GPRC5D single domain and humanized antibody thereof

Abstract

The invention provides the GPRC5D single domain and the humanized antibody thereof, the GPRC5D single domain and the humanized antibody thereof have relatively strong binding activity and reaction specificity, also have good species cross reactivity, are of great significance in research and development in different species models, and are beneficial to acceleration of a transformation process from a laboratory to clinic. The antibody sequence provided by the invention can be used for constructing CAR-T, and can be used for treating multiple myeloma.

Description

Technical Field

[0001] The present invention relates to a GPRC5D single domain and a humanized antibody thereof, and relates to the fields of genetic engineering and antibody technology. Background Art

[0002] Multiple myeloma (MM) is a malignant plasma cell disease, whose tumor cells originate from plasma cells in the bone marrow, and plasma cells are cells that develop from B lymphocytes to the final functional stage. Therefore, multiple myeloma can be classified as a type of B lymphocyte lymphoma. WHO classifies it as a type of B cell lymphoma, called plasma cell myeloma / plasmacytoma. It is characterized by abnormal proliferation of bone marrow plasma cells accompanied by excessive production of monoclonal immunoglobulins or light chains (M proteins). A very small number of patients may be non-secretory MM that does not produce M proteins. Multiple myeloma is often accompanied by multiple osteolytic lesions, hypercalcemia, anemia, and kidney damage. Because the production of normal immunoglobulins is suppressed, various bacterial infections are prone to occur.

[0003] Currently, MM is mainly treated through BCMA (B cell maturation antigen) target therapy, but for MM patients with BCMA negative or low BCMA expression, relapse still occurs after receiving targeted BCMA CAR-T cell therapy, and there is a problem of target escape. In order to alleviate the problem of BCMA escape-mediated relapse, it is key to find better specific MM targets (such as GPRC5D) or simultaneously target other antigen targets (such as CD3, CD19, CD38, GPRC5D, etc.), and GPRC5D is likely to become the next hot candidate target for the treatment of MM.

[0004] GPRC5D (G protein-coupled receptor C5 family subtype D) is an atypical class C orphan G protein-coupled receptor, which belongs to 7 transmembrane proteins. In recent years, GPRC5D has become an attractive target for the treatment of multiple myeloma (MM). This is mainly because GPRC5D is highly expressed on the surface of multiple myeloma cells, while its expression in normal tissues is very limited. Therefore, the present invention aims to provide antibody sequences targeting GPCR5D and their use in CAR-T and immune cell construction, thereby providing new treatment ideas for patients who are ineffective or relapsed with existing therapies. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide a GPRC5D single domain and a humanized antibody thereof, and an application thereof in CAR-T construction.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an antibody that binds to GPRC5D, the antibody comprising heavy chain complementary determining regions HCDR1-HCDR3, wherein: 1) HCDR1 consists of SEQ ID NO: 1; HCDR2 consists of SEQ ID NO: 2; HCDR3 consists of SEQ ID NO: 3; or 2) HCDR1 consists of SEQ ID NO: 5; HCDR2 consists of SEQ ID NO: 6; HCDR3 consists of SEQ ID NO: 7; or 3) HCDR1 consists of SEQ ID NO: 9; HCDR2 consists of SEQ ID NO: 10; HCDR3 consists of SEQ ID NO: 11; or 4) HCDR1 consists of SEQ ID NO: 13; HCDR2 consists of SEQ ID NO: 14; HCDR3 consists of SEQ ID NO: 15; or 5) HCDR1 consists of SEQ ID NO: 9; HCDR2 consists of SEQ ID NO: 20; HCDR3 consists of SEQ ID NO: 21; or 6) HCDR1 consists of SEQ ID NO: 23; HCDR2 consists of SEQ ID NO: 24; HCDR3 consists of SEQ ID NO: 25 NO:24; HCDR3 consists of

[0008] 7) HCDR1 consists of SEQ ID NO:27; HCDR2 consists of SEQ ID NO:28; HCDR3 consists of SEQ ID NO:25; or 8) HCDR1 consists of SEQ ID NO:30; HCDR2 consists of SEQ ID NO:31; HCDR3 consists of SEQ ID NO:32.

[0009] Further, the antibody comprises a heavy chain variable region, the sequence of which is as shown in any of the following groups: 1) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:4; 2) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:8; 3) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:12; 4) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:16; 5) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:22; 6) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:26; 7) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:29; 8) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:33.

[0010] Furthermore, the sequence of the heavy chain variable region is that part of the amino acids in the framework region of 4) are replaced with humanized amino acids.

[0011] Further, the sequence of the heavy chain variable region is as shown in any one of the following groups: 9) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 17; 10) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 18; 11) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 19.

[0012] Furthermore, the antibody also includes an Fc sequence.

[0013] Furthermore, the Fc sequence is shown in SEQ ID NO:35.

[0014] Furthermore, the antibody also includes an SP membrane exit signal.

[0015] Furthermore, the sequence of the SP membrane exit signal is shown in SEQ ID NO:34.

[0016] Furthermore, the antibody includes a single domain antibody, a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody or a bivalent antibody.

[0017] Furthermore, the antibody is a single domain antibody.

[0018] The second aspect of the present invention provides an antibody derivative, wherein the antibody derivative comprises any one of the following:

[0019] 1) The antibody according to the first aspect of the present invention and a detectable marker coupled thereto.

[0020] Preferably, the detectable label comprises at least one of a radioisotope, a metal nanomaterial, a fluorescein, a biotin, avidin, a biotin / avidin protein complex, a biotin / avidin protein complex, a chromophore, an electron-dense substance and an enzyme.

[0021] 2) A chimeric antigen receptor comprising the heavy chain variable region of the antibody according to the first aspect of the present invention.

[0022] 3) A pharmaceutical composition, comprising the antibody according to the first aspect of the present invention and a pharmaceutically acceptable carrier.

[0023] Furthermore, the chimeric antigen receptor also includes one or more of a hinge region, a transmembrane region, an intracellular signal transduction region, and a co-stimulatory signal domain.

[0024] Further, the hinge region is selected from the hinge regions of the following molecules: CD8, 4 1BB, IgG1, IgG4, PD 1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and variants thereof.

[0025] Further, the hinge region is CD8 Hinge.

[0026] Furthermore, the amino acid sequence of the CD8 Hinge is shown in SEQ ID NO:37.

[0027] Further, the transmembrane region is selected from the transmembrane regions of the following molecules: CD8, 4 1BB, IgG1, IgG4, PD 1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and their variants.

[0028] Furthermore, the transmembrane region is CD8 TM.

[0029] Furthermore, the amino acid sequence of the CD8 TM is shown in SEQ ID NO:38.

[0030] Further, the intracellular signal transduction region is selected from the intracellular signal transduction regions of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, CD278, CD21, CD22, FcεRI, FcRγ, FcRβ, CD4, CD5, CD8, CD79a, CD79b, DAP10, DAP12, CD66d and their variants.

[0031] Furthermore, the intracellular signal transduction region is CD3ζ.

[0032] Furthermore, the amino acid sequence of CD3ζ is shown in SEQ ID NO:40.

[0033] Further, the co-stimulatory signaling domain is selected from the co-stimulatory signaling domains of the following molecules: 41BB, HVEM, CD27, CD19, CD28, ICOS, CD4, CD8α, CD8β, CD40, OX40, DR3, CD2, GITR, CD30, TIM1, CD226, CD278 and their variants.

[0034] Furthermore, the co-stimulatory signaling domain is 4-1BB.

[0035] Furthermore, the amino acid sequence of the 4-1BB is shown in SEQ ID NO:39.

[0036] Furthermore, the chimeric antigen receptor also includes a suicide gene.

[0037] Furthermore, the suicide gene includes EGFRt.

[0038] Furthermore, the amino acid sequence of the EGFRt is shown in SEQ ID NO:43.

[0039] Furthermore, the suicide gene also includes a signal peptide of EGFRt.

[0040] Furthermore, the amino acid sequence of the EGFRt signal peptide is shown in SEQ ID NO:42.

[0041] Furthermore, the chimeric antigen receptor also includes a 2A peptide.

[0042] Furthermore, the 2A peptide is T2A.

[0043] Furthermore, the amino acid sequence of T2A is shown in SEQ ID NO:41.

[0044] Furthermore, the chimeric antigen receptor also includes a signal peptide.

[0045] Furthermore, the amino acid sequence of the signal peptide is shown in SEQ ID NO:36.

[0046] The third aspect of the present invention provides a nucleic acid molecule, a vector comprising the nucleic acid molecule, or a recombinant host cell comprising the nucleic acid molecule or the vector, wherein the nucleic acid molecule is capable of encoding the antibody described in the first aspect of the present invention and / or the antibody derivative described in the second aspect of the present invention.

[0047] Further, the nucleic acid molecule comprises the base sequence encoding HCDR1-3 in the heavy chain variable region of the antibody according to the first aspect of the present invention as shown in SEQ ID NO:45-55; and / or, the nucleic acid molecule comprises the base sequence of the antibody derivative as shown in SEQ ID NO:56-60.

[0048] Furthermore, the base sequences encoding HCDR1-3 are shown in SEQ ID NOs: 61-82, 94-96.

[0049] Furthermore, the nucleic acid molecule also comprises a base sequence encoding an Fc sequence as shown in SEQ ID NO:84.

[0050] Furthermore, the nucleic acid molecule also comprises a base sequence encoding an SP membrane exit signal as shown in SEQ ID NO:83.

[0051] Furthermore, the nucleic acid molecule also comprises a base sequence encoding CD8 Hinge as shown in SEQ ID NO:86.

[0052] Furthermore, the nucleic acid molecule also comprises a base sequence encoding CD8 TM as shown in SEQ ID NO:87.

[0053] Furthermore, the nucleic acid molecule also comprises a base sequence encoding CD3ζ as shown in SEQ ID NO:89.

[0054] Furthermore, the nucleic acid molecule also comprises a base sequence encoding 4-1BB as shown in SEQ ID NO:88.

[0055] Furthermore, the nucleic acid molecule also comprises a base sequence encoding EGFRt as shown in SEQ ID NO:92.

[0056] Furthermore, the nucleic acid molecule also comprises a base sequence encoding an export signal of EGFRt as shown in SEQ ID NO:91.

[0057] Furthermore, the nucleic acid molecule also comprises a base sequence encoding T2A as shown in SEQ ID NO:90.

[0058] Furthermore, the nucleic acid molecule also comprises a base sequence encoding a membrane release signal as shown in SEQ ID NO:85.

[0059] The fourth aspect of the present invention provides the use of the antibody according to the first aspect of the present invention, the antibody derivative according to the second aspect of the present invention and / or the nucleic acid molecule according to the third aspect of the present invention, wherein the use includes the following:

[0060] 1) Application in constructing CAR-T cells targeting GPRC5D; 2) Application in constructing bivalent antibodies targeting GPRC5D; 3) Application in diagnosing, treating GPRC5D-positive cancer progression, regression, and / or monitoring GPRC5D-positive cancer stability; 4) Application in preparing products for diagnosing, treating GPRC5D-positive cancer progression, regression, and / or monitoring GPRC5D-positive cancer stability; 5) Application in preparing products for detecting GPRC5D protein.

[0061] Furthermore, the bivalent antibody comprising the antibody targeting GPRC5D can also target T cell markers.

[0062] Furthermore, the T cell markers include CD3, CD19, and CD38.

[0063] Furthermore, the T cell marker is CD3.

[0064] Furthermore, the GPRC5D-positive cancers include multiple myeloma and plasma cell leukemia.

[0065] Furthermore, the GPRC5D-positive cancer is multiple myeloma.

[0066] Furthermore, the products include pharmaceutical compositions, test kits, nucleic acid chips, and nucleic acid membrane strips.

[0067] The fifth aspect of the present invention provides a method for preparing the antibody described in the first aspect of the present invention or the antibody derivative described in the second aspect of the present invention, wherein the method comprises artificial synthesis or preparation using genetic engineering technology.

[0068] The preparation using genetic engineering technology refers to culturing the recombinant host cell described in the third aspect of the present invention under conditions suitable for expressing antibodies.

[0069] Advantages and beneficial effects of the present invention: The GPRC5D antibody provided by the present invention has a specific amino acid sequence, has strong binding activity and reaction specificity, and also has good species cross-reactivity. This feature is of great significance for research and development in different species models, and helps to accelerate the transformation process from laboratory to clinic. The anti-GPRC5D antibody sequence can be used to construct chimeric antigen receptors (CAR) and bivalent antibodies (bispecific antibodies). The CAR constructed based on the antibody sequence can be transduced into T cells to create CAR-T cells targeting GPRC5D, which can be used to treat multiple myeloma. The bivalent antibody designed based on the antibody sequence can simultaneously target GPRC5D and T cells, thereby activating T cells and guiding them to attack tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is a diagram of mixed library sequence construction;

[0071] Figure 2 Schematic diagram of the construction of the pcDNA3.4-IgG1Fc vector;

[0072] Figure 3 This is a diagram for validation of GPRC5D antibody cell binding;

[0073] Figure 4 This is a graph showing the affinity validation of the purified GPRC5D antibody;

[0074] Figure 5 This is a graph showing the ability of the purified candidate GPRC5D antibody to bind to CHO-S-Mouse-GPRC5D;

[0075] Figure 6 Schematic diagram of the structure of the CAR-T vector;

[0076] Figure 7 This is the CAR-T killing rate detection chart of the GPRC5D candidate purified antibody;

[0077] Figure 8 This is the affinity verification diagram of humanized B05 antibody combined with CHO-S-GPRC5D cells by FACS;

[0078] Fig. 9 This is the affinity test diagram of humanized B05 antibody combined with CHO-S cells by FACS;

[0079] Fig.10 This is the affinity test diagram of humanized B05 antibody combined with CHO-Mouse GPRC5D cells;

[0080] Fig.11 This is the killing rate detection chart of GPRC5D humanized antibody CAR-T. DETAILED DESCRIPTION

[0081] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvement by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0082] In the context of the present invention, the term "antibody" is used in the broadest sense and specifically covers single domain antibodies, monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies and multispecific antibodies (e.g., diabodies) formed from at least two intact antibodies, so long as they exhibit the desired biological activity.

[0083] In the present invention, the term "single-domain antibodies" (sdAbs) is also called VHH antibodies or camelid antibodies, which are artificially designed antibody molecules. They are heavy-chain antibodies (HCAbs) that naturally lack light chains and are found in camelids such as alpacas and dromedaries, as well as cartilaginous fish such as sharks and rays. They include two constant regions (CH2 and CH3), a hinge region, and a variable heavy chain domain (VHH). Then, a single-domain antibody containing only one heavy chain variable region, i.e., a VHH antibody, is cloned. The crystal structure of VHH antibody is an oval of 4nm×2.5nm×3nm, and the molecular weight is only 1 / 10 of that of ordinary antibodies, about 12-14kDa. It is the smallest complete antigen-binding fragment, so it is also called a nanobody.

[0084] In the present invention, the term "CDR" refers to the "hypervariable region" or "complementarity determining region" of an antibody. Both the heavy chain variable region and the light chain variable region have three CDRs, which together constitute the antigen binding site of the antibody and can form precise complementarity with the antigenic determinant in terms of spatial structure. Examples of other antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from different antibody fragments. The position of CDR in the variable region is marked differently according to different numbering systems, and the numbering schemes include IMGT, Chothia, Kabat, etc. The IMGT numbering scheme is based on the amino acid sequence alignment of germline V genes; the Chothia numbering scheme is based on crystal structure; and the Kabat numbering scheme defines the variability parameter as the number of different amino acids at a given position divided by the frequency of the most amino acid at that position. In certain embodiments, the sequence of HCDR1-3 of the heavy chain variable region of the antibody described in the present invention can be obtained based on the full-length sequence of the above-mentioned heavy chain variable region according to the Kabat, IMGT, Chothia, AbM or Contact numbering system. The CDR sequence defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system is also within the scope of protection of the present invention.

[0085] In the present invention, the terms "binding", "targeting" refer to that the binding is selective for the antigen and can be distinguished from undesirable or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art.

[0086] In the present invention, the antibody sequence obtained by modification also belongs to the protection scope of the present invention. The term "modification" refers to any form of modification of the amino acid sequence, such as substitution, deletion, insertion and / or addition of amino acids. The term "substitution" refers to replacing one or more amino acids in the original amino acid sequence with different amino acids. The term "deletion" refers to the reduction of one or more amino acids in the original amino acid sequence. The term "insertion" or "addition" refers to a change in the amino acid sequence that results in the addition of one or more amino acids compared to the original amino acid sequence.

[0087] In the present invention, the antibody sequence obtained by modification and having 80% or more identity with the amino acid sequence of the antibody described in the first aspect of the present invention also belongs to the protection scope of the present invention. The term "identity" is also called "homology" and refers to an amino acid sequence whose sequence is at least 80% identical to the sequence provided by the present invention. In order to determine the sequence identity, sequence alignment can be performed in various ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE, Megalign (DNASTAR), Snapgene or DNAMAN software. Those skilled in the art can determine the appropriate parameters for alignment, including any algorithm required to achieve optimal alignment in the full-length sequences being compared.

[0088] "Humanization" of the present invention generally refers to humanizing non-human antibodies to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Generally, the CDR (or part thereof) of a humanized antibody is derived from a non-human antibody sequence, and the FR (or part thereof) is derived from a human antibody sequence. Optionally, a humanized antibody also includes at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody derived from HVR residues), for example, to restore or improve antibody specificity or affinity.

[0089] In certain embodiments, those skilled in the art can change the combination category and sequence of the signal peptide, the extracellular hinge region and the transmembrane domain, the co-stimulatory domain and the intracellular immune receptor tyrosine activation motif according to actual conditions or needs. Regardless of the form of the change, as long as the chimeric antigen receptor has the CDR sequence or the heavy chain variable region sequence of the heavy chain variable region of the above-mentioned humanized antibody of the present invention, it belongs to the protection scope of the present invention.

[0090] In the present invention, the term "nucleic acid molecule" refers to any polymer form having any length and consisting of ribonucleotides or deoxyribonucleotides. Generally, a nucleic acid molecule is a coding sequence, as used herein, which refers to a DNA sequence that is transcribed and translated into a polypeptide in a host cell when placed under the control of a suitable regulatory sequence. The boundaries of the coding sequence are determined by the start codon at the 5' (amino) end and the translation termination codon at the 3' (carboxyl) end. The coding sequence may include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even recombinant DNA sequences. The transcription termination sequence will generally be located at the 3' of the coding sequence.

[0091] In the present invention, a vector comprising the nucleic acid molecule of the present invention is provided. The term "vector" refers to a kind of artificial construct, which can deliver and preferably express one or more target genes or sequences in a host cell. The vector of the present invention can be a plasmid vector, a viral vector, etc. In some embodiments, the vector refers to a straight or circular nucleic acid molecule, which comprises a nucleic acid of the present invention that is operably connected to other segments of autonomous replication provided in a recombinant host cell, or an expression cassette according to a nucleic acid molecule. "Operably connected" means that the nucleic acid sequence of interest is connected to a regulatory sequence (e.g., in an in vitro transcription / translation system or when the vector is introduced into a host cell) in a manner that allows the expression of the nucleotide sequence. In the present invention, the term "recombinant host cell" refers to introducing the vector of the present invention into a host cell so that the recombinant host cell can transcribe the target nucleic acid sequence and / or translate the target protein.

[0092] In the present invention, the term "pharmaceutical composition" refers to a composition comprising at least one biologically active compound. The pharmaceutical composition of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or by implanted drug storage devices. In some embodiments, oral administration is preferred. The pharmaceutical composition of the present invention may contain any commonly used non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle. In some cases, a pharmaceutical acid, base or buffer may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. As long as the target tissue can be reached, the pharmaceutical composition of the present invention may be administered to the receptor by any route. The pharmaceutical composition of the present invention can also be used in combination with other drugs for treating, preventing, alleviating and / or relieving multiple spinal cord neoplasms. Other compounds for treating, preventing, alleviating and / or relieving multiple spinal cord neoplasms can be administered simultaneously with the main active ingredient (e.g., the antibody described in the first aspect of the present invention), or even administered simultaneously in the same composition. Other therapeutic compounds can also be administered separately in a separate composition or in a dosage form different from that of the main active ingredient.

[0093] In the present invention, the term "pharmaceutically acceptable carrier" refers to any pharmaceutical carrier that does not itself induce the production of antibodies that are harmful to the individual receiving the composition and can be administered without excessive toxicity. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids and amino acid copolymers. Such carriers are well known to those of ordinary skill in the art. The pharmaceutically acceptable carrier in the pharmaceutical composition can include fluids, such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting agents or emulsifiers, pH buffer substances, etc., can also be present in such vehicles.

[0094] In the present invention, the term "progression" refers to malignant changes in the disease, including growth, spread or metastasis of lesions (such as tumors). In some embodiments, the minimum value of the sum of all target lesion diameters measured during the entire experimental study is used as a reference, and the relative increase in the sum of diameters is at least 20% (if the baseline measurement value is the minimum, the baseline value is used as a reference); in addition, the absolute value of the sum of diameters must increase by at least 5 mm (the appearance of one or more new lesions is also considered as disease progression).

[0095] In the present invention, the term "regression" refers to a reduction in tumor volume or complete disappearance, whether spontaneous or as a result of treatment. In some embodiments, tumor regression is assessed by examining tissue samples to determine the extent of reduction in tumor cell populations and changes in tumor architecture.

[0096] In the present invention, the term "stability" means that the reduction degree of target lesions has not reached the level of disease remission, and the increase degree has not reached the level of disease progression, but is somewhere in between. The minimum value of the sum of diameters can be used as a reference for research.

[0097] The present invention is further described in detail below in conjunction with the accompanying drawings and examples. The experimental methods in the following examples are conventional methods unless otherwise specified, and the materials and reagents used in the following examples are commercially available unless otherwise specified. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Simple improvements to the present invention made according to the essence of the present invention all belong to the scope of protection claimed in the present invention.

[0098] Example 1 Construction of phage display library, selection of antibodies and validation

[0099] 1. Alpaca immunization to construct phage display library

[0100] Two alpacas were immunized 4 times in total. The immunogen was 293F-GPRC5D (human sequence), subcutaneous immunization, and the immune adjuvant was GERBU. After immunization, serum was isolated from the immunized alpacas, and ELISA and FACS tests were performed to verify. The peripheral blood of the alpacas was collected, PBMC cells were isolated, RNA was extracted, reverse transcribed into cDNA, and the single-domain antibody cloning primer combination was used to amplify the VHH sequence from the cDNA sample and subcloned into the phage display vector pDisplay. The SS320 Escherichia coli competent cells were electroporated to construct a single-domain antibody phage display library (mixed library of two alpacas). The mixed library sequence is shown in the figure. Figure 1 As shown in the figure, a library was constructed from two alpacas and 48 clones were sequenced. Two antibody sequences with one base missing and one empty sequence were removed, and the remaining sequences were all antibody sequences with good diversity.

[0101] 2. Antibody selection and validation

[0102] In the above-mentioned library, the selection protein / cell: GPRC5D-VLP protein / CHO-S-GPRC5D cell line and the negative screening protein / cell: VLP protein / CHO-S cell line were used for panning. The first and third rounds were VLP panning, and the second and fourth rounds were cell panning. The candidate clone sequences such as 1-B11, 2-D06, 1-G08, 1-G09, 2-A12, 2-B05, 2-B06, 2-B08 were screened and constructed into the pcDNA3.4-IgG1Fc antibody expression vector (such as Figure 2 After 293F transfection and expression, the supernatant was taken for binding verification with CHO-S-GPRC5D cells, and the effective binding cloned antibodies were arranged to detect FACS EC50 after antibody purification. The results showed that the candidate antibodies bound to CHO-S-GPRC5D cells, among which C07, F08, B05, A12 and B11 bound strongly.

[0103] 1) GPRC5D antibody cell binding verification: Flow cytometry specific parameters: 3×10 5 The primary antibody was GPRC5D transfection supernatant (100 μl / well), including positive antibody (10 μg / ml, 100 μl / well); the secondary antibody was PE-Goatanti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution), and the results were as follows Figure 3 shown.

[0104] 2) Validation of affinity of purified GPRC5D antibody: Flow cytometry specific parameters: CHO-S-GPRC5D cells (3×10 5 / well). The primary antibody was the GPRC5D target candidate antibody and positive antibody (starting from 30μg / ml, 3-fold gradient dilution 11 points, 100μl / well); the secondary antibody was PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution). The results are shown in Figure 4 , as shown in Table 1.

[0105] Table 1. Antibody affinity analysis

[0106]

[0107] 3) Binding test of GPRC5D candidate purified antibodies and CHO-S-Mouse-GPRC5D: 3×10 CHO-S cells overexpressing mouse GPRC5D were used. 5100 μl / well, GPRC5D candidate antibody + positive antibody (10 μg / ml, 100 μl / well) as primary antibody, PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution) as secondary antibody, flow cytometry detection. Figure 5 As shown, candidate antibodies B05 and F08 bind strongly to CHO-S-Mouse-GPRC5D cells.

[0108] Example 2 Construction of CAR-T cells using candidate antibody sequences and verification of killing ability

[0109] 1. CAR lentivirus preparation: The positive control CAR-T is BCMA replacing GPRC5D CAR (reference doi:10.1158 / 2643-3230.BCD-20-0020), and the pCDH-EF1α lentiviral expression plasmid is constructed by sequence synthesis, with the structure as follows Figure 6 The lentiviral system plasmid (pCDH-EF1α lentiviral expression plasmid, PsPAX2, pMD2.G three-plasmid system, mixed at a mass ratio of 3:2:1) was transfected into adherent 293T cells in the logarithmic growth phase, and the cell culture supernatant was harvested 48-72 hours after transfection. After concentration and filtration, the CAR lentivirus was obtained and stored at -80°C for later use.

[0110] 2. CAR-T cell construction: Peripheral blood mononuclear cells (PBMC) are isolated from human peripheral blood, and T cells are isolated using human CD3 / 28 magnetic beads. Virus transduction is performed within 72 hours of activation. The medium is changed 24 hours after transduction and cultured until the 8th day. The cells are collected by centrifugation and resuspended in normal saline. Flow cytometry is used to identify EGFRt molecules on the surface of T cells to ensure that the positive rate is greater than 30%. If it is lower, EGFR-PE primary antibody plus PE magnetic beads are used for enrichment and sorting. According to the CAR-T positive ratio, it is mixed with target cells (effective target ratio 1:1 and 5:1) for subsequent killing experiments to detect the killing rate. The results are as follows Figure 7 shown.

[0111] 3. Humanized sequence antibody affinity test: Based on the original antibody sequence information developed in the previous project, the homology model of the antibody is obtained through modeling and CDRs are analyzed The humanized antibody sequence is designed by analyzing the homology modeling results of the parent antibody. The amino acids similar to the surface residues of human antibodies are selected for replacement while maintaining the antibody activity and reducing heterology, and the humanized antibody sequence is designed. The humanized antibodies designed above are gene synthesized and subcloned into the pcDNA3.4-IgG1Fc expression vector. After the vector is verified by sequencing, the endotoxin-free plasmid is prepared and the humanized antibody is expressed by 293F cells, and its affinity is detected after incubation with CHO-S and CHO-S-GPRC5D.

[0112] 1) Affinity test results such as Figure 8 As shown, humanized B05 antibody was validated by FACS combined with CHO-S-GPRC5D cells. CHO-S-GPRC5D cells (3×10 5 / well), the primary antibody was GPRC5D target candidate antibody and positive antibody (starting from 30 μg / ml, 3-fold gradient dilution 10 points, 100 μl / well); the secondary antibody was PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution).

[0113] 2) Humanized B05 antibody FACS combined with CHO-S cells (negative cells) detection, using CHO-S cells (3×10 5 / well) primary antibody was GPRC5D target candidate antibody and positive antibody (starting from 30μg / ml, 3-fold gradient dilution 10 points, 100μl / well); secondary antibody was PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution). The results are shown in Fig. 9 shown.

[0114] 3) Humanized B05 antibody FACS combined with CHO-Mouse GPRC5D cells (mouse GPRC5D) detection, using CHO-Mouse GPRC5D cells (3×10 5 / well) primary antibody was GPRC5D target candidate antibody and positive antibody (starting from 30μg / ml, 3-fold gradient dilution 10 points, 100μl / well); secondary antibody was PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution). The results are shown in Fig.10 shown.

[0115] 4. The candidate B05HM2 and B05HM3 sequences were synthesized to construct the pCDH-EF1α lentiviral expression plasmid, and the lentiviral system plasmid (pCDH-EF1α lentiviral expression plasmid, PsPAX2, pMD2.G three-plasmid system, mixed at a mass ratio of 3:2:1) was transfected into adherent 293T cells in the logarithmic growth phase, and the cell culture supernatant was harvested for 48-72 hours of transfection. After concentration and filtration, the CAR lentivirus was obtained and stored at -80°C for standby use. Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood, and T cells were isolated using human CD3 / 28 magnetic beads, and viral transduction was performed within 72 hours of activation. After 24 hours of transduction, the medium was changed and cultured until the 8th day, and the cells were collected by centrifugation and resuspended in normal saline. Flow cytometry was used to identify EGFRt molecules on the surface of T cells to ensure that the positive rate was greater than 30%. If it was lower, EGFR-PE primary antibody plus PE magnetic beads were used for enrichment and sorting. The CAR-T positive ratio was mixed with the target cells (efficacy-target ratio 1:1 and 5:1) to conduct subsequent killing experiments and detect the killing rate. The results are as follows Fig.11 shown.

[0116] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. An antibody that binds to GPRC5D, characterized in that The antibody comprises heavy chain complementary determining regions HCDR1-HCDR3, wherein: 1) HCDR1 consists of SEQ ID NO: 1; HCDR2 consists of SEQ ID NO: 2; HCDR3 consists of SEQ ID NO: 3; or 2) HCDR1 consists of SEQ ID NO: 5; HCDR2 consists of SEQ ID NO: 6; HCDR3 consists of SEQ ID NO: 7; or 3) HCDR1 consists of SEQ ID NO: 9; HCDR2 consists of SEQ ID NO: 10; HCDR3 consists of SEQ ID NO: 11; or 4) HCDR1 consists of SEQ ID NO: 13; HCDR2 consists of SEQ ID NO: 14; HCDR3 consists of SEQ ID NO: 15; or 5) HCDR1 consists of SEQ ID NO: 9; HCDR2 consists of SEQ ID NO: 20; HCDR3 consists of SEQ ID NO: 21; or 6) HCDR1 consists of SEQ ID NO: 23; HCDR2 consists of SEQ ID NO: 24; HCDR3 consists of SEQ ID NO: 25; or 7) HCDR1 consists of SEQ ID NO: 27; HCDR2 consists of SEQ ID NO: 28; HCDR3 consists of SEQ ID NO: 25; or 8) HCDR1 consists of SEQ ID NO:30; HCDR2 consists of SEQ ID NO:31; and HCDR3 consists of SEQ ID NO:

32.

2. The antibody according to claim 1, characterized in that The antibody comprises a heavy chain variable region, the sequence of which is shown in any one of the following groups: 1) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 4; 2) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 8; 3) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 12; 4) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 16; 5) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 22; 6) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 26; 7) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 29; 8) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 33; Preferably, the sequence of the heavy chain variable region is that part of the amino acids in the framework region of 4) are replaced with humanized amino acids; Preferably, the sequence of the heavy chain variable region is as shown in any one of the following groups: 9) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 17; 10) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 18; 11) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 19; Preferably, the antibody further comprises an Fc sequence; Preferably, the Fc sequence is as shown in SEQ ID NO: 35; Preferably, the antibody further comprises an SP membrane exit signal; Preferably, the sequence of the SP membrane-exiting signal is shown as SEQ ID NO:

34.

3. The antibody according to claim 1 or 2, characterized in that The antibodies include single domain antibodies, monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies or bivalent antibodies; Preferably, the antibody is a single domain antibody.

4. An antibody derivative, characterized in that: The antibody derivative comprises any of the following: 1) The antibody according to any one of claims 1 to 3 and a detectable marker coupled thereto; Preferably, the detectable label comprises at least one of a radioisotope, a metal nanomaterial, a fluorescein, a biotin, avidin, a biotin / avidin complex, a biotin / avidin complex, a chromophore, an electron-dense substance, and an enzyme; 2) a chimeric antigen receptor comprising the heavy chain variable region of the antibody according to any one of claims 1 to 3; 3) A pharmaceutical composition comprising the antibody according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

5. The antibody derivative according to claim 4, characterized in that The chimeric antigen receptor further comprises one or more of a hinge region, a transmembrane region, an intracellular signal transduction region, and a co-stimulatory signaling domain; Preferably, the hinge region is selected from the hinge regions of the following molecules: CD8, 4 1BB, IgG1, IgG4, PD 1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and variants thereof; Preferably, the hinge region is CD8 Hinge; Preferably, the amino acid sequence of the CD8 Hinge is as shown in SEQ ID NO: 37; Preferably, the transmembrane region is selected from the transmembrane regions of the following molecules: CD8, 4 1BB, IgG1, IgG4, PD 1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and variants thereof; Preferably, the transmembrane region is CD8 TM; Preferably, the amino acid sequence of the CD8 TM is as shown in SEQ ID NO: 38; Preferably, the intracellular signal transduction region is selected from the intracellular signal transduction region of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, CD278, CD21, CD22, FcεRI, FcRγ, FcRβ, CD4, CD5, CD8, CD79a, CD79b, DAP10, DAP12, CD66d and variants thereof; Preferably, the intracellular signal transduction domain is CD3ζ; Preferably, the amino acid sequence of CD3ζ is as shown in SEQ ID NO:40; Preferably, the costimulatory signaling domain is selected from the costimulatory signaling domains of the following molecules: 4 1BB, HVEM, CD27, CD19, CD28, ICOS, CD4, CD8α, CD8β, CD40, OX40, DR3, CD2, GITR, CD30, TIM1, CD226, CD278 and variants thereof; Preferably, the co-stimulatory signaling domain is 4-1BB; Preferably, the amino acid sequence of 4-1BB is as shown in SEQ ID NO: 39; Preferably, the chimeric antigen receptor further comprises a suicide gene; Preferably, the suicide gene comprises EGFRt; Preferably, the amino acid sequence of EGFRt is as shown in SEQ ID NO:43; Preferably, the suicide gene further comprises a signal peptide of EGFRt; Preferably, the amino acid sequence of the signal peptide of EGFRt is as shown in SEQ ID NO:42; Preferably, the chimeric antigen receptor further comprises a 2A peptide; Preferably, the 2A peptide is T2A; Preferably, the amino acid sequence of T2A is shown in SEQ ID NO:41; Preferably, the chimeric antigen receptor further comprises a signal peptide; Preferably, the amino acid sequence of the signal peptide is shown in SEQ ID NO:

36.

6. A nucleic acid molecule, a vector comprising the nucleic acid molecule, or a recombinant host cell comprising the nucleic acid molecule or the vector, characterized in that: The nucleic acid molecule can encode the antibody according to any one of claims 1 to 3 and / or the antibody derivative according to claim 5.

7. The nucleic acid molecule, vector or recombinant host cell according to claim 6, characterized in that: The nucleic acid molecule comprises a base sequence encoding HCDR1-3 in the heavy chain variable region of the antibody according to any one of claims 1 to 3 as shown in SEQ ID NOs: 45-55; and / or, the nucleic acid molecule comprises a base sequence of an antibody derivative as shown in SEQ ID NOs: 56-60; Preferably, the base sequences encoding HCDR1-3 are shown in SEQ ID NOs: 61-82, 94-96; Preferably, the nucleic acid molecule further comprises a base sequence encoding an Fc sequence as shown in SEQ ID NO: 84; Preferably, the nucleic acid molecule further comprises a base sequence encoding an SP membrane exit signal as shown in SEQ ID NO: 83; Preferably, the nucleic acid molecule further comprises a base sequence encoding CD8 Hinge as shown in SEQ ID NO: 86; Preferably, the nucleic acid molecule further comprises a base sequence encoding CD8 TM as shown in SEQ ID NO: 87; Preferably, the nucleic acid molecule further comprises a base sequence encoding CD3ζ as shown in SEQ ID NO:89; Preferably, the nucleic acid molecule further comprises a base sequence encoding 4-1BB as shown in SEQ ID NO:88; Preferably, the nucleic acid molecule further comprises a base sequence encoding EGFRt as shown in SEQ ID NO:92; Preferably, the nucleic acid molecule further comprises a base sequence encoding a signal peptide of EGFRt as shown in SEQ ID NO:91; Preferably, the nucleic acid molecule further comprises a base sequence encoding T2A as shown in SEQ ID NO:90; Preferably, the nucleic acid molecule further comprises a base sequence encoding a signal peptide as shown in SEQ ID NO:

85.

8. Use of the antibody according to any one of claims 1 to 3, the antibody derivative according to claim 5 and / or the nucleic acid molecule according to any one of claims 6 to 7, characterized in that: The applications include the following: 1) Application in the construction of CAR-T cells targeting GPRC5D; 2) Application in constructing bivalent antibodies targeting GPRC5D; 3) Application in diagnosis, treatment of GPRC5D-positive cancer progression, regression, and / or monitoring of GPRC5D-positive cancer stability; 4) Use in the preparation of products for diagnosing, treating, progressing, regressing, and / or monitoring the stability of GPRC5D-positive cancers; 5) Application in the preparation of products for detecting GPRC5D protein.

9. The use according to claim 8, characterized in that: The bivalent antibody comprising targeting GPRC5D can also target T cell markers; Preferably, the T cell markers include CD3, CD19, and CD38; Preferably, the T cell marker is CD3; Preferably, the GPRC5D-positive cancer includes multiple myeloma and plasma cell leukemia; Preferably, the GPRC5D-positive cancer is multiple myeloma; Preferably, the products include pharmaceutical compositions, kits, nucleic acid chips, and nucleic acid membrane strips.

10. A method for preparing the antibody according to any one of claims 1 to 3 or the antibody derivative according to claim 5, said method comprising artificial synthesis or preparation by genetic engineering technology; The preparation by genetic engineering technology refers to culturing the recombinant host cell according to claim 6 under conditions suitable for expressing antibodies.

Images