CN115386010A - Chimeric antigen receptor targeting GPRC5D and application thereof - Google Patents

Abstract

The invention relates to the field of cell therapy, and further relates to a chimeric antigen receptor targeting a G protein coupled receptor C5 family subtype D (GPRC 5D), a preparation method and application thereof, in particular to application of the chimeric antigen receptor as an anti-cancer medicament. A chimeric antigen receptor targeting G protein-coupled receptor subtype C5D (GPRC 5D) comprising an extracellular antigen-binding domain comprising a GPRC5D antibody light chain variable region and a GPRC5D antibody heavy chain variable region. The invention combines a single chain antibody (scFv) for recognizing a tumor surface antigen GPRC5D with a T cell activation structural domain, so that a T cell expresses a chimeric antigen receptor, thereby recognizing and combining the tumor surface antigen, inducing the T cell to activate and secrete cytokines to further kill the tumor cell. Has obvious curative effect in treating B lymphocyte tumor and acute lymphocyte leukemia.

Description

Chimeric antigen receptor targeting GPRC5D and application thereof

The present invention claims priority from the chinese patent application filed on 23/5/2021 by the chinese patent office under the application number 202110561829.9 entitled "chimeric antigen receptor targeting GPRC5D and uses thereof," the entire contents of which are incorporated herein by reference.

Technical Field

The invention relates to the field of cell therapy, and further relates to a chimeric antigen receptor targeting a G protein coupled receptor C5 family subtype D (GPRC 5D) and a preparation method and application thereof.

Background

Chimeric Antigen Receptor (CAR) T cell therapy is a method in which a single chain antibody (scFv) recognizing a tumor surface antigen is combined with a T cell activation domain, so that T cells express the chimeric antigen receptor, thereby recognizing and binding the tumor surface antigen, inducing T cells to activate, secrete cytokines, and further kill tumor cells. Has obvious curative effect in the treatment of B lymphocyte tumor and acute lymphocyte leukemia [1].

The G protein-coupled receptor C5 family subtype D (GPRC 5D) was the orphan atypical, class C GPCR identified earlier than 2001 [2]. GPCR family group C group 5 receptors (GPRC 5 receptors) share 4 subtypes, namely GPRC5A, GPRC5B, GPRC C and GPRC5D, which are expressed induced by retinoic acid and are therefore also referred to as retinoic acid induced orphan G protein coupled Receptors (RAIG) [3]. GPRC5D is highly expressed in plasma cells of multiple myeloma, is not substantially expressed in normal tissues, and is expressed only in the hair follicle region having immune privilege [4]. Studies have reported that high expression of GPRC5D correlates with poor prognosis of multiple myeloma [5]. GPRC 5D-targeted CAR-T showed good therapeutic effect in mouse Multiple Myeloma (MM) model. As GPRC5D does not coincide with BCMA expression, GPRC5D CAR-T still has therapeutic effect for tumor recurrence models of BCMA loss [4]. In the GPRC5D positive mouse MM model, GPRC5D/CD3 dual antibodies recruit T cells and induce tumor regression [6].

GPRC5D is a promising surface antigen for MM immunotherapy, but most GPRC5D drugs are still in clinical trial or development phase at present, and no cell drugs targeting GPRC5D are on the market, so that there is a need to develop GPRC5D cell drugs with higher activity and therapeutic effect for therapeutic research and application of related diseases.

Reference documents:

1.Romero,D.,Initial results with liso-cel.Nat Rev Clin Oncol,2020.17(11):654-654.

2.Brauner-Osborne,H.,et al.,Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D.Biochim Biophys Acta,2001.1518(3):p.237-48.

3.Inoue,S.,T.Nambu,and T.Shimomura,The RAIG family member,GPRC5D,is associated with hard-keratinized structures.Journal of Investigative Dermatology,2004.122(3):p.565-573.

4.Smith,E.L.,et al.,GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells.Science Translational Medicine,2019.11(485).

5.Atamaniuk,J.,et al.,Overexpression of G protein-coupled receptor 5D in the bone marrow is associated with poor prognosis in patients with multiple myeloma.European Journal of Clinical Investigation,2012.42(9):p.953-960.

6.Pillarisetti,K.,et al.,A T-cell-redirecting bispecific G-protein-coupled receptor class 5member D x CD3 antibody to treat multiple myeloma.Blood,2020.135(15):p.1232-1243.

disclosure of Invention

A first object of the present invention is to provide a chimeric antigen receptor comprising an extracellular antigen-binding domain comprising a GPRC5D antibody light chain variable region and a GPRC5D antibody heavy chain variable region, wherein the GPRC5D antibody light chain variable region comprises any one of the following sequences: SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 39, SEQ ID No. 41 and SEQ ID No. 43;

the GPRC5D antibody heavy chain variable region comprises any one of the following sequences: SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 38, SEQ ID No. 40 and SEQ ID No. 42.

In a specific embodiment of the invention, the GPRC5D antibody heavy chain variable region sequence is SEQ ID No. 3 and the GPRC5D antibody light chain variable region sequence is SEQ ID No. 4, or the GPRC5D antibody heavy chain variable region sequence is SEQ ID No. 5 and the GPRC5D antibody light chain variable region sequence is SEQ ID No. 6, or the GPRC5D antibody heavy chain variable region sequence is SEQ ID No. 7 and the GPRC5D antibody light chain variable region sequence is SEQ ID No. 8, or the GPRC5D antibody heavy chain variable region sequence is SEQ ID No. 9 and the GPRC5D antibody light chain variable region sequence is SEQ ID No. 10, or the GPRC5D antibody heavy chain variable region sequence is SEQ ID No. 11 and the GPRC5D antibody light chain variable region sequence is SEQ ID No. 12, or the GPRC5D antibody heavy chain variable region sequence is SEQ ID No. 38 and the GPRC5D antibody heavy chain variable region sequence is SEQ ID No. 39, or GPRC5D antibody light chain variable region sequence is SEQ ID No. 40 and GPRC5D antibody light chain variable region sequence is SEQ ID No. 41 or GPRC5D antibody light chain variable region sequence is SEQ ID No. 40 and GPRC5D antibody light chain variable region No. 40.

In a particular embodiment of the invention, the chimeric antigen receptor further comprises a transmembrane domain and an intracellular domain.

In a specific embodiment of the present invention, a linker is comprised between the GPRC5D antibody heavy chain variable region and the GPRC5D antibody light chain variable region, preferably the linker is a linker peptide, preferably the sequence of the linker peptide is (GGGGS) n, wherein n is greater than or equal to 1, preferably 1, 2, 3, 4 or 5, more preferably n is 3;

alternatively, the sequence of the linker is EGKSSGSGSESKVD, KESGSVSSEQLAQFRSLD, GGRRGGGS, LRQRDGERP, LRQKDGGGSERP or GSTSGSGKPGSGEGSTKG.

In a particular embodiment of the invention, the extracellular antigen-binding domain further comprises a leader peptide and a hinge region.

In a particular embodiment of the invention, the leader peptide sequence is an IgG1 heavy chain signal polypeptide, a granulocyte-macrophage colony stimulating factor receptor 2 (GM-CSFR 2) signal peptide, or a CD8 α signal peptide, and/or the hinge region is CH2 and CH3 of CD8 α, igG1, or IgG4, CD28, or CD7.

In a particular embodiment of the invention, the transmembrane domain is any one or more of the group consisting of: the α, β or δ chain of the T cell receptor, CD3 epsilon, CD3 δ, CD4, CD5, CD8 α, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD 154 and PD1, and/or the intracellular domain is any one or more of the group: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134, CD137 (4-1 BB), CD3 ζ, CD150 (SLAMF 1), CD152 (CTLA 4), CD223 (LAG 3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70.

In a specific embodiment of the invention, the chimeric antigen receptor comprises the leader peptide of SEQ ID No. 44, the light chain variable region of the GPRC5D antibody, the linker peptide of SEQ ID No. 45, the heavy chain variable region of the GPRC5D antibody, the hinge region of SEQ ID No. 46, the CD8 α transmembrane domain of SEQ ID No. 47, the 4-1BB costimulatory signaling region of SEQ ID No. 48, and the CD3 zeta signaling domain of SEQ ID No. 49.

In certain embodiments, the intracellular domain of the chimeric antigen receptor described herein may comprise one or more costimulatory signaling domains, which may also be from a costimulatory molecule selected from the group consisting of: CARD11, CD2, CD3 ζ, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX 40), CD137 (4-1 BB), CD150 (SLAMF 1), CD152 (CTLA 4), CD223 (LAG 3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70.

In particular embodiments, the one or more co-stimulatory signaling domains are from a co-stimulatory molecule selected from the group consisting of: CD28, CD134, and CD137.

In further embodiments, the one or more co-stimulatory signaling domains are from a co-stimulatory molecule selected from the group consisting of: CD137 and CD3 ζ.

In further embodiments, the one or more co-stimulatory signaling domains are from CD28.

In particular embodiments, the one or more co-stimulatory signaling domains are from CD134.

In other embodiments, the one or more co-stimulatory signaling domains are from CD137.

The invention also provides the use of the aforementioned chimeric antigen receptor for the preparation of a medicament or pharmaceutical composition.

In a particular embodiment of the invention, the medicament or pharmaceutical composition is for the treatment of tumors.

In a particular embodiment of the invention, the neoplasm comprises multiple myeloma.

The invention also provides a polynucleotide encoding the chimeric antigen receptor described above.

In some embodiments, a polynucleotide encoding a CAR encompassed herein comprises an optimized Kozac sequence.

In other embodiments, the promoter operably linked to a polynucleotide encoding a CAR encompassed herein is selected from the group consisting of: cytomegalovirus immediate early gene promoter (CMV), elongation factor 1 α promoter (EF 1- α), phosphoglycerate kinase-1 Promoter (PGK), ubiquitin-C promoter (UBQ-C), cytomegalovirus enhancer/chicken β -actin promoter (CAG), polyoma enhancer/herpes simplex thymidine kinase promoter (MC 1), β -actin promoter (β -ACT), simian virus 40 promoter (SV 40), and myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substituted (MND) promoter.

The present invention also provides a vector comprising the aforementioned polynucleotide.

In certain embodiments, the vector is an expression vector.

In further embodiments, the vector is an episomal vector.

In particular embodiments, the vector is a viral vector.

In other embodiments, the vector is a retroviral vector.

In other embodiments, the vector is a lentiviral vector.

In further embodiments, the lentiviral vector is selected from the group consisting essentially of: human Immunodeficiency Virus (HIV), human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-madi virus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), equine Infectious Anemia Virus (EIAV), feline Immunodeficiency Virus (FIV), bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV).

In particular embodiments, the vector comprises a left (5 ') retroviral LTR, a Psi (Ψ) packaging signal, a central polypurine tract/DNA FLAP (cPPT/FLAP), a retroviral export element, a promoter operably linked to a polynucleotide encoding a CAR contemplated herein, and a right (3') retroviral LTR.

In certain embodiments, the promoter of the 5' LTR is replaced with a heterologous promoter.

In other embodiments, the heterologous promoter is a Cytomegalovirus (CMV) promoter, a Rous Sarcoma Virus (RSV) promoter, or an simian Virus 40 (SV 40) promoter.

In particular embodiments, the 5'LTR or 3' LTR is a lentiviral LTR.

In particular embodiments, the 3' ltr comprises one or more modifications.

In some embodiments, the 3' ltr comprises one or more deletions.

In certain embodiments, the 3' LTR is a self-inactivating (SIN) LTR.

The invention also provides an immune effector cell comprising the aforementioned polynucleotide or the aforementioned vector.

In a particular embodiment of the invention, the immune effector cell is a T cell.

In various embodiments, an immune effector cell is provided comprising a vector encompassed herein. In various embodiments, the immune effector cell is transduced by a vector encompassed herein.

In other embodiments, the immune effector cell is selected from the group consisting of: t lymphocytes, macrophages, and Natural Killer (NK) cells.

In various embodiments, a method of generating an immune effector cell comprising a CAR contemplated herein is provided, comprising introducing into an immune effector cell a vector comprising a polynucleotide encoding the CAR.

In other embodiments, the immune effector cell is a PD1 gene knockout cell.

In certain embodiments, the sgRNA targeting PD1, the Cas9 protein, and the double-stranded DNA containing the GPRC5D CAR sequence are introduced into the T cell by electrotransfer to achieve precise insertion of the CAR element at the PD1 gene site of the T cell, thereby obtaining the GPRC5D non-viral PD1 site-directed integration CAR-T cell and enhancing the effect of targeted tumor killing.

In addition, sgrnas targeting other genes in T cells can be introduced into the T cells in the above manner, so as to obtain CAR-T cells targeting the site-specific integration of GPRC5D on other genes, thereby improving the effect of targeted tumor killing.

The invention also provides a pharmaceutical composition comprising the chimeric antigen receptor, polynucleotide, vector or host cell as described above.

In a specific embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

In a specific embodiment of the present invention, the pharmaceutically acceptable adjuvant is one or more of citric acid, sodium hydroxide, sodium dihydrogen phosphate, disodium hydrogen phosphate, mannitol tween 20, tween 60, tween 80, sodium chloride, and water for injection.

In one embodiment, the CAR comprises an amino acid sequence as set forth in SEQ ID NO 3 and SEQ ID NO 4, or SEQ ID NO 5 and SEQ ID NO 6, or SEQ ID NO 7 and SEQ ID NO 8, or SEQ ID NO 9 and SEQ ID NO 10, or SEQ ID NO 11 and SEQ ID NO 12, or SEQ ID NO 38 and SEQ ID NO 39, or SEQ ID NO 40 and SEQ ID NO 41, or SEQ ID NO 42 and SEQ ID NO 43.

In various embodiments, a polynucleotide is provided that encodes a CAR encompassed herein.

In various specific embodiments, a polynucleotide encoding a CAR is provided, wherein the polynucleotide sequence is set forth in SEQ ID NO 50 and SEQ ID NO 51, or SEQ ID NO 52 and SEQ ID NO 53, or SEQ ID NO 54 and SEQ ID NO 55, or SEQ ID NO 56 and SEQ ID NO 57, or SEQ ID NO 58 and SEQ ID NO 59, or SEQ ID NO 60 and SEQ ID NO 61, or SEQ ID NO 62 and SEQ ID NO 63, or SEQ ID NO 64 and SEQ ID NO 65.

In various certain embodiments, there is provided a vector comprising a polynucleotide encoding a CAR encompassed herein or a polynucleotide as set forth in SEQ ID NO 50 and SEQ ID NO 51, or SEQ ID NO 52 and SEQ ID NO 53, or SEQ ID NO 54 and SEQ ID NO 55, or SEQ ID NO 56 and SEQ ID NO 57, or SEQ ID NO 58 and SEQ ID NO 59, or SEQ ID NO 60 and SEQ ID NO 61, or SEQ ID NO 62 and SEQ ID NO 63, or SEQ ID NO 64 and SEQ ID NO 65.

In various embodiments, there is provided a method of treating a B cell related condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising GPRC5D CAR T cells as encompassed herein and optionally a pharmaceutically acceptable excipient. In other embodiments, the B cell related condition is multiple myeloma, non-hodgkin's lymphoma, B cell proliferation with uncertain malignant potential, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorder, immune modulation disorder, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, chagas' disease, ge Lei Fushi disease (Grave's disease), wegener's granulomatosis, polyarteritis nodosa, sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, a related vasculitis, goodpasture's disease, kawasaki disease (kasaki disease), autoimmune hemolytic anemia and rapidly progressive nephropathies, primary or immune cell-related glomerulopathies, or monoclonal immune globulin-related diseases with undetermined significance.

In other embodiments, the B cell related condition is a B cell malignancy.

In certain embodiments, the B cell malignancy is Multiple Myeloma (MM) or non-hodgkin's lymphoma (NHL).

In certain embodiments, the MM is selected from the group consisting of: multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, igD myeloma, sclerosteous myeloma, solitary plasmacytoma, and extramedullary plasmacytoma.

In some embodiments, the NHL is selected from the group consisting of: burkitt's lymphoma (Burkittlymphoma), chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, and mantle cell lymphoma.

In particular embodiments, the B cell related condition is a plasma cell malignancy.

In other embodiments, the B cell related condition is an autoimmune disease.

In a further embodiment, the autoimmune disease is systemic lupus erythematosus.

In certain embodiments, the B cell related condition is rheumatoid arthritis.

In particular embodiments, the B cell related condition is idiopathic thrombocytopenic purpura or myasthenia gravis or autoimmune hemolytic anemia.

Drawings

FIG. 1 is a schematic diagram of a chimeric antigen receptor targeting GPRC5D target in example 4 of the present invention;

FIG. 2 is a graph of the T-cell positivity of the chimeric antigen receptor targeting the GPRC5D target in example 7 of the present invention;

FIG. 3 is a graph showing the in vitro anti-tumor effect of murine chimeric antigen receptor T cells targeting GPRC5D target in example 8 of the present invention;

FIG. 4 is a graph of the in vitro anti-tumor effect of humanized chimeric antigen receptor T cells targeting GPRC5D target in example 9 of the present invention;

FIG. 5 is a graph showing in vitro amplification rate and survival rate results for GPRC5D chimeric antigen receptor T cells and positive control PD1-GPRC5D-CART (BMK) in example 10 of the present invention;

FIG. 6 is a flow cytogram of CAR positivity for GPRC5D chimeric antigen receptor T cells and positive control PD1-GPRC5D-CART (BMK) in example 10 of the present invention;

FIG. 7 is a graph showing the results of cell killing rate of LDH killing of GPRC5D chimeric antigen receptor T cells and positive control PD1-GPRC5D-CART (BMK) in example 10 of the present invention;

FIG. 8 is a graph showing the results of cell killing rates of Luciferase killing of GPRC5D chimeric antigen receptor T cells and positive control PD1-GPRC5D-CART (BMK) in example 10 of the present invention;

FIG. 9 is a graph showing the results of the release amount of cytokines targeted to GPRC5D chimeric antigen receptor T cells and positive control PD1-GPRC5D-CART (BMK) in example 10 of the present invention.

Detailed Description

Example 1 preparation of human GPRC5D expression vector and Stable transgenic cell line

Obtaining a base sequence (NM-018654.1) of a CDS region of a coding human GPRC5D gene from an NCBI database, designing a PCR primer 1 (SEQ ID NO: 1) and a primer 2 (SEQ ID NO: 2), selecting a multiple myeloma cell line MM.1S (purchased from Nanjing model animal research institute) for high expression of GPRC5D and cDNA of NCI-H929 (ATCC) as templates for PCR amplification, treating a PCR product and a vector by using a restriction endonuclease, transfecting Escherichia coli DH5 alpha after T4 ligase connection, and picking out a single clone for sequencing. The sequence correctness is verified by sequencing. Successfully constructed strains expressing the plasmid of interest were cultured in medium and plasmid DNA was extracted using a kit (purchased from Tiangen Biochemical technology Ltd.). 293T cells with good logarithmic growth phase were selected and the three plasmids psPAX2 (from Clonetech), pMD2.G (from Clonetech) and pLVX-huGPRC5D-IRES-ZSGreen1 target plasmid were co-transfected with PEI as transfection reagent. Virus fluid was collected at 48 and 72 hours post-transfection, virus was filtered through a 0.45 μ M syringe filter, and cells of interest were infected at MOI = 10. Fluid changes were made 48 hours after viral infection. The constructed cell strain is used for detecting the expression condition of the hGPRC5D through a flow cytometry analyzer, and the construction of the hGPRC5D stable expression cell strain is successful.

EXAMPLE 2 preparation of monoclonal antibodies and screening of antibodies

The mice are immunized by using the cell strain stably expressed by the hGPRC5D, the canthus blood collection is carried out on the mice, and the serum titer of the mice is detected by a flow cytometry analyzer. Selecting the mice with highest titer and stable continuous immunization titer for two times, and injecting the mice into the abdominal cavity before constructing the antibody library for one-time impact immunization. Reference is made to the literature (Krebber, A., bornhauser, S., burmester, J., honegger, A., willuda, J., bosshard, H.R., and Pl ü ckthun, A. (1997) Reable cloning of functional antibody variant from antibodies and dispersed cell polypeptides using a amplified a purified a mutated phase display system.journal of immunological methods 8978 zft 8978-55.) A library of mouse immune phages is constructed, mice are treated, spleens are taken and gently triturated, cells are collected, total RNA of the cells is extracted after lysis using cell lysates, reverse transcription is obtained, variable regions of the antibodies are amplified using mouse specific anti-body weight primers and light chain primers, and variable regions of the antibodies are briefly cloned into a phage display vector. And performing NGS sequencing, limiting dilution method counting storage capacity, PCR detection cloning positive rate and the like on the constructed phage display library to evaluate the diversity and effectiveness of the antibody library. Preparing phage for the phage display library with qualified quality control, performing enrichment panning on the pretreated phage supernatant by using cells stably expressed by hGPRC5D, washing away unbound phage by DPBS, eluting the phage bound to the cells by 0.1M HCl-Glycine, neutralizing the eluate by Tris-HCl, infecting Escherichia coli in logarithmic phase with the phage, and preparing the phage for the next panning. And (3) selecting an escherichia coli monoclonal infected by the phage with the elutriation termination, inoculating the escherichia coli monoclonal into a 96-well plate, preparing a single-chain antibody (scFv) by IPTG induction, and detecting the binding condition of the scFv and the GPRC5D high-expression cell line by a flow cytometry analyzer. HTS0370, HTS0372, HTS0373, HTS0374, and HTS0375 scFv single chain antibodies were optimized for subsequent studies by binding activity assays. After sequencing, the VH and VL sequences and CDR sequences of these 5 antibodies were as follows.

Example 3 humanization of murine GRPC5D antibody

Murine antibodies HTS0370 and HTS0375 were humanized by CDR grafting procedures, i.e., the VH and VK base sequences of HTS0370 and HTS0375 antibodies were analyzed using the IMGT/V-QUEST tool (http:// www.imgt.org/IMGT _ vquest/input) to determine the CDR regions of the antibody light and heavy chains. The amino acid sequences of HTS0370 and HTS0375 antibodies were analyzed using the Igblast tool (https:// www.ncbi.nlm.nih.gov/IgBlast /) to obtain the human germline VH and VK sequences of the highest homology for both antibodies. The CDRs of HTS0370 and HTS0375 antibodies were grafted into the framework regions of selected VH and VK human germline sequences, i.e. humanized antibody sequences. HTS0370 and HTS0375 humanized heavy chain sequences are synthesized into whole genes and then are respectively cloned into a vector containing an IgG1 heavy chain constant region base sequence in a homologous recombination mode to obtain humanized antibody heavy chain expression plasmids; HTS0370 and HTS0375 humanized light chain sequence complete genes are synthesized and cloned into expression vectors respectively in a homologous recombination mode, and plasmids are prepared by a conventional method. Mammalian cells with log-extended period were inoculated into a cell culture flask for culture, PEI co-transfected with humanized light chain plasmid and humanized heavy chain, cell supernatant after transfection was collected, centrifuged and filtered using a filter, protein a medium was used to purify antibody and the antibody was replaced into PBS ph7.2 buffer by dialysis method. The binding of the obtained humanized antibody to GPRC5D overexpressing cell line and endogenously expressing cells was detected by flow cytometry. HTS0370 high-activity humanized antibodies HTS0370Z22 and HTS0370Z23 are obtained by screening, HTS0375 high-activity humanized antibody HTS0375Z56 is obtained, and VH and VL sequences of the antibodies are as follows.

Name of antibody	VH amino acid sequence	VL amino acid sequence
			HTS0370Z22	SEQ ID NO:38	SEQ ID NO:39
HTS0370Z23	SEQ ID NO:40	SEQ ID NO:41
			HTS0375Z56	SEQ ID NO:42	SEQ ID NO:43

Name of antibody	VH nucleotide sequence	VL nucleotide sequence
			HTS0370Z22	SEQ ID NO:60	SEQ ID NO:61
HTS0370Z23	SEQ ID NO:62	SEQ ID NO:63
			HTS0375Z56	SEQ ID NO:64	SEQ ID NO:65

Example 4 construction of chimeric antigen receptor vectors targeting GPRC5D targets

And constructing a chimeric antigen expression vector for the high-activity murine GPRC5D antibody and a corresponding humanized antibody. The Leader (Leader, SEQ ID NO: 44), the light chain variable region of the GPRC5D antibody, the Linker (Linker, SEQ ID NO: 45), the heavy chain variable region of the GPRC5D antibody, the Hinge region (Hinge, SEQ ID NO: 46), the CD8 α transmembrane domain (TM, SEQ ID NO: 47), the 4-1BB costimulatory signaling region (SEQ ID NO: 48), and the CD3 zeta signaling domain (SEQ ID NO: 49) were synthesized in whole genes. The sequences are connected in sequence to obtain chimeric antigen receptor expression cassettes which are respectively named as: the expression cassette structure of the 0370 chimeric antigen receptor, the 0372 chimeric antigen receptor, the 0373 chimeric antigen receptor, the 0374 chimeric antigen receptor, the 0375 chimeric antigen receptor, the 0370Z22 chimeric antigen receptor, the 0370Z23 chimeric antigen receptor and the 0375Z56 chimeric antigen receptor is shown in figure 1. Introducing a Kozac sequence (with the sequence of gccacc) at the forefront end of each expression cassette, synthesizing the sequence of the chimeric antigen receptor expression cassette by the whole gene, connecting the sequence to an empty vector pCDH-EF1-MSC-copGFP (purchased from Changshoubao Biotechnology limited) through an XbaI/SalI enzyme cutting site to obtain a chimeric antigen receptor expression vector, and verifying the correctness by sequencing. Extracting plasmids with plasmid extraction kit (endotoxin-free plasmid extraction kit of Beijing Tiangen Biochemical technology Co., ltd.) to obtain plasmids pCDH-EF1-CAR-GPRC5D-0370-copGFP,

pCDH-EF1-CAR-GPRC5D-0372-copGFP、pCDH-EF1-CAR-GPRC5D-0373-copGFP、

pCDH-EF1-CAR-GPRC5D-0374-copGFP、pCDH-EF1-CAR-GPRC5D-0375-copGFP、

pCDH-EF1-CAR-GPRC5D-0370Z22-copGFP、

pCDH-EF1-CAR-GPRC5D-0370Z23-copGFP、

pCDH-EF1-CAR-GPRC5D-0375Z56-copGFP, ready for infection. The plasmid extraction method is carried out according to the instruction.

Example 5 preparation and concentration of viruses targeting chimeric antigen receptors for GPRC5D targets

Cells were transfected by PEI method. The 293T cells were trypsinized 24 hours before transfection, the 4E6 293T cells were plated in a 10cm cell culture dish, and the cells were cultured in DMEM medium containing 10% FBS for not more than 24 hours, and were transfected when the cells reached a density of 60-80%.

The method comprises the following specific steps:

(1) Placing the plasmid, PEI and DMEM culture medium at room temperature for 5min;

(2) Adding DMEM 450 μ L into 1.5mL EP tube, adding PEI 50 μ L (1 μ g/μ L), mixing, and standing at room temperature for 5min;

(3) Mu.g of the desired plasmid (pCDH-EF 1-CAR-GPRC 5D-0370-copGFP),

pCDH-EF1-CAR-GPRC5D-0372-copGFP、

pCDH-EF1-CAR-GPRC5D-0373-copGFP、

pCDH-EF1-CAR-GPRC5D-0374-copGFP、

pCDH-EF1-CAR-GPRC5D-0375-copGFP、

pCDH-EF1-CAR-GPRC5D-0370Z22-copGFP、

pCDH-EF1-CAR-GPRC5D-0370Z23-copGFP、

Or pCDH-EF1-CAR-GPRC5D-0375Z 56-copGFP), 10 mug of psPAX2 and 5 mug of pMD2.G, adding DMEM to 500 mug L, mixing evenly, and standing for 5min at room temperature;

(4) Adding the prepared PEI-DMEM solution in the step (2) into the DMEM containing the plasmids obtained in the step (3), uniformly mixing, and standing at room temperature for 20min; obtaining a DNA/PEI mixture;

(5) Slowly dropping 1mL of DNA/PEI mixture into a 293T cell culture dish, gently mixing uniformly, and incubating for 6-8h at 37 ℃ in an incubator;

(6) Discarding the original culture medium, replacing the fresh culture medium, and putting the culture medium into a 37 ℃ incubator for continuous incubation;

(7) After the culture medium is replaced for 48 hours, collecting the culture medium, adding 10mL of fresh culture medium into each dish to continue culturing, and collecting the supernatant again after 24 hours to be mixed with the supernatant collected for 48 hours;

(8) Centrifuging at 4000g for 10min at 4 ℃ to remove cell debris;

(9) The resulting supernatant was filtered through a 0.45 μm filter;

(10) Subjecting the filtered supernatant to tangential flow filtration;

(11) Transferring the virus supernatant subjected to tangential flow filtration into an ultracentrifuge tube, centrifuging at 25000rpm for 2 hours, carrying out heavy suspension on the virus precipitate obtained after ultracentrifuge by using a serum-free culture medium, and gently blowing and beating until the virus precipitate is completely dissolved to obtain virus liquid;

(12) Subpackaging the virus solutions, and storing in a refrigerator at-80 deg.C.

293T cells were digested and counted, cell suspensions were prepared in DMEM medium containing 10% FBS, cell density was adjusted to 4E5/mL, and 0.5mL of cell suspension was added to each well of a 24-well culture plate. After the cells are cultured for 8 hours in an adherent way, 1 mu L, 10 mu L, 20 mu L, 30 mu L and 50 mu L of virus solution diluted by 100 times are infected, the solution is changed after 24 hours, and the positive rate of 293T cells is detected by a flow cytometry after 48 hours. The virus titer was calculated to be 1E8 TU.

Example 6 chimeric antigen receptor T cell preparation targeting GPRC5D target

Approximately 25mL of peripheral blood was collected using an anticoagulation tube, following a 1: adding the mixture into lymphocyte separating medium according to the volume ratio of 1, carrying out gradient centrifugation for 25min, taking leucoderma cells after centrifugation, and washing twice by using DPBS to obtain the PBMC of the human peripheral blood mononuclear cells. Resuspending PBMC, adjusting density to 1E 5/. Mu.L, adding CD4/CD8 magnetic beads 10. Mu.L each into 50. Mu.L cell suspension, and separating by magnetic pole to obtain CD4 ⁺ CD8 ⁺ T cells were cultured by adding AIM-V complete medium (purchased from Gibco) containing 10% of FBS, and PBMC was activated with anti-human CD3/CD28 antibody (purchased from America whirlpool Biotech) at an IL-2 concentration of 200IU/mL. After 24 hours of activation, the medium was changed and the culture was continued using complete medium. After 48 hours of culture, T cell density was adjusted to 1E6/mL, infected with virus fluid MOI =10, and fluid changes were performed after 24 hours to obtain T cells expressing chimeric antigen receptors targeting human GPRC5D antigen.

Example 7 chimeric antigen receptor T cell CAR expression Positive Rate detection targeting GPRC5D target

In the culture process, T cells 72 hours after virus infection are taken, centrifuged, resuspended and adjusted to the cell density of 1E6/mL, the anti-Fab antibody is diluted according to the proportion of 1. The results show that CAR is highly expressed on the surface of T cells, and the results are shown in figure 2.

Example 8 murine chimeric antigen receptor T targeting GPRC5D target in vitro anti-tumor Effect

Taking T cells expressing murine GPRC5D chimeric antigen receptors 72 hours after infection and GPRC5D positive myeloma cells NCI-H929, counting and adjusting the cell density to 1E6/mL, and co-culturing according to an effective-target ratio 5:1, namely: t cells 1x10 ⁶ ，NCI-H929 2x10 ⁵ Control cells were CD4 untreated with viral infection ⁺ CD8 ⁺ T cells, scored as Ctrl cells. The mixed cultured cells were labeled with the Antibody APC-conjugated Human BCMA Antibody at 0 hour, 24 hours and 48 hours (0 hour, 24 hours and 48 hours), respectively, the flow analysis cytometry detected the proportion of NCI-H929 cells in total cells, and the target cells in HTS0370, HTS0372, HTS0373, HTS0374 and HTS0375 test groups were reduced to 4.00%, 1.64%, 2.93%, 5.56% and 4.40% respectively at 48 hours, and the in vitro anti-tumor effect of the murine chimeric antigen receptor T cells was significant, and the results are shown in FIG. 3.

Example 9 humanized chimeric antigen receptor T targeting GPRC5D target in vitro anti-tumor Effect

Taking T cells expressing humanized GPRC5D chimeric antigen receptor 72 hours after infection and GPRC5D positive myeloma cells NCI-H929, counting and adjusting the cell density to be 1E6/mL, and co-culturing according to an effective target ratio 1:2, namely: t cells 1x10 ⁶ ，NCI-H929 2x10 ⁶ Control cells were CD4 untreated with viral infection ⁺ CD8 ⁺ T cells, scored as Ctrl cells. The ratio of NCI-H929 cells in total cells was detected by flow cytometry using Antibody APC-conjugated Human BCMA Antibody labeled mixed cultured cells at 0 hr, 24 hr, 48 hr and 72 hr, respectively, and the ratio of target cells in HTS0370, HTS0370Z22, HTS0370Z23 and HTS0375Z56 in experimental groups of HTS0370, HTS0370Z23 and HTS0375Z56 was reduced to 5.33%, 6.95%, 9.85% and 6.89% respectively, and the humanized chimeric antigen receptor T cells showed significant anti-tumor effects in vitro, with the most effective humanized sequences HTS0370Z22 and HTS0375Z56, as shown in FIG. 4.

Example 10 non-viral PD1 site-directed integration targeting GPRC5D chimeric antigen receptor T cell preparation and functional evaluation

This example prepared targeting GPRC5D non-viral PD1 site-directed integration chimeric antigen receptor T cells by electroporation (PD 1-GPRC5D-CART (Z22) used the single chain antibody sequence HTS0370Z22 provided in example 3 of the present invention, PD1-GPRC5D-CART (BMK) used the antibody sequence of US20210393689A1 patent (VL amino acid sequence SEQ ID NO:66 vh amino acid sequence SEQ ID NO: 67) as a positive control). Reference Lonza electrotransfer kit (V4 XP-302)4) The instructions formulate a buffer and resuspend T cells, co-incubate sgRNA targeting PD1 (targeting sequence of sgRNA SEQ ID NO: 68), cas9 protein, and DNA template containing a targeting GPRC5D chimeric antigen receptor element, and add to perform electrotransformation. After the electric conversion, the cells are put into an incubator for continuous culture. The in vitro amplification rates and survival rates of PD1-GPRC5D-CART (BMK) and PD1-GPRC5D-CART (Z22) were examined on days 1,3, 5, and 7 after electrotransformation (FIG. 5). Cells were harvested on day 7 after electroporation and the flow analyzer tested for CAR positivity for PD1-GPRC5D-CART (BMK) and PD1-GPRC5D-CART (Z22) (fig. 6). FIG. 6 results show that the CAR element integration rates in PD1-GPRC5D-CART (BMK) and PD1-GPRC5D-CART (Z22) were 5.08%, 7.09%, respectively. PD1-GPRC5D-CART (BMK), PD1-GPRC5D-CART (Z22) and Untreated T cells (Untreated T) on the 7 th day after the electricity taking transfer are co-cultured according to the effective target ratio of 1:9, 1:3 and 1:1 and tumor target cells K562-GPCR5D over-expressing GPRC5D, and then the cells are cultured according to an LDH killing detection kit (CytoTox)

Non-Radioactive cytoxicity Assay, promega, G1780) protocol and calculate cell killing rate (fig. 7). The results in FIG. 7 show that both PD1-GPRC5D-CART (BMK) and PD1-GPRC5D-CART (Z22) exhibit significant killing effect compared to untreated T cells, and that the anti-tumor effect of PD1-GPRC5D-CART (Z22) is superior to that of the positive control PD1-GPRC5D-CART (BMK). Co-culturing PD1-GPRC5D-CART (BMK) and PD1-GPRC5D-CART (Z22) on the 7 th day after electricity taking transfer and tumor target cells K562-GPCR5D over-expressing GPRC5D according to the effective target ratio of 1:9 and 1:3, and performing Luciferase killing detection according to a Luciferase killing detection kit (Bright-Glo) ^TM Luciferase Assay System, promega, E2620) protocol and calculate cell killing rate (FIG. 8). The results of fig. 8 show that both PD1-GPRC5D-CART (BMK) and PD1-GPRC5D-CART (Z22) showed significant killing effect, and the antitumor effect of PD1-GPRC5D-CART (Z22) was superior to that of the positive control PD1-GPRC5D-CART (BMK). The PD1-GPRC5D-CART (BMK), PD1-GPRC5D-CART (Z22) and Untreated T cells (Untreated T) on the 7 th day after the electricity transfer are co-cultured with tumor target cells K562-GPCR5D over-expressing GPRC5D according to the effective target ratio 1:1, cell supernatants are collected and then respectively treated according to a Human IL-2ELISA detection kit (ELISA MAXTM Deluxe Set Human IL-2, biolegend, 431804) and Human TNF-Alpha ELISA test kit (ELISA MAXTM Deluxe Set Human TNF-alpha, biolegend, 430204), human IFN-gamma ELISA test kit (ELISA MAXTM Deluxe Set Human IFN-gamma, biolegend, 430104) instructions for operation and calculation of the release amount of each cytokine (FIG. 9). The results in figure 9 show that both PD1-GPRC5D-CART (BMK) and PD1-GPRC5D-CART (Z22) are able to significantly secrete relevant cytokines compared to untreated T cells. PD1-GPRC5D-CART (Z22) shows higher release amount on cytokines IL-2, TNF-alpha and IFN-gamma compared with PD1-GPRC5D-CART (BMK). Therefore, the results of the above examples show that PD1-GPRC5D-CART (Z22) has a better killing effect than the current positive control PD1-GPRC5D-CART (BMK).

Sequence listing

<110> Shanghai Yao Biotechnology Ltd

<120> GPRC 5D-targeting chimeric antigen receptor and use thereof

<160> 68

<170> SIPOSequenceListing 1.0

<210> 1

<211> 52

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

agcgaattct ctagacaatt gcttgccgcc accatgtaca aggactgcat cg 52

<210> 2

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cgggggatcc ctcgagtgat catactcctc ctgcatcttg 40

<210> 3

<211> 127

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Trp Met Glu Trp Val Lys Gln Arg Pro Gly Leu Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Leu Pro Gly Ser Ser Tyr Thr Asn Tyr Asn Asp Lys Phe

50 55 60

Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Lys Gly Gly Pro Ile Tyr Tyr Gly Asn Arg Pro Phe Tyr Tyr

100 105 110

Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120 125

<210> 4

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Asn Ile Met Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Gln Ile

35 40 45

Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 5

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Arg Thr Ile Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Ala Leu Arg Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Leu Thr Val Ser Ser

115

<210> 6

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Asn

100 105

<210> 7

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ala Gly Tyr

20 25 30

Thr Val Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Thr Tyr Thr Pro Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Gly Phe Tyr Arg Tyr Asp Phe Asp Phe Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 8

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Met Lys

100 105

<210> 9

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Val Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Thr Tyr Thr Pro Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Gly Phe Tyr Arg Tyr Asp Phe Asp Phe Trp Gly Gln Gly

100 105 110

Thr Ser Leu Thr Val Ser Ser

115

<210> 10

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser

20 25 30

Leu Asn Trp Leu Gln Gln Glu Pro Asp Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys

100 105

<210> 11

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Arg Thr Ile Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Ala Leu Arg Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Leu Thr Val Ser Ser

115

<210> 12

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Ser Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Lys Val Ser Val Thr Cys Lys Ala Ser Gln Ser Val Tyr Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ser Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Asn Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Val

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 13

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Gly Tyr Thr Phe Ser Ser Tyr Trp

1 5

<210> 14

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Ile Leu Pro Gly Ser Ser Tyr Thr

1 5

<210> 15

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Ala Lys Lys Gly Gly Pro Ile Tyr Tyr Gly Asn Arg Pro Phe Tyr Tyr

1 5 10 15

Ala Met Asp Tyr

20

<210> 16

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Gly Tyr Ser Phe Thr Gly Tyr Thr

1 5

<210> 17

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Ile Asn Pro Tyr Asn Gly Arg Thr

1 5

<210> 18

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Ala Arg Val Ala Leu Arg Tyr Ala Met Asp Tyr

1 5 10

<210> 19

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Gly Tyr Ser Phe Ala Gly Tyr Thr

1 5

<210> 20

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Ile Asn Pro Tyr Asn Gly Gly Thr

1 5

<210> 21

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Thr Arg Gly Gly Phe Tyr Arg Tyr Asp Phe Asp Phe

1 5 10

<210> 22

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Gly Tyr Ser Phe Thr Gly Tyr Thr

1 5

<210> 23

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Ile Asn Pro Tyr Asn Gly Gly Thr

1 5

<210> 24

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Thr Arg Gly Gly Phe Tyr Arg Tyr Asp Phe Asp Phe

1 5 10

<210> 25

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Gly Tyr Ser Phe Thr Gly Tyr Thr

1 5

<210> 26

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Ile Asn Pro Tyr Asn Gly Arg Thr

1 5

<210> 27

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Ala Arg Val Ala Leu Arg Tyr Ala Met Asp Tyr

1 5 10

<210> 28

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

Gln Ser Val Ser Asn Asp

1 5

<210> 29

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

Gln Gln Asp Tyr Ser Ser Pro Leu Thr

1 5

<210> 30

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

Gln Asn Val Gly Thr Asn

1 5

<210> 31

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 31

Gln Gln Tyr Asn Ser Tyr Pro Leu Thr

1 5

<210> 32

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

Gln Glu Ile Ser Gly Tyr

1 5

<210> 33

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 33

Leu Gln Tyr Ala Ser Tyr Pro Phe Thr

1 5

<210> 34

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 34

Gln Asp Ile Gly Ser Ser

1 5

<210> 35

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

Leu Gln Tyr Ala Ser Ser Pro Tyr Thr

1 5

<210> 36

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 36

Gln Ser Val Tyr Thr Asn

1 5

<210> 37

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 37

Gln Gln Tyr Asn Ser Tyr Pro Val Thr

1 5

<210> 38

<211> 127

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 38

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Leu Pro Gly Ser Ser Tyr Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Lys Gly Gly Pro Ile Tyr Tyr Gly Asn Arg Pro Phe Tyr Tyr

100 105 110

Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 39

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 39

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Ser Asn Asp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 40

<211> 127

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 40

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Leu Pro Gly Ser Ser Tyr Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Lys Gly Gly Pro Ile Tyr Tyr Gly Asn Arg Pro Phe Tyr Tyr

100 105 110

Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 41

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 41

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 42

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 42

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Arg Thr Ile Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Ala Leu Arg Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 43

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 43

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Tyr Thr Asn

20 25 30

Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ser Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Val

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 44

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 45

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatct 45

<210> 46

<211> 135

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 47

<211> 72

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 48

<211> 126

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 49

<211> 336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 50

<211> 381

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

gaggttcagc tgcagcagtc tggagctgag ctgatgaagc ctggggcctc agtgaagata 60

tcctgcaagg ctactggcta cacattcagt agctactgga tggagtgggt aaagcagagg 120

cctggacttg gccttgagtg gattggacag attttacctg gaagtagtta tactaactac 180

aatgacaaat tcaagggcaa ggccacattc actgcagata catcctccaa cacagcctac 240

atgcaactca gcagcctgac atctgaggac tctgccgtct attactgtgc aaaaaagggg 300

ggcccgatat actatggtaa ccgtcctttt tactatgcta tggactactg gggtcaagga 360

acctcagtca ccgtctcgag t 381

<210> 51

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

aacattatga tgacacagac tcccaaattc ctgcttgtat cagcaggaga cagggttacc 60

ataacctgca aggccagtca gagtgtgagt aatgatgtag cttggtacca acagaagcca 120

gggcagtctc ctaaactgca gatatactat gcatccaatc gctacactgg agtccctgat 180

cgcttcactg gcagtggata tgggacggat ttcactttca ccatcagcac tgtgcaggct 240

gaagacctgg cagtttattt ctgtcagcag gattatagct ctccgctcac gttcggtgct 300

gggacaaagt tggaaataaa a 321

<210> 52

<211> 354

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

gaggtccagc tgcaacagtc tggacctgag ctggtgaagc ctggagcttc aatgaagata 60

tcctgcaagg cttctggtta ctcattcact ggctacacca tgaactgggt gaaacagagc 120

catggaaaga accttgagtg gattggactt attaatcctt ataatggtcg tactatatac 180

aaccagaagt tcaagggcaa ggccacatta actgtagaca agtcatccag cacggcctac 240

atggagctcc tcagtctgac gtccgaggac tctgcagtct attactgtgc aagagtggca 300

ttacggtatg ctatggacta ctggggtcaa ggcacctctc tcacagtctc gagt 354

<210> 53

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

gacattgtga tgactcagtc tcaaaaattc atgtccacat cagtaggaga cagggtcagc 60

gtcacctgca aggccagtca gaatgtgggt actaatgtag cctggtatca acagaaacca 120

gggcaatctc ctaaagcact gatttactcg gcatcctacc ggtacagtgg agtccctgat 180

cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct 240

gaagacttgg cagagtattt ctgtcagcaa tataacagct atcctctcac gttcggtgct 300

ggcaccaagc tggaaatcaa t 321

<210> 54

<211> 357

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 54

gaggtccagc ttcagcagtc tggacctgag ctggtgaagc ctggagcttc aatgaagatt 60

tcctgcaagg cttctggtta ctcattcgct ggctacaccg tgaactgggt gaagcagagc 120

catggcaaga accttgagtg gattggactt attaatcctt acaatggtgg tactacctac 180

accccgaagt tcaaggacaa ggccacatta actgtagaca agtcatccag cacagcctat 240

atggagctcc tcagtctgac atctgaggac tctgcagtct actactgtac aagagggggt 300

ttctataggt acgactttga cttctggggc caaggaacct cagtcaccgt ctcgagt 357

<210> 55

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

gacatcctga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggaaattagt ggttacttaa gttggcttca gcagaaacca 120

gatggaacta ttaaacgcct gatctacgcc gcatccactt tagattctgg tgtcccaaaa 180

aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240

gaagattttg cagactatta ctgtctacaa tatgctagtt atccattcac gttcggctcg 300

gggaccaagc tggaaatgaa a 321

<210> 56

<211> 357

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 56

caggttcagc tgcagcagtc tggacctgag ctggtgaagc ctggaacttc aatgaagatt 60

tcctgcaagg cttctggtta ctcattcact ggctacaccg tgaactgggt gaagcagagc 120

catggcaaga accttgagtg gattggactt attaatcctt acaatggtgg tactacctac 180

accccgaagt tcaaggacaa ggccacatta actgtagaca agtcatccag cacagcctat 240

atggaactcc tcagtctgac atctgaggac tctgcagtct actactgtac aagagggggt 300

ttctataggt acgactttga cttttggggc caaggcacct ctctcacagt ctcgagt 357

<210> 57

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

gaaatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggacattggt agtagcttaa actggcttca gcaggaacca 120

gatggaacta ttaaacgcct gatctacgcc acatccagtt tagattctgg tgtccccaaa 180

aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240

gaagattttg tagactatta ctgtctacaa tatgctagtt ctccgtacac gttcggaggg 300

gggaccaagc tggaaatgaa a 321

<210> 58

<211> 354

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 58

gaggttcagc tgcagcagtc tggacctgag ctggtgaagc ctggagcttc aatgaagata 60

tcctgcaagg cttctggtta ctcattcact ggctacacca tgaactgggt gaaacagagc 120

catggaaaga accttgagtg gattggactt attaatcctt ataatggtcg tactatatac 180

aaccagaagt tcaagggcaa ggccacatta actgtagaca agtcatccag cacggcctac 240

atggagctcc tcagtctgac gtctgaggac tctgcagtct attactgtgc aagagtggca 300

ttacggtatg ctatggacta ctggggtcaa ggcacctctc tcacagtctc gagt 354

<210> 59

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 59

agcattgtga tgacccagtc tcaaaaattc atgtccacat cagtaggaga caaagtcagc 60

gtcacctgca aggccagtca gagtgtgtat actaatgttg cctggtatca acagaaacca 120

gggcaatctc ctaaatcact gatttactcg gcatcctacc gatacaatgg ggtccctgat 180

cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct 240

gaagacttgg cagagtattt ctgtcagcaa tataacagct atccggtcac gttcggtgct 300

gggacaaaat tggaaataaa a 321

<210> 60

<211> 381

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 60

caagtccagt tggtccagag tggggctgaa gtaaaaaagc caggcgcaag tgtcaaagtt 60

tcctgtaaag ccactggtta tacattttct agctactgga tgcattgggt acggcaagcg 120

ccgggacaag gtttagagtg gatggggata atccttcctg gctccagcta tacctcgtat 180

gcgcaaaaat tccaaggacg ggtgaccatg actagggata caagcacctc cacagtgtac 240

atggaactct ctagcctgcg tagcgaggat acagccgtgt attattgtgc aaaaaaaggc 300

ggccccattt actatgggaa tagacccttt tattacgcaa tggattattg gggccaggga 360

accactgtga cagtctcgag t 381

<210> 61

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 61

gatatcgtga tgacgcaatc acctgacagt ctggccgtta gcctgggcga acgcgccaca 60

attaattgta aggccagcca aagcgtatca aacgacttgg cctggtatca gcaaaaaccg 120

ggccagccgc cgaaattgct tatttattat gcatcaacac gtgaaagcgg agttcccgac 180

cggttcagcg gctctggatc gggaactgat ttcacgctga cgatatcttc tctccaggca 240

gaggacgtcg ccgtttatta ttgccaacag gattactcct cacccctgac ttttggcggc 300

ggcaccaaag tcgagatcaa g 321

<210> 62

<211> 381

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 62

caagtccagt tggtccagag tggggctgaa gtaaaaaagc caggcgcaag tgtcaaagtt 60

tcctgtaaag ccactggtta tacattttct agctactgga tgcattgggt acggcaagcg 120

ccgggacaag gtttagagtg gatggggata atccttcctg gctccagcta tacctcgtat 180

gcgcaaaaat tccaaggacg ggtgaccatg actagggata caagcacctc cacagtgtac 240

atggaactct ctagcctgcg tagcgaggat acagccgtgt attattgtgc aaaaaaaggc 300

ggccccattt actatgggaa tagacccttt tattacgcaa tggattattg gggccaggga 360

accactgtga cagtctcgag t 381

<210> 63

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 63

gatatacagt tgacccagtc accgagcttc ctgagcgcca gcgttggcga ccgagtgacc 60

attacgtgta aagcgagtca atcggtgtcc aacgatttgg cgtggtatca acagaagcct 120

ggaaaggccc ctaaactcct gatatactac gcaagcacat tacaaagcgg cgtcccatcc 180

cgtttctctg ggagcggcag tggcactgag ttcactctta ccatcagcag tctccaaccg 240

gaagacttcg ccacctacta ctgtcagcaa gactactcta gtccgctgac atttggcggc 300

ggtacgaagg tggaaattaa g 321

<210> 64

<211> 354

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 64

caagtgcaat tggtgcaaag tggcgcagaa gttaaaaagc caggtagtag cgttaaagtg 60

tcatgcaagg cgagcggcta ctcatttact ggttacacga tcagctgggt gagacaagca 120

ccgggtcaag gactcgaatg gatggggctg atcaaccctt acaatggcag gacaatctac 180

aaccaaaagt tcaaaggaag agtgacaatc accgcggaca aatcaaccag tacggcgtac 240

atggaacttt caagtctgcg atctgaagat acggctgtat actactgcgc ccgtgtcgcc 300

ttaagatatg ctatggatta ctgggggcaa gggactaccg tgacagtctc gagt 354

<210> 65

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 65

gatatccaaa tgactcagtc accgtcgagc ctaagcgcct ccgtgggcga ccgggttacc 60

ataacatgca aagcctctca aagcgtatac acgaatttgg cttggttcca acagaaaccc 120

ggtaaggctc cgaaatctct gatctacagc gccagctcac tgcaatctgg tgtaccaagt 180

cgcttctccg ggtccgggtc cgggacagac tttacgctga cgatttcgtc attgcagccc 240

gaggactttg ccacgtacta ttgccaacag tataacagct acccggtaac tttcgggcaa 300

ggaaccaaag ttgaaatcaa g 321

<210> 66

<211> 110

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 66

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn Pro

85 90 95

Pro Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 67

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 67

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val His Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser His

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Asp Ser Thr Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Gly Gln Trp Lys Tyr Tyr Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 68

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 68

cgactggcca gggcgcctgt 20

Claims (11)

1. A chimeric antigen receptor comprising an extracellular antigen-binding domain comprising a GPRC5D antibody light chain variable region and a GPRC5D antibody heavy chain variable region, wherein the GPRC5D antibody light chain variable region comprises any one of the following group of sequences: SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 39, SEQ ID No. 41 and SEQ ID No. 43;

the GPRC5D antibody heavy chain variable region comprises any one of the following sequences: SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 38, SEQ ID No. 40 and SEQ ID No. 42.

2. The chimeric antigen receptor of claim 1, wherein the GPRC5D antibody heavy chain variable region sequence is SEQ ID No 3 and the GPRC5D antibody light chain variable region sequence is SEQ ID No 4, or the GPRC5D antibody heavy chain variable region sequence is SEQ ID No 5 and the GPRC5D antibody light chain variable region sequence is SEQ ID No 6, or the GPRC5D antibody heavy chain variable region sequence is SEQ ID No 7 and the GPRC5D antibody light chain variable region sequence is SEQ ID No 8, or the GPRC5D antibody heavy chain variable region sequence is SEQ ID No 9 and the GPRC5D antibody light chain variable region sequence is SEQ ID No 10, or GPRC5D antibody heavy chain variable region sequence is SEQ ID No 11 and GPRC5D antibody light chain variable region sequence is SEQ ID No 12, or GPRC5D antibody heavy chain variable region sequence is SEQ ID No 38 and GPRC5D antibody heavy chain variable region sequence is SEQ ID No 39, or GPRC5D antibody light chain variable region sequence is SEQ ID No 43 and GPRC5D antibody light chain variable region sequence is SEQ ID No 40.

3. The chimeric antigen receptor of claim 1 or 2, further comprising a transmembrane domain and an intracellular domain.

4. The chimeric antigen receptor of any one of claims 1-3, wherein the extracellular antigen-binding domain further comprises a leader peptide and a hinge region.

5. The chimeric antigen receptor of any one of claims 1 to 4, which comprises a leader peptide as shown in SEQ ID No. 44, a GPRC5D antibody light chain variable region, a linker peptide as shown in SEQ ID No. 45, a GPRC5D antibody heavy chain variable region, a hinge region as shown in SEQ ID No. 46, a CD8 a transmembrane domain as shown in SEQ ID No. 47, a 4-1BB costimulatory signaling region as shown in SEQ ID No. 48, and a CD3 zeta signaling domain as shown in SEQ ID No. 49.

6. Use of the chimeric antigen receptor of any one of claims 1-5 for the preparation of a medicament or pharmaceutical composition, preferably for the treatment of tumors, more preferably said tumors comprise multiple myeloma.

7. A polynucleotide encoding the chimeric antigen receptor of any one of claims 1-6.

8. A vector comprising the polynucleotide of claim 7.

9. An immune effector cell comprising the polynucleotide of claim 7 or the vector of claim 8, preferably the immune effector cell is a T cell.

10. The immune effector cell of claim 9, wherein the immune effector cell is a PD1 gene knockout cell.

11. A pharmaceutical composition comprising the chimeric antigen receptor of any one of claims 1-6, the polynucleotide of claim 7, the vector of claim 8, or the host cell of claim 9.

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