CAR-T cell targeting GPRC5D as well as preparation method and application of CAR-T cell

Abstract

The invention discloses a GPRC5D-targeted CAR-T cell and a preparation method and application thereof.The novel specific GPRC5D-targeted scFv sequence is firstly provided, codon optimization is conducted on the scFv sequence, then the scFv sequence is constructed into a second-generation CAR carrier, the CAR-T cell is prepared, the CAR-T cell can efficiently kill GPRC5D positive cells, a basis is provided for design of other GPRC5D-targeted immune effector cell therapies in the future, and the CAR-T cell has the advantages of being high in specificity, high in specificity, good in specificity and high in specificity. It is hopeful to provide a new method for the treatment of diseases or conditions associated with GPRC5D expression.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to CAR-T cells targeting GPRC5D and a preparation method and application thereof. Background Art

[0002] GPRC5D is a C-type 7-channel transmembrane receptor protein, belonging to the G protein-coupled receptor family and a member of the GPRC5 subfamily. As an orphan receptor, GPRC5D is expressed at a low level in normal human tissues, usually only in hair follicle cells, and specifically in malignant tumor cells such as myeloma, lymphoma, and leukemia. Currently, most GPRC5D drugs are still in the clinical trial stage or research and development stage. Therefore, it is necessary to develop GPRC5D cell drugs with higher activity and therapeutic effects for the treatment and application of related diseases.

[0003] Chimeric antigen receptor T cell (CAR-T) therapy is currently an important method for treating refractory and recurrent hematological tumors. However, this therapy lacks tumor-specific target antigens and is prone to off-target effects, leading to loss of humoral immune function and severe cytokine release syndrome (CRS). Therefore, target selection is an important research topic in CAR-T cell therapy. Summary of the invention

[0004] In view of this, in order to make up for the shortcomings of the prior art, the purpose of the present invention is to provide a CAR-T cell targeting GPRC5D and a preparation method and application thereof. The CAR-T cell targeting GPRC5D can specifically recognize and kill multiple myeloma, and is expected to provide a basis for the design of other immune effector cell therapies targeting GPRC5D in the future, and is expected to provide a new method for the treatment of diseases or conditions related to GPRC5D expression.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The first aspect of the present invention provides an antibody specifically targeting GPRC5D antigen, the antibody comprising: 1) CDR1 of the heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 1-2; or 2) CDR2 of the heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:3-4; or 3) CDR3 of the heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:5-6; or 4) CDR1 of the light chain variable region whose amino acid sequence is shown in SEQ ID NO:7-8; or 5) CDR2 of the light chain variable region having an amino acid sequence as shown in SEQ ID NO:9; or 6) CDR3 of the light chain variable region having an amino acid sequence as shown in SEQ ID NO: 10-11.

[0006] Furthermore, the amino acid sequence of the heavy chain variable region of the antibody comprises a combination of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, or a combination of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6.

[0007] Furthermore, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:12-13 and SEQ ID NO:16.

[0008] Furthermore, the amino acid sequence of the light chain variable region of the antibody comprises a combination of SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:10 or a combination of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:11.

[0009] Furthermore, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:14-15 and SEQ ID NO:17.

[0010] In the present invention, the term "antigen-binding fragment" generally refers to a polypeptide fragment of an antibody that binds to an antigen or competes with an intact antibody (i.e., the intact antibody from which they are derived) for antigen binding (i.e., specific binding). In certain embodiments, the antigen-binding fragment includes but is not limited to VHH, Fab, Fab', F(ab')2 and Fv fragments.

[0011] Furthermore, the antigen binding fragment is a Fv fragment.

[0012] Furthermore, the Fv fragment is scFv.

[0013] In the present invention, the terms "specificity", "targeting", "binding" refer to that the binding is selective for the antigen and can be distinguished from undesired or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art.

[0014] In certain embodiments, the sequences of CDR1-3 of the antibody variable region described in the present invention can be obtained based on the above-mentioned amino acid sequence according to the Kabat, IMGT, Chothia, AbM or Contact numbering system. The CDR sequences defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system are also within the scope of protection of the present invention.

[0015] In the present invention, the term "antibody" is used in the broadest sense and specifically covers antigen-binding fragments, monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, and multispecific antibodies (e.g., diabodies) formed from at least two intact antibodies, so long as they exhibit the desired biological activity.

[0016] In the present invention, the antibody sequence obtained by modification and having at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence of the antibody described in the first aspect of the present invention also belongs to the protection scope of the present invention. The term "modification" refers to any form of modification of the amino acid sequence, such as substitution, deletion, insertion and / or addition of amino acids. The term "substitution" refers to replacing one or more amino acids in the original amino acid sequence with different amino acids. The term "deletion" refers to reducing one or more amino acids in the original amino acid sequence. The term "insertion" or "addition" refers to the addition of one or more amino acids compared to the original amino acid sequence caused by the change in the amino acid sequence. The term "identity", also known as "homology", refers to an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to a sequence provided by the present invention. To determine sequence identity, sequence alignment can be performed in various ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE, Megalign (DNASTAR), Snapgene or DNAMAN software, etc. Those skilled in the art can determine appropriate parameters for alignment, including any algorithm required to achieve optimal alignment in the full-length sequence being compared.

[0017] In certain embodiments, the antibodies of the present invention are obtained by artificial synthesis. Methods for artificially synthesizing antibodies are known in the art, for example, the antibodies of the present invention are obtained by direct amino acid synthesis. In certain embodiments, the antibodies of the present invention are obtained by genetic engineering expression. Genetic engineering expression systems include prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Among them, prokaryotic cell expression systems include Escherichia coli expression systems. Eukaryotic cell expression systems include zymocyte expression systems, insect cell expression systems, and mammalian cell expression systems.

[0018] The second aspect of the present invention provides a chimeric antigen receptor targeting GPRC5D, wherein the chimeric antigen receptor comprises an extracellular antigen binding domain consisting of the antibody described in the first aspect of the present invention.

[0019] Furthermore, the antibody targeting the GPRC5D antigen is composed of a heavy chain variable region and a light chain variable region. In one embodiment, the heavy chain variable region and the light chain variable region may be directly connected. In one embodiment, the heavy chain variable region and the light chain variable region may be connected by at least one linker, and the arrangement may be VH-linker-VL or VL-linker-VH.

[0020] Further, the linker includes but is not limited to glycine polymer (G)n, glycine-serine polymer, glycine-alanine polymer, alanine-serine polymer, LRQKD(GGGS)2ERP, DGGGS, LRQRDGERP, TGEKP, KESGSVSSEQLAQFRSLD, EGKSSGSGSESKVD, GGRRGGGS, GGRR, LRQKDGGGSERP or (GGGGS)n. In one embodiment, the linker is (GGGGS)n, and in a specific embodiment of the present invention, the linker is (GGGGS)3.

[0021] Furthermore, the amino acid sequence of the scFv is shown in SEQ ID NO:18.

[0022] Furthermore, the chimeric antigen receptor also includes the following components: a transmembrane domain, a co-stimulatory domain, and a primary signaling domain.

[0023] Further, the transmembrane domain is selected from the transmembrane domains of the following molecules: CD8, CD8α, CD28, CD4, CD28, CD3ζ, CD137 / 4-1BB, CD134 / OX40, ICOS, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD154, TCRα, and TCRβ.

[0024] Furthermore, the transmembrane domain is selected from the transmembrane region of CD8.

[0025] Furthermore, the amino acid sequence of the transmembrane region of CD8 corresponds to the partial sequence shown in SEQ ID NO:19.

[0026] Furthermore, the co-stimulatory domain is selected from the co-stimulatory domains of the following molecules: CD28, CD137 / 4-1BB, CD134 / OX40, CD27, ICOS, B7-H3, 2B4, DAP10, DNAM1, and DAP12.

[0027] Furthermore, the co-stimulatory domain is selected from the co-stimulatory domain of CD137 / 4-1BB.

[0028] Furthermore, the amino acid sequence of the costimulatory domain of CD137 / 4-1BB is shown in SEQ ID NO:20.

[0029] Furthermore, the primary signaling domain includes human CD3ζ and human FcεRIγ.

[0030] Further, the primary signaling domain is selected from human CD3ζ.

[0031] Furthermore, the amino acid sequence of human CD3ζ is shown in SEQ ID NO:21.

[0032] Furthermore, the chimeric antigen receptor also includes one or more of the following components: a detection tag, a cleavage protein, a signal peptide, and a hinge region.

[0033] Furthermore, the detection labels include protein labels, fluorescent dyes, colloidal gold, chemiluminescent markers, and chemiluminescent catalysts.

[0034] Furthermore, the detection tag is selected from a protein tag.

[0035] Furthermore, the detection tags include RQR8, EGFRt, CD20, c-MYC, 6×His, GST, Fc, Flag, and HA.

[0036] Furthermore, the protein tag is selected from RQR8.

[0037] Furthermore, the amino acid sequence of RQR8 corresponds to the partial sequence shown in SEQ ID NO:22.

[0038] Furthermore, the cleavage protein comprises cleavage protein 2A or furin.

[0039] Furthermore, the cleavage protein 2A includes cleavage protein T2A, cleavage protein P2A, cleavage protein E2A, and cleavage protein F2A.

[0040] Furthermore, the cleavage protein F2A is selected from FMD-2A.

[0041] Furthermore, the amino acid sequence of the FMD-2A corresponds to the partial sequence shown in SEQ ID NO:22.

[0042] Furthermore, the fluorescent dyes include total protein dyes, phosphorylated protein and glycosylated protein dyes propidium iodide (PI), DAPI, Hoechst 33342, thiazole orange, and acridine orange.

[0043] Furthermore, the chemiluminescent markers include luminol and its derivatives, isoluminol and its derivatives, acridinium esters and their derivatives, adamantane, rare earth elements, and bipyridine ruthenium complexes.

[0044] Furthermore, the chemiluminescent catalyst includes horseradish peroxidase and alkaline phosphatase.

[0045] Furthermore, the signal peptide also includes CD8α signal peptide, T2A, GMCSF signal peptide, EGFR D Ⅲ~D Ⅳ. In one embodiment, the signal peptide is selected from CD8α signal peptide.

[0046] Furthermore, the sequence of the CD8α signal peptide is shown in SEQ ID NO:23.

[0047] Further, the hinge region includes the hinge region of CD8, CD28, CD34, OX40, CD3ε, IgG1, IgG4, 4-1BB, PD-1, IL-2 receptor, IL-7 receptor, IL-11 receptor molecules. In one embodiment, the hinge region is the CD8 hinge region.

[0048] In the present invention, the term "chimeric antigen receptor" (CAR) generally refers to a fusion protein comprising an extracellular domain capable of binding to an antigen and at least one intracellular domain. CAR is an engineered receptor that can implant any specific receptor into immune effector cells, especially T cells. In CAR, the scFv fragment of a monoclonal antibody that specifically recognizes a tumor antigen can be implanted into a T cell or a NK cell. A nucleic acid encoding CAR can be introduced into a T cell, a NK cell, or a NK T cell using, for example, a retroviral vector. In this way, a large number of cancer-specific T cells, NK cells, or NK T cells can be generated for adoptive cell transfer.

[0049] In certain embodiments, those skilled in the art can change the combination type and sequence of the transmembrane domain, co-stimulatory domain, primary signaling domain, detection tag, cleavage protein, signal peptide, and hinge region according to actual conditions or needs. Regardless of the form of the change, as long as the chimeric antigen receptor has the CDR sequence of the above-mentioned antibody of the present invention, it belongs to the protection scope of the present invention.

[0050] The third aspect of the present invention provides an antibody derivative, which comprises the antibody according to the first aspect of the present invention and a detectable marker coupled thereto.

[0051] Furthermore, the detectable markers include fluorescent dyes, colloidal gold, chemiluminescent markers, chemiluminescent catalysts, and radioactive nuclides.

[0052] Furthermore, the fluorescent dyes include total protein dyes, phosphorylated protein and glycosylated protein dyes propidium iodide (PI), DAPI, Hoechst 33342, thiazole orange, and acridine orange.

[0053] Furthermore, the chemiluminescent markers include luminol and its derivatives, isoluminol and its derivatives, acridinium esters and their derivatives, adamantane, rare earth elements, and bipyridine ruthenium complexes.

[0054] Furthermore, the chemiluminescent catalyst includes horseradish peroxidase and alkaline phosphatase.

[0055] Furthermore, the radionuclides include 67 Ga, 68 Ga, 18 F. 52 Fe, 62 Cu, 64 Cu, 67 Cu, 86 Y. 90 Y. 89 Zr, 120 I. 123 I. 13 N. 15 O. 186 Re, 110 In, 111 In.

[0056] A fourth aspect of the present invention provides a biomaterial comprising: (I) a nucleic acid molecule encoding the antibody of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, or the antibody derivative of the third aspect of the present invention; or (II) a vector comprising the nucleic acid molecule described in (I); or (III) a recombinant host cell comprising the nucleic acid molecule described in (I) and / or the vector described in (II); or (IV) A CAR-T cell comprising the chimeric antigen receptor described in the second aspect of the present invention.

[0057] Furthermore, the nucleic acid molecule further comprises a promoter, and / or a restriction enzyme cleavage site located after the promoter, and / or a kozak sequence located after the restriction enzyme cleavage site.

[0058] Furthermore, the nucleic acid molecule sequence encoding the antibody corresponds to part or all of SEQ ID NO:25.

[0059] Furthermore, the nucleic acid molecule sequence encoding the chimeric antigen receptor corresponds to part or all of SEQ ID NO:26.

[0060] In the present invention, the term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence.

[0061] Those skilled in the art can use standard molecular biology techniques to obtain nucleic acid molecules of the present invention. Once the DNA fragments encoding the heavy chain variable region and the light chain variable region are obtained, these DNA fragments are further operated by standard recombinant DNA techniques, such as to convert the variable region genes into full-length antibody chain genes, Fab fragment genes or scFv genes. In these operations, the DNA fragments encoding the heavy chain variable region and the light chain variable region are operably connected to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. Wherein, the term "operably connected" is intended to mean connecting two DNA fragments when used herein so that the amino acid sequences encoded by the two DNA fragments remain in the same reading frame (in-frame).

[0062] In some embodiments, the type of the carrier is not limited, for example, plasmid, phagemid, phage derivative, animal virus and clay, can be changed according to the host cell to be introduced. Viral vector technology is well known in the art and described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals, and introduction can be by standard techniques, for example, infection, transfection, transduction or transformation. Examples of gene transfer modes include, for example, naked DNA, CaPO4 precipitation, DEAE polydextrose, electroporation, protoplast fusion, liposome transfection, cell microinjection and viral vectors. Viruses that can be used as carriers include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. In addition, in order to evaluate the expression of target proteins (for example, monoclonal antibodies), the carrier introduced into the cell may also include any one or two of selectable marker genes or reporter genes, so as to identify and select expression cells from the cell populations that seek to be transfected or infected by viral vectors.

[0063] In some embodiments, the vector can be an expression vector, a cloning vector or an integration vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences and promoters that can be used to regulate the expression of the desired nucleic acid sequence. Integration vectors contain components that integrate the target sequence into the cell genome. These vectors can be used to transform appropriate host cells to enable them to express proteins. The vector usually contains sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequence usually includes one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a multi-linker region and a selectable marker element for inserting a nucleic acid encoding a monoclonal antibody to be expressed.

[0064] The recombinant host cells of the present invention refer to any cell type suitable for transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a nucleic acid molecule of the present invention. Host cells include any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication.

[0065] Furthermore, the recombinant host cells include prokaryotic cells and eukaryotic cells.

[0066] Furthermore, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasmas, chlamydia, and rickettsia.

[0067] Furthermore, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells.

[0068] Furthermore, the mammalian cells include immune cells, Chinese hamster ovary (CHO) cells, 293T cells or 293F cells.

[0069] Further, the immune cells include T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages or any combination thereof. In one embodiment, the immune cells are T cells.

[0070] The fifth aspect of the present invention provides a composition, which comprises the antibody described in the first aspect of the present invention, the chimeric antigen receptor described in the second aspect, the antibody derivative described in the third aspect, and the biomaterial described in the fourth aspect.

[0071] Furthermore, the composition also includes a pharmaceutically acceptable carrier and / or excipient.

[0072] The pharmaceutically acceptable carrier and / or adjuvant of the present invention include any material suitable for humans and / or mammals without excessive adverse side effects (e.g., toxicity, irritation, and allergic reactions), i.e., with a reasonable benefit / risk ratio. The pharmaceutically acceptable carrier and / or adjuvant are used to help the stability of the formulation as needed or to help improve its activity or its biological effectiveness or to produce an acceptable taste or smell in the case of oral administration. The pharmaceutically acceptable carrier and / or adjuvant include diluents, adhesives, surfactants, humectants, adsorption carriers, lubricants, and / or disintegrants. Among them, diluents include but are not limited to lactose, sodium chloride, glucose, urea, starch, and water; adhesives include but are not limited to starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginate, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methylcellulose; surfactants include but are not limited to polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride of stearic acid, and hexadecanol; humectants include but are not limited to glycerol and starch; adsorption carriers include but are not limited to starch, lactose, bentonite, silica gel, kaolin, and bentonite; lubricants include but are not limited to zinc stearate, glyceride monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose acid ester, sodium lauryl sulfate, magnesium lauryl sulfate, and magnesium lauryl sulfate.

[0073] The sixth aspect of the present invention provides a kit, which comprises the antibody described in the first aspect of the present invention, the chimeric antigen receptor described in the second aspect, the antibody derivative described in the third aspect, the biomaterial described in the fourth aspect, and the composition described in the fifth aspect.

[0074] In some embodiments, the kit may further include a sample diluent, a washing solution, a color developing solution, and a stop solution.

[0075] A seventh aspect of the present invention provides any of the following methods: 1) A method for producing the antibody of the first aspect of the present invention, the method comprising culturing the recombinant host cell in the biological material of the fourth aspect of the present invention so that the recombinant host cell can express the antibody of the first aspect of the present invention; 2) A method for preparing a recombinant host cell in the biological material according to the fourth aspect of the present invention, the preparation method comprising introducing the nucleic acid molecule or vector according to the fourth aspect of the present invention into a host cell; 3) A method for in vitro targeted killing of cells expressing GPRC5D for non-therapeutic purposes, the method comprising: Contacting the GPRC5D-expressing cells with the antibody described in the first aspect, the chimeric antigen receptor described in the second aspect, the antibody derivative described in the third aspect, the biomaterial described in the fourth aspect, the composition described in the fifth aspect, or the kit described in the sixth aspect of the present invention, and observing the killing of the GPRC5D-expressing cells; 4) A method for detecting GPRC5D, the method comprising: The sample to be tested is contacted with the antibody described in the first aspect, the chimeric antigen receptor described in the second aspect, the antibody derivative described in the third aspect, the biological material described in the fourth aspect, the composition described in the fifth aspect, or the kit described in the sixth aspect of the present invention to form an antigen-antibody complex, and the complex is detected, wherein detection of the complex indicates the presence of GPRC5D in the sample to be tested.

[0076] Further, the cells expressing GPRC5D include adrenocortical carcinoma cells, bladder urothelial carcinoma cells, breast cancer cells, cervical squamous cell carcinoma cells, endocervical adenocarcinoma cells, bile duct carcinoma cells, colon adenocarcinoma cells, lymphoid tumor cells, esophageal carcinoma cells, glioblastoma multiforme cells, head and neck squamous cell carcinoma cells, renal chromophobe carcinoma cells, renal clear cell carcinoma cells, renal papillary cell carcinoma cells, acute myeloid leukemia cells, brain low-grade glioma cells, hepatocellular carcinoma cells, mesothelial carcinoma cells, ovarian cancer cells, pancreatic cancer cells, pheochromocytoma cells One or more of the following: tumor and paraganglioma cells, prostate cancer cells, colorectal cancer cells, malignant sarcoma cells, melanoma cells, gastric cancer cells, testicular germ cell tumor cells, thyroid cancer cells, thymic cancer cells, endometrial cancer cells, uterine sarcoma cells, uveal melanoma cells, myeloma cells, acute lymphoid leukemia cells, chronic lymphoid leukemia cells, chronic myeloid leukemia cells, lymphoma cells, lung cancer cells (such as lung adenocarcinoma cells, lung squamous cell carcinoma cells), sarcoma cells, anal cancer cells, melanoma cells, retinoblastoma cells.

[0077] Furthermore, the cells expressing GPRC5D are selected from lymphoma cells or myeloma cells.

[0078] Furthermore, the myeloma cells are multiple myeloma cells.

[0079] Furthermore, the multiple myeloma cells are selected from MM.1S, NCI-H929, and RPMI-8226.

[0080] Furthermore, the multiple myeloma cells are MM.1S.

[0081] The eighth aspect of the present invention provides any one of the following applications: 1) Use of the antibody of the first aspect, the chimeric antigen receptor of the second aspect, the antibody derivative of the third aspect, the biomaterial of the fourth aspect, and the composition of the fifth aspect of the present invention in the preparation of a drug for diagnosing and / or treating a disease or condition associated with GPRC5D expression; 2) Use of the antibody described in the first aspect, the chimeric antigen receptor described in the second aspect, the antibody derivative described in the third aspect, the biomaterial described in the fourth aspect, the composition described in the fifth aspect, and the kit described in the sixth aspect of the present invention in targeting and killing cells expressing GPRC5D in vitro; 3) Use of the antibody described in the first aspect, the chimeric antigen receptor described in the second aspect, the antibody derivative described in the third aspect, the biomaterial described in the fourth aspect, the composition described in the fifth aspect, and the kit described in the sixth aspect of the present invention in detecting GPRC5D; Further, the diseases or conditions associated with GPRC5D expression include adrenal cortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, endocervical adenocarcinoma, bile duct cancer, colon adenocarcinoma, lymphoid tumors, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma, mesothelial carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic cancer, endometrial cancer, uterine sarcoma, uveal melanoma, myeloma, acute lymphoid leukemia, chronic lymphoid leukemia, chronic myeloid leukemia, lymphoma, lung cancer (lung adenocarcinoma, lung squamous cell carcinoma), sarcoma, anal cancer, melanoma, retinoblastoma and one or more.

[0082] Furthermore, the disease or condition associated with GPRC5D expression is selected from lymphoma or myeloma.

[0083] Furthermore, the myeloma is multiple myeloma.

[0084] The present invention has the following advantages and beneficial effects: The present invention provides antibodies and CAR-T cells targeting GPRC5D, and preparation methods and applications thereof. The CAR-T cells can efficiently kill MM.1S cells, provide a basis for the design of other immune effector cell therapies targeting GPRC5D, and provide a new method for detecting GPRC5D, diagnosing or treating diseases or conditions related to GPRC5D expression, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 These are the results of GPRC5D antibody screening and humanization, where 1A is the result of the antibody-cell binding ability test; 1B is the result of the antibody humanization.

[0086] Figure 2 The figures are the expression of GPRC5D in different hematological tumor cells, among which, 2A is the expression of GPRC5D outside the cell membrane of 8 hematological tumor cells detected by flow cytometry; 2B is the expression of GPRC5D at the mRNA level in 8 hematological tumor cells detected by qPCR; 2C is the result of agarose gel electrophoresis to verify the RT-PCR product.

[0087] Figure 3Figure 3 is a diagram of the construction of GPRC5D CAR-T cells, where 3A is a schematic diagram of the molecular construction of GPRC5D CAR-T cells; 3B is a diagram of the PCR results of the GPRC5D CAR fragment; 3C is a diagram of the results of agarose gel electrophoresis to verify pHAGE and the GPRC5D CAR fragment; 3D is a diagram of the sequencing results of the homologous recombinant bacterial solution; 3E is a diagram of the proliferation and cell viability changes of GPRC5D CAR-T cells after infection with the CAR virus; 3F is a diagram of the positive rate detection results of GPRC5D CAR-T cells after infection with the CAR virus.

[0088] Figure 4 Figure 4 is a graph showing the killing results of GPRC5D CAR-T cells against multiple myeloma cells MM.1S, where 4A is a graph showing the gating strategy for flow cytometry analysis on FlowJo; and 4B is a graph showing the killing results of GPRC5D CAR-T cells against multiple myeloma cells MM.1S at different effector-target ratios. DETAILED DESCRIPTION

[0089] The present invention is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute limitations of the present invention in any way. The experimental methods used in the following examples are conventional methods unless otherwise specified; the reagents, biological materials, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0090] Example 1 GPRC5D antibody screening and humanization 1. Experimental methods (1) Construct a GPRC5D overexpression lentiviral expression vector, transfect the CHO-S cell line, and screen the stable cell line CHO-S-GPRC5D cell line that overexpresses the target gene for subsequent mouse immunization.

[0091] (2) CHO-S-GPRC5D cell line was used to immunize 5 CD-1 mice and 5 BALB / c mice. The immunization strategy was as follows: First immunization: 5*10^ 6 / mouse intraperitoneal injection, Freund's complete adjuvant; second immunization: CHO-S-GPRC5D cells 5*10^ 6 / mouse, intraperitoneal injection, Freund's incomplete adjuvant; three immunizations: CHO-S-GPRC5D cells 5*10^ 6 / mouse, intraperitoneal injection, Freund's incomplete adjuvant; four immunizations: CHO-S-GPRC5D cells 5*10^ 6 / mouse, intraperitoneal injection, Freund's incomplete adjuvant.

[0092] Five CD-1 mice were immunized with GPRC5D adenovirus. First immunization: 1*10^ 8 PFU / mouse, intramuscular injection, Freund's complete adjuvant; second immunization: 1*10^ 8 PFU / mouse, intramuscular injection, Freund's incomplete adjuvant; three immunizations: 1*10^ 8 PFU / mouse, intramuscular injection, Freund's incomplete adjuvant; four immunizations: 1*10^ 8 PFU / mouse, intramuscular injection, Freund's incomplete adjuvant.

[0093] (3) Titer detection by flow cytometry. Cells: 300,000 / well; primary antibody: immune serum 1:1000, 100ul / well; secondary antibody: PE anti mouse IgG. Select mice No. 28 and No. 35 with high titer for fusion.

[0094] (4) Use a multifunctional microplate reader to detect the titer and select clones with high fluorescence intensity for flow cytometry. Sequence clones with high fluorescence intensity.

[0095] (6) The heavy chain variable regions and light chain variable regions of the two antibodies were connected via (G4S)3, and the binding ability of the antibodies to CHO-S-GPRC5D was detected by flow cytometry.

[0096] (7) The heavy chain variable region amino acids and light chain variable region amino acids of antibody No. 2 with better binding activity were humanized.

[0097] Experimental Results (1) Flow cytometry results (Table 1, Table 2) showed that the titers of mice No. 28 and No. 35 were higher.

[0098] Table 1 Results of titer detection of CHO-S-GPRC5D cell line immunized mice

[0099] Table 2 Results of titer detection of GPRC5D adenovirus immunized mice

[0100] (2) After fusion, 6 clones were selected for sequencing, and 2 antibody sequences were obtained. The amino acid sequence of the heavy chain variable region of antibody No. 1 is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14; the amino acid sequence of the heavy chain variable region of antibody No. 2 is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 15.

[0101] (3) Flow cytometry results ( Figure 1 A) shows that both antibodies can bind to CHO-S-GPRC5D cells.

[0102] (4) The results of humanization transformation are as follows Figure 1 As shown in B, the amino acid sequence of the modified heavy chain variable region and the amino acid sequence of the light chain variable region are shown in SEQ ID NO: 16 and 17, respectively.

[0103] Example 2 Detection of GPRC5D expression in multiple myeloma cell lines by flow cytometry and PCR 1. Experimental methods Flow cytometry and PCR were used to detect the expression of GPRC5D in hematological tumor cells: MM.1S, NCI-H929, RPMI-8226, U266, K562, NALM-6, DB and Jurkat cells.

[0104] 2. Experimental results The results showed that multiple myeloma cells MM.1S, NCI-H929, and RPMI-8226 were all GPRC5D-positive tumor cells, and the rest of the cells tested were all GPRC5D-negative cells ( Figure 2 A. Figure 2 B. Figure 2 C). The above results confirmed that GPRC5D was highly expressed in MM.1S, expressed in NCI-H929, and lowly expressed in RPMI-8226.

[0105] Example 3 Construction of GPRC5D CAR-T cells 1. Experimental methods 1) The scFv sequence targeting GPRC5D was codon optimized using a T cell-specific codon optimization algorithm, and then the heavy chain and light chain were connected using a G4S linker to obtain a scFv sequence targeting GPRC5D and optimized for T cell specific codons. The optimized sequence is shown in SEQ ID NO: 25. The above scFv sequence was constructed into a second-generation CAR vector, using RQR8 as a detection tag and 41BB as a co-stimulatory molecule to construct Figure 3 The CAR structure shown in A, the constructed plasmid was verified by agarose gel electrophoresis and sequenced by the company. The constructed CAR nucleotide sequence and amino acid sequence are shown in SEQ ID NO: 26 and SEQ ID NO: 24, respectively.

[0106] 2) CAR vectors were used to package lentivirus, and CAR-T cells were infected and constructed at MOI = 5 and 10, respectively. The proliferation and viability of GPRC5D CAR-T cells were monitored after infection. The CAR-T cell positive rate was detected on the 7th, 9th and 11th day after CAR virus infection.

[0107] 2. Experimental results 1) Agarose gel electrophoresis verification results ( Figure 3 B. Figure 3 C) and the sequencing results of the company ( Figure 3 D) Shows that the pHAGE-GPRC5D CAR plasmid was successfully constructed.

[0108] 2) The results showed that the proliferation and viability of GPRC5D CAR-T were good ( Figure 3 E), CAR can be stably expressed on T cells, and the CAR-T cell positive rate is high, which can be stably maintained at more than 70% in the later stage ( Figure 3 F).

[0109] Example 4 Detection of the killing function of GPRC5D CAR-T cells 1. Experimental methods GPRC5D-positive multiple myeloma cells MM.1S were used as target cells to evaluate the ability of the constructed GPRC5D CAR-T cells to kill multiple myeloma cells. Cell Tracker Deep Red-APC was used to stain MM.1S live cells before co-culture, and the stained MM.1S were co-cultured with T cells at effector-target ratios of 1:5, 1:10, and 1:15. After 24 h, flow cytometry ( Figure 4 A) Detection of the proportion of T cells and tumor cells in living cells after co-culture of CAR-T cells and MM.1S.

[0110] 2. Experimental results The results showed that after 24 h of co-culture, when the effector-target ratio (E:T) was 1:5, the proportion of tumor cells decreased from 72.9% to 12.3%; when the effector-target ratio (E:T) was 1:10, the proportion of tumor cells decreased from 85.9% to 54.3%; when the effector-target ratio (E:T) was 1:15, the proportion of tumor cells decreased from 90.5% to 76.6% ( Figure 4 B). This shows that GPRC5D CAR-T cells have a significant killing effect on MM.1S cells, and as the effector-target ratio increases, the killing becomes more obvious, with a clear dose-dependency.

[0111] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. An antibody specifically targeting GPRC5D antigen, characterized in that: The antibodies include: 1) CDR1 of the heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 1-2; or 2) CDR2 of the heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:3-4; or 3) CDR3 of the heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:5-6; or 4) CDR1 of the light chain variable region whose amino acid sequence is shown in SEQ ID NO:7-8; or 5) CDR2 of the light chain variable region having an amino acid sequence as shown in SEQ ID NO:9; or 6) CDR3 of the light chain variable region having an amino acid sequence as shown in SEQ ID NO: 10-11; Preferably, the amino acid sequence of the heavy chain variable region of the antibody comprises a combination of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, or a combination of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6; Preferably, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 12-13, SEQ ID NO: 16; Preferably, the amino acid sequence of the light chain variable region of the antibody comprises a combination of SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:10 or a combination of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:11; Preferably, the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO:14-15 and SEQ ID NO:

17.

2. The antibody according to claim 1, characterized in that The antibody includes an antigen-binding fragment, a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody or a multivalent antibody; Preferably, the antigen-binding fragments include single-domain antibodies, Fab, Fab', F(ab')2 and Fv fragments; Preferably, the antigen-binding fragment is an Fv fragment; Preferably, the Fv fragment is a scFv.

3. A chimeric antigen receptor targeting GPRC5D, characterized in that The chimeric antigen receptor comprises an extracellular antigen binding domain consisting of the antibody of any one of claims 1-2; Preferably, the amino acid sequence of the scFv is as shown in SEQ ID NO: 18; Preferably, the chimeric antigen receptor further comprises the following components: a transmembrane domain, a co-stimulatory domain, and a primary signaling domain; Preferably, the transmembrane domain is selected from the transmembrane domains of the following molecules: CD8, CD8α, CD28, CD4, CD28, CD3ζ, CD137 / 4-1BB, CD134 / OX40, ICOS, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD154, TCRα, TCRβ; Preferably, the transmembrane domain is selected from the transmembrane region of CD8; Preferably, the amino acid sequence of the transmembrane region of CD8 corresponds to the partial sequence shown in SEQ ID NO: 19; Preferably, the costimulatory domain is selected from the costimulatory domains of the following molecules: CD28, CD137 / 4-1BB, CD134 / OX40, CD27, ICOS, B7-H3, 2B4, DAP10, DNAM1, DAP12; Preferably, the costimulatory domain is selected from the costimulatory domain of CD137 / 4-1BB; Preferably, the amino acid sequence of the costimulatory domain of CD137 / 4-1BB is as shown in SEQ ID NO: 20; Preferably, the primary signaling domain includes human CD3ζ and human FcεRIγ; Preferably, the primary signaling domain is selected from human CD3ζ; Preferably, the amino acid sequence of human CD3ζ is shown in SEQ ID NO: 21; Preferably, the chimeric antigen receptor further comprises one or more of the following components: a detection tag, a cleavage protein, a signal peptide, a hinge region; Preferably, the detection tags include RQR8, EGFRt, CD20, c-MYC, 6×His, GST, Fc, Flag, and HA; Preferably, the detection tag is selected from RQR8; Preferably, the amino acid sequence of RQR8 corresponds to the partial sequence shown in SEQ ID NO: 22; Preferably, the cleavage protein comprises cleavage protein 2A or furin; Preferably, the cleavage protein 2A includes cleavage protein T2A, cleavage protein P2A, cleavage protein E2A, and cleavage protein F2A; Preferably, the cleavage protein F2A is selected from FMD-2A; Preferably, the amino acid sequence of FMD-2A corresponds to the partial sequence shown in SEQ ID NO:

22.

4. An antibody derivative, characterized in that: The antibody derivative comprises the antibody according to any one of claims 1 to 2 and a detectable marker coupled thereto.

5. The antibody derivative according to claim 4, characterized in that The detectable label comprises at least one of a radioisotope, a metal nanomaterial, a fluorescein, a biotin, avidin, a biotin / avidin protein complex, a biotin / avidin protein complex, a chromophore, an electron-dense substance, and an enzyme.

6. A biomaterial comprising: (I) a nucleic acid molecule encoding the antibody according to any one of claims 1 to 2, the chimeric antigen receptor according to claim 3, or the antibody derivative according to any one of claims 4 to 5; or (II) a vector comprising the nucleic acid molecule described in (I); or (III) a recombinant host cell comprising the nucleic acid molecule described in (I) and / or the vector described in (II); or (IV) A CAR-T cell comprising the chimeric antigen receptor of claim 3; Preferably, the nucleic acid molecule further comprises a promoter, and / or a restriction enzyme cleavage site located after the promoter, and / or a kozak sequence located after the restriction enzyme cleavage site; Preferably, the nucleic acid molecule sequence encoding the antibody corresponds to part or all of SEQ ID NO: 25; Preferably, the nucleic acid molecule sequence encoding the chimeric antigen receptor corresponds to part or all of SEQ ID NO:

26.

7. A composition comprising the antibody according to any one of claims 1 to 2, the chimeric antigen receptor according to claim 3, the antibody derivative according to any one of claims 4 to 5, and the biomaterial according to claim 6; Preferably, the composition further comprises pharmaceutically acceptable and / or auxiliary materials.

8. A kit comprising the antibody according to any one of claims 1-2, the chimeric antigen receptor according to claim 3, the antibody derivative according to any one of claims 4-5, and the biomaterial according to claim 6.

9. Any of the following methods: 1) A method for producing the antibody according to any one of claims 1 to 2, characterized in that: The method comprises culturing the recombinant host cell in the biological material of claim 6 so that it can express the antibody of any one of claims 1-2; 2) A method for preparing a recombinant host cell in the biological material of claim 6, characterized in that the preparation method comprises introducing the nucleic acid molecule or vector of claim 6 into a host cell; 3) A method for in vitro targeted killing of cells expressing GPRC5D for non-therapeutic purposes, the method comprising: Contacting the GPRC5D-expressing cell with the antibody according to any one of claims 1 to 2, the chimeric antigen receptor according to claim 3, the antibody derivative according to any one of claims 4 to 5, the biomaterial according to claim 6, the composition according to claim 7, or the kit according to claim 8, and observing the killing of the GPRC5D-expressing cell; 4) A method for detecting GPRC5D, the method comprising: Contacting the test sample with the antibody according to any one of claims 1 to 2, the chimeric antigen receptor according to claim 3, the antibody derivative according to any one of claims 4 to 5, the biomaterial according to claim 6, the composition according to claim 7, or the kit according to claim 8, thereby forming an antigen-antibody complex, and detecting the complex, wherein detection of the complex indicates the presence of GPRC5D in the test sample; Preferably, the cells expressing GPRC5D include adrenocortical carcinoma cells, bladder urothelial carcinoma cells, breast cancer cells, cervical squamous cell carcinoma cells, endocervical adenocarcinoma cells, bile duct carcinoma cells, colon adenocarcinoma cells, lymphoid tumor cells, esophageal carcinoma cells, glioblastoma multiforme cells, head and neck squamous cell carcinoma cells, renal chromophobe carcinoma cells, renal clear cell carcinoma cells, renal papillary cell carcinoma cells, acute myeloid leukemia cells, brain low-grade glioma cells, hepatocellular carcinoma cells, mesothelial carcinoma cells, ovarian cancer cells, pancreatic cancer cells, pheochromocytoma cells. Tumor and paraganglioma cells, prostate cancer cells, rectal cancer cells, malignant sarcoma cells, melanoma cells, gastric cancer cells, testicular germ cell tumor cells, thyroid cancer cells, thymic cancer cells, endometrial cancer cells, uterine sarcoma cells, uveal melanoma cells, myeloma cells, acute lymphoid leukemia cells, chronic lymphoid leukemia cells, chronic myeloid leukemia cells, lymphoma cells, lung cancer cells (such as lung adenocarcinoma cells, lung squamous cell carcinoma cells), sarcoma cells, anal cancer cells, melanoma cells, retinoblastoma cells, one or more; Preferably, the GPRC5D-expressing cells are selected from lymphoma cells or myeloma cells; Preferably, the myeloma cells are multiple myeloma cells; Preferably, the multiple myeloma cells are selected from MM.1S, NCI-H929, RPMI-8226; Preferably, the multiple myeloma cells are MM.1S.

10. Any of the following applications: 1) Use of the antibody according to any one of claims 1 to 2, the chimeric antigen receptor according to claim 3, the antibody derivative according to any one of claims 4 to 5, the biomaterial according to claim 6, or the composition according to claim 7 in the preparation of a drug for diagnosing and / or treating a disease or condition associated with GPRC5D expression; 2) Use of the antibody according to any one of claims 1 to 2, the chimeric antigen receptor according to claim 3, the antibody derivative according to any one of claims 4 to 5, the biomaterial according to claim 6, the composition according to claim 7, or the kit according to claim 8 in targeting and killing cells expressing GPRC5D in vitro; 3) Use of the antibody according to any one of claims 1 to 2, the chimeric antigen receptor according to claim 3, the antibody derivative according to any one of claims 4 to 5, the biomaterial according to claim 6, the composition according to claim 7, and the kit according to claim 8 in detecting GPRC5D; Preferably, the disease or condition associated with GPRC5D expression comprises one or more of adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, endocervical adenocarcinoma, bile duct cancer, colon adenocarcinoma, lymphoid tumors, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma, mesothelial carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic cancer, endometrial carcinoma, uterine sarcoma, uveal melanoma, myeloma, acute lymphoid leukemia, chronic lymphoid leukemia, chronic myeloid leukemia, lymphoma, lung cancer (lung adenocarcinoma, lung squamous cell carcinoma), sarcoma, anal cancer, melanoma, retinoblastoma; Preferably, the disease or disorder associated with GPRC5D expression is selected from lymphoma or myeloma; Preferably, the myeloma is multiple myeloma.

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