WO2022247756A1 - Chimeric antigen receptor targeting gprc5d, and use thereof - Google Patents

Abstract

Provided are a chimeric antigen receptor targeting G protein-coupled receptor C5 family subtype D (GPRC5D), and a preparation method and use therefor, especially the use thereof as an anti-cancer drug. The chimeric antigen receptor targeting GPRC5D includes extracellular antigen-binding domain. The extracellular antigen-binding domain includes a GPRC5D antibody light chain variable region and a GPRC5D antibody heavy chain variable region. A single-chain antibody (ScFv) that recognizes the tumor surface antigen GPRC5D is bound to a T cell activation domain and expressed in T cells, so as to recognize and bind the tumor surface antigen, induce T cell activation, secrete cytokines to kill tumor cells, which has a significant effect in the treatment of B lymphoma and acute lymphoblastic leukemia.

Description

Chimeric antigen receptors targeting GPRC5D and uses thereof

The present invention claims the priority of the Chinese patent application with the application number 202110561829.9 and the application title "Chimeric Antigen Receptor Targeting GPRC5D and Its Use" submitted to the China Patent Office on May 23, 2021, the entire contents of which are incorporated by reference incorporated in this application.

technical field

The invention relates to the field of cell therapy, and further, the invention relates to a chimeric antigen receptor targeting G protein-coupled receptor C5 family subtype D (GPRC5D) and a preparation method and application thereof.

Background technique

Chimeric antigen receptor (chimeric antigen receptor, CAR) T cell therapy is to combine single-chain antibody (scFv) that recognizes tumor surface antigen with T cell activation domain, so that T cells express chimeric antigen receptor, so as to recognize and Bind to tumor surface antigens, induce T cell activation, secrete cytokines and kill tumor cells. It is effective in the treatment of B lymphocytoma and acute lymphoblastic leukemia [1].

G protein-coupled receptor C5 family subtype D (GPRC5D) is the first orphan SARS class C GPCR identified in 2001 [2]. There are four subtypes of GPCR family C group 5 receptors (GPRC5 receptors), namely GPRC5A, GPRC5B, GPRC5C and GPRC5D, which are induced by retinoic acid, so they are also called retinoic acid-induced orphan G protein-coupled receptors. body (RAIG) [3]. GPRC5D is highly expressed in the plasma cells of multiple myeloma, basically not expressed in normal tissues, and only expressed in the hair follicle area with immune pardon [4]. Studies have reported that high expression of GPRC5D is associated with poor prognosis in multiple myeloma [5]. CAR-T targeting GPRC5D showed good therapeutic effect in mouse multiple myeloma (MM) model. Since the expressions of GPRC5D and BCMA do not overlap, GPRC5D CAR-T still has a therapeutic effect on tumor recurrence models with BCMA loss [4]. In a GPRC5D-positive mouse model of MM, GPRC5D/CD3 bi-antibody recruited T cells and induced tumor regression [6].

GPRC5D is a surface antigen that is expected to be used for MM immunotherapy, but most GPRC5D drugs are still in the clinical trial stage or research and development stage, and no cell-based drugs targeting GPRC5D have been marketed. Therefore, it is necessary to develop more active and therapeutic drugs. The GPRC5D cell drug with effective effect can be used for the treatment research and application of related diseases.

references:

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. .

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

Contents of the invention

The first object of the present invention is to provide a chimeric antigen receptor comprising an extracellular antigen binding domain, the extracellular antigen binding domain comprising a GPRC5D antibody light chain variable region and a GPRC5D antibody heavy chain variable region , wherein, the light chain variable region of the GPRC5D antibody 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 heavy chain variable region of the GPRC5D antibody 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 present invention, the heavy chain variable region sequence of the GPRC5D antibody is SEQ ID No: 3 and the light chain variable region sequence of the GPRC5D antibody is SEQ ID No: 4, or the heavy chain variable region sequence of the GPRC5D antibody is The 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 The 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 light chain variable region sequence is SEQ ID No: 39, or the GPRC5D antibody heavy chain variable region sequence is SEQ ID No: 40 and the GPRC5D antibody light chain variable region sequence is SEQ ID No: 41, or the GPRC5D antibody heavy chain variable region sequence is SEQ ID No: 42 and the GPRC5D antibody light chain variable region sequence is SEQ ID No: 43.

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

In a specific embodiment of the present invention, the heavy chain variable region of the GPRC5D antibody and the variable region of the light chain of the GPRC5D antibody comprise a linker, the linker is preferably a connecting peptide, and the sequence of the connecting peptide is preferably (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 specific embodiment of the present invention, the extracellular antigen-binding domain further comprises a leader peptide and a hinge region.

In a specific embodiment of the present invention, the leader peptide sequence is IgG1 heavy chain signal polypeptide, granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2) signal peptide or CD8α signal peptide, and/or all The hinge region is CD8α, CH2 and CH3 of IgG1 or IgG4, CD4, CD28 or CD7.

In a specific embodiment of the present invention, the transmembrane domain is any one or more of the following group: α, β or δ chain of T cell receptor, CD3ε, CD3δ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD 154 and PD1, and/or the intracellular domain is any of the following group One or more: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134, CD137 (4-1BB), CD3ζ, CD150 (SLAMF1), CD152 (CTLA4), CD223 ( LAG3), CD270(HVEM), CD273(PD-L2), CD274(PD-L1), CD278(ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70.

In a specific embodiment of the present invention, the chimeric antigen receptor comprises a leader peptide shown in SEQ ID No: 44, a GPRC5D antibody light chain variable region, a connecting peptide shown in SEQ ID No: 45, a GPRC5D anti- Body weight chain variable region, hinge region shown in SEQ ID No:46, CD8α transmembrane domain shown in SEQ ID No:47, 4-1BB co-stimulatory signal transduction region shown in SEQ ID No:48 and SEQ ID CD3ζ signaling domain shown in No:49.

In certain embodiments, the intracellular domain of the chimeric antigen receptor described herein may comprise one or more co-stimulatory signaling domains, and the one or more co-stimulatory signaling domains may also be derived from A co-stimulatory molecule selected from the group consisting of: CARD11, CD2, CD3ζ, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1 ), CD152(CTLA4), CD223(LAG3), CD270(HVEM), CD273(PD-L2), CD274(PD-L1), CD278(ICOS), DAP10, LAT, NKD2C SLP76, TRIM and ZAP70.

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

In further embodiments, the one or more co-stimulatory signaling domains are from co-stimulatory molecules selected from the group consisting of CD137 and CD3ζ.

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

In certain embodiments, said 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 present invention also provides the use of the aforementioned chimeric antigen receptor for preparing medicine or pharmaceutical composition.

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

In a specific embodiment of the invention, said tumor comprises multiple myeloma.

The present invention also provides a polynucleotide encoding the aforementioned chimeric antigen receptor.

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

In other embodiments, said promoter operably linked to a polynucleotide encoding a CAR contemplated herein is selected from the group consisting of: cytomegalovirus immediate early gene promoter (CMV), elongation factor 1 alpha promoter promoter (EF1-α), phosphoglycerate kinase-1 promoter (PGK), ubiquitin-C promoter (UBQ-C), cytomegalovirus enhancer/chicken β-actin promoter (CAG), multiple Oncovirus enhancer/herpes simplex thymidine kinase promoter (MC1), β-actin promoter (β-ACT), Simian virus 40 promoter (SV40) 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 other embodiments, the vector is an episomal vector.

In specific 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 additional 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-maedi virus (visna-maedi virus, VMV) virus, goat arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunization Deficiency Virus (BIV) and Simian Immunodeficiency Virus (SIV).

In specific 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, operably linked to a coding The promoter and right (3') retroviral LTR of the polynucleotide of the CAR contemplated herein.

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 a Simian Virus 40 (SV40) promoter.

In certain embodiments, said 5'LTR or 3'LTR is a lentiviral LTR.

In certain embodiments, said 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 present invention also provides an immune effector cell comprising the aforementioned polynucleotide or the aforementioned vector.

In a specific embodiment of the present invention, said immune effector cells are T cells.

In various embodiments, there is provided an immune effector cell comprising a vector contemplated herein. In various embodiments, the immune effector cells are transduced with a vector contemplated herein.

In other embodiments, the immune effector cells are selected from the group consisting of T lymphocytes, macrophages, and natural killer (NK) cells.

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

In other embodiments, the immune effector cells are PD1 knockout cells.

In some embodiments, sgRNA targeting PD1, Cas9 protein, and double-stranded DNA containing the GPRC5D CAR sequence are introduced into the T cells by electroporation to achieve precise insertion of the CAR element at the PD1 gene site of the T cell, thereby Obtain targeted integration of CAR-T cells targeting GPRC5D non-viral PD1 to improve the effect of targeting and killing tumors.

It should be noted that sgRNAs targeting other genes in T cells can also be introduced into the T cells through the above method to obtain CAR-T cells that target GPRC5D and integrate at other genes, and improve the effect of targeting and killing tumors .

The present invention also provides a pharmaceutical composition, which comprises the aforementioned chimeric antigen receptor, polynucleotide, vector or host cell.

In a specific embodiment of the present invention, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

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

In one embodiment, CAR comprises such as 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 Amino acid sequences set forth in SEQ ID NO:42 and SEQ ID NO:43.

In various embodiments, a polynucleotide encoding a CAR contemplated herein is provided.

In various specific embodiments, there is provided a polynucleotide encoding a CAR, wherein said 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:60 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 contemplated herein or such as 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 A polynucleotide set forth in 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 said subject a therapeutically effective amount of a composition comprising Contemplated GPRC5D CAR T cells and optionally a pharmaceutically acceptable excipient. In other embodiments, the B-cell-associated condition is multiple myeloma, non-Hodgkin's lymphoma, B-cell proliferation of uncertain malignant potential, lymphomatoid granulomatosis, post-transplantation lymphoproliferative disorder, immunomodulatory Conditions, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas' disease, Grave's disease, Wegener's meat Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, ANCA-associated vasculitis , Goodpasture's disease, Kawasaki disease, autoimmune hemolytic anemia and rapidly progressive glomerulonephritis, heavy chain disease, primary or immune cell-associated amyloid Monoclonal gammopathy of degenerative or undetermined significance.

In other embodiments, said B cell-associated 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 overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma , Solitary skeletal plasmacytoma and extramedullary plasmacytoma.

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

In specific embodiments, said B cell-associated condition is a plasma cell malignancy.

In other embodiments, said B cell-associated condition is an autoimmune disease.

In additional embodiments, the autoimmune disease is systemic lupus erythematosus.

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

In specific embodiments, said B cell-associated condition is idiopathic thrombocytopenic purpura or myasthenia gravis or autoimmune hemolytic anemia.

Description of drawings

Figure 1 is a schematic diagram of a chimeric antigen receptor targeting a GPRC5D target in Example 4 of the present invention;

Figure 2 is a positive rate diagram of chimeric antigen receptor T cells targeting the GPRC5D target in Example 7 of the present invention;

Figure 3 is an in vitro anti-tumor effect diagram of murine chimeric antigen receptor T cells targeting the GPRC5D target in Example 8 of the present invention;

Figure 4 is an in vitro anti-tumor effect diagram of humanized chimeric antigen receptor T cells targeting the GPRC5D target in Example 9 of the present invention;

Fig. 5 is a result graph of in vitro expansion rate and survival rate of targeting GPRC5D chimeric antigen receptor T cells and positive control PD1-GPRC5D-CART (BMK) in Example 10 of the present invention;

Fig. 6 is the flow cytogram of CAR positive rate of targeting GPRC5D chimeric antigen receptor T cells and positive control PD1-GPRC5D-CART (BMK) in Example 10 of the present invention;

Fig. 7 is a result graph of the cell killing rate of LDH killing targeting GPRC5D chimeric antigen receptor T cells and positive control PD1-GPRC5D-CART (BMK) in Example 10 of the present invention;

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

9 is a graph showing the release of cytokines targeting GPRC5D chimeric antigen receptor T cells and the positive control PD1-GPRC5D-CART (BMK) in Example 10 of the present invention.

Detailed ways

Example 1. Preparation of human GPRC5D expression vector and stable transfection cell line

Obtain the nucleotide sequence (NM_018654.1) encoding the CDS region of the human GPRC5D gene from the NCBI database, design PCR primer 1 (SEQ ID NO: 1) and primer 2 (SEQ ID NO: 2), and select multiples with high expression of GPRC5D The myeloma cell line MM.1S (purchased from Nanjing Institute of Model Animals), and the cDNA of NCI-H929 (ATCC) were used as templates for PCR amplification, and the PCR products and vectors were treated with restriction endonucleases, and T4 ligase was ligated and transfected. Escherichia coli DH5α was transfected, and single clones were picked for sequencing. The correctness of the sequence was verified by sequencing. The successfully constructed bacterial strain expressing the target plasmid was cultivated in the culture medium and the plasmid DNA was extracted using a kit (purchased from Tiangen Biochemical Technology Co., Ltd.). 293T cells in the logarithmic growth phase in good growth state were selected, and the three plasmids psPAX2 (purchased from Clonetech), pMD2.G (purchased from Clonetech) and pLVX-huGPRC5D-IRES-ZSGreen1 plasmids of interest were co-transfected using the transfection reagent PEI. The virus liquid was collected 48 hours and 72 hours after transfection, filtered through a 0.45 μM syringe filter, and infected with the target cells according to MOI=10. The medium was changed 48 hours after virus infection. The constructed cell line was detected by flow cytometry to detect the expression of hGPRC5D, and the stable expression cell line of hGPRC5D was successfully constructed.

Example 2. Preparation of Monoclonal Antibodies and Screening of Antibodies

Cell lines stably expressing hGPRC5D were used to immunize mice, blood was collected from the canthus of mice, and the serum titer of mice was detected by flow cytometry. Select the mouse with the highest titer and two consecutive immune titers that tend to be stable, and perform a shock immunization by intraperitoneal injection before building the antibody library. Reference literature (Krebber, A., Bornhauser, S., Burmester, J., Honegger, A., Willuda, J., Bosshard, H.R., and Plückthun, A. (1997). Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. Journal of immunological methods 201,35-55.) method to construct mouse immune phage library, in short, treat mice, take spleen and gently grind, collect cells, use After the cell lysate was lysed, the total RNA of the cells was extracted, reverse-transcribed to obtain cDNA, and the variable region gene of the antibody was amplified by using mouse-specific antibody heavy chain variable region primers and light chain variable region primers, and cloned into phage displayed on the carrier. The constructed phage display library was subjected to NGS sequencing, limited dilution method to count the library capacity, and PCR to detect the positive rate of clones to evaluate the diversity and effectiveness of the antibody library. Prepare phages from quality-controlled phage display libraries, use cells stably expressing hGPRC5D to enrich and pan the pretreated phage supernatants, wash unbound phages with DPBS, and then elute them with 0.1M HCl-Glycine to bind to the cells The phages on the plate were then neutralized with Tris-HCl to neutralize the eluate, and the phages were used to infect Escherichia coli in the logarithmic growth phase, and the phages were prepared for the next round of panning. The Escherichia coli monoclonal clones infected with phages that terminated the panning were selected, inoculated in 96-well plates, induced by IPTG to prepare single-chain antibodies (scFv), and the combination of scFv and GPRC5D high-expression cell lines was detected by flow cytometry. Through binding activity analysis, HTS0370, HTS0372, HTS0373, HTS0374 and HTS0375 scFv single chain antibodies were selected for subsequent research. After sequence determination, the VH and VL sequences of the five antibodies and their CDR sequences are as follows.

Antibody heavy chain CDR sequence list HCDR1 HCDR2 HCDR3 HTS0370 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15 HTS0372 SEQ ID NO:16 SEQ ID NO:17 SEQ ID NO:18 HTS0373 SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21 HTS0374 SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 HTS0375 SEQ ID NO:25 SEQ ID NO:26 SEQ ID NO:27

Antibody light chain CDR sequence list LCDR1 LCDR2 LCDR3 HTS0370 SEQ ID NO:28 YAS SEQ ID NO:29 HTS0372 SEQ ID NO: 30 SAS SEQ ID NO: 31 HTS0373 SEQ ID NO:32 AAS SEQ ID NO:33 HTS0374 SEQ ID NO:34 ATS SEQ ID NO:35 HTS0375 SEQ ID NO:36 SAS SEQ ID NO:37

Example 3. Humanization of murine GRPC5D antibody

According to the CDR transplantation method, the murine antibodies HTS0370 and HTS0375 were humanized, that is, the VH and VK bases of the HTS0370 and HTS0375 antibodies were analyzed using the IMGT/V-QUEST tool (http://www.imgt.org/IMGT_vquest/input) Sequence to determine the CDR regions of the antibody light and heavy chains. Using the IgBlast tool (https://www.ncbi.nlm.nih.gov/igblast/) to analyze the amino acid sequences of HTS0370 and HTS0375 antibodies, the most homologous human germline VH and VK sequences of the two antibodies were obtained. The CDRs of the HTS0370 and HTS0375 antibodies were grafted into the framework regions of selected VH and VK human germline sequences, which were humanized antibody sequences. The HTS0370 and HTS0375 humanized heavy chain sequences were synthesized and cloned into vectors containing the IgG1 heavy chain constant region base sequence by homologous recombination to obtain humanized antibody heavy chain expression plasmids; HTS0370 and HTS0375 After the whole gene synthesis of the humanized light chain sequence is cloned into the expression vector by means of homologous recombination, the plasmid is prepared by a conventional method. Mammalian cells in the logarithmic growth phase were inoculated into cell culture flasks for culture, PEI co-transfected humanized light chain plasmid and humanized heavy chain, collected the transfected cell supernatant, centrifuged and filtered with a filter, Protein Antibody was purified in medium A and replaced by dialysis into PBS pH7.2 buffer. The combination of the obtained humanized antibody with the GPRC5D overexpression cell line and endogenous expression cells was detected by flow cytometry. HTS0370 highly active humanized antibodies HTS0370Z22 and HTS0370Z23 were screened, and HTS0375 highly active humanized antibody HTS0375Z56 was obtained. The VH and VL sequences of the antibodies are as follows.

Antibody name 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

Antibody name 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 vector targeting GPRC5D target

A chimeric antigen expression vector was constructed for the highly active murine GPRC5D antibody and the corresponding humanized antibody. Whole gene synthesis leader peptide (Leader, SEQ ID NO:44), GPRC5D antibody light chain variable region, connecting peptide (Linker, SEQ ID NO:45), GPRC5D antibody heavy chain variable region, hinge region (Hinge, SEQ ID NO:46), CD8α transmembrane domain (TM, SEQ ID NO:47), 4-1BB co-stimulatory signaling domain (SEQ ID NO:48) and CD3ζ signaling domain (SEQ ID NO:49). The above sequences were ligated sequentially to obtain chimeric antigen receptor expression cassettes, named respectively: 0370 chimeric antigen receptor expression cassette, 0372 chimeric antigen receptor expression cassette, 0373 chimeric antigen receptor expression cassette, 0374 chimeric antigen receptor expression cassette 0375 chimeric antigen receptor expression cassette, 0370Z22 chimeric antigen receptor expression cassette, 0370Z23 chimeric antigen receptor expression cassette and 0375Z56 chimeric antigen receptor expression cassette. The structure of the expression cassette is shown in Figure 1. A Kozac sequence (sequence gccacc) was introduced at the front end of each expression cassette, and after the sequence of the chimeric antigen receptor expression cassette was synthesized, the empty vector pCDH-EF1-MSC-copGFP (purchased The chimeric antigen receptor expression vector was obtained from Changsha Youbao Biotechnology Co., Ltd., and it was verified to be correct by sequencing. Use a plasmid extraction kit (Beijing Tiangen Biochemical Technology Co., Ltd. endotoxin-free plasmid extraction kit) to extract plasmids, and 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 can be carried out according to the instructions.

Example 5. Preparation and Concentration of Viruses Targeting GPRC5D Targeted Chimeric Antigen Receptors

Cells were transfected by PEI method. Trypsinize 293T cells 24 hours before transfection, spread 4E6 293T cells in a 10cm cell culture dish, and culture the cells in DMEM medium containing 10% FBS, no more than 24 hours, when the cells reach 60-80% density can be transfected.

Specific steps are as follows:

(1) Place the plasmid, PEI, and DMEM medium at room temperature for 5 minutes;

(2) Put 450 μL of DMEM into a 1.5 mL EP tube, then add 50 μL of PEI (1 μg/μL) to mix, and let stand at room temperature for 5 minutes;

(3) Take 10 μg of the target plasmid (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,

or pCDH-EF1-CAR-GPRC5D-0375Z56-copGFP), 10 μg psPAX2, 5 μg pMD2.G, add DMEM to 500 μL, mix well, and stand at room temperature for 5 minutes;

(4) Add the prepared PEI-DMEM solution of step (2) into the plasmid-containing DMEM obtained in step (3), mix well, and let stand at room temperature for 20 minutes; obtain a DNA/PEI mixture;

(5) Slowly drop 1mL DNA/PEI mixture into a 293T cell culture dish, mix gently, and incubate in a 37°C incubator for 6-8 hours;

(6) Discard the original medium, replace with fresh medium, and put it into a 37°C incubator to continue incubation;

(7) After changing the medium for 48 hours, collect the medium, then add 10 mL of fresh medium to each dish to continue the culture, collect the supernatant again after 24 hours, and mix it with the supernatant collected for 48 hours;

(8) Centrifuge at 4000g for 10min at 4°C to remove cell debris;

(9) supernatant obtained by filtering with a 0.45 μm filter;

(10) performing tangential flow filtration on the filtered supernatant;

(11) Transfer the virus supernatant after tangential flow filtration into an ultracentrifuge tube, centrifuge at 25,000 rpm for 2 hours, resuspend the virus pellet obtained after ultracentrifugation with serum-free medium, and gently blow until completely dissolved to obtain virus fluid;

(12) Aliquot each virus solution and store in a -80°C refrigerator.

Digest and count 293T cells, make cell suspension with DMEM medium containing 10% FBS, adjust cell density to 4E5/mL, add 0.5 mL of cell suspension to each well of 24-well culture plate. After 8 hours of adherent cell culture, 1 μL, 10 μL, 20 μL, 30 μL, and 50 μL of virus solution diluted 100 times were infected, and the medium was changed after 24 hours, and the positive rate of 293T cells was detected by flow cytometry after 48 hours. After calculation, the virus titer was 1E8TU.

Example 6. Preparation of chimeric antigen receptor T cells targeting GPRC5D target

Use an anticoagulant tube to collect about 25 mL of peripheral blood, add it to the lymphocyte separation medium at a volume ratio of 1:1, and centrifuge for 25 minutes in a gradient. After centrifugation, take the buffy coat cells and wash them twice with DPBS to obtain human peripheral blood mononuclear cells. PBMCs. Resuspend PBMC, adjust the density to 1E5/μL, add 10 μL each of CD4/CD8 magnetic beads to 50 μL cell suspension, obtain CD4 ⁺ CD8 ⁺ T cells by magnetic pole separation, add AIM-V complete medium containing 10% FBS ( purchased from Gibco) and cultured, and activated PBMC with anti-human CD3/CD28 antibody (purchased from Miltenyi Biotechnology Co., Ltd.) with an IL-2 concentration of 200 IU/mL. After 24 hours of activation, the medium was changed, and the complete medium was used to continue culturing. After culturing for 48 hours, adjust the T cell density to 1E6/mL, infect with virus liquid at MOI=10, and change the liquid after 24 hours to obtain T cells expressing chimeric antigen receptors targeting human GPRC5D antigen.

Example 7. Detection of positive rate of CAR expression in chimeric antigen receptor T cells targeting GPRC5D

During the culture process, take the T cells 72 hours after virus infection, centrifuge, resuspend and adjust the cell density to 1E6/mL, dilute the anti-Fab antibody at a ratio of 1:100, incubate on ice for 30 minutes, wash once with DPBS, After resuspension, the positive rate of CAR-GPRC5D was detected by flow cytometer. The results showed that CAR was highly expressed on the surface of T cells, the results are shown in Figure 2.

Example 8. In vitro anti-tumor effect of murine chimeric antigen receptor T targeting GPRC5D

Take T cells expressing murine GPRC5D chimeric antigen receptor and GPRC5D-positive myeloma cells NCI-H929 72 hours after infection, count and adjust the cell density to 1E6/mL, and co-culture according to the effect-to-target ratio of 5:1, namely: T cells 1x10 ⁶ , NCI-H929 2x10 ⁵ , and control cells were CD4 ⁺ CD8 ⁺ T cells without virus infection, which were designated as Ctrl cells. At 0 hours, 24 hours and 48 hours (0h, 24h and 48h), the mixed cultured cells were labeled with the antibody APC-conjugated Human BCMA Antibody, and the proportion of NCI-H929 cells in the total cells was detected by flow cytometry, 48 hours , the target cells in the HTS0370, HTS0372, HTS0373, HTS0374 and HTS0375 experimental groups were reduced to 4.00%, 1.64%, 2.93%, 5.56% and 4.40%, respectively, and the mouse-derived chimeric antigen receptor T cells had a significant anti-tumor effect in vitro, see the results image 3.

Example 9. In vitro anti-tumor effect of humanized chimeric antigen receptor T targeting GPRC5D

Take T cells expressing humanized GPRC5D chimeric antigen receptor 72 hours after infection and GPRC5D-positive myeloma cells NCI-H929, count and adjust the cell density to 1E6/mL, and co-culture according to the effect-to-target ratio of 1:2, that is : T cells 1x10 ⁶ , NCI-H929 2x10 ⁶ , control cells were CD4 ⁺ CD8 ⁺ T cells without virus infection treatment, which were recorded as Ctrl cells. At 0 hours, 24 hours, 48 hours and 72 hours, the mixed cultured cells were labeled with the antibody APC-conjugated Human BCMA Antibody, and the proportion of NCI-H929 cells in the total cells was detected by flow cytometry. At 72 hours, the target cells were in HTS0370, HTS0370Z22, HTS0370Z23, and HTS0375Z56 experimental groups were reduced to 5.33%, 6.95%, 9.85%, and 6.89%, respectively, and the anti-tumor effect of humanized chimeric antigen receptor T cells was remarkable in vitro, and the best effect was humanized sequence HTS0370Z22 and HTS0375Z56, the results are shown in Figure 4.

Example 10. Preparation and functional evaluation of non-viral PD1 site-specific integration targeting GPRC5D chimeric antigen receptor T cells

Non-Radioactive Cytotoxicity Assay,Promega,G1780)说明书操作并计算细胞杀伤率(图7)。图7结果显示相比于未处理的T细胞，PD1-GPRC5D-CART(BMK)和PD1-GPRC5D-CART(Z22)均表现出显著的杀伤效果，PD1-GPRC5D-CART(Z22)的抗肿瘤效果要优于阳性对照PD1-GPRC5D-CART(BMK)。取电转后第7天的PD1-GPRC5D-CART(BMK)和PD1-GPRC5D-CART(Z22)与过表达GPRC5D的肿瘤靶细胞K562-GPCR5D按效靶比1:9、1:3共培养，按照Luciferase杀伤检测试剂盒(Bright-Glo ^TM Luciferase Assay System,Promega,E2620)说明书操作并计算细胞杀伤率(图8)。图8结果显示PD1-GPRC5D-CART(BMK)和PD1-GPRC5D-CART(Z22)均表现出显著的杀伤效果，PD1-GPRC5D-CART(Z22)的抗肿瘤效果要优于阳性对照PD1-GPRC5D-CART(BMK)。取电转后第7天的PD1-GPRC5D-CART(BMK)、PD1-GPRC5D-CART(Z22)和未处理T细胞(Untreated T)按效靶比1:1与过表达GPRC5D的肿瘤靶细胞K562-GPCR5D共培养，收集细胞上清液后分别按照人源IL-2ELISA检测试剂盒 (ELISA MAXTM Deluxe Set Human IL-2,Biolegend,431804)、人源TNF-αELISA检测试剂盒(ELISA MAXTM Deluxe Set Human TNF-α,Biolegend,430204)、人源IFN-γELISA检测试剂盒(ELISA MAXTM Deluxe Set Human IFN-γ,Biolegend,430104)说明书操作并计算各细胞因子的释放量(图9)。图9结果显示相比于未处理的T细胞，PD1-GPRC5D-CART(BMK)和PD1-GPRC5D-CART(Z22)均能显著地分泌相关细胞因子。PD1-GPRC5D-CART(Z22)相对于PD1-GPRC5D-CART(BMK)在细胞因子IL-2、TNF-α、IFN-γ上均表现出更高的释放量。因此，从上述实施例结果显示PD1-GPRC5D-CART(Z22)较目前阳性对照PD1-GPRC5D-CART(BMK)具有更好的杀伤效果。 In this example, the chimeric antigen receptor T cells targeting GPRC5D non-viral PD1 site-specific integration (PD1-GPRC5D-CART (Z22) were prepared by electroporation using the single-chain antibody sequence HTS0370Z22 provided in Example 3 of the present invention, PD1-GPRC5D-CART (BMK) uses the antibody sequence (VL amino acid sequence SEQ ID NO: 66; VH amino acid sequence SEQ ID NO: 67) in the US20210393689A1 patent as a positive control). Prepare the buffer and resuspend T cells according to the instructions of the Lonza electroporation kit (V4XP-3024), and combine the sgRNA targeting PD1 (sgRNA targeting sequence SEQ ID NO:68), Cas9 protein and chimeric antigen receptor containing targeting GPRC5D The DNA template of the element was added after co-incubation for electroporation. After electroporation, the cells were placed in an incubator to continue culturing. The in vitro expansion rate and survival rate of PD1-GPRC5D-CART (BMK) and PD1-GPRC5D-CART (Z22) were detected on day 1, day 3, day 5, and day 7 after electroporation (Figure 5). The cells were collected on the 7th day after electroporation, and the CAR positive rates of PD1-GPRC5D-CART (BMK) and PD1-GPRC5D-CART (Z22) were detected by flow cytometry (Figure 6). The results in Figure 6 show that the integration rates of CAR elements in PD1-GPRC5D-CART (BMK) and PD1-GPRC5D-CART (Z22) were 5.08% and 7.09%, respectively. Take PD1-GPRC5D-CART (BMK), PD1-GPRC5D-CART (Z22) and untreated T cells (Untreated T) on the 7th day after electroporation according to the effect-target ratio of 1:9, 1:3, 1:1 and the treated cells. Tumor target cells expressing GPRC5D K562-GPCR5D co-cultured, according to the LDH killing detection kit (CytoTox

Non-Radioactive Cytotoxicity Assay, Promega, G1780) manual operation and calculation of cell killing rate (Figure 7). The results in Figure 7 show that compared with untreated T cells, both PD1-GPRC5D-CART (BMK) and PD1-GPRC5D-CART (Z22) exhibited significant killing effects, and the anti-tumor effect of PD1-GPRC5D-CART (Z22) It is better than the positive control PD1-GPRC5D-CART (BMK). The PD1-GPRC5D-CART (BMK) and PD1-GPRC5D-CART (Z22) on the 7th day after electroporation were co-cultured with the tumor target cell K562-GPCR5D overexpressing GPRC5D according to the effect-to-target ratio of 1:9 and 1:3. The Luciferase Killing Assay Kit (Bright-Glo ^TM Luciferase Assay System, Promega, E2620) was operated according to the instructions and the cell killing rate was calculated ( FIG. 8 ). The results in Figure 8 show that both PD1-GPRC5D-CART (BMK) and PD1-GPRC5D-CART (Z22) showed significant killing effects, and the anti-tumor effect of PD1-GPRC5D-CART (Z22) was better than that of the positive control PD1-GPRC5D- CART (BMK). PD1-GPRC5D-CART (BMK), PD1-GPRC5D-CART (Z22) and untreated T cells (Untreated T) on the 7th day after electroporation were compared with tumor target cells K562- GPCR5D was co-cultured, and after the cell supernatant was collected, the human IL-2 ELISA detection kit (ELISA MAXTM Deluxe Set Human IL-2, Biolegend, 431804) and the human TNF-αELISA detection kit (ELISA MAXTM Deluxe Set Human TNF -α, Biolegend, 430204), human IFN-γ ELISA detection kit (ELISA MAXTM Deluxe Set Human IFN-γ, Biolegend, 430104) instructions to operate and calculate the release of each cytokine (Figure 9). The results in Figure 9 show that compared with untreated T cells, both PD1-GPRC5D-CART (BMK) and PD1-GPRC5D-CART (Z22) can significantly secrete related cytokines. Compared with PD1-GPRC5D-CART (BMK), PD1-GPRC5D-CART (Z22) showed higher release of cytokines IL-2, TNF-α, and IFN-γ. Therefore, the results from the above examples show that PD1-GPRC5D-CART (Z22) has a better killing effect than the current positive control PD1-GPRC5D-CART (BMK).

Claims (11)

A chimeric antigen receptor comprising an extracellular antigen-binding domain, the 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 The variable region contains 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 heavy chain variable region of the GPRC5D antibody 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. The chimeric antigen receptor according to 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 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 It is SEQ ID No: 38 and the GPRC5D antibody light chain variable region sequence is SEQ ID No: 39, or the GPRC5D antibody heavy chain variable region sequence is SEQ ID No: 40 and the GPRC5D antibody light chain variable region sequence The region sequence is SEQ ID No: 41, or the heavy chain variable region sequence of the GPRC5D antibody is SEQ ID No: 42 and the light chain variable region sequence of the GPRC5D antibody is SEQ ID No: 43. The chimeric antigen receptor of claim 1 or 2, further comprising a transmembrane domain and an intracellular domain. The chimeric antigen receptor of any one of claims 1-3, the extracellular antigen binding domain further comprising a leader peptide and a hinge region. The chimeric antigen receptor according to any one of claims 1-4, said chimeric antigen receptor comprising a leader peptide shown in SEQ ID No: 44, GPRC5D antibody light chain variable region, SEQ ID No: Linker peptide shown in 45, GPRC5D antibody heavy chain variable region, hinge region shown in SEQ ID No:46, CD8α transmembrane domain shown in SEQ ID No:47, 4- 1BB co-stimulatory signaling domain and the CD3ζ signaling domain shown in SEQ ID No:49. The use of the chimeric antigen receptor according to any one of claims 1-5 for the preparation of a medicament or a pharmaceutical composition, preferably, the medicament or the pharmaceutical composition is used for treating tumors, more preferably, the Tumors include multiple myeloma. A polynucleotide encoding the chimeric antigen receptor according to any one of claims 1-6. A vector comprising the polynucleotide according to claim 7. An immune effector cell comprising the polynucleotide of claim 7 or the carrier of claim 8, preferably, the immune effector cell is a T cell. The immune effector cell according to claim 9, said immune effector cell is a PD1 gene knockout cell. A pharmaceutical composition comprising the chimeric antigen receptor according to any one of claims 1-6, the polynucleotide according to claim 7, the carrier according to claim 8 or the polynucleotide according to claim 9 host cell.

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