GPRC5D-targeted single-domain antibody, chimeric antigen receptor and application of GPRC5D-targeted single-domain antibody and chimeric antigen receptor

Abstract

The invention relates to the technical field of immunotherapy, and provides a GPRC5D-targeted single-domain antibody, a chimeric antigen receptor and application of the GPRC5D-targeted single-domain antibody and the chimeric antigen receptor. Specifically, the invention provides a GPRC5D-targeted single-domain antibody, a GPRC5D-targeted chimeric antigen receptor (CAR) constructed by using the single-domain antibody, and an engineered immune effector cell containing the CAR. The invention also provides a single-domain antibody targeting GPRC5D, a chimeric antigen receptor and an application of the engineered immune effector cell containing the CAR in preparation of drugs for diagnosis, prevention and / or treatment of diseases or symptoms related to GPRC5D expression.

Description

[0001] This application is a divisional application of a patent application with an application date of November 14, 2023, application number 202311506682.9, and invention name “A single-domain antibody, chimeric antigen receptor and its application targeting GPRC5D”. Technical Field

[0002] The present invention belongs to the field of biomedicine, and more specifically, relates to a single domain antibody specifically targeting GPRC5D, a chimeric antigen receptor targeting GPRC5D constructed by the single domain antibody, an engineered immune effector cell comprising such a chimeric antigen receptor, and uses thereof in diagnosing, preventing and / or treating diseases related to GPRC5D expression. Background Art

[0003] Multiple myeloma (MM) is a malignant disease caused by abnormal proliferation of clonal plasma cells. It is classified as a B-cell related malignancy. In many countries, it is the second most common hematological malignancy, accounting for about 2% of all cancer deaths. Common clinical symptoms of multiple myeloma include manifestations of myeloma-related organ dysfunction, namely "CRAB" symptoms (calcium elevation, renal impairment, anemia, bone disease) and related manifestations such as secondary amyloidosis. Currently, the preferred treatment for multiple myeloma is autologous hematopoietic stem cell transplantation (ASCT) (see Chinese Medical Association Hematology Branch, Chinese Medical Association Hematology Branch. Chinese Multiple Myeloma Diagnosis and Treatment Guidelines (Revised in 2022) [J]. Chinese Journal of Internal Medicine, 2022, 61(5): 480-487). Traditional treatments for multiple myeloma, such as chemotherapy, radiotherapy, stem cell transplantation, bone marrow transplantation, etc., although current treatments usually relieve the disease, almost all patients will eventually relapse. For patients with newly diagnosed multiple myeloma, commonly used first-line treatment drugs include proteasome inhibitors, immunomodulatory drugs and alkylating agents. For most patients, commonly used first-line treatments can stabilize the patient's condition for 3-5 years, but a small number of patients show primary drug resistance at the time of initial treatment, and the condition cannot be effectively controlled. For most newly diagnosed patients who are effectively treated, they will inevitably enter the relapse / refractory stage after a period of disease stabilization. Therefore, there are still unmet medical needs for a considerable number of patients with multiple myeloma, especially those with relapsed / refractory multiple myeloma.

[0004] When selecting therapeutic targets, unlike other B-cell malignancies, CD19 expression is observed in only 2% of myeloma patients, and common extracellular immune phenotypic markers in myeloma (CD138, CD38, and CD56) are co-expressed on other basic cell types. Currently, BCMA is the main target in immunotherapy for multiple myeloma, but there are still a considerable number of multiple myeloma patients with BCMA-negative or low BCMA expression, as well as BCMA-negative relapses due to antigen immune escape after BCMA-targeted therapy. Therefore, there is still a large unmet demand for immunotherapy methods based on new therapeutic targets that can be effectively used to treat multiple myeloma.

[0005] GPRC5D (G protein-coupled receptor, class C, group 5, member D), that is, G protein-coupled receptor family C group 5 member D, is a member of the G protein-coupled receptor family. G protein-coupled receptors are membrane protein receptors that play an important role in the human body. As a new G protein-coupled receptor drug target, GPRC5D has not yet found its endogenous ligand and is an orphan receptor. Existing studies have shown that GPRC5D is specifically highly expressed in multiple myeloma cells and is almost not expressed in normal tissues. The unique expression of GPRC5D in the plasma cell lineage also makes it potentially an ideal target for anti-myeloma treatment. Studies have also shown that in the tumor recurrence model caused by BCMA antigen loss, GPRC5D-targeted CAR-T cells have anti-tumor activity. In alleviating the problem of tumor recurrence caused by BCMA escape, GPRC5D will be a highly potential specific therapeutic target.

[0006] Single-domain antibodies (SdAbs) differ from traditional four-chain antibodies because they have a single monomer antibody variable domain. For example, camelids and sharks produce antibodies that naturally lack light chains, which are called heavy chain-only antibodies, or heavy chain antibodies (HCAbs) for short. The antigen-binding fragment in each arm of the camelid heavy chain antibody has a single heavy chain variable domain, which is called a heavy chain single-domain antibody (VHH). This type of VHH can have a high affinity for antigens without the help of light chains. Camelid-derived VHH antibodies are known as the smallest functional antigen-binding fragments, with a molecular weight of only about 15kD, and are therefore also called nanobodies (Nbs). VHH antibodies have the natural advantages of good solubility, high stability, strong penetration, and wide binding epitopes. Since their discovery, they have gradually attracted the attention of researchers in the field of immunotherapy, and various types of research on them have become increasingly mature. In terms of application, it has gradually entered the clinical research stage for blood system malignancies, autoimmune diseases, viral infections, etc., and has shown great advantages in the treatment of malignant tumors, autoimmune diseases, and anti-infection.

[0007] As an immunotherapy strategy, T cells modified with chimeric antigen receptors (CARs) are receiving widespread attention and application in tumor treatment, especially in hematological malignancies. The chimeric antigen receptor is a recombinant polypeptide construct. Its principle is to modify through genetic engineering so that T cells express receptor structures (e.g., single-chain antibodies) that can specifically recognize tumor cell surface antigens, and after the receptor specifically binds to tumor cell surface antigens, activate its downstream immune co-stimulatory factors and T cells, thereby activating T cells to secrete related cytokines and specifically kill tumor cells, and is not restricted by the major histocompatibility complex (MHC) that is specific to the target tumor antigen. Since this process directs T cells to the target in a non-MHC-restricted manner, it bypasses the main mechanism of tumor escape. The representative structure of CAR consists of four parts: an extracellular antigen binding domain (usually a single-chain antibody with antigen recognition function), a hinge region, a transmembrane domain, and an intracellular signal transduction domain. At present, according to whether and how many co-stimulatory molecules are added to the intracellular signal transduction domain, the classic CAR structure is usually divided into the first generation (no co-stimulatory molecules), the second generation (including one co-stimulatory molecule) and the third generation (including two co-stimulatory molecules). So far, the second generation CAR structure is the most widely used in marketed products and clinical research stages. Since CAR-T cell manufacturing technology requires the use of single-chain antibodies with good binding activity and efficient binding epitopes, one of the key technical parts of CAR-T cell therapy is to screen high-affinity antibodies with good specificity, strong binding force and effective binding epitopes.

[0008] Therefore, there is still a wide demand for screening and developing improved single-domain antibodies targeting GPRC5D, chimeric antigen receptors targeting GPRC5D constructed using the same, and engineered immune effector cells. In particular, the development of single-domain antibodies specifically targeting GPRC5D suitable for more effective and efficient CAR-T cell therapy. Summary of the invention

[0009] The object of the present invention is to provide a single-domain antibody targeting GPRC5D and its application. The structure of the single-domain antibody targeting GPRC5D described in the present invention is a natural single-chain structure, which has the advantages of small molecular weight, high solubility, high stability, low immunogenicity, high tissue permeability, and no need for additional folding and assembly steps or linker optimization and transformation, making it a promising alternative to scFv single-chain antibodies with larger molecular weight, and after the single-domain antibody is constructed into CAR-T cells, it has significant specific tumor cell killing ability.

[0010] According to the first aspect of the present disclosure, a single domain antibody targeting GPRC5D is provided. The single domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions, wherein CDR1 comprises any one of the amino acid sequences shown in SEQ ID NOs: 1-18, wherein CDR2 comprises any one of the amino acid sequences shown in SEQ ID NOs: 19-33, wherein CDR3 comprises any one of the amino acid sequences shown in SEQ ID NOs: 34-50.

[0011] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions, wherein CDR1 is an amino acid sequence as shown in any one of SEQ ID NOs: 1-18, CDR2 is an amino acid sequence as shown in any one of SEQ ID NOs: 19-33, and CDR3 is an amino acid sequence as shown in any one of SEQ ID NOs: 34-50.

[0012] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions, wherein CDR1, CDR2 and CDR3 comprise the amino acid sequences shown in Table 1.

[0013] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions, wherein CDR1, CDR2 and CDR3 have the amino acid sequences shown in Table 1.

[0014] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:1, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:19, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:34.

[0015] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:1, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:20, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:35.

[0016] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:2, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:21, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:36.

[0017] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:3, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:21, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:37.

[0018] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:4, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:21, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:37.

[0019] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:5, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:21, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:37.

[0020] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:2, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:22, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:37.

[0021] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:2, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:22, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:38.

[0022] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:6, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:22, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:38.

[0023] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:7, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:22, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:38.

[0024] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:8, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:23, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:39.

[0025] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:9, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:24, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:40.

[0026] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 10, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 25, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 41.

[0027] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:11, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:26, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:42.

[0028] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 12, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 26, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 43.

[0029] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 13, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 27, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 44.

[0030] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 14, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 28, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 45.

[0031] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 15, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 29, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 46.

[0032] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 15, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 30, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 47.

[0033] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 16, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 31, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 48.

[0034] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 17, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 32, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 49.

[0035] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 18, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 33, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 50.

[0036] The technical solution disclosed by the present invention, wherein the specific amino acid sequence information shown by SEQ ID NO: 1-50 is as shown in Table 1.

[0037] Table 1 SEQ ID NO: Amino acid sequence 1 GIIFGSSV(CDR1) 2 GLTFSSYA(CDR1) 3 GFTFSSYA(CDR1) 4 GRTFSAYA(CDR1) 5 GLAFGSYA(CDR1) 6 GRTFTNYA(CDR1) 7 GFAFNVYA(CDR1) 8 GRTFANYA(CDR1) 9 GGTFSSYA(CDR1) 10 GLTFSSYT(CDR1) 11 GRTFSSYA(CDR1) 12 GRTFSNYA(CDR1) 13 GRTISSLA (CDR1) 14 GRTITTYT(CDR1) 15 GRTFSSYS(CDR1) 16 GRTASIYR(CDR1) 17 GRSFKNYG(CDR1) 18 GFTFSTSV(CDR1) 19 IGYGGNT(CDR2) 20 VGYDGST(CDR2) 21 ISPNGAGT(CDR2) 22 ISPNGAAT(CDR2) 23 INPNGAAT(CDR2) 24 ISPNGGAT(CDR2) 25 ISQNGVNT(CDR2) 26 INYSTGT(CDR2) 27 INWSGRQT(CDR2) 28 ISEGT(CDR2) 29 ITRRGSST(CDR2) 30 TTRRGSDV(CDR2) 31 ITWVGSRT(CDR2) 32 IKWSDGRT(CDR2) 33 HSWRGTST(CDR2) 34 NARRRINY(CDR3) 35 NARRRIDY (CDR3) 36 AADQIHYSRISYSRMDY(CDR3) 37 AADQVHYSRISYSRMDY(CDR3) 38 AADQMHYSRISYSGHNMDY(CDR3) 39 ASHQMHYSRILYSGHNMDY (CDR3) 40 AADHVHYSRFSYSAMDY(CDR3) 41 AASPRFFARSYSYTAYDY(CDR3) 42 AASRGLRLLTRASEYTY(CDR3) 43 AASRGLRLLTRASEYSY(CDR3) 44 AADTGLLLSTMSQHYDY(CDR3) 45 AASRGLRRLVTPMNYNY(CDR3) 46 AAALVLGPTDSQDRYPY(CDR3) 47 AAALVLGPTDSQDRYQY(CDR3) 48 AAGFrTTLTEYTS(CDR3) 49 ALDAQPRRILDLKLTSAYDY(CDR3) 50 VKSRGIVAGPLGASSVSN(CDR3)

[0038] The single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1, CDR2 and CDR3 are determined according to the IMGT numbering scheme, the Kabat numbering scheme, the AbM numbering scheme, the Chothia numbering scheme or the Contact numbering scheme.

[0039] The single-domain antibody targeting GPRC5D provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1, CDR2 and CDR3 are determined according to the IMGT numbering scheme.

[0040] The single domain antibodies or CDR regions thereof provided by the present invention include CDRs defined by the above numbering schemes, other known numbering schemes, and CDRs determined by other methods. For example, those skilled in the art will appreciate that CDR regions determined by any numbering scheme or any method, as long as they are the same as the CDR regions of the present invention, or include the CDR regions of the present invention, fall within the scope of protection of the present invention.

[0041] The single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to any one of the amino acid sequences shown in Table 2 or SEQ ID NOs: 51-72.

[0042] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:51.

[0043] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:52.

[0044] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:53.

[0045] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:54.

[0046] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:55.

[0047] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:56.

[0048] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:57.

[0049] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:58.

[0050] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:59.

[0051] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:60.

[0052] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:61.

[0053] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:62.

[0054] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:63.

[0055] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:64.

[0056] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:65.

[0057] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:66.

[0058] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:67.

[0059] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:68.

[0060] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:69.

[0061] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:70.

[0062] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:71.

[0063] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:72.

[0064] The single-domain antibody targeting GPRC5D provided by the present invention comprises an amino acid sequence as shown in Table 2 or any one of SEQ ID NOs: 51-72.

[0065] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:51.

[0066] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:52.

[0067] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:53.

[0068] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:54.

[0069] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:55.

[0070] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:56.

[0071] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:57.

[0072] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:58.

[0073] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:59.

[0074] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:60.

[0075] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:61.

[0076] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:62.

[0077] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:63.

[0078] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:64.

[0079] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:65.

[0080] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:66.

[0081] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:67.

[0082] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:68.

[0083] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:69.

[0084] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:70.

[0085] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:71.

[0086] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:72.

[0087] The single-domain antibody targeting GPRC5D provided by the present invention is any one of the amino acid sequences shown in Table 2 or in SEQ ID NOs: 51-72.

[0088] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:51.

[0089] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:52.

[0090] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:53.

[0091] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:54.

[0092] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:55.

[0093] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:56.

[0094] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:57.

[0095] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:58.

[0096] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:59.

[0097] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:60.

[0098] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:61.

[0099] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:62.

[0100] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:63.

[0101] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:64.

[0102] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:65.

[0103] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:66.

[0104] In some embodiments, the single domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:67.

[0105] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:68.

[0106] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:69.

[0107] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:70.

[0108] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:71.

[0109] In some embodiments, the single-domain antibody targeting GPRC5D provided by the present invention has an amino acid sequence as shown in SEQ ID NO:72.

[0110] The technical solution disclosed by the present invention, wherein the specific amino acid sequence information shown in SEQ ID NO:51-72 is as shown in Table 2.

[0111] Table 2

[0112] According to a second aspect of the present disclosure, a chimeric antigen receptor is provided, wherein the chimeric antigen receptor comprises the single domain antibody targeting GPRC5D according to the present invention.

[0113] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an extracellular antigen binding domain, and the extracellular antigen binding domain comprises a single domain antibody targeting GPRC5D as described in the present invention.

[0114] In some embodiments, the chimeric antigen receptor provided by the present invention may also include one or more of the following structures: a linker (eg, a peptide linker), a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory signaling domain, and an intracellular signaling domain.

[0115] In some embodiments, the chimeric antigen receptor provided by the present invention comprises: (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) intracellular signaling domain; Wherein, the extracellular antigen binding domain comprises the single-domain antibody targeting GPRC5D described in the first aspect of the present disclosure.

[0116] In some embodiments, the chimeric antigen receptor provided by the present invention comprises: (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) intracellular signaling domain; Wherein, the extracellular antigen binding domain comprises a single domain antibody targeting GPRC5D, and the single domain antibody comprises CDR1, CDR2 and CDR3 regions, wherein the CDR1, CDR2 and CDR3 regions comprise the CDR1, CDR2 and CDR3 regions of the single domain antibody targeting GPRC5D as described in the first aspect of the present disclosure.

[0117] In some embodiments, the chimeric antigen receptor provided by the present invention comprises: (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) intracellular signaling domain; Wherein, the extracellular antigen binding domain comprises a single domain antibody targeting GPRC5D, and the single domain antibody comprises CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the CDR1, CDR2 and CDR3 regions of the single domain antibody targeting GPRC5D as described in the first aspect of the present disclosure.

[0118] In some embodiments, the chimeric antigen receptor provided by the present invention comprises: (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) intracellular signaling domain; wherein the extracellular antigen binding domain comprises a single domain antibody targeting GPRC5D; wherein the single domain antibody comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to any one of the amino acid sequences shown in SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 or SEQ ID NO:72.

[0119] In some embodiments, the chimeric antigen receptor provided by the present invention comprises: (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) intracellular signaling domain; wherein the extracellular antigen binding domain comprises a single domain antibody targeting GPRC5D; wherein the single domain antibody comprises any one of the amino acid sequences shown in SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 or SEQ ID NO:72.

[0120] In some embodiments, the chimeric antigen receptor provided by the present invention comprises: (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) intracellular signaling domain; wherein the extracellular antigen binding domain comprises a single domain antibody targeting GPRC5D; wherein the single domain antibody is any one of the amino acid sequences shown in SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 or SEQ ID NO:72.

[0121] In some embodiments, the present invention provides a chimeric antigen receptor, wherein the transmembrane domain is derived from CD8α, CD28, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, TCRα, TCRβ, TCRγ, TCRδ, TCRζ, OX40, ICOS, LAG-3, 2B4, BTLA, CTLA-4, PD-1, or any combination thereof.

[0122] In some embodiments, the present invention provides a chimeric antigen receptor wherein the transmembrane domain is derived from CD8α.

[0123] In some embodiments, the present invention provides a chimeric antigen receptor, wherein the transmembrane domain comprises an amino acid sequence as shown in SEQ ID NO:75.

[0124] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the transmembrane domain is an amino acid sequence as shown in SEQ ID NO:75.

[0125] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the intracellular signaling domain comprises a major intracellular signaling domain of an immune effector cell (e.g., a T cell). In some embodiments, the intracellular signaling domain is derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d, or any combination thereof.

[0126] In some embodiments, the present invention provides a chimeric antigen receptor wherein the intracellular signaling domain is derived from CD3ζ.

[0127] In some embodiments, the present invention provides a chimeric antigen receptor, wherein the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:77.

[0128] In some embodiments, the present invention provides a chimeric antigen receptor, wherein the intracellular signaling domain is an amino acid sequence as shown in SEQ ID NO:77.

[0129] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the intracellular signaling domain further comprises a costimulatory signal domain. In some embodiments, the costimulatory signal domain is derived from MHC class I molecules, BTLA and Toll ligand receptors. In some embodiments, the costimulatory signal domain is derived from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD134 (OX40), CD2, CD7, CD27, CD28, CD30, CD40, CD83, ICAM, 4-1BB (CD137), CD276 (B7-H3), CD278 (ICOS), GITR, LIGHT, HVEM (LIGHTR), BTLA, CD8α, LFA-1, NKG2C, LAT, SLP-76, DAP10, PD-1, TRIM, ZAP70 ligands or any combination thereof.

[0130] In some embodiments, the present invention provides a chimeric antigen receptor, wherein the co-stimulatory signaling domain is derived from 4-1BB (CD137).

[0131] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the co-stimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO:76.

[0132] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the co-stimulatory signaling domain is an amino acid sequence as shown in SEQ ID NO:76.

[0133] In some embodiments, the chimeric antigen receptor provided by the present invention further comprises a hinge region located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain.

[0134] In some embodiments, the present invention provides a chimeric antigen receptor, wherein the hinge region is derived from CD8α, CD28, CD137, IgG4, IgG1, or any combination thereof.

[0135] In some embodiments, the present invention provides a chimeric antigen receptor wherein the hinge region is derived from CD8α.

[0136] In some embodiments, the present invention provides a chimeric antigen receptor, wherein the hinge region comprises the amino acid sequence shown in SEQ ID NO:74.

[0137] In some embodiments, the present invention provides a chimeric antigen receptor, wherein the hinge region is an amino acid sequence as shown in SEQ ID NO:74.

[0138] In some embodiments, the chimeric antigen receptor provided by the present invention further comprises a signal peptide located at the N-terminus of the chimeric antigen receptor polypeptide.

[0139] In some embodiments, the present invention provides a chimeric antigen receptor, wherein the signal peptide is derived from HLA-A, CD8α, CD4, CD33, CD137, GM-CSFRα, IgG1, Igκ, IL-2 or any combination thereof.

[0140] In some embodiments, the present invention provides a chimeric antigen receptor wherein the signal peptide is derived from HLA-A.

[0141] In some embodiments, the present invention provides a chimeric antigen receptor, wherein the signal peptide is derived from CD8α.

[0142] In some embodiments, the present invention provides a chimeric antigen receptor, wherein the signal peptide comprises the amino acid sequence shown in SEQ ID NO:73.

[0143] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the signal peptide is the amino acid sequence shown in SEQ ID NO:73.

[0144] The technical solution disclosed by the present invention, wherein the specific amino acid sequence information shown in SEQ ID NO:73-77 is as shown in Table 3.

[0145] Table 3

[0146] In some embodiments, the chimeric antigen receptor polypeptide provided by the present invention comprises, from N-terminus to C-terminus, an HLA-A signal peptide or a CD8α signal peptide, an extracellular antigen binding domain, a CD8α hinge region, a CD8α transmembrane domain, a co-stimulatory signaling domain derived from 4-1BB (CD137), and an intracellular signaling domain derived from CD3ζ.

[0147] The chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in Table 4 or to the amino acid sequence shown in SEQ ID NO:78-99.

[0148] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:78.

[0149] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:79.

[0150] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:80.

[0151] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:81.

[0152] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:82.

[0153] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:83.

[0154] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:84.

[0155] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:85.

[0156] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:86.

[0157] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:87.

[0158] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:88.

[0159] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:89.

[0160] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:90.

[0161] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:91.

[0162] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:92.

[0163] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:93.

[0164] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:94.

[0165] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:95.

[0166] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:96.

[0167] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:97.

[0168] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:98.

[0169] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:99.

[0170] The chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in Table 4 or as shown in SEQ ID NO: 78-99.

[0171] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:78.

[0172] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:79.

[0173] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:80.

[0174] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:81.

[0175] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:82.

[0176] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:83.

[0177] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:84.

[0178] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:85.

[0179] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:86.

[0180] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:87.

[0181] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:88.

[0182] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:89.

[0183] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:90.

[0184] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:91.

[0185] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:92.

[0186] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:93.

[0187] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:94.

[0188] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:95.

[0189] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:96.

[0190] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:97.

[0191] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:98.

[0192] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:99.

[0193] The chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in Table 4 or as shown in SEQ ID NO: 78-99.

[0194] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:78.

[0195] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:79.

[0196] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:80.

[0197] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:81.

[0198] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:82.

[0199] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:83.

[0200] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:84.

[0201] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:85.

[0202] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:86.

[0203] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:87.

[0204] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:88.

[0205] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:89.

[0206] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:90.

[0207] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:91.

[0208] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:92.

[0209] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:93.

[0210] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:94.

[0211] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:95.

[0212] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:96.

[0213] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:97.

[0214] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:98.

[0215] In some embodiments, the chimeric antigen receptor provided by the present invention has an amino acid sequence as shown in SEQ ID NO:99.

[0216] The technical solution disclosed by the present invention, wherein the specific amino acid sequence information shown in SEQ ID NO:78-99 is as shown in Table 4.

[0217] Table 4

[0218]

[0219]

[0220]

[0221]

[0222]

[0223] According to the third aspect of the present disclosure, an isolated nucleic acid is provided, wherein the isolated nucleic acid encodes the chimeric antigen receptor of the present invention.

[0224] In some embodiments, the isolated nucleic acid provided by the invention is a DNA form or an RNA form. In some embodiments, the DNA provided by the invention comprises cDNA, genomic DNA or artificially synthesized DNA. In some embodiments, the DNA provided by the invention is a single-stranded DNA or a double-stranded DNA. In some embodiments, the DNA provided by the invention is a coding strand DNA or a non-coding strand DNA.

[0225] The isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in Table 5 or comprises the nucleic acid sequence shown in SEQ ID NO: 100-121.

[0226] In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO:100.

[0227] In some embodiments, the isolated nucleic acid provided by the present invention comprises a nucleic acid sequence as shown in SEQ ID NO:101.

[0228] In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO:102.

[0229] In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO:103.

[0230] In some embodiments, the isolated nucleic acid provided by the present invention comprises a nucleic acid sequence as shown in SEQ ID NO:104.

[0231] In some embodiments, the isolated nucleic acid provided by the present invention comprises a nucleic acid sequence as shown in SEQ ID NO:105.

[0232] In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO:106.

[0233] In some embodiments, the isolated nucleic acid provided by the present invention comprises a nucleic acid sequence as shown in SEQ ID NO:107.

[0234] In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO:108.

[0235] In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO:109.

[0236] In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO:110.

[0237] In some embodiments, the isolated nucleic acid provided by the present invention comprises a nucleic acid sequence as shown in SEQ ID NO:111.

[0238] In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO:112.

[0239] In some embodiments, the isolated nucleic acid provided by the present invention comprises a nucleic acid sequence as shown in SEQ ID NO:113.

[0240] In some embodiments, the isolated nucleic acid provided by the present invention comprises a nucleic acid sequence as shown in SEQ ID NO:114.

[0241] In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO:115.

[0242] In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO:116.

[0243] In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO:117.

[0244] In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO:118.

[0245] In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO:119.

[0246] In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO:120.

[0247] In some embodiments, the isolated nucleic acid provided by the present invention comprises a nucleic acid sequence as shown in SEQ ID NO:121.

[0248] The technical solution disclosed by the present invention, wherein the specific nucleic acid sequence information shown in SEQ ID NO:100-121 is as shown in Table 5.

[0249] Table 5

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256] According to a fourth aspect of the present disclosure, a vector is provided, wherein the vector comprises the isolated nucleic acid described in the present invention.

[0257] In some embodiments, the vector is suitable for replication and integration in eukaryotic cells, such as mammalian cells. In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof.

[0258] In some embodiments, the vector is contained in a host cell.

[0259] In some embodiments, the host cell is a killer cell.

[0260] In some embodiments, the killer cell is a T cell or a NK cell.

[0261] In some embodiments, the NK cells are primary NK cells.

[0262] In some embodiments, the T cells are peripheral blood T lymphocytes or umbilical cord blood T lymphocytes.

[0263] According to a fifth aspect of the present disclosure, an engineered immune effector cell is provided, which comprises the chimeric antigen receptor or the isolated nucleic acid described in the present invention.

[0264] In some embodiments, the engineered immune effector cells provided by the present invention can be selected from T cells, B cells, NK cells, macrophages, dendritic cells, immune effector cells differentiated from induced pluripotent stem cells (iPSCs), or any combination thereof.

[0265] In some embodiments, the engineered immune effector cells provided by the present invention are T cells.For example, T cells can be selected from CD4+ / CD8+T cells, CD4+ / CD8-T cells, CD4- / CD8+T cells, CD4- / CD8-T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+T cells (such as cytotoxic T cells), tumor infiltrating cells, memory T cells, immature T cells, γδ-T cells, αβ-T cells, NKT cells, DNT cells (double negative T cells).

[0266] In some embodiments, the T cells are CD4+ / CD8- T cells, CD4- / CD8+ T cells, CD4+ / CD8+ T cells, CD4- / CD8- T cells, or a combination thereof.

[0267] In some embodiments, T cells produce cytokines such as IL-2, IFN-γ and / or TNF-α after expressing chimeric antigen receptors and binding to target cells (e.g., GPRC5D+ tumor cells). In some embodiments, CD8+ T cells lyse antigen-specific target cells after expressing chimeric antigen receptors and binding to target cells.

[0268] In some embodiments, the immune effector cells are NK cells. In some embodiments, the immune effector cells can be established cell lines, such as NK-92 cells.

[0269] In some embodiments, immune effector cells can be differentiated from stem cells, such as iPSCs.

[0270] According to a sixth aspect of the present disclosure, a pharmaceutical composition is provided, comprising the GPRC5D single domain antibody, a chimeric antigen receptor, an engineered immune effector cell and one or more pharmaceutically acceptable excipients and / or carriers according to the present invention.

[0271] Excipients and / or carriers are also commonly referred to as adjuvants. Pharmaceutically acceptable excipients and / or carriers include, but are not limited to, fillers, adhesives, disintegrants, coatings, adsorbents, antiadhesives, glidants, antioxidants, flavoring agents, colorants, sweeteners, solvents, cosolvents, buffers, chelating agents, surfactants, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delay agents, stabilizers and tension regulators, diluents, solubilizers, emulsifiers, preservatives and / or adjuvants. Those skilled in the art will appreciate that pharmaceutically acceptable excipients and / or carriers are nontoxic or substantially nontoxic to the recipient at the dosage and concentration adopted, and are pharmacologically and / or physiologically compatible with the active ingredient of the present invention, such as not affecting its viability or efficacy. In some embodiments, the pharmaceutical composition may contain a material for improving, maintaining or retaining, for example, pH, permeability, viscosity, clarity, color, isotonicity, smell, sterility, stability, dissolution or release rate, absorption or penetration of the composition. The optimal pharmaceutical composition can be determined depending on the intended route of administration, mode of delivery, and desired dosage.Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed., 1990).

[0272] The pharmaceutical composition of the present invention is provided in the form of a sterile preparation in some embodiments. Sterilization is achieved by filtering through a sterile filtration membrane. When the composition is freeze-dried, this method can be used for sterilization before or after freeze-drying, reconstitution, and dilution. Sterile preparations are such as isotonic aqueous solutions, suspensions, emulsions, dispersions, etc. In some embodiments, the pharmaceutical composition of the present invention, wherein the pharmaceutically acceptable excipients and / or carriers include but are not limited to: one or more of saline, buffer, glucose, water, glycerol, ethanol, and polyols. Compositions for parenteral administration can be stored in a lyophilized form or in a solution. For example, physiological saline or an aqueous solution containing glucose and other adjuvants is prepared by conventional methods. Parenteral compositions are usually placed in containers with sterile access holes, such as intravenous solution bags or vials with stoppers that can be pierced by injection needles. Alternatively, the composition can be selected for inhalation or delivery through the digestive tract (such as oral). The preparation of the pharmaceutically acceptable composition is within the technical scope of the art. Other pharmaceutical compositions will also be apparent to those skilled in the art, including formulations containing antibodies or engineered immune effector cells in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery methods (such as liposome carriers, bioerodible microparticles or porous beads, and depot injections) are also well known to those skilled in the art. Once formulated, the pharmaceutical composition is stored in a sterile vial in the form of a solution, suspension, gel, emulsion, viscous composition, solid, crystal, or lyophilized powder. The formulation can be stored in a ready-to-use form or reconstituted and processed for use prior to administration.

[0273] Pharmaceutical composition can be used in any convenient way, including by spraying, injection, swallowing, infusion, implantation or transplantation. Pharmaceutical composition of the present invention can be injected by intravenous, intraperitoneal, intracerebral (in the brain parenchyma), intraventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional approach, and can also be used by sustained release system or by implantation device. In some embodiments, use is completed by parenteral. Parenteral delivery methods include local, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.

[0274] The therapeutically effective amount of the pharmaceutical composition containing the single domain antibody or engineered immune effector cell targeting GPRC5D of the present invention to be used will depend on, for example, the extent and goal of treatment. It will be appreciated by those skilled in the art that the appropriate dosage level for treatment will vary depending in part on the delivered molecule, indication, route of administration, and patient condition (body weight, body surface or organ size) and / or condition (age and general health). In certain embodiments, the clinician may titrate the dose and change the route of administration to obtain the best therapeutic effect.

[0275] The frequency of administration will depend on the pharmacokinetic parameters of the single domain antibody or engineered immune effector cell targeting GPRC5D in the formulation used. Clinicians typically administer the pharmaceutical composition until the dose that achieves the desired effect is reached. The pharmaceutical composition can therefore be administered as a single dose, or administered over time as two or more doses (which may or may not contain the same amount of the desired molecule), or administered by continuous infusion via an implant device or catheter.

[0276] According to the seventh aspect of the present disclosure, the present invention provides a use of a single domain antibody, a chimeric antigen receptor, an engineered immune effector cell, or a pharmaceutical composition targeting GPRC5D as described above in the preparation of a medicament for diagnosing, preventing and / or treating a disease or condition.

[0277] The present invention also provides a method for treating a subject with a disease associated with GPRC5D expression, comprising administering to the subject an effective amount of the engineered immune effector cell or pharmaceutical composition according to the present invention. Therefore, the present invention provides a single domain antibody, chimeric antigen receptor, engineered immune effector cell, or pharmaceutical composition targeting GPRC5D according to the present invention for use in the preparation of a medicament for diagnosing, preventing and / or treating a disease or condition.

[0278] In some embodiments, the disease or disorder comprises a disease or disorder associated with GPRC5D expression.

[0279] In some embodiments, the disease or disorder is a cancer, malignancy, autoimmune and / or inflammatory disease associated with GPRC5D expression.

[0280] In some embodiments, the disease or disorder associated with GPRC5D expression is a GPRC5D expression-associated malignancy, a GPRC5D expression-associated autoimmune disease and / or an inflammatory disease.

[0281] In some embodiments, the disease or disorder is systemic lupus erythematosus, rheumatoid arthritis, Waldenstrom's Macroglobulinemia, marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal large B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, Burkitt's lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin's lymphoma, high grade B-cell lymphoma, and / or multiple myeloma.

[0282] In some embodiments, the disease or disorder is multiple myeloma.

[0283] The single domain antibody, nanobody or heavy chain antibody of the present invention can be prepared by conventional methods in the art, such as phage display technology well known in the art. Alternatively, various antibodies of the present invention can be expressed in other cell lines. Suitable mammalian host cells can be transformed with sequences encoding various antibodies of the present invention. Transformation can be carried out by any known method, for example, including packaging the polynucleotide in a virus (or a viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Host mammalian cell lines that can be used for expression are well known in the art, such as a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma (HepG2) cells, etc. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with substantial GPRC5D binding properties.

[0284] The chimeric antigen receptor of the present invention can be prepared by conventional methods in the art, see, for example, Park et al, Trends Biotechnol., 29:550-557, 2011; Grupp et al, N Engl J Med., 368:1509-1518, 2013; Han et al, J. Hematol. Oncol., 6:47, 2013.

[0285] As is well known to those skilled in the art, due to the degeneracy of the genetic code, a very large number of nucleic acids can be produced, all of which encode the chimeric antigen receptor of the present invention. Thus, in the case where a specific amino acid sequence has been identified, one skilled in the art can produce any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the encoded protein. Therefore, the present invention also relates to polynucleotides that hybridize with the above-mentioned polynucleotide sequences and have at least 70%, preferably at least 80%, and more preferably at least 90% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions.

[0286] The full-length nucleic acid sequences or fragments thereof of various chimeric antigen receptors of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequences by artificial synthesis, especially when the fragment length is short. Generally, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.

[0287] The present invention also relates to nucleic acid constructs, such as expression vectors and recombinant vectors, comprising the above-mentioned nucleic acid sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins. The vectors usually contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequences (collectively referred to as "flanking sequences" in certain embodiments) usually include one or more of the following nucleotide sequences: promoters, one or more enhancer sequences, replication origins, transcription termination sequences, complete intron sequences containing donor and acceptor splice sites, leader sequences encoding for polypeptide secretion, ribosome binding sites, polyadenylation sequences, multiple linker regions for inserting nucleic acids encoding antibodies to be expressed, and selectable marker elements.

[0288] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the host cells after proliferation by conventional methods. The biomolecules (nucleic acids, polypeptides, etc.) involved in the present invention include biomolecules in isolated form. At present, the DNA sequence encoding the polypeptide of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the polypeptide sequence of the present invention by chemical synthesis.

[0289] In some embodiments, the nucleic acid encoding the chimeric antigen receptor is operably linked to a constitutive promoter. A constitutive promoter allows a heterologous gene (also referred to as a transgene) to be constitutively expressed in a host cell. Exemplary constitutive promoters of the present invention include, but are not limited to, cytomegalovirus (CMV) promoter, human elongation factor-1α (hEF1α) promoter, ubiquitin C (UbiC) promoter, phosphoglycerol kinase (PGK) promoter, simian virus 40 (SV40) early promoter, and chicken β-actin coupled to CMV early enhancer (CAGG) promoter. The efficiency of such constitutive promoters in driving transgenic expression has been extensively compared in a large number of studies. For example, Michael C. Milone et al. (Molecular Therapy, 17(8): 1453-1464 (2009)) compared the efficiency of CMV, hEF1α, UbiC and PGK in driving chimeric antigen receptor expression in human primary T cells, and concluded that the hEF1α promoter not only induced the highest level of transgene expression, but also maintained the best in CD4 and CD8 human T cells. In some embodiments, the nucleic acid encoding the chimeric antigen receptor is operably linked to the hEF1α promoter.

[0290] In some embodiments, the nucleic acid encoding the chimeric antigen receptor is operably linked to an inducible promoter. An inducible promoter is a regulated promoter. An inducible promoter can be induced by one or more conditions, such as physical conditions, inducers, etc.

[0291] In some embodiments, the expression of endogenous genes in engineered mammalian cells and / or subjects receiving the pharmaceutical composition is induced under induction conditions. In some embodiments, the induction conditions are selected from: an inducing agent, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox state, tumor environment, and the activation state of engineered mammalian cells.

[0292] Viral vector technology is well known in the art and is described in Sambrook et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. (2001), as well as other virology and molecular biology manuals.

[0293] Many virus-based systems have been developed in the prior art for transferring genes into mammalian cells. Heterologous nucleic acids can be inserted into vectors and packaged in retroviral particles using techniques known in the art. Recombinant viruses can then be isolated in vitro or in vitro and delivered to engineered mammalian cells. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying immunomodulatory agents (e.g., immune checkpoint inhibitors) coding sequences and / or self-inactivating lentiviral vectors carrying chimeric antigen receptors can be packaged with protocols known in the art. In some embodiments, lentiviral vectors are used. Using methods known in the art, the resulting lentiviral vectors can be used to transduce mammalian cells (e.g., primary human T cells).

[0294] Can use any known molecular cloning method in the art nucleic acid is cloned into vector, comprise for example use restriction endonuclease site and one or more selective markers.In some embodiments, nucleic acid is operably connected with promoter.Prior art has disclosed multiple promoters for gene expression in mammalian cell, and any promoter known in the art can be used for the present invention.Promoter can be further divided into constitutive promoter or regulated promoter, for example inducible promoter.

[0295] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with CaCl 2 Another method is to use MgCl 2 In addition, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0296] The obtained transformant can be cultivated by conventional methods to express the polypeptide encoded by the nucleic acid molecule of the present invention. Depending on the host cell used, the culture medium used in the cultivation can be selected from various conventional culture media, such as serum-containing culture medium or serum-free culture medium. Cultivate under conditions suitable for host cell growth. After the host cell grows to an appropriate cell density, induce the selected promoter with a suitable method (such as temperature conversion or chemical induction), and the cell is cultivated for a period of time.

[0297] The polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

[0298] The engineered immune effector cells of the present invention are prepared by introducing chimeric antigen receptors into immune effector cells (eg, T cells).

[0299] The nucleic acid sequence encoding the chimeric antigen receptor can be introduced into immune effector cells using conventional methods known in the art (such as by transfection, transduction, transformation, etc.).

[0300] Methods for introducing vectors or isolated nucleic acids into immune effector cells are known in the art. The described vectors can be transferred into immune effector cells by physical, chemical or biological methods.

[0301] Physical methods for introducing vectors into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for preparing cells containing vectors and / or exogenous nucleic acids are well known in the art and are described in, for example, Sambrook, J., Fritsch, EF and Maniatis, T. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. and other virology and molecular biology manuals. In some embodiments, the vector is introduced into immune effector cells by electroporation.

[0302] Biological methods for introducing vectors into immune effector cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian cells (e.g., human cells).

[0303] Chemical methods for introducing vectors into immune effector cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro delivery vehicle is a liposome.

[0304] In some embodiments, the vector further comprises a selection marker gene or a reporter gene to select cells expressing the chimeric antigen receptor from a population of host cells transfected by a lentiviral vector. Both the selection marker and the reporter gene may be flanked by appropriate regulatory sequences for expression in host cells. For example, the vector may contain transcription and translation terminators, initiation sequences, and promoters for regulating expression of nucleic acid sequences.

[0305] Reporter genes can be used to identify cells that may be transfected and to evaluate the function of regulatory sequences. In general, reporter genes are genes that are not present in or expressed by receptor organisms or tissues, and they encode polypeptides that express some easily detectable properties, such as enzyme activity. The expression of reporter genes is measured at the appropriate time after DNA is introduced into receptor cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein (see Kumiko Ui-Tei.FEBS Letters, 479: 79-82 (2000)). Suitable expression systems are known in the art and can be prepared or commercially obtained using known techniques. Confirming that there are other methods for encoding CAR nucleic acids in engineered immune effector cells includes: molecular biological test methods well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemical assays, such as detecting the presence or absence of specific peptides; immunological methods, such as ELISA.

[0306] In some embodiments, the nucleic acid molecule encoding any CAR described herein can be prepared by conventional methods (e.g., in vitro transcription) and then introduced into immune effector cells by known methods such as mRNA electroporation (see Peter MRabinovich. Human Gene Therapy, 17: 1027-1035 (2006)).

[0307] In some embodiments, the transduced or transfected immune effector cells are cultured in vitro after the introduction of a vector or isolated nucleic acid. In some embodiments, the transduced or transfected immune effector cells are cultured to proliferate for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14 days. In some embodiments, the transduced or transfected immune effector cells can be further evaluated or screened to select engineered immune effector cells.

[0308] Terminology explanation:

[0309] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0310] The term "sequence identity" as used herein means the extent to which two (nucleotide or amino acid) sequences have the same residue at the same position in the alignment, and is usually expressed as a percentage. Preferably, the identity is determined over the entire length of the sequence being compared. Therefore, two copies having exactly the same sequence have 100% identity. It is known to those skilled in the art that some algorithms can be used to determine sequence identity, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197) and Clustal W. In addition, sequence analysis software can be used for determination, such as the computer program BLAST using default parameters, especially BLASTP or TBLASTN.

[0311] As used herein, the term "derived from" indicates a relationship between the two, and generally refers to the structural similarity between the two. For example, in the case of an intracellular signaling domain derived from CD3ζ, the intracellular signaling domain retains sufficient CD3ζ structure so that it has the desired function, i.e., the ability to generate a signal under appropriate conditions. It does not imply or include a limitation on a specific process for generating an intracellular signaling domain, for example, it does not mean that in order to provide an intracellular signaling domain, one must start with a CD3ζ sequence and delete unwanted sequences, or impose mutations, to arrive at the intracellular signaling domain.

[0312] The term "antibody" used in the present invention refers to a protein or polypeptide sequence derived from an immunoglobulin molecule, which specifically binds to an antigen. It includes, for example, monoclonal antibodies (including complete antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (such as bispecific antibodies), double antibodies and single-chain molecules, and antibody fragments or synthetic polypeptides carrying one or more CDR sequences that can exhibit the desired biological activity, especially antigen-binding fragments, such as Fab, F(ab')2 and Fv. In some embodiments of the present invention, the terms "immunoglobulin (Ig)" and "antibody" are used interchangeably.

[0313] The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of the heavy or light chain of an antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are usually the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen binding site.

[0314] Heavy chain antibodies are antibodies derived from camelids or cartilaginous fish. Compared with 4-chain antibodies, heavy chain antibodies lack light chains and heavy chain constant region 1 (CH1), and only contain 2 heavy chains composed of variable regions (VHH) and other constant regions, and the variable regions are connected to the constant regions through a hinge region-like structure. Each heavy chain of camelid heavy chain antibodies contains 1 variable region (VHH) and 2 constant regions (CH2 and CH3), and each heavy chain of cartilaginous fish heavy chain antibodies contains 1 variable region and 5 constant regions (CH1-CH5). The antigen-binding fragments of heavy chain antibodies include VHH and single-chain heavy chain antibodies. By fusing with the constant region of human IgG Fc, heavy chain antibodies can have CH2 and CH3 of human IgG Fc.

[0315] The term "antigen" or "Ag" as used herein refers to a molecule that causes an immune response. An immune response may involve the production of antibodies or the activation of specific immunocompetent cells or both. The skilled person will appreciate that virtually any macromolecule, including all proteins or peptides, may serve as an antigen. In addition, antigens may be derived from recombinant or genomic DNA. The skilled person will appreciate that any DNA comprising a nucleotide sequence or a partial nucleotide sequence encoding a protein that causes an immune response thus encodes an "antigen". Antigens may be synthetically produced or may be derived from a biological sample, or may be a macromolecule other than a polypeptide. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or fluids having other biological components.

[0316] The term "antigen-binding fragment" or "antibody fragment" used in the present invention refers to at least a portion of a complete antibody or a recombinant variant thereof, generally comprising the antigen binding site of the complete antibody and thus retaining the ability to bind to the antigen. Examples of antibody fragments include, but are not limited to, Fab, scFv, the heavy chain variable region (VH) of an antibody, linear antibodies, single domain antibodies, nanobodies, natural ligands of antigens or functional fragments thereof, etc.

[0317] The term "functional variant" or "functional fragment" used in the present invention refers to a variant that substantially comprises the amino acid sequence of the parent but contains at least one amino acid modification (e.g., substitution, deletion or insertion) compared to the parent amino acid sequence, provided that the variant retains the biological activity of the parent amino acid sequence. In some embodiments, the amino acid modification is a conservative modification.

[0318] Single domain antibodies (sdAb) can have the same or different sources and have the same or different sizes.Exemplary sdAbs include, but are not limited to, heavy chain variable domains (e.g., VHH) from heavy chain antibodies only, natural binding molecules without light chains, single domains (e.g., VH or VL) derived from conventional 4-chain antibodies, human single domain antibodies produced by transgenic mice or rats expressing human heavy chain fragments, and non-derived from antibody engineering domains and single domain scaffolds.Any sdAb known in the art or disclosed by the present invention, including single domain antibodies disclosed by the present invention, can be used to construct CAR described herein.sdAb can be derived from any species, including but not limited to mice, rats, humans, camels, llamas, lampreys, sharks, goats, rabbits, and cattle.The single domain antibodies of the present invention also include naturally occurring single domain antibody molecules from species other than camelids and sharks.

[0319] In the present invention, "single domain antibody", "single domain antibody targeting GPRC5D", "heavy chain single domain antibody", "VHH", and "nanoantibody" are used interchangeably, all referring to single domain antibodies that specifically recognize and bind to GPRC5D. Generally, a single domain antibody contains three CDR regions and four FR regions. A single domain antibody is the smallest functional antigen-binding fragment. Usually, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.

[0320] The term "complementarity determining region" or "CDR" as used herein refers to the sequence of amino acids within the variable region of an antibody that imparts antigen specificity and binding affinity. Generally, there are three CDRs (e.g., HCDR1, HCDR2, and HCDR3) in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, and LCDR3) in each light chain variable region.

[0321] Portions of chimeric antigen receptors comprising antibodies or antibody fragments thereof can exist in a variety of forms, for example, where the antigen binding domain is expressed as part of a polypeptide chain (including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), or, for example, a human or humanized antibody), see Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426.

[0322] "Antigen binding domain" refers to the part of the chimeric antigen receptor that specifically binds to the target antigen. The chimeric antigen receptor can use its antigen binding properties to direct T cells and / or other immune cells to the selected target. The antigen binding domain can typically include an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), but it does not have to include both variable regions. For example, it can include a Fab fragment with antigen binding activity, a Fab' fragment, a single Fv fragment such as scFv, a single domain antibody, etc.

[0323] "Transmembrane domain" is used to connect the extracellular domain and intracellular domain of chimeric antigen receptor. Transmembrane domain can be natural or synthetic, and can also be derived from any membrane-bound protein or transmembrane protein. Non-limiting examples of transmembrane domain include, TCRα, TCRβ, TCRγ, TCRδ, TCRζ, CD28, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152, CD154, OX40, ICOS, LAG-3, 2B4, BTLA, CTLA-4, PD-1.

[0324] "Intracellular signaling domain" refers to a functional part of a protein, i.e., a functional part of an intracellular signaling domain that is sufficient to transduce effector function signals. The intracellular signaling domain is responsible for the intracellular primary signaling after the antigen binding region binds to the antigen, thereby causing the activation of immune cells and immune responses. In other words, the intracellular signaling domain is responsible for activating at least one of the normal effector functions of immune cells expressing CAR. For example, the effector function of T cells can be cytolytic activity or auxiliary activity, including the secretion of cytokines. In one embodiment, the intracellular signaling domain included in the chimeric antigen receptor of the present invention can be a cytoplasmic sequence of a T cell receptor and a co-receptor, which works together to induce signal transduction after antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences with the same or similar functions. The intracellular signaling domain can include many immunoreceptor tyrosine-based activation motifs (ITAMs). Non-limiting examples of intracellular signaling domains include, but are not limited to, FcRγ, FcRβ, TCRζ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, DAP12, or CD66d.

[0325] "Hinge region" refers to any oligopeptide or polypeptide used to connect the transmembrane domain and the antigen binding domain. Specifically, the hinge region is used to provide greater flexibility and accessibility for the antigen binding domain. The hinge region can contain up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. The hinge region can be derived in whole or in part from natural molecules, such as in whole or in part from the extracellular region of CD8 or CD28, or in whole or in part from an antibody constant region. Alternatively, the hinge region can be a synthetic sequence corresponding to a naturally occurring hinge sequence, or can be a fully synthetic hinge sequence.

[0326] Different domains of CAR can also be fused to each other through peptide linkers. This depends on the structure and / or functional characteristics of single domain antibodies and / or various domains, and each peptide linker in CAR can have the same or different lengths and / or sequences. Those skilled in the art can independently select and optimize each peptide linker. In some embodiments, the peptide linker is composed of amino acids connected together by peptide bonds, wherein the amino acids are selected from 20 naturally occurring amino acids: glycine, alanine, valine, leucine, isoleucine, serine, cysteine, threonine, methionine, proline, phenylalanine, tyrosine, tryptophan, histidine, lysine, arginine, aspartic acid, glutamic acid, asparagine and glutamine. As understood by those skilled in the art, one or more of these amino acids can be glycosylated. In some embodiments, the peptide linker comprises flexible residues (eg, glycine and serine) so that adjacent domains can move freely relative to each other. For example, a glycine-serine doublet can be a suitable peptide linker.

[0327] The peptide linker can have any suitable length. In some embodiments, the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acid lengths. In some embodiments, the peptide linker is no more than about 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or less amino acid lengths. In some embodiments, the peptide linker has a length of about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

[0328] The peptide linker can have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of only a heavy chain antibody can be used as a linker. See, for example, WO1996 / 34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymers (G) n, glycine-serine polymers (e.g., (GS) n, (GSG) n, (GGGS) n, and (GGGGS) n, wherein n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.

[0329] "Costimulatory signal domain" can be an intracellular functional signal transduction domain from a costimulatory molecule, which includes the entire intracellular portion of the costimulatory molecule, or a functional fragment thereof."Costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., proliferation) of a T cell. Non-limiting examples of costimulatory domains include, but are not limited to, intracellular regions of the following proteins: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD134 (OX40), CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CDS, ICAM, CD137 (4-1BB) , CD276(B7-H3), CD278(ICOS), GITR, BAFFR, LIGHT, HVEM(LIGHTR), BTLA, KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp 30. NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, ITGB7, NKG2D, N KG2C, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD16 0(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD28-OX40, CD28-4-1BB, DAP10, PD-1, TRIM, ZAP70 ligand, or any combination thereof.

[0330] "Signal peptide" can make the chimeric antigen receptor be expressed in cells (such as T cells) so that the nascent protein is directed to the endoplasmic reticulum and then to the cell surface. Generally, the core of the signal peptide can contain a long hydrophobic amino acid segment, which has a tendency to form a single α-helix. The signal peptide guides the transport and / or secretion of the translated protein across the membrane. At the end of the signal peptide, there is usually an amino acid segment that is recognized and cut by a signal peptidase. The signal peptidase can cut during or after the translocation to produce a free signal peptide and a mature protein. Then, the free signal peptide is digested by a specific protease. The signal peptide can also be referred to as a targeting signal, a transit peptide, a positioning signal or a signal sequence. For example, a signal sequence can be a co-translational or post-translational signal peptide.

[0331] "Immune effector cell" refers to an immune cell that can perform immune effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC). For example, immune effector cells can be T cells, macrophages, dendritic cells, monocytes, NK cells and / or NKT cells, or are derived from stem cells, such as adult stem cells, embryonic stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. When the immune effector cell is a T cell, the T cell can be any T cell, such as a T cell cultured in vitro, such as a primary T cell, or a T cell from a T cell line cultured in vitro such as Jurkat, SupT1, etc., or a T cell obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from infection sites, ascites, pleural effusion, spleen tissue and tumors. T cells can also be concentrated or purified. T cells can be at any developmental stage, including but not limited to, CD4+ / CD8+ T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), CD4- / CD8-T cells, tumor infiltrating cells, memory T cells, naive T cells, γδ-T cells, αβ-T cells, etc.

[0332] The terms "patient", "subject", "individual" and "object" used in the present invention are used interchangeably herein and include any organism, preferably an animal, more preferably a mammal (e.g., rats, mice, dogs, cats, rabbits, etc.), and most preferably a human.

[0333] "Treatment" refers to the use of the treatment methods described herein to achieve at least one positive therapeutic effect (e.g., a decrease in the number of cancer cells, a decrease in tumor volume, a decrease in the rate of cancer cell infiltration into peripheral organs, or a decrease in the rate of tumor metastasis or tumor growth) in a subject. Treatment methods that effectively treat a patient can be adjusted based on a variety of factors (e.g., the patient's disease state, age, weight, and the ability of the therapy to stimulate an anti-cancer response in the subject). BRIEF DESCRIPTION OF THE DRAWINGS

[0334] Figure 1 The expression rate of GPRC5D CAR molecules on each group of prepared CD4+CAR-T cells is shown.

[0335] Figure 2 The expression rate of GPRC5D CAR molecules on each group of prepared CD8+CAR-T cells is shown.

[0336] Figure 3 The proliferation times of the prepared CD4+CAR-T cells in each group after 3 days of culture are shown.

[0337] Figure 4 The proliferation times of the prepared CD8+CAR-T cells in each group after 3 days of culture are shown.

[0338] Figure 5 The results show that after 13 days of culture, the CAR-T cells in each group were SCM +T CM The proportion.

[0339] Figure 6 The figure shows the proportion of Tim3-expressing CAR-T cells in each group of prepared CAR-T cells after 13 days of culture.

[0340] Figure 7 The expression of surface GPRC5D antigen of various target cells, namely Raji-1F7 cells, Raji-BE6 cells and Raji-CF10 cells, is shown.

[0341] Figure 8 The results of flow cytometry analysis of the in vitro killing rate of Raji-1F7 cells by effector cells of each group on day 1 are shown when the effector-target ratio (E:T) is 1:3.

[0342] Fig. 9 The results of flow cytometry analysis of the in vitro killing rate of Raji-1F7 cells by effector cells of each group on day 3 are shown when the effector-target ratio (E:T) is 1:3.

[0343] Fig.10The results of flow cytometry analysis of the in vitro killing rate of Raji-BE6 cells by effector cells of each group on day 1 are shown when the effector-target ratio (E:T) is 1:3.

[0344] Fig.11 The results of flow cytometry analysis of the in vitro killing rate of Raji-BE6 cells by effector cells of each group on day 3 are shown when the effector-target ratio (E:T) is 1:3.

[0345] Fig.12 The results of flow cytometry analysis of the in vitro killing rate of Raji-CF10 cells by effector cells of each group on day 3 are shown when the effector-target ratio (E:T) is 1:3.

[0346] Fig.13 The results show that when the effector-target ratio (E:T) is 1:3, target cells (Raji-1F7) and CAR-T cells of each group were mixed and cultured in vitro for 6 days, among which T SCM +T CM The proportion.

[0347] Fig.14 It shows the proportion of CAR-T cells expressing Tim3 after target cells (Raji-1F7) and each group of CAR-T cells were mixed and cultured in vitro for 6 days when the effector-target ratio (E:T) was 1:3.

[0348] Fig.15 The figure shows the proliferation multiples of CD4+CAR-T cells after target cells (Raji-1F7) and effector cells of each group were mixed and cultured in vitro for 9 days when the effector-target ratio (E:T) was 1:3.

[0349] Fig.16 The figure shows the proliferation multiples of CD8+CAR-T cells after target cells (Raji-1F7) and effector cells of each group were mixed and cultured in vitro for 9 days when the effector-target ratio (E:T) was 1:3.

[0350] Fig.17 The figure shows the proliferation multiples of CD4+CAR-T cells after target cells (Raji-BE6) and effector cells of each group were mixed and cultured in vitro for 9 days when the effector-target ratio (E:T) was 1:3.

[0351] Fig.18 The figure shows the proliferation multiples of CD8+CAR-T cells after target cells (Raji-BE6) and effector cells of each group were mixed and cultured in vitro for 9 days when the effector-target ratio (E:T) was 1:3.

[0352] Fig.19 The results of LDH release assay of in vitro killing rate of MM.1S cells by effector cells of each group on day 1 are shown when the effector-target ratio (E:T) is 1:3.

[0353] Fig. 20 The results of the IL-2 release experiment when the effector cells of each group killed the target cells (Raji-1F7) when the effector-target ratio (E:T) was 1:3 were shown.

[0354] Fig.21 The results of the TNF-α release experiment when the effector cells of each group killed the target cells (Raji-1F7) when the effector-target ratio (E:T) was 1:3 were shown.

[0355] Fig. 22 The results of the IFN-γ release experiment when the effector cells of each group killed the target cells (Raji-1F7) when the effector-target ratio (E:T) was 1:3 were shown. DETAILED DESCRIPTION

[0356] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions in the art or according to the product specifications.

[0357] Example 1. Preparation of single domain antibody (VHH) targeting GPRC5D

[0358] (1) Animal immunization and immune response tests

[0359] Adult healthy alpacas were selected as immunization subjects, and 10 mL of blood was collected before immunization as a negative serum control. The immunization cycle was 14 days / time, for a total of 6 times. The immunization process stimulated B cells to express antigen-specific nanoantibodies. The first immunization was to inoculate 1×10 CHOK1 human GPRC5D cells 7 The cells were emulsified with an equal volume of complete Freund's adjuvant (CFA) and injected subcutaneously. For the 2nd to 6th immunization, the same number of CHOK1 human GPRC5D cells were emulsified with an equal volume of incomplete Freund's adjuvant (IFA) and injected subcutaneously. After immunization, peripheral blood was collected and serum titers were detected by FACS. Cells in the logarithmic growth phase were washed by centrifugation, resuspended in PBS, and viable cells were counted by trypan blue staining. The cell density was adjusted to 4×10 using 1% horse serum / PBS. 6 Afterwards, 50 μL of cell suspension (containing 2×10 5 cells) into an EP tube, add serum, and incubate at 4°C for 1 hour. After incubation, add washing solution, centrifuge and remove the supernatant, resuspend the cells with 50 μL of diluted secondary antibody goat anti-alpaca IgG H&L conj-488 (NBbiolab, Cat#S001A488, Lot#20211120), incubate at 4°C for 1 hour, wash the cells twice, resuspend and place at 4°C to detect serum titer. When the titer reaches 1×105 After reaching the level, 50 mL of peripheral blood was collected for library construction.

[0360] (2) Construction of antibody phage library

[0361] 1) RNA extraction and reverse transcription

[0362] After the animal is immunized, the peripheral blood is collected and the lymphocytes are separated. Total RNA is extracted using RNAiso Plus reagent. PrimeScript TM The extracted RNA was reverse transcribed into cDNA using II 1st Strand cDNA Synthesis Kit (Takara, Catalog No.: 6210A). The operation steps are shown in the instruction manual.

[0363] 2) PCR amplification

[0364] Specific antibody fragments were amplified from reverse transcribed cDNA using PCR. The PCR product was cut from gel and a single VHH gene fragment of about 750 bp in size was recovered and purified. The VHH gene fragment was purified using a DNA product purification kit Gel Extraction Kit (OMEGA, catalog number: D2500-01), and the obtained fragment was used as the target gene fragment.

[0365] The PCR primer sequences are as follows: First round upstream primer: 5'CTTGGTGGTCCTGGCTGC 3' First round downstream primer: 5'GGTACGTGCTGTTGAACTGTTCC 3' Second round upstream primer: 5'CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC 3' Second round downstream primer: 5'CATGCCATGACTCGCGGCCGGCCTGGCCGCTGGGGTCTTCGCTGTGGTGCG3'.

[0366] 3) Enzyme digestion

[0367] The vector and target gene fragment were digested separately using restriction endonuclease SfiI. The digestion was carried out overnight at 50°C. After the digestion was completed, the target gene fragment was ligated to the pComb3XSS phage plasmid vector at a ligation molar ratio of VHH:Vector=3:1 using T4 DNA ligase (NEB, catalog number: M0202L) to construct a recombinant plasmid.

[0368] 4) Electroconversion

[0369] The recombinant plasmid was purified and electroporated into electroporated TG1 competent cells 10 times. The electroporation parameters were as follows: electroporator BIO-RAD, Gene Pulser Xcell Total System, voltage 2.5KV, electroporation cup 2mm. Immediately after electroporation, 1mL 2YT medium (preheated at 37°C) was added to the electroporation cup, the electroporation product was aspirated and the electroporation cup was washed with 2YT medium, a total of 100mL of liquid was obtained, and it was revived at 37°C and 180rpm for 45min as the revived product, from which 100μL was taken out and gradiently diluted to 10 -3 and 10 -4 The remaining resuscitation products were centrifuged, resuspended in 8 mL 2YT medium, and then plated. The next day, there were 165 clones on the plate for determining the number of transformants in the library. The library capacity was determined, and the insertion rate and library diversity of the constructed library were verified by colony PCR.

[0370] (3) Packaging of phage libraries

[0371] Inoculate the phage library into 2×300mL 2YT+A(Amp)+G(Glu) medium (Amp: 100μg / mL, Glu: 1%) until the initial OD600 is 0.1-0.2. Culture at 37°C and 230rpm until the OD600 is above 0.8. Add helper phage M13KO7 (helper phage number: bacterial number = 20:1) according to the OD value and mix well. After standing at 37°C for 30min, continue to shake at 180rpm at 37°C for 30min. Then centrifuge at 5000rpm for 10min, discard the supernatant, resuspend the precipitate with an equal volume of 2YT+A(Amp)+K(Kan) medium (Amp: 100μg / mL, Kan: 50μg / mL), and culture overnight at 30°C and 220rpm. After the overnight culture was centrifuged at 4°C and 10,000 rpm for 20 min, the supernatant was collected and transferred to a new centrifuge tube, and centrifuged at 4°C and 10,000 rpm for 20 min to collect the supernatant. Then, add PEG8000 / NaCl solution with a volume of 1 / 5 of the supernatant, mix well, and precipitate on ice for at least 2 hours. Centrifuge at 4°C and 10,000 rpm for 20 minutes to remove the supernatant. Add 1mL 1×PBS to suspend the precipitate, and then add 1 / 5 volume of PEG8000 / NaCl solution for secondary precipitation for 1 hour. After the secondary precipitation, centrifuge at 4°C and 12,000 rpm for 10 min, remove the supernatant, and add 1×PBS to resuspend the precipitate according to the amount of the precipitate. Then add 100% glycerol until the final concentration of the liquid is 50%, mix well and store at -80°C. Take 10μL of the library phage and dilute it gradiently with 2YT medium, starting from 10 -8 and 10 -9Take 10 μL from the tube and add it to 90 μL of TG1 bacterial solution, mix gently. Let stand at 37°C for 15 min, spread on Amp-resistant plates, and culture at 37°C overnight. -9 There were 264 clones on the titer plate, and the phage library titer was 2.64×10 13 cfu / mL(264×10 9 ×100).

[0372] (4) Phage screening

[0373] 1) Cell selection

[0374] GPRC5D (5×10 6 Cells were centrifuged at 500g for 5min, and the cell pellet was resuspended and washed with 5% serum-PBS. This was repeated twice to wash the GPRC5D cells. 500μL OVA blocking solution was added to the cell EP tube and blocked with slight shaking at 4°C for 1h. The phage library was diluted with 5% serum-PBS and 3% BSA until the BSA concentration was 2%, and blocked at 37°C as the phage library dilution solution for later use. After the GPRC5D cells were blocked and centrifuged, the supernatant was removed and 2×10 11 The cfu phage library dilution was gently shaken at 4°C for 1 hour and then centrifuged to remove unbound phages, and the cells were washed 6 times with 5% serum-PBS. 100 μL of Gly-HCl eluent was added and enriched at 37°C for 8 minutes to elute the specifically bound phages. The eluent was transferred to a sterile centrifuge tube and immediately neutralized with 10 μL of Tris-HCl neutralization buffer to obtain the panning eluate. 10 μL was taken for gradient dilution to determine the titer and calculate the panning recovery rate.

[0375] 2) Amplification of the library after panning

[0376] The panning eluate was mixed with 20 mL of E. coli TG1 culture in the early logarithmic growth period, and then 1 mL of 20% glucose was added after standing at 37°C for 30 min, and cultured at 220 rpm for 30 min. M13K07 phage and 4 μL Amp were added at a ratio of cell: phage = 1:20, and then stood at 37°C for 30 min, and 20 mL of 2YT medium was added, and cultured at 220 rpm for 30 min. The culture was centrifuged at 4°C and 5000 rpm for 10 min, and the obtained cell pellet was resuspended in 50 mL of 2×YT+A+K liquid culture medium and cultured overnight at 30°C and 250 rpm. The overnight culture was centrifuged at 4°C and 10000 rpm for 20 min, and the supernatant was transferred to a new centrifuge tube and 1 / 5 volume of PEG / NaCl solution of the supernatant was added. After mixing, it was allowed to stand at 4°C for at least 2 h. After standing, centrifuge at 4°C and 10,000 rpm for 20 minutes, remove the supernatant, resuspend the precipitate in 1 mL PBS, add 1 / 5 volume of PEG / NaCl solution, mix well, and stand at 4°C for at least 1 hour. After standing, centrifuge again at 4°C and 12,000 rpm for 2 minutes, remove the supernatant, and resuspend the precipitate with 200 μL PBS to obtain the amplified product. Determine the titer for the next round of panning or analysis.

[0377] The panning conditions were changed according to Table 6 below, and the second and third rounds of cell panning were performed to gradually screen the phages. The panning conditions for each round were as follows.

[0378] Table 6 Affinity panning conditions

[0379] (5) Monoclonal ELISA test

[0380] 293T human GPRC5D cells and 293T cells were cultured until the entire plate was filled. Washed twice with PBS, 100 μL of 4% paraformaldehyde was added, and the cells were fixed at 25°C for 20-30 min. Washed twice with PBS, then 300 μL of 5% skim milk was added to each well, blocked at 37°C for 1 h, washed once with PBST, 50 μL of phage culture supernatant and 50 μL of 5% skim milk were added to each well, and incubated at 37°C for 1 h. After incubation, washed 5 times with PBST, and then 100 μL of horseradish peroxidase-labeled anti-M13 antibody (diluted with PBS at a ratio of 1:10000) was added to each well, and incubated at 37°C for 1 h. Washed the plate 6 times with PBST, 100 μL of TMB colorimetric solution was added to each well for color development, reacted at 37°C for 7 min, and 50 μL / well of stop solution was added to stop the reaction, and the optical density was measured at 450 nm. The positive clones were sequenced to obtain the gene sequence of the VHH single domain antibody.

[0381] Example 2. Construction of chimeric antigen receptor targeting GPRC5D and expression in immune cells

[0382] (1) Construction of GPRC5D-CAR

[0383] Each group of GPRC5D-targeted CAR nucleotide sequences (SEQ ID NO: 100-121) were designed and artificially synthesized. Each group of sequences contained HLA-A signal peptide (SEQ ID NO: 73), GPRC5D-VHH extracellular antigen binding domain (SEQ ID NO: 51-72), CD8α hinge region (SEQ ID NO: 74), CD8α transmembrane domain (SEQ ID NO: 75), 4-1BB (CD137) co-stimulatory signal domain (SEQ ID NO: 76) and CD3ζ intracellular signal transduction domain (SEQ ID NO: 77) encoding nucleotide sequences, which were used to express the complete GPRC5D-CAR polypeptide molecules (SEQ ID NO: 78-99) of each experimental group. The GPRC5D-CAR nucleotide sequence was inserted into the multiple cloning site of the lentiviral expression vector pK1 by homologous recombination to obtain pK1-GPRC5D-CAR, and the lentiviral expression vector sequence was successfully constructed by electrophoresis and sequencing results.

[0384] In addition, the positive control CAR was constructed in the same manner, using the antibody sequence GC5B602 disclosed in the patent document CN109715667A (see VL amino acid sequence SEQ NO ID: 58 and VH amino acid sequence SEQ ID NO: 88 in CN109715667A) as the antigen binding domain of the positive control CAR. The complete amino acid sequence of the positive control CAR is shown in SEQ ID NO: 122 and its encoding nucleic acid sequence is shown in SEQ ID NO: 123.

[0385] Amino acid sequence of positive control CAR: AVMAPRTLLLLLSGALALTQTWADIQMTQSPSSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFG QGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNGDTNYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARVA LRVALDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:122) The nucleic acid sequence of the positive control CAR:

[0386] (2) Packaging of lentiviral vectors

[0387] 293T cells were revived and cultured in DMEM medium containing 10% FBS. After 2-3 generations of cell expansion, 4×10 4 Pieces / cm 2 The density was inoculated into the 2-layer cell factory. Plasmid transfection was performed 3 days after cell inoculation. After adding 40 mL of Opti-MEM to a sterile 50 mL centrifuge tube for plasmid transfection, the viral packaging vector and the viral envelope vector were added according to the ratio of pK1-GPRC5D-CAR:pLP1:pLP2:pLP-VSVG=5:4:3:1, and 800 μL of PEI transfection reagent was added, mixed immediately, and incubated at room temperature for 15 min. Then the plasmid / vector / transfection reagent complex was added dropwise to the culture flask of 293T cells. After 24 h, the viral supernatant was collected into a 50 mL centrifuge tube, centrifuged at 250 g for 5 min, and the supernatant was filtered with a 0.45 μm filter after centrifugation. The filtered supernatant was ultracentrifuged (25000 g, 4 ° C, 3 h) to obtain concentrated GPRC5D-CAR lentivirus. After centrifugation, the supernatant was discarded, and the lentivirus was resuspended with PBS precooled at 4°C. The resuspended GPRC5D-CAR lentivirus solution was aliquoted and stored at -80°C for later use.

[0388] (3) T cell recovery and activation

[0389] The frozen cord blood was taken out and thawed in a 38°C water bath. The cord blood was transferred to a 50 mL centrifuge tube, and an appropriate amount of RPMI1640 medium was added. After mixing, the samples were counted and centrifuged at 300 g for 5 min. After centrifugation, the lower layer of cells was collected and resuspended in complete medium (RPMI1640 medium containing 10% FBS, supplemented with glutamine, 2-mercaptoethanol, IL-7 (10 ng / mL), IL-15 (10 ng / mL), and IL-2 (200 U / mL)) to a T lymphocyte density of 1×10 6 / mL, add activation antibodies Anti-human CD3 antibody and Anti-human CD28 antibody according to the resuspension volume, where Anti-human CD3 antibody is used at a concentration of 0.15μg / mL and Anti-human CD28 antibody is used at a concentration of 0.625μg / mL. Place T cells in a 37℃, 5% CO 2 Culture in an incubator.

[0390] (4) T cell sorting and purification

[0391] After 2 days of T cell activation, 20 μL of sample was mixed and added with 10 μL of diluted fluorescent labeled antibody, stained for 10 min, diluted 10 times with PBS, and counted using flow cytometry, recording the density of CD45+, CD3+, CD4+, and CD8+ T cells, and observing the expression of activation markers CD69 and CD25 molecules. Record the cell volume and confirm the total number of T cells. Transfer the cell suspension to a centrifuge tube and centrifuge (500g, 5min), discard the supernatant and collect the lower layer of cells; add MACS Buffer for washing, centrifuge again (500g, 5min), collect the lower layer of cells, and resuspend the cells with an appropriate amount of MACS Buffer. The amount of CD4+ magnetic beads (Miltenyi Biotec, 130-045-101) and CD8+ magnetic beads (Miltenyi Biotec, 130-045-201) used was calculated according to the cell amount and the magnetic beads manual, and CD4+ and CD8+ magnetic beads were used for sorting according to the instructions of the magnetic beads to obtain sorted T cells.

[0392] (5) Preparation of CAR-T cells targeting GPRC5D

[0393] The cell density of the sorted T cells was adjusted to 400 cells / μL for plating, and the volume of each well was 500 μL. According to the actual number of cells in each well, GPRC5D-CAR lentivirus solution was added, mixed and incubated at 37°C and 5% CO. 2 The cells were cultured in an incubator, and the expression rate of GPRC5D-CAR molecules on the surface of CD4+ and CD8+ T cells and the proliferation of cells were detected after 3 days of culture. FITC anti-human GPRC5D antibody (FITC-Labeled Human GPRC5D Protein, Flag, His Tag (Nanodisc, Acro) was used to detect the expression rate of positive control CAR molecules, and the antibody GPD-HF2D7 & Alexa 488AffiniPure Goat Anti-Alpaca IgG, VHH domain, (Jackson ImmunoResearch, 128-545-230) was used to detect the expression rate of CAR molecules in each experimental group. The expression rates of GPRC5D-CAR molecules in CD4+ and CD8+ T cells were as follows: Figure 1 and 2 As shown in Figure 2, it shows that the CAR molecule constructed by the present invention is successfully expressed on T cells with a high expression rate. The proliferation of CD4+ and CD8+ T cells is shown in Figure 2. Figure 3 and Figure 4As shown, it is shown that the CAR-T cells obtained after the CAR molecules constructed in the present invention are transduced into T cells still have good proliferation ability. The proliferation of CAR-T cells was observed every three days after plating and fresh complete medium was added. After 13 days of culture, the T cells in CAR-T cells were detected. SCM +T CM The proportion of CAR-T cells expressing Tim3, T SCM +T CM The test results are as follows Figure 5 As shown, the detection results of Tim3 expression are as follows Figure 6 The experimental results show that the CAR-T cells constructed by the present invention can still maintain a high proportion of T SCM +T CM The phenotype indicates that it has stronger persistence in vivo. CAR-T cells were harvested after 19 days of culture and used for subsequent in vitro killing experiments. The constructed CAR-T cell groups were G1 (SEQ ID NO: 89), G2 (SEQ ID NO: 78), G3 (SEQ ID NO: 94), G4 (SEQ ID NO: 86), G5 (SEQ ID NO: 99), G6 (SEQ ID NO: 88), G7 (SEQ ID NO: 79), G8 (SEQ ID NO: 83), G9 (SEQ ID NO: 97), G10 (SEQ ID NO: 92), G11 (SEQ ID NO: 91), G12 (SEQ ID NO: 95), G13 (SEQ ID NO: 81), G14 (SEQ ID NO: 98), G15 (SEQ ID NO: 96), G16 (SEQ ID NO: 82), G17 (SEQ ID NO: 80), G18 (SEQ ID NO: 85), G19 (SEQ ID NO: 93), G20 (SEQ ID NO: 87), G21 (SEQ ID NO: 84), G22 (SEQ ID NO: 86), G23 (SEQ ID NO: 87), G24 (SEQ ID NO: 88), G25 (SEQ ID NO: 89), G26 (SEQ ID NO: 91), G27 (SEQ ID NO: 92), G28 (SEQ ID NO: 93), G29 (SEQ ID NO: 94), G30 (SEQ ID NO: 95), G31 (SEQ ID NO: 96), G32 (SEQ ID NO: 97), G33 (SEQ ID NO: 98), G34 (SEQ ID NO: 99), G35 (SEQ ID NO: 99), G36 (SEQ ID NO: 91), G37 (SEQ ID NO: 91), G38 (SEQ ID NO: 91), G39 (SEQ ID NO: 92), G40 (SEQ ID NO: NO:90), the negative control T cell group was UnT (untransduced CAR), and the positive control CAR-T cell group was GC5B602 (SEQ ID NO:122).

[0394] Stem cell-like memory T cells (T SCM Central memory T cells (T cells) are a subset of T cells that have the ability to self-renew and generate effector T cells. They have the ability to survive in the body for a long time and play a role in maintaining long-term anti-tumor ability. CM ) is T SCM The latter stage of differentiation is usually analyzed by T SCM +T CMThe in vivo persistence and therapeutic potential of effector T cells can be judged by analyzing the percentage of CD45RO-CD62L+T cells and CD45RO+CD62L+T cells. SCM +T CM The higher the proportion of CD45RO:APC / Cyanine7 anti-human CD45RO,Biolegend,B350148; CD62L:PE / Cyanine7 anti-human CD62L,Biolegend,B373155), the stronger the persistence of CAR-T cells in the body. SCM +T CM It will also be beneficial for its use in in vivo therapy.

[0395] The present invention analyzes T cells expressing Tim3 (Brilliant Violet 421 TM The exhaustion of T cells was determined by the percentage of T cells expressing anti-human CD366, Biolegend, B355191). The higher the proportion of CAR-T cells expressing Tim3, the lower the effector function of CAR-T cells.

[0396] Example 3. Verification of tumor cell killing effect of CAR-T cells targeting GPRC5D

[0397] (1) Determination of GPRC5D antigen expression on the target cell surface

[0398] Human multiple myeloma cells (MM.1S) cells (purchased from ATCC, CRL-2974), Raji cells (purchased from ATCC, CCL-86) and Raji GPRC5D positive cells were used as target cells, respectively, and the above cells were cultured in RPMI 1640 medium.

[0399] Preparation of Raji GPRC5D positive cells: obtain lentivirus expressing GPRC5D antigen and inoculate 293T cells at 2×10 5 / cm 2The cells were inoculated at a density of 100 μg / mL in a T-shaped container, and 293T cells were transiently transfected with the plasmid. After adding 40 mL of Optim-MEM to a sterile 50 mL centrifuge tube for plasmid transfection, the viral packaging vector and viral envelope vector were added according to the ratio of pK1-GPRC5D:pLP1:pLP2:pLP-VSVG=5:4:3:1, and 800 μL of PEI transfection reagent, mix immediately, incubate at room temperature for 15 minutes, add the plasmid / vector / transfection reagent complex dropwise to 293T cells, collect the virus supernatant into a 50mL centrifuge tube after 24 hours, centrifuge at 250g for 5 minutes, filter the supernatant with a 0.45μm filter after centrifugation, ultracentrifuge the filtered supernatant (25000g, 4℃, 3h) to obtain concentrated GPRC5D lentivirus, discard the supernatant after centrifugation, resuspend the lentivirus with PBS precooled at 4℃, aliquot the resuspended GPRC5D lentivirus solution, and store it at -80℃ for later use. Normally proliferating Raji cells were cultured at 4×10 4 The cells were plated in a 96-well plate at a density of 0.008 cells / well, and GPRC5D lentivirus solution was added at MOI=25, and the volume was supplemented to 200 μL by fresh growth medium; on the third day after virus infection, the GPRC5D antigen expression of the cells was detected. When the GPRC5D expression of the cells was stable for more than 5 generations and the infection efficiency was higher than 90%, monoclonal plating was performed in a 96-well plate using the limiting dilution method, and the cell density was adjusted to 0.008 cells / μL, and the plating volume per well was 100 μL. The growth state of the monoclonality was observed at 8h, D1, D7, and D14 after plating. After confirming that Raji cells were monoclonal, the cells were cultured for more than 2 months, and then according to the instructions of the GPRC5D fluorescent labeled antibody, the Raji monoclonal cells were stained in the dark and the GPRC5D antigen expression on the cell surface was detected by flow cytometry. When the GPRC5D antigen expression was determined, the Raji GPRC5D positive cell line was obtained. According to the instructions of the GPRC5D fluorescently labeled antibody (Human GPRC5D PE-conjugated Antibody, R&D SYSTEMS, FAB6300RP), Raji-1F7 cells, Raji-BE6 cells, and Raji cells (i.e., Raji-CF10 cells) were stained in the dark for 10 minutes, and then the expression of GPRC5D antigen on the cell surface was detected by flow cytometry. The results are shown in the figure. Figure 7 The results showed that GPRC5D antigen was highly expressed in Raji-1F7 cells (Raji GPRC5D positive cells), lowly expressed in Raji-BE6 cells (Raji GPRC5D positive cells), and not expressed in Raji-CF10 cells (Raji cells).

[0400] (2) Flow cytometry determination of the in vitro killing effect of CAR-T cells targeting GPRC5D

[0401] Target cells (9×10 5 / well) Raji-1F7, Raji-BE6, Raji-CF10, and according to the effector-target ratio (E:T) of 1:3, the amount of effector cells to be added was calculated based on the amount of CD8+CAR-T cells in CAR-T cells, and the CAR-T cells constructed in Example 2 were added to the 24-well plate according to the corresponding effector cell amount. The negative control UnT group without CAR transduction also added the same amount of UnT cells and target cells according to the corresponding effector-target ratio. All groups were supplemented with complete culture medium to 500 μL / well, and the well plate was placed at 37°C, 5% CO 2 The cells were cultured in an incubator, and the amount of Raji cells in each well was detected by flow cytometry after 1 day and 3 days of culture, and the killing rate was calculated. The results are shown in Figure 8 , 9 , 10, 11 and Fig.12 The killing experiment results show that the CAR-T cells constructed by the present invention have significant killing ability for GPRC5D-positive target cells; the CAR-T cells constructed by the present invention have almost no killing ability for GPRC5D-negative target cells, indicating that they have specific killing ability.

[0402] The calculation formula is: killing rate % = target cell reduction / target cell plated cell number × 100%.

[0403] (3) Post-killing proliferation of CAR-T cells targeting GPRC5D, memory T cell subsets, and CAR-T cell exhaustion assay

[0404] Six days after plating in the in vitro killing experiment of item (2) of Example 3, 20 μL of the culture fluid of each group of CAR-T cells with target cells being Raji-1F7 and an effector-target ratio of 1:3 was collected, 10 μL of staining solution was added, and 170 μL of DPBS was added after staining in the dark for 7 minutes. The cells of each group were detected by flow cytometry. SCM +T CM and Tim3 expression, the results are as follows Fig.13 and Fig.14 The results show that the CAR-T cells constructed by the present invention can still maintain a high proportion of T cells after killing. SCM +T CM , indicating that it has stronger persistence in vivo. After the in vitro killing experiment was plated, flow cytometry was performed every 3 days to measure the proliferation of each group of CAR-T cells, and the cumulative proliferation times of CD4+ and CD8+ T cells in different positive target cell groups (effect-target ratio 1:3) after the killing experiment were counted on the 9th day. The results are as follows Fig.15 , 16 , 17 and 18. The experimental results show that the CAR-T cells constructed by the present invention not only have specific killing ability, but also have good proliferation ability after killing.

[0405] (4) In vitro killing effect of CAR-T cells targeting GPRC5D: lactate dehydrogenase (LDH) release assay

[0406] According to the effect-target ratio (E:T) of 1:3, 6×10 4 Target cells MM.1S are added to a 96-well plate, and the amount of effector cells to be added is calculated based on the amount of CD8+CAR-T cells in the CAR-T cells. The CAR-T cells constructed in Example 2 after 17 days of culture are added to the 96-well plate according to the corresponding amount of effector cells. The final density is 300 target cells / μL, 100 CD8+CAR-T cells / μL, and the total system of each well is 200μL. The negative control UnT group without CAR transduction also adds the same amount of UnT cells and target cells according to the corresponding effector-target ratio. At the same time, target cell spontaneous release control wells, target cell maximum release control wells, effector cell spontaneous release wells, background wells (200μL of culture medium) and volume correction wells (200μL of culture medium + 20μL of lysate) are set, and 5 sets of duplicate wells are set for each group. The 96-well plate is placed at 37°C and 5% CO 2 After incubation in the incubator for 24 h, the cells were taken out and centrifuged at 600 g for 10 min. 50 μL of the supernatant was transferred to a new 96-well plate and assayed according to the LDH cytotoxicity assay kit (CytoTox The degree of target cell damage was determined by detecting the activity of LDH in the cell culture supernatant of each group.

[0407] The calculation formula is: Cytotoxicity = (OD value of experimental group - OD value of spontaneous release well of effector cells - OD value of spontaneous release well of target cells) / (OD value of maximum release well of target cells - OD value of spontaneous release control well of target cells) * 100%; OD value of experimental group = measured OD value of experimental group - OD value of background well; OD value of effector cell spontaneous release well = measured OD value of effector cell spontaneous release well - OD value of background well; OD value of target cell spontaneous release control well = measured OD value of target cell spontaneous release well - OD value of background well; The OD value of the target cell maximum release well = the measured OD value of the target cell maximum release control well - the OD value of the volume correction well.

[0408] The culture medium used in the above experiment was: RPMI 1640+10% FBS+0.1% 2-mercaptoethanol+1% L-Glu.

[0409] The results are as follows Fig.19 As shown, UnT cells had no obvious killing effect on MM.1S target cells, while all experimental group GPRC5D CAR-T cells and positive control group CAR-T cells constructed by the present invention had obvious killing effect on MM.1S target cells, indicating that GPRC5D CAR-T cells can specifically kill MM.1S myeloma cell line.

[0410] (5) Cytokine release assay of CAR-T cells targeting GPRC5D

[0411] 18 hours after plating in the in vitro killing experiment of item (2) of Example 3, the supernatant of the CAR-T cell culture fluid of each group was collected and tested by CBA detection kit LEGENDplex TM HU Th Cytokine Panel (12-plex) w / FP V02 (biolegend, catalog number 741027) was used to detect the release of cytokines (IL-2, TNF-α, IFN-γ) of CAR-T cells in each group. The results are shown in the figure. Fig. 20 , 21 , 22. The experimental results show that the CAR-T cells constructed by the present invention can secrete a large amount of inflammatory cytokines (IL-2, TNF-α, INF-γ) during the killing process, indicating that they have significant killing ability.

[0412] The specific steps are as follows: 18h after plating in the in vitro killing experiment, the supernatant of the CAR-T cell culture fluid of each group was collected. After the capture microspheres (Beads) were restored to room temperature, vortexed for 2min to fully mix them, and prepared into 15μL per sample for standby. 20xWashBuffer was restored to room temperature to fully dissolve the salts therein, and it was prepared into 1x Wash Buffer with ultrapure water for standby. 250μL Assay Buffer was used to dissolve and mix the standard, and it was gradiently diluted, diluted 4 times according to the dilution factor, and diluted 6 times to obtain 6 gradient concentration points to establish a cell supernatant standard curve. Add 15uL of AssayBuffer, Beads, and standard to the EP tube, mix well, and prepare the standard well. Add 15μL of Assay Buffer, Beads, and each sample to the EP tube, mix well, and prepare the sample well. The standard well and sample well were shaken at 900rpm and incubated at room temperature for 2h under light-proof conditions. The sample was vacuum filtered, and 200 μL 1xWash buffer was added to each EP tube for shaking and mixing, and the washing was repeated once. 15 μL detection antibody and 15 μL Assay buffer were added to each EP tube, and the mixture was mixed by pipetting. Under the condition of protection from light, the mixture was shaken at 900 rpm and incubated at room temperature for 1 hour in the dark. Then 15 μL streptavidin-phycoerythrin (SA-PE) was added to each EP tube, and the mixture was shaken at 900 rpm under the condition of protection from light, and the mixture was incubated at room temperature for 0.5 hours in the dark to allow the capture microspheres (Beads), the target analyte to bind to the biotinylated detection antibody and SA-PE. 200 uL 1xWash buffer was added to each EP tube for secondary washing, and the mixture was shaken and mixed, and the washing was repeated once. 200 μL 1xWash buffer was added to each tube, and the mixture was tested in the dark. Each sample was shaken for 90 seconds before testing, and the flow cytometer parameters selected FSC, SSC, PE and APC channels to detect the release of cytokines IL-2, TNF-α and IFN-γ respectively.

Claims

1. A single domain antibody targeting GPRC5D, characterized in that: The single domain antibody comprises CDR1, CDR2 and CDR3; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 1, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 19, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 34; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 1, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 20, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 35; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:2, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:21, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:36; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:3, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:21, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:37; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:4, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:21, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:37; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:5, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:21, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:37; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:2, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:22, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:37; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:2, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:22, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:38; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:6, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:22, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:38; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:7, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:22, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:38; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:8, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:23, wherein CDR3 is the amino acid sequence shown in SEQ ID NO:39; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:9, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:24, wherein CDR3 is the amino acid sequence shown in SEQ ID NO:40; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 10, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 25, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 41; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 11, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 26, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 42; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 12, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 26, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 43; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 13, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 27, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 44; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 14, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 28, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 45; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 15, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 29, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 46; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 15, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 30, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 47; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 16, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 31, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 48; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 17, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 32, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 49; or Wherein CDR1 is the amino acid sequence shown in SEQ ID NO:18, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:33, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:

50.

2. The single domain antibody according to claim 1, characterized in that The single-domain antibody comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to any one of the amino acid sequences shown in SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 or SEQ ID NO:

72.

3. The single domain antibody according to claim 1, characterized in that The single-domain antibody comprises any one of the amino acid sequences shown in SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 or SEQ ID NO:

72.

4. Use of the single-domain antibody targeting GPRC5D according to any one of claims 1 to 3 in preparing a chimeric antigen receptor.

5. A chimeric antigen receptor, characterized in that Include (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) intracellular signaling domain; Wherein, the extracellular antigen binding domain comprises a single domain antibody targeting GPRC5D as described in any one of claims 1-3.

6. The chimeric antigen receptor according to claim 5, characterized in that wherein the transmembrane domain is derived from CD8α, CD28, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, TCRα, TCRβ, TCRγ, TCRδ, TCRζ, OX40, ICOS, LAG-3, 2B4, BTLA, CTLA-4, PD-1 or any combination thereof, preferably, the transmembrane domain is derived from CD8α; and / or wherein the intracellular signaling domain is derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d or any combination thereof, preferably, the intracellular signaling domain is derived from CD3 ζ; and / or wherein the intracellular signaling domain further comprises a co-stimulatory signaling domain, and the co-stimulatory signaling domain is derived from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD134 (OX40), CD2, CD7, CD27, CD28, CD30, CD40, CD83, ICAM, 4-1BB (CD137), CD276 (B7-H3), CD278 (ICOS), GITR, LIGHT, HVEM (LIGHTR), BTLA, CD8α, LFA-1, NKG2C, LAT, SLP-76, DAP10, PD-1, TRIM, ZAP70 ligand or any combination thereof, preferably, the co-stimulatory signaling domain is derived from 4-1BB (CD137).

7. The chimeric antigen receptor according to claim 5, characterized in that It further comprises a hinge region located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain, preferably, the hinge region is derived from CD8α, CD28, CD137, IgG4, IgG1 or any combination thereof; and / or it further comprises a signal peptide located at the N-terminus of the chimeric antigen receptor polypeptide, preferably, the signal peptide is derived from HLA-A, CD8α, CD4, CD33, CD137, GM-CSFRα, IgG1, Igκ, IL-2 or any combination thereof.

8. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to any one of the amino acid sequences set forth in SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98 or SEQ ID NO:99; or the chimeric antigen receptor comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to any one of the amino acid sequences set forth in SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ Any amino acid sequence shown in IDNO:96, SEQ ID NO:97, SEQ ID NO:98 or SEQ ID NO:

99.

9. An isolated nucleic acid, characterized in that The invention comprises a nucleic acid sequence encoding a chimeric antigen receptor as described in claims 5 to 8; preferably, the isolated nucleic acid comprises any one of the nucleic acid sequences shown in SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120 or SEQ ID NO:

121.

10. A carrier, characterized in that The vector comprises the isolated nucleic acid of claim 9.

11. An engineered immune effector cell, characterized in that: Comprising the chimeric antigen receptor according to any one of claims 5 to 8, the isolated nucleic acid according to claim 9, or the vector according to claim 10; preferably, the immune effector cells are selected from T cells, NKT cells, DNT cells, NK cells, B cells, macrophages, dendritic cells, immune effector cells differentiated from induced pluripotent stem cells, or any combination thereof.

12. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the single domain antibody according to any one of claims 1 to 3, the chimeric antigen receptor according to any one of claims 5 to 8, or the engineered immune effector cell according to claim 11, and one or more pharmaceutically acceptable excipients and / or carriers.

13. Use of a single domain antibody targeting GPRC5D as claimed in any one of claims 1 to 3, a chimeric antigen receptor as claimed in any one of claims 5 to 8, an engineered immune effector cell as claimed in claim 11 or a pharmaceutical composition as claimed in claim 12 in the preparation of a medicament for diagnosing, preventing and / or treating a disease or condition associated with GPRC5D expression; preferably, the disease or condition associated with GPRC5D expression is a malignant tumor associated with GPRC5D expression, an autoimmune disease and / or an inflammatory disease associated with GPRC5D expression; more preferably, the malignant tumor associated with GPRC5D expression is selected from From marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal large B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, Burkitt's lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin's lymphoma, high-grade B-cell lymphoma, Waldenstrom's macroglobulinemia and / or multiple myeloma, wherein the autoimmune disease and / or inflammatory disease associated with GPRC5D expression is selected from systemic lupus erythematosus, and / or rheumatoid arthritis.

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