any one of SEQ ID NO: 4-7
- the amino acid sequence of CDR2 ⁇ comprises Any of SEQ ID NOs: 8-11 or comprising at least 80% homology with the amino acid sequence shown in any of SEQ ID NOs: 8-11
- the amino acid sequence of CDR3 ⁇ comprising SEQ ID NOs: 12-17 Any of or comprising at least 80% homology to the amino acid sequence set forth in any of SEQ ID NOs: 12-17.
- the ⁇ -chain polypeptide comprises any of the following groups of CDR1 ⁇ -CDR3 ⁇ :
- the third aspect of the present invention provides a ⁇ -chain polypeptide that binds to CMV pp65, the ⁇ -chain polypeptide comprises CDR1 ⁇ , CDR2 ⁇ and/or CDR3 ⁇ .
- the amino acid sequence of CDR1 ⁇ comprises any one of SEQ ID NOs: 18-22 or comprises at least 80% homology with the amino acid sequence shown in any one of SEQ ID NOs: 18-22
- the amino acid sequence of CDR2 ⁇ comprises Any of SEQ ID NOs: 23-27 or comprising at least 80% homology to the amino acid sequence shown in any of SEQ ID NOs: 23-27
- the amino acid sequence of CDR3 ⁇ comprising SEQ ID NOs: 28-33 Any of or comprising at least 80% homology to the amino acid sequence set forth in any of SEQ ID NOs: 28-33.
- the ⁇ chain polypeptide comprises any of the following groups of CDR1 ⁇ -CDR3 ⁇ :
- the fourth aspect of the present invention provides a T cell antigen receptor that specifically binds to CMV pp65.
- the binding epitope of CMV pp65 comprises any one, two or a combination of three of SEQ ID NOs: 1-3.
- the T cell antigen receptor specifically binds to the antigen peptide from the cytomegalovirus (CMV) phosphoprotein pp65 through the presentation of major histocompatibility complex molecules (MHC).
- CMV cytomegalovirus
- MHC major histocompatibility complex molecules
- the T cell antigen receptor comprises at least one ⁇ chain variable region and/or ⁇ chain variable region.
- the T cell antigen receptor is an ⁇ heterodimer.
- the T cell antigen receptor comprises CDR1 ⁇ -CDR3 ⁇ of ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of ⁇ chain.
- the amino acid sequence of CDR3 ⁇ comprises any one of SEQ ID NO: 12-17 or comprises at least 80% homology with the amino acid sequence shown in any one of SEQ ID NO: 12-17.
- the amino acid sequence of CDR3 ⁇ comprises any one of SEQ ID NOs: 28-33 or comprises at least 80% homology with the amino acid sequence shown in any one of SEQ ID NOs: 28-33.
- the amino acid sequence of CDR1 ⁇ comprises any one of SEQ ID NOs: 4-7 or comprises at least 80% homology with the amino acid sequence shown in any one of SEQ ID NOs: 4-7.
- the amino acid sequence of CDR2 ⁇ comprises any one of SEQ ID NOs: 8-11 or comprises at least 80% homology with the amino acid sequence shown in any one of SEQ ID NOs: 8-11.
- the amino acid sequence of CDR1 ⁇ comprises any one of SEQ ID NO: 18-22 or comprises at least 80% homology with the amino acid sequence shown in any one of SEQ ID NO: 18-22.
- the amino acid sequence of CDR2 ⁇ comprises any one of SEQ ID NO: 23-27 or comprises at least 80% homology with the amino acid sequence shown in any one of SEQ ID NO: 23-27.
- the CDR1 ⁇ -CDR3 ⁇ and CDR1 ⁇ -CDR3 ⁇ comprise any of the following groups:
- the amino acid sequence of its beta chain comprises any one of SEQ ID NOs: 247-252 or at least 80% of the amino acid sequence shown in any one of SEQ ID NOs: 247-252 homology.
- the amino acid sequence of its ⁇ chain comprises any one of SEQ ID NOs: 253-258 or at least 80% of the amino acid sequence shown in any one of SEQ ID NOs: 253-258 homology.
- the ⁇ chain is directly or indirectly connected to the amino acids of the ⁇ chain, preferably indirectly, more preferably through fp2A (furin-SGSG-p2A).
- amino acid sequence of fp2A comprises SEQ ID NO:259.
- the connecting sequence of the ⁇ chain and the ⁇ chain can be ⁇ chain, fp2A and ⁇ chain, or, ⁇ chain, fp2A and ⁇ chain.
- the amino acid sequence thereof comprises any one of SEQ ID NO: 36, 38, 40, 42, 44 or 46 or comprises and SEQ ID NO: 36, 38, 40, 42, 44 or 46 any of the indicated amino acid sequences have at least 80% homology.
- the fifth aspect of the present invention provides an antibody or an antigen-binding fragment thereof, which specifically binds to CMV pp65.
- the binding epitope of CMV pp65 comprises any one, two or three combinations of SEQ ID NOs: 1-3.
- the antibody or antigen-binding fragment thereof comprises CDR H1-CDR H3 of the heavy chain and/or CDR L1-CDR L3 of the light chain.
- amino acid sequence of the light chain of the antibody or its antigen-binding fragment comprises any one of SEQ ID NO: 247-252 or the amino acid sequence shown in any one of SEQ ID NO: 247-252 have at least 80% homology.
- amino acid sequence of the heavy chain of the antibody or its antigen-binding fragment comprises any one of SEQ ID NO: 253-258 or the amino acid sequence shown in any one of SEQ ID NO: 253-258 have at least 80% homology.
- the antibody or antigen-binding fragment thereof is a single-domain antibody or a single-chain antibody scFv.
- the heavy chain of the antibody or its antigen-binding fragment is directly or indirectly connected to the amino acid of the light chain, preferably indirectly, more preferably via p2A.
- described p2A is fp2A, and its amino acid sequence comprises SEQ ID NO:259.
- connection sequence of the heavy chain and the light chain can be heavy chain, fp2A and light chain, or light chain, fp2A and heavy chain.
- the antibody or antigen-binding fragment thereof may further comprise Fab, Fab', Fab'-SH, Fv, scFv, (Fab')2, single domain antibody, diabody (dAb) or linear antibody Fragment.
- the amino acid sequence of the antibody or antigen-binding fragment thereof comprises any one of SEQ ID NOs: 36, 38, 40, 42, 44 or 46 or comprises and SEQ ID NO:
- the amino acid sequences shown in any of 36, 38, 40, 42, 44 or 46 have at least 80% homology.
- the sixth aspect of the present invention provides a nucleic acid encoding the above-mentioned CDR.
- the seventh aspect of the present invention provides a nucleic acid encoding the ⁇ chain of the T cell antigen receptor of the present invention or the above-mentioned ⁇ chain polypeptide.
- the nucleic acid sequence encoding the beta chain of the T cell antigen receptor comprises any one of SEQ ID NOs: 260-265 or is at least 80% identical to the nucleic acid sequence shown in any one of SEQ ID NOs: 260-265 origin.
- the eighth aspect of the present invention provides a nucleic acid encoding the ⁇ chain of the T cell antigen receptor of the present invention or the above-mentioned ⁇ chain polypeptide.
- the nucleic acid sequence encoding the alpha chain of the T cell antigen receptor comprises any one of SEQ ID NOs: 266-271 or is at least 80% identical to the nucleic acid sequence shown in any one of SEQ ID NOs: 266-271 origin.
- the ninth aspect of the present invention provides a nucleic acid encoding the T cell antigen receptor or antibody or antigen-binding fragment thereof of the present invention.
- a nucleic acid sequence encoding fp2A is used to connect the ⁇ chain and the ⁇ chain encoding the T cell antigen receptor. Further preferably, the nucleic acid sequence encoding fp2A comprises SEQ ID NO: 272.
- the nucleic acid sequence encoding the T-cell antigen receptor comprises any one of SEQ ID NO: 37, 39, 41, 43, 45 or 47 or comprises and SEQ ID NO: 37, 39, 41, 43, 45 or 47 Any of the indicated nucleic acid sequences have at least 80% homology.
- the nucleic acid sequence encoding the antibody or antigen-binding fragment thereof comprises any one of SEQ ID NO: 37, 39, 41, 43, 45 or 47 or a
- the nucleic acid sequences shown in any of 47 have at least 80% homology.
- the nucleic acid sequence of the present invention may be single-stranded or double-stranded, and may be DNA or RNA.
- the nucleic acid sequence may be codon-optimized.
- the codon optimization includes changing a large number of rare codons used by viruses and the like into corresponding mammalian codons and/or removing mRNA unstable motifs and/or cryptic splice sites.
- a tenth aspect of the present invention provides an expression vector, the expression vector comprising any one of the nucleic acids of the present invention.
- the expression vector can be expressed in vivo or in vitro or in vitro. Further preferably, the expression vector is continuously expressed at a high level in cells in vivo.
- the expression vector may be a prokaryotic expression vector or a retroviral vector.
- the prokaryotic expression vector is Escherichia coli series.
- the expression vector is pET-26b or pET28a+.
- Rous Sarcoma Virus (RSV), Lentivirus, Human Immunodeficiency Virus (HIV), Murine Leukemia Virus (MLV), Equine Infectious Anemia Virus (EIAV), Mouse Breast Cancer Virus (MMTV), Fujinami Sarcoma virus (FuSV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine leukemia virus (Mo-MLV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV) ), avian myelocytosis virus 29 (MC29) or avian myeloblastosis virus (AEV) and so on.
- the expression vector is a lentiviral expression vector.
- the expression vector is pHAGE-IRES-RFP.
- the connecting sequence of ⁇ chain, ⁇ chain and backbone vector in the expression vector is promoter, ⁇ chain, fp2A, ⁇ chain, IRES and RFP sequence.
- the eleventh aspect of the present invention provides a host cell, the host cell comprising the nucleic acid or expression vector of any one of the present invention.
- the host cell may be eukaryotic or prokaryotic. More preferably, the host cells are yeast cells, 293 cells, CHO cells, Escherichia coli and the like.
- the host cell is Stbl3, BL21 or transetta.
- the twelfth aspect of the present invention provides an immune cell that expresses the CDR, ⁇ chain polypeptide, ⁇ chain polypeptide, T cell antigen receptor or antibody or antigen-binding fragment thereof of the present invention.
- the immune cells comprise one or more of the heterologous nucleic acid sequences described in any of the present invention.
- the immune cells include but are not limited to stem cells and lymphocytes (including T cells and B cells). Further, the immune cells are B cells, and the B cells express the above-mentioned antibody or antigen-binding fragment thereof.
- the immune cells are T cells, and the T cell antigen receptor structure of the T cells is as defined above.
- the T cells can be CD4+T, CD8+T and the like.
- the immune cells are derived from T cells isolated from the subject.
- the immune cells are T cells from a CMV seronegative donor.
- the thirteenth aspect of the present invention provides a method for preparing an immune cell, comprising adding a nucleic acid encoding the above-mentioned CDR, ⁇ -chain polypeptide, ⁇ -chain polypeptide, antibody or antigen-binding fragment thereof, or the T cell antigen receptor The sequences were transfected into immune cells for expression.
- the immune cells include but are not limited to stem cells and lymphocytes (including T cells and B cells). Further, the immune cells are B cells, and the B cells express the above-mentioned antibody or antigen-binding fragment thereof.
- the immune cells are T cells, and the T cell antigen receptor structure of the T cells is as defined above.
- a guide targeting endogenous TCR can be constructed into a lentiviral vector, and co-transfected with a packaging plasmid and a transfection reagent to T cells.
- a fourteenth aspect of the present invention provides a method for preparing recombinant T cells, comprising the following steps:
- step 3 Deliver the nucleic acid obtained in step 1) to the primary T cells described in step 2) to obtain T cells expressing any of the CDRs, ⁇ -chain polypeptides, ⁇ -chain polypeptides, antibodies or antigen-binding fragments thereof of the present invention Recombinant T cells with antigen receptors.
- the T cells are selected from hematopoietic stem cells or peripheral blood lymphocyte (PBL)-derived T cells.
- PBL peripheral blood lymphocyte
- a fifteenth aspect of the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof or a T cell antigen receptor, comprising the following steps:
- step (3) transforming the expression vector obtained in step (2) into a host cell, and then inducing its expression;
- the positive T cells specifically bind to the cytomegalovirus (CMV) phosphoprotein pp65 antigen peptide presented by MHC.
- CMV cytomegalovirus
- the cytomegalovirus (CMV) phosphoprotein pp65 antigen peptide complex presented by MHC is a monomeric or multimeric complex.
- the sixteenth aspect of the present invention provides a multimeric complex comprising the T cell antigen receptor according to any one of the present invention.
- the multimeric complex further includes a monomer, a biotin molecule, and a streptavidin molecule or avidin molecule, wherein the monomer includes the ⁇ chain extracellular region of the MHC molecule and the ⁇ 2m chain and An antigenic peptide, the monomer is coupled to the biotin molecule, the biotin molecule is bound to the streptavidin or avidin molecule.
- the C-terminus of the extracellular region of the ⁇ chain of the MHC molecule is connected to an avi-tag sequence.
- the extracellular region of the ⁇ chain of the MHC molecule does not contain a signal peptide sequence. And add the M amino acid in front of the mature peptide sequence.
- the ⁇ 2m chain does not contain a signal peptide sequence.
- two amino acids M and A are added in front of the mature peptide sequence.
- the ⁇ 2m chain does not contain a signal peptide and two amino acids, preferably M and A, are added before the mature peptide sequence.
- the antigenic peptide comprises any one or a combination of two or more of SEQ ID NOs: 1-3.
- the multimeric complex comprises:
- T cell antigen receptor preferably comprising any one of SEQ ID NO: 36, 38, 40, 42, 44 or 46 or comprising any one of SEQ ID NO: 36, 38, 40, 42, 44 or 46
- One of the indicated amino acid sequences has at least 80% homology.
- the monomer includes an antigenic peptide, an ⁇ -chain extracellular region of an MHC molecule whose C-terminal is connected to the avi-tag sequence, and a ⁇ 2m chain that does not contain a signal peptide;
- the antigenic peptide is selected from SEQ ID NO: Any one or a combination of two or more of 1-3;
- Streptavidin molecule or avidin molecule wherein, the monomer is coupled to the biotin molecule, and the biotin molecule binds to the streptavidin or avidin.
- the MHC molecules are MHC class I molecules or MHC class II molecules. More preferably, the MHC molecules are MHC class I molecules.
- the MHC molecules are selected from HLA-A*0201, HLA-A*2402 and HLA-A*1101.
- the amino acid sequence of the ⁇ chain of the MHC molecule is any one of SEQ ID NO: 48, 50 or 52 or any one of SEQ ID NO: 48, 50 or 52
- the amino acid sequences shown have at least 80% homology.
- the nucleotide sequence of the ⁇ chain of the MHC molecule is such as any one of SEQ ID NO: 49, 51 or 53 or any one of SEQ ID NO: 49, 51 or 53
- One of the indicated nucleotide sequences has at least 80% homology.
- the amino acid sequence of the ⁇ 2m chain of the MHC molecule such as the amino acid sequence shown in any one of SEQ ID NO: 54 or any one of SEQ ID NO: 54, has at least 80 % homology.
- the nucleotide sequence of the ⁇ 2m chain of the MHC molecule as shown in any one of SEQ ID NO: 55 or the amino acid sequence shown in any one of SEQ ID NO: 55, has At least 80% homology.
- the monomer further includes chemical modification, mutation, insertion and/or deletion of at least one amino acid.
- the ⁇ chain extracellular region of the MHC molecule and the ⁇ 2m chain are non-covalently bound.
- the multimeric complex comprises at least one monomer.
- each monomer is coupled to at least one biotin molecule.
- the seventeenth aspect of the present invention provides a preparation method of any of the multimeric complexes of the present invention, comprising the following steps:
- step II Refolding the antigen peptide, the ⁇ 2m chain obtained in step I) and the extracellular region of the ⁇ chain of the MHC molecule connected with the avi-tag sequence at the C-terminus to prepare a monomer;
- step III biotinylating the monomer prepared in step II) to obtain a biotinylated monomer
- step IV reacting the biotinylated monomer obtained in step III) with fluorescently labeled streptavidin or avidin to prepare an antigen peptide-MHC molecule tetramer;
- step IV Co-incubating the antigen peptide-MHC molecule tetramer obtained in step IV) with T cells to form a T cell antigen receptor and antigen peptide-MHC molecule tetramer complex.
- the nucleotide sequence of the extracellular region of the ⁇ chain of the MHC molecule and the nucleotide sequence of the ⁇ 2m chain of the MHC molecule that encode the C-terminal connection of the avi-tag sequence are respectively cloned, and after being connected to the carrier, Transform into expression bacteria for culture, add inducer, and extract inclusion bodies.
- the expressing bacteria are cultured until the OD 600 value is between 0.2-0.4.
- the final molar concentration of the inducer after adding is 0.5-1 mM.
- the expression is induced for 4-6h.
- the step II) comprises the renaturation and folding of the ⁇ 2m chain, that is, the antigen peptide, the ⁇ 2m chain of the MHC molecule, and the extracellular region of the MHC molecule ⁇ chain with the C-terminal connected to the avi-tag sequence are sequentially added to the dilution buffer in a light-proof water bath,
- the renaturation and folding of the ⁇ 2m chain includes denaturation of inclusion bodies, addition of protease inhibitors, and then dialysis.
- the molar ratio of the antigenic peptide, the ⁇ 2m chain without the signal peptide and the ⁇ chain connected to the C-terminal of the avi-tag sequence is (30-50):(2-2.5):1. More preferably, it is 40:2:1.
- the step II) further comprises a step of purifying the monomer.
- the biotinylation in the step III) is to combine the monomer with BiomixA and BiomixB under the catalysis of BirA enzyme.
- the step III) also includes a step of purifying the biotinylated monomer.
- the molar ratio of the reaction of the monomer and streptavidin in the step IV) is (4-7):1.
- the eighteenth aspect of the present invention provides an application of the above-mentioned multimeric complex in the preparation, screening or detection of the antibody or antigen-binding fragment thereof or T cell antigen receptor of the present invention.
- a nineteenth aspect of the present invention provides a method for preparing a T cell antigen receptor, comprising the following steps:
- step II folded the ⁇ chain extracellular region and ⁇ 2m chain of the MHC molecule obtained in step I) with the avi-tag sequence connected to the C-terminus to prepare a monomer;
- step III biotinylating the monomer prepared in step II) to obtain a biotinylated monomer
- step IV reacting the biotinylated monomer obtained in step III) with fluorescently labeled streptavidin or avidin to prepare an antigen peptide-MHC molecule tetramer;
- step IV) Co-incubate the antigen peptide-MHC molecule tetramer obtained in step IV) with T cells to form a T cell antigen receptor and antigen peptide-MHC molecule tetramer complex, and separate and obtain T cell antigen receptor.
- the twentieth aspect of the present invention provides any one of the CDRs of the present invention, any one of the ⁇ chain polypeptides of the present invention, any one of the ⁇ chain polypeptides of the present invention, and any one of the T cells of the present invention.
- Antigen receptor, antibody or antigen-binding fragment thereof of any of the present invention nucleic acid of any of the present invention, expression vector of the present invention, host cell of the present invention, immune cell of the present invention .
- the CMV-related disease is selected from neonatal CMV inclusion body disease, acutely acquired CMV infection or diseases caused by CMV infection in immunocompromised persons.
- the CMV-related disease is selected from liver, spleen or central nervous system disease, disability, infectious mononucleosis, skeletal muscle pain, CMV retinitis, gastrointestinal CMV or brain inflammation and so on.
- the twenty-first aspect of the present invention provides any of the CDRs of the present invention, the ⁇ chain polypeptide of any of the present invention, the ⁇ chain polypeptide of any of the present invention, and the T of any of the present invention.
- Cellular antigen receptors, antibodies or antigen-binding fragments thereof of any of the present invention, nucleic acids of any of the present invention, expression vectors of the present invention, host cells of the present invention, immunizations of the present invention Use of cells, multimeric complexes of any one of the present invention in T cell labeling, detection, cell sorting or activation.
- a twenty-second aspect of the present invention provides a pharmaceutical composition comprising any of the following groups:
- the pharmaceutical composition may further comprise pharmaceutically acceptable adjuvants.
- the pharmaceutical composition can also be used together with other therapeutic agents.
- the therapeutic agent may be an immunomodulatory agent.
- a twenty-third aspect of the present invention provides a kit, the kit comprising any of the following groups:
- a twenty-fourth aspect of the present invention provides a method for detecting cytomegalovirus (CMV) phosphoprotein pp65, the method comprising combining a sample to be detected with the antibody or antigen-binding fragment thereof or T cell of the present invention The antigen receptor is contacted and then the complex formed by the cytomegalovirus (CMV) phosphoprotein pp65 and the antibody or its antigen binding fragment or T cell antigen receptor is detected.
- CMV cytomegalovirus
- the detection of cytomegalovirus (CMV) phosphoprotein pp65 is to detect the presence or content of cytomegalovirus (CMV) phosphoprotein pp65.
- the presence indicates presence or absence, and the content may be the expression amount or protein concentration.
- the antibody or antigen-binding fragment thereof or T cell antigen receptor includes a detectable label.
- the marker may be His and/or HA.
- the method for detecting cytomegalovirus (CMV) phosphoprotein pp65 described in the present invention is not a method for diagnosing diseases.
- the sample to be tested is not an organism or its ex vivo tissue or cell, and secondly, even if the cytomegalovirus (CMV) phosphoprotein pp65 is present in the organism or contains a certain concentration or expression level of the cytomegalovirus (CMV) phosphoprotein pp65 It's not a sure thing, it's just a possibility.
- the twenty-fifth aspect of the present invention provides a method for treating and/or preventing CMV-related diseases, the method comprising administering to an individual an effective amount of the antibody or antigen-binding fragment thereof of the present invention, the T Cellular antigen receptor, the nucleic acid, the expression vector, the host cell, the immune cell or the pharmaceutical composition.
- the method includes the step of adoptively transferring T cells expressing the T cell antigen receptor of the present invention to a subject.
- the method comprises localizing the T cell antigen receptor of the present invention in the vicinity of a disease associated with CMV (preferably a tumor or metastatic tumor) to increase the efficacy of the toxin or immunostimulatory agent.
- a disease associated with CMV preferably a tumor or metastatic tumor
- CMV reactivation after organ transplantation (eg kidney, liver, pancreas, intestine, cornea), tissue transplantation, cell transplantation (islet cells, limbal stem cells) or stem cell therapy.
- organ transplantation eg kidney, liver, pancreas, intestine, cornea
- tissue transplantation eg kidney, liver, pancreas, intestine, cornea
- cell transplantation islet cells, limbal stem cells
- stem cell therapy eg.g stem cells
- the T cells expressing the T cell antigen receptor of the present invention are derived from a subject.
- the T cells expressing the T cell antigen receptor of the present invention are derived from the same donor as the hematopoietic stem cells, organs, tissues, cells or stem cells.
- the twenty-sixth aspect of the present invention provides a method for diagnosing a disease associated with CMV, the method comprising sampling, contacting the sample with the antibody or antigen-binding fragment thereof or T cell antigen receptor of the present invention, The complexes formed by CMV pp65 with antibodies or antigen-binding fragments thereof or T-cell antigen receptors are then detected.
- the antibody or antigen-binding fragment thereof or T-cell antigen receptor includes a detectable label.
- the TCR of the present invention can specifically recognize the corresponding CMV pp65 antigen peptide-MHC molecular complex, activate TCR T cells, and then produce high levels of cytokines IFN ⁇ , IL2, TNF ⁇ , and both in vivo and in vitro tests significantly kill tumor cells.
- the "T cell antigen receptor" of the present invention is a molecule capable of recognizing a peptide when presented by an MHC molecule or its tetramer.
- the "tumor” in the present invention includes but is not limited to pancreatic cancer, liver cancer, colon cancer, rectal cancer, gastric cancer, lymphoma, basal cell carcinoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, uterine cancer Endometrial cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma , melanoma, myelodysplastic syndrome, and sarcoma.
- the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia;
- the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma , T-cell lymphoma, and Waldenstrom macroglobulinemia;
- the sarcoma is selected from the group consisting of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhab
- CMV-related diseases include diseases caused by CMV pp65 infection, including neonatal CMV inclusion body disease, which can seriously affect the liver, spleen and central nervous system and disability. Also included are acute acquired CMV infection, similar to infectious mononucleosis, including fever, skeletal muscle pain, and more. Also included are immunocompromised people, such as people who have had an organ transplant, or people with HIV whose infection can lead to CMV retinitis, gastrointestinal CMV, and encephalitis, to name a few.
- the "antigen-binding fragments" of the present invention include, but are not limited to: Fab fragments, which have VL, CL, VH and CH1 domains; Fab' fragments, which have one or more cysteines at the C-terminus of the CH1 domain Fab fragments of residues; Fd fragments, which have VH and CH1 domains; Fd' fragments, which have VH and CH1 domains and one or more cysteine residues C-terminal to the CH1 domain; Fv fragments, which have antibodies The VL and VH domains of a single arm of a Bivalent fragments of 'fragments; single-chain antibody molecules (e.g., single-chain Fv; scFv); "diabodies" with two antigen-binding sites comprising a variable domain (VL) linked to a light chain in the same polypeptide chain a heavy chain variable domain (VH); a "linear antibody” comprising a pair of tandem Fd segments (VH-CH1-VH
- CDRs as used in the present invention are short fragments of immunoglobulins (Ig) or T cell antigen receptors (TCRs) that bind epitopes alone or in combination with other CDRs.
- the immunoglobulin can be an antibody, and the CDRs correspond to the complementarity determining regions in the variable sequence of the antibody. For each variable region, there are three CDRs in each variable region of the heavy and light chains, which are referred to as CDR1, CDR2, and CDR3 of the heavy or light chain, respectively.
- the CDRs are present in the ⁇ chain or the ⁇ chain, and there are three CDRs in the ⁇ chain or the ⁇ chain, which are called CDR1, CDR2 and CDR2 of the ⁇ chain or ⁇ chain, respectively.
- CDR3 The exact boundaries of these CDRs are defined differently from system to system.
- the system described by Kabat et al (Kabat et al, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides unambiguous residue numbering for antibody variable regions system, but also provides residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs.
- Each complementarity determining region may comprise amino acid residues from a "complementarity determining region" as defined by Kabat. Chothia et al. ( Chothia & Lesk, J. Mol. Biol, 196: 901-917 (1987) and Chothia et al., Nature 342: 877-883 (-1989)) found that certain sub-portions within the Kabat CDRs adopt nearly identical peptide backbone conformations , although there is a large diversity at the amino acid sequence level. These subsections are called L1, L2 and L3 or H1, H2 and H3, respectively, where “L” and "H” represent the light and heavy chain regions, respectively.
- the “antibody variable region” refers to the light and heavy chains of the antibody molecule comprising the Part of the amino acid sequence of the complementarity determining regions (CDRs, ie CDR1, CDR2 and CDR3) and framework regions (FR).
- VH refers to the variable domain of the heavy chain.
- VL refers to the variable domain of the light chain.
- the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides, but still have the activity described in the present invention.
- Prevention in the context of the present invention refers to all acts of suppressing symptoms or delaying the stress of a particular symptom by administering the product of the present invention.
- Diagnosing means to ascertain whether a patient has a disease or condition in the past, at the time of diagnosis or in the future, or to ascertain the progression or possible future progression of a disease, or to assess a patient's response to treatment.
- Treatment means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease, but not necessarily all disease-related signs, Complete elimination of a symptom, disorder, or disorder, and refers to therapeutic interventions that improve the signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.
- an "effective amount” as used herein refers to the amount or dose of the product of the present invention that provides the desired treatment or prevention following administration to a patient or organ in single or multiple doses.
- the "product" of the present invention includes, but is not limited to, the antibody or its antigen-binding fragment of the present invention, the T cell antigen receptor, the nucleic acid, the expression vector, the host cell, Said immune cells or said multimeric complex, and other auxiliary or synergistic reagents with the above-mentioned products.
- the "product" in the present invention can be a pharmaceutical composition such as a kit, a chip, an antibody conjugate or a multifunctional antibody.
- the "subject" in the present invention includes, but is not limited to, human or non-human mammals.
- the non-human mammals include but are not limited to mice, rats, monkeys, pigs or rabbits and the like.
- the "homology" in the present invention means that in terms of using amino acid sequence or nucleotide sequence, those skilled in the art can adjust the sequence according to actual work needs without changing the main structure or function of the original sequence.
- the used sequence has (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% compared with the specific sequence described in the present invention %, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43% , 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60 %, 70%
- SEQ ID NO: 12, 13, 14, 15, 16 or 17 can be adjusted according to actual work needs, including one or more changes such as substitution, deletion and/or insertion of one or more amino acids, etc., truncated, or lengthened at one or both ends, as long as it maintains more than 80% homology and retains the ability to bind to pp65 epitope:MHC complexes or pp65 epitope:MHC molecule tetramers.
- the alterations can be substitutions, additions or deletions. It can be substitution between amino acids of the same polarity, substitution between amino acids of the same charge, substitution between uncharged amino acids, substitution between aliphatic amino acids, substitution between aromatic amino acids, or between non-polar amino acids. Substitutions between amino acids or substitutions between amino acids of side chain moieties with related properties.
- the basic side chain includes but is not limited to lysine, arginine or histidine.
- Acidic side chains include, but are not limited to, aspartic acid or glutamic acid.
- Uncharged amino acids include, but are not limited to, aspartyl, glutamine, serine, threonine, or tyrosine.
- Non-polar side chains include, but are not limited to, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, or cysteine .
- the said at least 80% includes but is not limited to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
- Figure 1 SDS PAGE detection results of antigenic peptide-MHC monomers, where M is protein Marker, and bands 1 and 2 are monomers;
- Figure 2 The detection results of tetramer fishing specific T cells, the first row is the comparison results of self-developed A0201-NLV tetramer and commercial tetramer fishing specific T cells, and the second row is the self-developed A0201-NLV tetramer The comparison results of the development of A2402-QYD tetramer and the commercialized tetramer to capture specific T cells;
- Figure 3 The positive rate of C44TCR-T cells detected by A1101-ATV tetramer
- Figure 4 Schematic diagram of the connection of TCR ⁇ chain and ⁇ chain in pHAGE vector, wherein the sequence of connection is promoter, ⁇ chain, furin-p2A, ⁇ chain, IRES and RFP sequence;
- Figure 5 Flow cytometry detection of TCR membrane surface expression
- Figure 5A is the membrane surface expression of HLA-A*A0201NLVPMVATV-specific TCR (C2, C22, C29)
- Figure 5B is HLA-A*A2402QYDPVAALF-specific TCR ( C27, C30) membrane surface expression
- Figure 5C shows the membrane surface expression of HLA-A*A1101ATVQGQNLK-specific TCR (C44, C45), among which, BV421 is one of the series of fluorescein, and the full name of BV is Brilliant Violet ;
- Figure 6 Flow cytometric detection of the affinity of TCR binding to the CMV pp65 tetramer probe.
- Figure 6A is the affinity detection result of HLA-A*A0201 NLVPMVATV-specific TCR (C2, C22, C29) binding to CMV pp65 tetramer probe
- Figure 6B is HLA-A*A2402 QYDPVAALF-specific TCR (C27, C30) Affinity detection result of binding to CMV pp65 tetramer probe
- Figure 6C is the affinity detection result of HLA-A*A1101 ATVQGQNLK-specific TCR (C44, C45) binding to CMV pp65 tetramer probe;
- Figure 7 Release levels of C2-TCRT, C22-TCRT, C29-TCR T cytokines IL2 (Figure 7B), TNF ⁇ ( (Figure 7C) and IFN ⁇ ( ( Figure 7D) and target cell luciferase levels (Figure 7 7A) detection result diagram, wherein, C2, C22, C29 are the TCR prepared by embodiment 2, NE represents blank control group, NT represents only T cell group, Raji is the Raji cell that does not transfer pp65, 1G4 represents for NY-ESO -1 specific TCR;
- FIG. 8 The detection results of the release levels of C27-TCR T, C30-TCR T cytokines IL2 (Figure 8B), TNF ⁇ (Figure 8C) and IFN ⁇ ( Figure 8D) and the level of target cell luciferase (Figure 8A), wherein, C27 and C30 are TCRs prepared in Example 2, NE represents blank control group, NT represents only T cell group, Raji represents Raji cells without pp65 transfection, and 1G4 represents TCR specific for NY-ESO-1;
- Figure 9 The detection results of C44-TCR T and C45-TCR T cytokine IFN ⁇ release levels (Figure 9B) and target cell luciferase levels (Figure 9A), wherein C44 and C45 are TCRs prepared in Example 2 , NE represents blank control group, NT represents only T cell group, Raji represents Raji cells without pp65 transfection, and 1G4 represents TCR specific for NY-ESO-1;
- FIG. 10 Evaluation of C22-TCR inhibiting tumor growth in mice in an animal model of lymphoma
- FIG 11 The lymphoma animal model, the statistical results of the survival rate of mice in the no T cell injection group (PBS), the control T cell injection group (NC), and the CMV TCR-T injection group (C22-TCR);
- Figure 12 Lymphoma animal model, statistical results of mouse body weight in no T cell injection group (PBS), control T cell injection group (NC), and CMV TCR-T injection group (C22-TCR);
- Figure 13 Lymphoma animal model, statistical results of TCR T cell-specific proliferation in mice of no T cell injection group (PBS), control T cell injection group (NC), and CMV TCR-T injection group (C22-TCR), The results showed that the results of the PBS group and the NC group were both 0, and the 6 broken lines in Figure 13 were the statistical results of the specific proliferation of TCR T cells in 6 different mice in the C22-TCR group;
- Figure 14 Evaluation of C27-TCR inhibiting tumor growth in mice in an animal model of lymphoma
- FIG. 15 Lymphoma animal model, statistical results of tumor growth of mice in no T cell injection group (PBS), control T cell injection group (NC), and CMV TCR-T injection group (C27-TCR);
- FIG. 16 Lymphoma animal model, statistical results of TCR T cell-specific proliferation in mice in no T cell injection group (PBS), control T cell injection group (NC), and CMV TCR-T injection group (C27-TCR);
- Figure 17 Evaluation of the inhibition of tumor growth by C45-TCR in mice in an animal model of lymphoma
- FIG. 18 Lymphoma animal model, statistical results of tumor growth in mice of no T cell injection group (PBS), control T cell injection group (NC), and CMV TCR-T injection group (C45-TCR);
- FIG 19 Lymphoma animal model, statistical results of TCR T cell-specific proliferation in mice of no T cell injection group (PBS), control T cell injection group (NC), and CMV TCR-T injection group (C45-TCR).
- HLA-A*0201 (its amino acid sequence is as shown in SEQ ID NO: 48, nucleotide sequence is as shown in SEQ ID NO: 49), HLA-A*2402 (its amino acid sequence is as shown in SEQ ID NO: 49), HLA-A*2402 (its amino acid sequence is as shown in SEQ ID NO: 49) of expression sequence optimization NO: 50, the nucleotide sequence is shown in SEQ ID NO: 51) and HLA-A*1101 (its amino acid sequence is shown in SEQ ID NO: 52, and the nucleotide sequence is shown in SEQ ID NO: 53) ) of the ⁇ chain and ⁇ 2m chain (the amino acid sequence is shown in SEQ ID NO: 54, and the nucleotide sequence is shown in SEQ ID NO: 55).
- the structure of the ⁇ chain is that the extracellular region sequence of the corresponding HLA type ⁇ chain is linked with the Avi-tag sequence, and the BamHI restriction site is separated to provide a biotinylation site.
- the ⁇ 2m chain has the signal peptide sequence removed and two amino acids (M and A) added in front of the mature peptide sequence.
- the expression vector is PET28a+, and the expression strain is transetta or BL21.
- the IPTG concentration was 0.5 mM, and the expression was induced for 4 h. ⁇ -chain and ⁇ 2m-chain protein inclusion bodies were extracted.
- HLA-A*0201 type corresponds to the antigenic epitope NLVPMVATV (SEQ ID NO: 1)
- HLA-A*2402 type corresponds to the antigenic epitope QYDPVAALF (SEQ ID NO: 2)
- HLA-A*1101 type corresponds to the epitope ATVQGQNLK (SEQ ID NO: 3).
- step 3 Folding and purification of pMHC I monomers: adding the antigenic peptide described in step 2), the ⁇ 2m chain renatured protein and the ⁇ chain protein described in corresponding step 1) to the reduction system in order at a molar ratio of 40:2:1 , the folding reaction is 72h.
- the obtained product was purified by Superdex75 10/300GL column.
- the purified product was collected and biotinylated with avidity kit, purified again to obtain the biotinylated monomer, and the purity of the monomer was detected by gel electrophoresis.
- step 4) Combine the biotinylated monomer described in step 3) with APC-labeled streptavidin to obtain corresponding tetramers, which are named A0201-NLVPMVATV-tetramers (referred to as A0201-NLV tetramers for short) ), A2402-QYDPVAALF-tetramer (referred to as A2402-QYD tetramer), A1101-ATVQGQNLK-tetramer (referred to as A1101-ATV tetramer).
- A0201-NLVPMVATV-tetramers referred to as A0201-NLV tetramers for short
- A2402-QYDPVAALF-tetramer referred to as A2402-QYD tetramer
- A1101-ATVQGQNLK-tetramer referred to as A1101-ATV tetramer.
- PBMC peripheral blood mononuclear cells
- TCR-T cells prepare a cell suspension, and the cell density is 1 ⁇ 10 6 cells/mL.
- the SDS PAGE detection results of the monomers are shown in Figure 1.
- Figure 1 As shown in Figure 1, after renaturation and HPLC purification, the obtained monomer clearly shows the heavy chain (the extracellular region of the ⁇ chain with the Avi-tag sequence connected to the C-terminus) and the light chain (the ⁇ 2m chain with the signal peptide region removed) The protein of the corresponding size, and the purity is high.
- the constructed tetramers were co-incubated with corresponding HLA-type PBMC cells to fish for specific T cells.
- the self-developed tetramer has obvious advantages over the commercial tetramer (MBL company) in fishing for specific T cells.
- the self-developed A0201-NLV tetramer for sorting specific T cells is twice as high as the commercial tetramer (MBL, TS-0010-2C).
- the sorting efficiency of different batches is similar, showing extremely high sorting efficiency and stability.
- C22TCR and C29TCR were fished with this A0201-NLV tetramer.
- the commercial A2402-QYD (MBL, TS-0020-2C) tetramer did not detect the corresponding specific T cells, while the self-developed tetramer could detect 0.02-0.05% of the positive population, showing that the self-developed tetramer High sensitivity in sorting specific T cells and high stability across batches of data.
- C27TCR and C30TCR were fished with this A2402-QYD tetramer.
- the TCR (C44TCR) corresponding to the A1101 type can be recognized by both the self-developed and commercialized tetramers (MBL, TS-M012-1) of the same epitope (see Figure 3), and the positive population of the commercialized tetramers accounted for 90.5% of infected cells, self-developed tetramer-positive population accounted for 91% of infected cells. See Figure 5 for the C45TCR results.
- HLA-A*0201-NLVPMVATV-tetramer, HLA-A*2402-QYDPVAALF-tetramer, HLA-A*1101-ATVQGQNLK-tetramer purchased from MBL company or self-made by our company, according to Product instructions and peripheral blood staining, T cells positive for tetramer staining were subjected to flow single cell sorting, and cDNA was obtained by reverse transcription ( IV Reverse Transcriptase, Invitrogen). According to the principle of multiplex PCR, the variable region fragment of TCR ⁇ gene was amplified by two rounds of PCR (KOD-Plus-Neo, TOYOBO).
- the reverse transcription primer is: TRBC1-TCAGGCAGTATCTGGAGTCATTG (SEQ ID NO: 136)
- PCR amplification primers are:
- Upstream primer 1 T cell receptor beta variable region--TRBV_F1 (see SEQ ID NOs: 56-95)
- Upstream primer 2 T cell receptor beta variable region--TRBV_F2 (see SEQ ID NOs: 96-135)
- Downstream Primer 1 T cell receptor beta constant region--TRBC2-GCACCTCCTTCCCATTCACC (SEQ ID NO: 137)
- Downstream Primer 2 T cell receptor beta constant region-TRBC3-GCTTCTGATGGCTCAAACACAG (SEQ ID NO: 138)
- one round of PCR system was 20 ⁇ L, the annealing temperature was 60° C., and the reaction was performed for 30 cycles.
- the second round PCR system is 30 ⁇ L, the annealing temperature is 60°C, and the reaction is carried out for 30 cycles.
- the second round of PCR products were then run on agarose gel, and the bands of the corresponding size were cut and recovered (Tiangengum recovery kit), and sent for sequencing, and the sequencing primer was the downstream primer 2.
- the beta-strand nucleotide sequences of C2, C22, C27, C29, C30, C44, and C45 are shown in SEQ ID NOs: 277, 260-265.
- the reverse transcription primer is: T cell receptor alpha constant region-TRAC1-CGACCAGCTTGACATCACAG (SEQ ID NO: 139)
- PCR amplification primers are:
- Upstream primer 3 T cell receptor alpha variable region-TRAV_F1 (see SEQ ID NOs: 142-186)
- Upstream primer 4 T cell receptor alpha variable region-TRAV_F2 (see SEQ ID NOs: 187-228)
- Downstream primer 3 T cell receptor alpha constant region-TRAC2-GTTGCTCTTGAAGTCCATAGACCTC (SEQ ID NO: 140)
- Downstream primer 4 T cell receptor alpha constant region-TRAC3-CAGGGTCAGGGTCTGGATA (SEQ ID NO: 141)
- one round of PCR system was 20 ⁇ L, the annealing temperature was 60° C., and the reaction was performed for 30 cycles.
- the second round PCR system is 30 ⁇ L, the annealing temperature is 60°C, and the reaction is carried out for 30 cycles.
- the sequencing primer is the downstream primer 4.
- TCR ⁇ , fp2A, TCR ⁇ were amplified by overlap-PCR (KOD-Plus-Neo, TOYOBO) with long primers (including fp2A sequence) to obtain TCR ⁇ -fp2A-TCR ⁇ fragments, named C22, C27, C29, C30, C44 or C45, respectively .
- the amplified primers include upstream primer 5 (Table 1), downstream primer 5, upstream primer 6 (Table 2), and downstream primer 6.
- Downstream primer 6 agggatcctctagactcgagctagcTCAGCTGGACCACAGCCGCA (SEQ ID NO: 242).
- primer 5 and primer 6 were used to amplify TCR ⁇ and TCR ⁇ respectively, the PCR system was 50 ⁇ L, the annealing temperature was 60° C., and the reaction was performed for 30 cycles.
- the PCR product was run and recovered (Tiangengum recovery kit), 1 ⁇ L of the recovered product was taken as a template, and the upstream primer 5 and the downstream primer 6 were used for overlap PCR, the PCR system was 50 ⁇ L, the annealing temperature was 60 ° C, and the reaction was carried out for 30 cycles. . Run agarose gel to obtain a band of about 1800 bp, which is recovered by cutting the gel.
- the lentiviral vector pHAGE-IRES-RFP was double digested with NotI and NheI.
- the digestion system was 40 ⁇ L, containing 1.5 ⁇ L of NotI and NheI respectively, 2-3 ⁇ g of plasmid, digested at 37°C for 6 h, and then added 1 ⁇ L of alkaline phosphatase (NEB) to the system for 1 h to reduce the self-ligation of the plasmid.
- the excised plasmid was recovered by gel running, and the concentration was measured by nanodrop, which was used as a backbone for plasmid construction.
- the TCR was ligated with the linearized pHAGE-IRES-RFP vector after enzymatic cleavage through overlap (see Figure 4), transformed into Stbl3 strain, and cultured on LB plate containing ampicillin for 12 -16h, pick a single clone for sequencing, and the sequencing primers are the primers seq-pHAGE-F and seq-pHAGE-R on the pHAGE carrier, and the downstream primer 4.
- C22 (its nucleotide sequence is shown in SEQ ID NO: 37, and its amino acid sequence is shown in SEQ ID NO: 36), C27 (its nucleotide sequence is shown in SEQ ID NO: 39) shown, its amino acid sequence is shown in SEQ ID NO: 38), C29 (its nucleotide sequence is shown in SEQ ID NO: 41, and its amino acid sequence is shown in SEQ ID NO: 40), C30 (its nucleotide sequence is shown in SEQ ID NO: 40) The sequence is shown in SEQ ID NO: 43, and its amino acid sequence is shown in SEQ ID NO: 42), C44 (its nucleotide sequence is shown in SEQ ID NO: 45, and its amino acid sequence is shown in SEQ ID NO: 44) ), C45 (its nucleotide sequence is shown in SEQ ID NO: 47, its amino acid sequence is shown in SEQ ID NO: 46), C2 (its nucleotide sequence is shown in SEQ ID NO:
- the prepared C2 was used as a control, which differed from C22 by only a few amino acids in the CDR region, as shown in Table 3 for details.
- TRA_oligo1-CACCGTCTCTCAGCTGGTACACGGC SEQ ID NO: 243
- TRA_oligo2-AAACGCCGTGTACCAGCTGAGAGAC SEQ ID NO: 244
- TRB_oligo1-CACCGGGCTCAAACACAGCGACCTC SEQ ID NO: 244
- TRB_oligo2-AAACGAGGTCGCTGTGTTTGAGCCC SEQ ID NO: 246
- the synthetic guide sequences of ⁇ chain and ⁇ chain were constructed into sgRNA-LentiCRISPR-puro and sgRNA-LentiCRISPR-BSD lentiviral vectors, respectively, and co-transfected 293T with the packaging plasmids psPAX2, pMD2.G and PEI transfection reagents in a certain proportion 48h and 72h cell culture supernatants were collected, and the two concentrated viruses were simultaneously infected with the human JurkatT cell line. After 48 hours of infection, appropriate concentrations of puromycin and blasticidin were used to kill the cells until all the cells in the control group of the two drugs died. Surviving cells were cultured by flow sorting single cells into 96-well plates. For the obtained monoclonal cell line, the expression of TCR ⁇ chain and ⁇ chain antibody was used to identify its expression respectively, and the cell line deficient in both chains was the obtained endogenous TCR knockout Jurkat T cells, named JC5.
- pHAGE-TCR plasmids such as C22 and C27 constructed in Example 2 were mixed with the packaging plasmids psPAX2, pMD2.G and the transfection reagent PEI respectively according to a certain ratio to transfect 293T cells.
- Three days after infection, cells were stained with anti-human CD3 and anti-human TCR ⁇ flow antibodies, and cells with the same TCR expression level were sorted and cultured, which was the JC5-TCR cell line.
- the specific C27-TCR and C30-TCR for CMV pp65HLA-A*A2402 QYDPVAALF and the specific C44-TCR and C45-TCR for CMV pp65HLA-A*A1101 ATVQGQNLK can be correctly expressed and displayed on the outside of the cell membrane, and are tetrameric with the corresponding
- the bulk probe has a certain affinity. To sum up, the results show that the TCRs prepared in the examples of the present application have good affinity.
- Example 4 Construction of human primary TCR T cells and in vitro functional detection
- PBMCs mononuclear cells
- the cells were isolated according to the EasySep Human T cell isolation kit (stem cell technologies) product instructions, obtained T cells from PBMC by negative selection, resuspended the cells to 1 ⁇ 10 6 cells/mL in 1640 complete medium containing 100 U/mL IL2, and cultured in anti-CD3/CD28 antibody packs. activated in a petri dish. After 48 hours of activation, the TCR-loaded virus particles (prepared by Example 3) were used to infect T cells with a lentiviral system.
- the infection method was centrifugation at 1500 rpm for 2 hours at 32°C.
- the infection was terminated by changing the medium and continued to be cultured in a 37°C cell incubator.
- TCR-positive cells can be sorted out by flow cytometry to obtain TCRT cells (including the above-mentioned C2, C22, C27, C29, C30, C44 and C45).
- the virus particles loaded with pp65-PURO, HLA-A*0201-BSD, HLA-A*2402-BSD, HLA-A*1101-BSD and luciferase-GFP were infected with the logarithmic growth phase using the lentivirus system.
- Raji cells Through drug screening and flow sorting, Raji cells stably expressing pp65, HLA-A*0201/2402/1101 molecules and luciferase-GFP were obtained, which were named Raji-A0201-pp65-luciferase, Raji- A2402-pp65-luciferase, Raji-A1101-pp65-luciferase.
- Raji/HLA-A0201/pp65 and Raji cells without pp65 were co-cultured with 1G4-TCRT, TCR-C2T, TCR-C22T, TCR-C29T cells in a ratio of 1:1. After 24 hours of culture, cells and supernatants were collected respectively, and the activation of TCR-C22T and TCR-C29T cells and the death of target cells were preliminarily detected.
- TCR-C22T and TCR-C29T Judging from the release levels of extracellular cytokines TNF ⁇ , IL2 and IFN ⁇ (see Figure 7), after co-incubating TCR-C22T and TCR-C29T with target cells, compared with the control group 1G4-TCRT and TCR-C2T, significantly cause the activation of T cells.
- the amount of luciferase released from target cells after lysis can reflect the death of target cells (see Figure 7).
- TCR-C22T and TCR-C29T prepared in the examples of the present application have significant the effect of killing target cells.
- the experimental results prove that the TCR-C22T and TCR-C29T cells constructed in the embodiment of the present invention can be specifically activated by CMV pp65 antigen peptide-presenting cells, and can significantly kill target cells.
- the CDR3 hypervariable regions of TCR ⁇ chain and ⁇ chain have the greatest impact on the function of TCR.
- Existing experiments have confirmed that even if the CDR3 hypervariable regions are highly similar and differ by only one amino acid, their functions will be very different.
- the amino acids after the first position of the CDR3 region of the ⁇ chain and ⁇ chain of TCR E141 were sequentially mutated to alanine and constructed on a lentiviral vector to prepare corresponding TCR-T cells. It was co-cultured with target cells 1:1, and the amount of luciferase and the secretion of cytokines TNF ⁇ , IL2 and IFN ⁇ were detected.
- the unmutated E141 can effectively eliminate target cells after encountering HLA matched-Raji, and specifically produce a large amount of cytokine IL2.
- a single amino acid mutation is sufficient to completely inactivate TCR T cells (for details, see patent CN202010373100.4).
- the mutated TCR although only one amino acid differs, is sufficient to completely inactivate TCR-T cells. It can be seen that although the target is the same, only one amino acid difference can make two nearly identical TCRs have completely different technical effects, let alone multiple amino acids.
- Raji/HLA2402/pp65 and Raji cells without pp65 transfection were co-cultured with 1G4-TCRT, TCR-C27T, and TCR-C30T cells in a ratio of 1:1. After co-culture for 24 hours, The cells and supernatant were collected respectively, and the activation of TCR-C27T and TCR-C30T cells and the inactivation of target cells were preliminarily detected.
- TCR-C27T and TCR-C30T cells Judging from the release levels of extracellular cytokines TNF ⁇ , IL2 and IFN ⁇ (see Figure 8), after co-incubating TCR-C27T and TCR-C30T cells with target cells, compared with the control group 1G4-TCRT, TCR-C27T and TCR-C30T cells can significantly induce T activation of cells. And the amount of luciferase released after lysis from target cells can reflect that TCR-C27T and TCR-C30T can significantly kill target cells (see Figure 8).
- the experimental results prove that the TCR-C27T and TCR-C30T cells constructed in the embodiment of the present invention can be specifically activated by CMV pp65 antigen peptide-presenting cells, and can significantly kill target cells.
- Raji/HLA1101/pp65 and Raji cells without pp65 transfection were co-cultured with 1G4-TCRT, TCR-C44T, and TCR-C45T cells in a ratio of 1:1. After co-cultivation for 24 hours, The cells and supernatant were collected respectively, and the activation of TCR-C44T and TCR-C45T cells and the inactivation of target cells were preliminarily detected. Judging from the release level of extracellular cytokine IFN ⁇ (see Figure 9), after co-incubating TCR-C44T and TCR-C45T cells with target cells, compared with the control group 1G4-TCRT, it can significantly induce T cell activation.
- TCR-C44T and TCR-C45T can significantly kill target cells (see Figure 9).
- the experimental results prove that the TCR-C44T and TCR-C45T cells constructed in the embodiment of the present invention can be specifically activated by CMV pp65 antigen peptide-presenting cells, and can significantly kill target cells.
- mice 3 ⁇ 10 5 Raji-HLA-A*1101/0201/2402-pp65-luciferase tumor cells were inoculated into 5-6 weeks old NOD/Scid IL-2R ⁇ null (NCG) female mice through the tail vein to construct a lymphoma model (See Figures 10, 14, and 17), recorded as the first day, and on the sixth day, the mice were divided into 3 groups, A: PBS injection group (injected with equal volume of PBS); B: control NC cell injection group (NC T cells); C: CMV TCR T injection group (C22/C27/C45-TCR T cells), mice in group B were injected with 1 ⁇ 10 7 NC T cells through the tail vein, and mice in group C were injected with 1 ⁇ 10 NC T cells through the tail vein.
- A PBS injection group (injected with equal volume of PBS)
- B control NC cell injection group (NC T cells)
- C CMV TCR T injection group (C22/C27/C45-TCR T cells)
- mice in group B were
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Abstract
Description
TCR | 上游引物5 |
C2 | atttcaggtgtcgtgaagcggccgcgccaccATGGGCCCCCAGCTCCT(SEQ ID NO:279) |
C22 | atttcaggtgtcgtgaagcggccgcgccaccATGAGCATCGGCCTCCT(SEQ ID NO:229) |
C27 | atttcaggtgtcgtgaagcggccgcgccaccATGAGCATCGGCCTCCT(SEQ ID NO:230) |
C29 | atttcaggtgtcgtgaagcggccgcgccaccATGGACTCCTGGACCTT(SEQ ID NO:231) |
C30 | atttcaggtgtcgtgaagcggccgcgccaccATGGGCACCAGGCTCCT(SEQ ID NO:232) |
C44 | atttcaggtgtcgtgaagcggccgcgccaccATGGGCCCCGGGCTCCT(SEQ ID NO:233) |
C45 | atttcaggtgtcgtgaagcggccgcgccaccATGGGCTCCAGGCTGCT(SEQ ID NO:234) |
Claims (49)
- 一种与CMV pp65结合的决定簇互补区(CDR),其特征在于,所述的CDR选自SEQ ID NO:4-33中的一种或两种以上的组合。
- 根据权利要求1所述的CDR,其特征在于,所述的CDR包括CDR1α-CDR3α和/或CDR1β-CDR3β,其中,CDR1α的氨基酸序列包含SEQ ID NO:4-7中的任一种或包含与SEQ ID NO:4-7中任一种所示氨基酸序列具有至少80%同源性,CDR2α的氨基酸序列包含SEQ ID NO:8-11中的任一种或包含与SEQ ID NO:8-11中任一种所示氨基酸序列具有至少80%同源性,CDR3α的氨基酸序列包含SEQ ID NO:12-17中的任一种或包含与SEQ ID NO:12-17中任一种所示氨基酸序列具有至少80%同源性;CDR1β的氨基酸序列包含SEQ ID NO:18-22中的任一种或包含与SEQ ID NO:18-22中任一种所示氨基酸序列具有至少80%同源性,CDR2β的氨基酸序列包含SEQ ID NO:23-27中的任一种或包含与SEQ ID NO:23-27中任一种所示氨基酸序列具有至少80%同源性,CDR3β的氨基酸序列包含SEQ ID NO:28-33中的任一种或包含与SEQ ID NO:28-33中任一种所示氨基酸序列具有至少80%同源性。
- 一种与CMV pp65结合的α链多肽,其特征在于,所述的α链多肽包含CDR1α、CDR2α、和/或CDR3α,其中,CDR1α的氨基酸序列包含SEQ ID NO:4-7中的任一种或包含与SEQ ID NO:4-7中任一种所示氨基酸序列具有至少80%同源性,CDR2α的氨基酸序列包含SEQ ID NO:8-11中的任一种或包含与SEQ ID NO:8-11中任一种所示氨基酸序列具有至少80%同源性,CDR3α的氨基酸序列包含SEQ ID NO:12-17中的任一种或包含与SEQ ID NO:12-17中任一种所示氨基酸序列具有至少80%同源性。
- 一种与CMV pp65结合的β链多肽,其特征在于,所述的β链多肽包含CDR1β、CDR2β和/或CDR3β,其中,CDR1β的氨基酸序列包含SEQ ID NO:18-22中的任一种或包含与SEQ ID NO:18-22中任一种所示氨基酸序列具有至少80%同源性,CDR2β的氨基酸序列包含SEQ ID NO:23-27中的任一种或包含与SEQ ID NO:23-27中任一种所示氨基酸序列具有至少80%同源性,CDR3β的氨基酸序列包含SEQ ID NO:28-33中的任一种或包含与SEQ ID NO:28-33中任一种所示氨基酸序列具有至少80%同源性。
- 一种抗体或其抗原结合片段,其特征在于,所述的抗体或其抗原结合片段特异性结合CMV pp65,所述的抗体或其抗原结合片段包含重链的CDR H1-CDR H3和/或轻链的CDR L1-CDR L3,其中,CDR H3的氨基酸序列包含SEQ ID NO:12-17中的任一种或包含与SEQ ID NO:12-17中任一种所示氨基酸序列具有至少80%同源性,CDR L3的氨基酸序列包含SEQ ID NO:28-33中的任一种或包含与SEQ ID NO:28-33中任一种所示氨基酸序列具有至少80%同源性。
- 根据权利要求8所述的抗体或其抗原结合片段,其特征在于,CDR H1的氨基酸序列包含SEQ ID NO:4-7中的任一种或包含与SEQ ID NO:4-7中任一种所示氨基酸序列具有至少80%同源性,CDR H2的氨基酸序列包含SEQ ID NO:8-11中的任一种或包含与SEQ ID NO:8-11中任一种所示氨基酸序列具有至少80%同源性,CDR L1的氨基酸序列包含SEQ ID NO:18-22中的任一种或包含与SEQ ID NO:18-22中任一种所示氨基酸序列具有至少80%同源性,CDR L2的氨基酸序列包含SEQ ID NO:23-27中的任一种或包含与SEQ ID NO:23-27中任一种所示氨基酸序列具有至少80%同源性。
- 根据权利要求8或9所述的抗体或其抗原结合片段,其特征在于,CMV pp65的结合表位包含SEQ ID NO:1-3中的任一种、两种或三种的组合。
- 根据权利要求8-11任一所述的抗体或其抗原结合片段,其特征在于,其轻链的氨基酸序列包含SEQ ID NO:247-252中的任一种或包含与SEQ ID NO:247-252任一种所示氨基酸序列具有至少80%同源性。
- 根据权利要求8-12任一所述的抗体或其抗原结合片段,其特征在于,其重链的氨基酸序列包含SEQ ID NO:253-258中的任一种或包含与SEQ ID NO:253-258任一种所示氨基酸序列具有至少80%同源性。
- 根据权利要求8-13任一所述的抗体或其抗原结合片段,其特征在于,所述的抗体或其抗原结合片段为单域抗体或单链抗体scFv。
- 根据权利要求8-14任一所述的抗体或其抗原结合片段,其特征在于,所述的抗体或其抗原结合片段的氨基酸序列包含SEQ ID NO:36、38、40、42、44或46中的任一种或包含与SEQ ID NO:36、38、40、42、44或46任一种所示氨基酸序列具有至少80%同源性。
- 一种T细胞抗原受体,其特征在于,所述的T细胞抗原受体特异性结合CMV pp65,其中,所述的T细胞抗原受体包含α链的CDR1α-CDR3α和/或β链的CDR1β-CDR3β,其中,CDR3α的氨基酸序列包含SEQ ID NO:12-17中的任一种或包含与SEQ ID NO:12-17中任一种所示氨基酸序列具有至少80%同源性,CDR3β的氨基酸序列包含SEQ ID NO:28-33中的任一种或包含与SEQ ID NO:28-33中任一种所示氨基酸序列具有至少80%同源性。
- 根据权利要求16所述的T细胞抗原受体,其特征在于,CDR1α的氨基酸序列包含SEQ ID NO:4-7中的任一种或包含与SEQ ID NO:4-7中任一种所示氨基酸序列具有至少80%同源性,CDR2α的氨基酸序列包含SEQ ID NO:8-11中的任一种或包含与SEQ ID NO:8-11中任一种所示氨基酸序列具有至少80%同源性,CDR1β的氨基酸序列包含SEQ ID NO:18-22中的任一种或包含与SEQ ID NO:18-22中任一种所示氨基酸序列具有至少80%同源性,CDR2β的氨基酸序列包含SEQ ID NO:23-27中的任一种或包含与SEQ ID NO:23-27中任一种所示氨基酸序列具有至少80%同源性。
- 根据权利要求16-17任一所述的T细胞抗原受体,其特征在于,CMV pp65的结合表位包含SEQ ID NO:1-3中的任一种、两种或三种的组合。
- 根据权利要求16-19任一所述的T细胞抗原受体,其特征在于,其β链的氨基酸序列包含SEQ ID NO:247-252中的任一种或包含与SEQ ID NO:247-252任一种所示氨基酸序列具有至少80%同源性。
- 根据权利要求16-20任一所述的T细胞抗原受体,其特征在于,其α链的氨基酸序列包含SEQ ID NO:253-258中的任一种或包含与SEQ ID NO:253-258任一种所示氨基酸序列具有至少80%同源性。
- 根据权利要求16-21任一所述的T细胞抗原受体,其特征在于,其α链与β链的氨基酸直接连接或间接连接,优选为间接连接,更优选通过fp2A连接。
- 根据权利要求16-22任一所述的T细胞抗原受体,其特征在于,其氨基酸序列包含SEQ ID NO:36、38、40、42、44或46中的任一种或包含与SEQ ID NO:36、38、40、42、44或46任一种所示氨基酸序列具有至少80%同源性。
- 一种编码权利要求1-3任一所述的CDR的核酸。
- 一种编码权利要求16-23任一所述的T细胞抗原受体的β链或权利要求6-7任一所述的β链多肽的核酸。
- 根据权利要求25所述的核酸,其特征在于,所述核酸的序列包含SEQ ID NO:260-265中任一种或包含与SEQ ID NO:260-265中任一种所示核酸序列具有至少80%同源性。
- 一种编码权利要求16-23任一所述的T细胞抗原受体的α链或权利要求4-5任一所述的α链多肽的核酸。
- 根据权利要求27所述的核酸,其特征在于,所述核酸的序列包含SEQ ID NO:266-271中任一种或包含与SEQ ID NO:266-271中任一种所示核酸序列具有至少80%同源性。
- 一种编码权利要求16-23任一所述的T细胞抗原受体或权利要求8-15任一所述的抗体或其抗原结合片段的核酸。
- 根据权利要求29所述的核酸,其特征在于,所述核酸的序列包含SEQ ID NO:37、39、41、43、45或47中任一种或包含与SEQ ID NO:37、39、41、43、45或47中任一种所示核酸序列具有至少80%同源性。
- 一种表达载体,其特征在于,所述的表达载体包含权利要求24-30任一所述的核酸。
- 一种宿主细胞,其特征在于,所述的宿主细胞包含权利要求24-30任一所述的核酸或权利要求31所述的表达载体。
- 一种免疫细胞,其特征在于,所述的免疫细胞表达权利要求1-3任一所述的CDR、权利要求4-5任一所述的α链多肽、权利要求6-7任一所述的β链多肽、权利要求8-15任一所述的抗体或其抗原结合片段或者权利要求16-23任一所述的T细胞抗原受体。
- 根据权利要求33所述的免疫细胞,其特征在于,所述的免疫细胞包含一个或多个权利要求24-30任一所述的异源核酸序列。
- 根据权利要求33或34所述的免疫细胞,其特征在于,所述的免疫细胞选自T细胞或干细胞。
- 根据权利要求33-35任一所述的免疫细胞,其特征在于,所述的免疫细胞分离自受试者的T细胞衍生。
- 一种免疫细胞的制备方法,其特征在于,所述的制备方法包括将编码权利要求1-3任一所述的CDR、权利要求4-5任一所述的α链多肽、权利要求6-7任一所述的β链多肽、权利要求8-15任一所述的抗体或其抗原结合片段或者权利要求16-23任一所述的T细胞抗原受体的核酸序列转 染至免疫细胞中表达获得。
- 根据权利要求37所述的制备方法,其特征在于,所述的制备方法还包括敲除细胞内源性TCR的步骤。
- 一种重组T细胞的制备方法,其特征在于,包括如下步骤:1)从阳性T细胞克隆得到权利要求24-30任一所述的核酸;2)分离、培养原代T细胞;3)将步骤1)得到的核酸递送至步骤2)所述的原代T细胞中,获得表达权利要求1-3任一所述的CDR、权利要求4-5任一所述的α链多肽、权利要求6-7任一所述的β链多肽、权利要求8-15任一所述的抗体或其抗原结合片段或者权利要求16-23任一所述的T细胞抗原受体的重组T细胞。
- 一种T细胞抗原受体的制备方法,其特征在于,包括如下步骤:(1)从阳性T细胞克隆得到权利要求24-30任一所述的核酸;(2)将步骤(1)得到的核酸连接至载体骨架,获得表达载体;(3)将步骤(2)获得的表达载体转化至宿主细胞,然后诱导其表达;(4)获得T细胞抗原受体。
- 一种多聚体复合物,其特征在于,所述多聚体复合物包含权利要求16-23任一所述的T细胞抗原受体。
- 根据权利要求41所述的多聚体复合物,其特征在于,所述多聚体复合物还包括单体,生物素分子,以及链霉亲和素分子或亲和素分子,其中,所述单体包括MHC分子α链胞外区和β2m链及抗原肽,所述单体与所述生物素分子偶联,所述生物素分子与所述链霉亲和素或亲和素分子结合。
- 根据权利要求42所述的多聚体复合物,其特征在于,所述的抗原肽包含SEQ ID NO:1-3中的任一种或两种以上的组合。
- 根据权利要求42或43所述的多聚体复合物,其特征在于,所述的MHC分子选自HLA-A*0201、HLA-A*2402和HLA-A*1101。
- 一种权利要求41-44任一所述的多聚体复合物的制备方法,其特征在于,包括如下步骤:I)表达和纯化在C端连接avi-tag序列的MHC分子α链胞外区和β2m链;II)将抗原肽、步骤I)获得的β2m链和在C端连接avi-tag序列的MHC分子α链胞外区复性折叠,制备单体;III)将步骤II)制备的单体生物素化,获得生物素化的单体;IV)将步骤III)获得的生物素化的单体与带荧光标记的链霉亲和素或亲和素反应,制备抗原肽-MHC分子四聚体;V)将步骤IV)得到的抗原肽-MHC分子四聚体与T细胞共孵育,形成T细胞抗原受体与抗原肽-MHC分子四聚体复合物。
- 权利要求1-3任一所述的CDR、权利要求4-5任一所述的α链多肽、权利要求6-7任一所述的β链多肽、权利要求8-15任一所述的抗体或其抗原结合片段、权利要求16-23任一所述的T细胞抗原受体、权利要求24-30任一所述的核酸、权利要求31所述的表达载体、权利要求32所述的宿主细胞、权利要求33-36任一所述的免疫细胞、权利要求41-44任一所述的多聚体复合物在制备诊断或治疗肿瘤或与CMV相关疾病的产品中的应用。
- 权利要求1-3任一所述的CDR、权利要求4-5任一所述的α链多肽、权利要求6-7任一所述的β链多肽、权利要求8-15任一所述的抗体或其抗原结合片段、权利要求16-23任一所述的T 细胞抗原受体、权利要求24-30任一所述的核酸、权利要求31所述的表达载体、权利要求32所述的宿主细胞、权利要求33-36任一所述的免疫细胞、权利要求41-44任一所述的多聚体复合物在T细胞标记、检测、细胞分选或活化中的应用。
- 一种药物组合物,所述的药物组合物包含下列任一组:i)权利要求1-3任一所述的CDR;ii)权利要求4-5任一所述的α链多肽;iii)权利要求6-7任一所述的β链多肽;iv)权利要求8-15任一所述的抗体或其抗原结合片段;v)权利要求16-23任一所述的T细胞抗原受体;vi)权利要求24-30任一所述的核酸;vii)权利要求31所述的表达载体;viii)权利要求32所述的宿主细胞;ix)权利要求33-36任一所述的免疫细胞;或x)权利要求41-44任一所述的多聚体复合物。
- 一种试剂盒,所述的试剂盒包含下列任一组:i)权利要求1-3任一所述的CDR;ii)权利要求4-5任一所述的α链多肽;iii)权利要求6-7任一所述的β链多肽;iv)权利要求8-15任一所述的抗体或其抗原结合片段;v)权利要求16-23任一所述的T细胞抗原受体;vi)权利要求24-30任一所述的核酸;vii)权利要求31所述的表达载体;viii)权利要求32所述的宿主细胞;ix)权利要求33-36任一所述的免疫细胞;或x)权利要求41-44任一所述的多聚体复合物。
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WO2024131372A1 (zh) * | 2022-12-23 | 2024-06-27 | 上海市第一人民医院 | 靶向巨细胞病毒pp65的TCR和表达其的T细胞及应用 |
WO2024139780A1 (zh) * | 2022-12-26 | 2024-07-04 | 上海市第一人民医院 | 靶向巨细胞病毒pp65的T细胞受体和表达其的T细胞及应用 |
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CN113880953A (zh) | 2022-01-04 |
KR20230061336A (ko) | 2023-05-08 |
CN113880937A (zh) | 2022-01-04 |
US20230257447A1 (en) | 2023-08-17 |
CN113881680A (zh) | 2022-01-04 |
JP2023532108A (ja) | 2023-07-26 |
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EP4163297A4 (en) | 2024-05-22 |
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