GPRC5D single domain and its humanized antibody

Abstract

The present invention provides a single GPRC5D domain and a humanized antibody thereof, which exhibits strong binding activity and reaction specificity, as well as good species cross-reactivity. This is of great significance for research and development in different species models, helping to accelerate the transition from laboratory to clinic. The antibody sequence provided by the present invention can be used to construct CAR-T, which can be used to treat 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 disorder in which the tumor cells originate from plasma cells in the bone marrow. Plasma cells are the final functional stage of B lymphocyte development. Therefore, multiple myeloma can be classified as a type of B-cell lymphoma. The WHO classifies it as a type of B-cell lymphoma, termed 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 protein). A very small number of patients may have non-secretory MM that does not produce M protein. Multiple myeloma is often accompanied by multiple osteolytic lesions, hypercalcemia, anemia, and renal damage. Because the production of normal immunoglobulins is suppressed, various bacterial infections are prone to occur.

[0003] Currently, the primary approach for treating MM is to target BCMA (B cell maturation antigen). However, for MM patients with BCMA-negative or low BCMA expression, relapse can occur even after receiving BCMA-targeted CAR-T cell therapy, posing a risk of target escape. To mitigate BCMA escape-mediated relapse, finding more specific MM targets (such as GPRC5D) or simultaneously targeting other antigenic targets (such as CD3, CD19, CD38, and GPRC5D) is crucial. GPRC5D is likely to become the next hot candidate target for treating MM.

[0004] GPRC5D (G protein-coupled receptor C5 family subtype D) is an atypical class C orphan G protein-coupled receptor that belongs to the 7-transmembrane protein class. In recent years, GPRC5D has become an attractive target for the treatment of multiple myeloma (MM). This is primarily due to the high expression of GPRC5D 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 application in the construction of CAR-T and immune cells, thereby providing new treatment ideas for patients who are refractory to existing therapies or whose disease relapses. Summary of the Invention

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

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

[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 NO:24; HCDR3 consists of

[0008] 7) HCDR1 consists of SEQ ID NO: 27; HCDR2 consists of SEQ ID NO: 28; and 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.

[0009] Further, the antibody comprises a heavy chain variable region, the sequence of which is as 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.

[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] Furthermore, 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 further comprises 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-exiting 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 label coupled thereto.

[0020] Preferably, the detectable label comprises at least one of a radioisotope, a metal nanomaterial, fluorescein, biotin, avidin, a biotin / avidin complex, a biotin / avidin 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 further includes one or more of a hinge region, a transmembrane region, an intracellular signal transduction region, and a co-stimulatory signaling domain.

[0024] Furthermore, 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] Furthermore, the hinge region is CD8 Hinge.

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

[0027] Furthermore, 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.

[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] Furthermore, 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 variants thereof.

[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] Furthermore, the costimulatory signaling domain is selected from the costimulatory 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 variants thereof.

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

[0035] Furthermore, the amino acid sequence of 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 further comprises 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 can encode 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 further comprises a base sequence encoding an Fc sequence as shown in SEQ ID NO: 84.

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

[0051] Furthermore, the nucleic acid molecule further 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 EGFRt export signal as shown in SEQ ID NO:91.

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

[0058] Furthermore, the nucleic acid molecule further 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 a 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 comprises 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 the cell binding validation diagram of GPRC5D antibody;

[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 purified candidate GPRC5D antibodies to bind to CHO-S-Mouse-GPRC5D;

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

[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 bound to CHO-S-GPRC5D cells by FACS;

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

[0079] Figure 10 This is a graph showing the affinity test of humanized B05 antibody using FACS combined with CHO-Mouse GPRC5D cells;

[0080] Figure 11 This is a graph showing the killing rate of GPRC5D humanized antibody CAR-T. DETAILED DESCRIPTION

[0081] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art and do not constitute 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), also known as VHH antibodies or camelid antibodies, are artificially designed antibody molecules. They are heavy-chain antibodies (HCAbs) naturally lacking light chains and 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). Single-domain antibodies containing only a single heavy chain variable domain, or VHH antibodies, are then cloned. The crystal structure of VHH antibodies is an oval shape measuring 4nm × 2.5nm × 3nm. Their molecular weight is only 1 / 10 that of ordinary antibodies, approximately 12-14kDa, making them the smallest complete antigen-binding fragment and, therefore, also known as nanobodies.

[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 common amino acid at that position. In certain embodiments, the sequences of HCDR1-3 of the heavy chain variable region of the antibodies 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 sequences defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system are also within the scope of protection of the present invention.

[0085] In the present invention, the terms "binding" and "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 sequences obtained by modification also fall within the scope of protection 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, antibody sequences that have been modified to have 80% or more identity with the amino acid sequence of the antibody described in the first aspect of the present invention also fall within the scope of protection of the present invention. The term "identity" is also known as "homology" and refers to an amino acid sequence that is at least 80% identical to the sequence provided by the present invention. In order to determine sequence identity, sequence alignment can be performed in various ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE, Megalign (DNASTAR), Snapgene or DNAMAN software. Those skilled in the art can determine 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 a non-human antibody 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, while the FR (or part thereof) is derived from a human antibody sequence. Optionally, the humanized antibody also comprises at least a portion of a human constant region. In some embodiments, some FR residues in the 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, extracellular hinge region and transmembrane domain, co-stimulatory domain and intracellular immunoreceptor tyrosine activation motif according to actual conditions or needs. Regardless of the form of change, as long as the chimeric antigen receptor has the CDR sequence or heavy chain variable region sequence of the heavy chain variable region of the above-mentioned humanized antibody of the present invention, it falls within the scope of protection 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 an appropriate regulatory sequence. The boundaries of the coding sequence are determined by the start codon at the 5' (amino) end and the translation stop 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' place of the coding sequence.

[0091] In the present invention, a vector comprising the nucleic acid molecules of the present invention is provided. The term "vector" refers to a kind of artificial construct, which can be delivered 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 the nucleic acid of the present invention that is operably connected to other segments of the autonomous replication provided in the recombinant host cell, or according to the expression cassette of the nucleic acid molecule. "Operably connected" means that the nucleic acid sequence of interest is connected to a regulatory sequence (for example, 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] As used herein, the term "pharmaceutical composition" refers to a composition comprising at least one biologically active compound. The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. In some embodiments, oral administration is preferred. The pharmaceutical compositions of the present invention may contain any conventional non-toxic pharmaceutically acceptable carrier, adjuvant, or vehicle. In some cases, pharmaceutically acceptable acids, bases, or buffers 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. The pharmaceutical compositions of the present invention may be administered to the recipient by any route that reaches the target tissue. The pharmaceutical compositions of the present invention may also be used in combination with other drugs for treating, preventing, alleviating, and / or alleviating multiple myeloma. These other compounds for treating, preventing, alleviating, and / or alleviating multiple myeloma may 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. The other therapeutic compounds may 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 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. may also be present in such vehicles.

[0094] In the present invention, the term "progression" refers to a malignant change in a disease, including growth, spread, or metastasis of a lesion (e.g., a tumor). In some embodiments, the relative increase in the sum of diameters of target lesions measured throughout the entire study is at least 20% (if the baseline measurement is the smallest, the baseline value is used as the reference). In addition, the absolute increase in the sum of diameters must be at least 5 mm (the appearance of one or more new lesions is also considered disease progression).

[0095] In the present invention, the term "regression" refers to a decrease 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 degree of reduction of target lesions has not reached the level of disease remission, and the degree of increase 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 will be further described in detail below with reference to the accompanying drawings and examples. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. The following examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Simple modifications to the present invention based on the essence of the present invention fall within the scope of protection claimed in the present invention.

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

[0099] 1. Construction of phage display library by alpaca immunization

[0100] Two alpacas were immunized four times in total. The immunogen was 293F-GPRC5D (human sequence), and the immunization was subcutaneous. The immune adjuvant was GERBU. After immunization, serum was isolated from the immunized alpacas. After verification by ELISA and FACS, peripheral blood was collected from the alpacas, PBMC cells were isolated, RNA was extracted, and reverse transcribed into cDNA. 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 as follows: Figure 1 A library was constructed from two alpacas, and 48 clones were sequenced. After removing two antibody sequences with one base missing and one empty sequence, the remaining sequences were all antibody sequences, indicating 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, and 2-B08 were screened and constructed into the pcDNA3.4-IgG1Fc antibody expression vector (such as Figure 2 After expression in 293F cells, the supernatant was tested for binding to CHO-S-GPRC5D cells. Effectively binding cloned antibodies were purified and analyzed by FACS EC50. The results showed that the candidate antibodies bound to CHO-S-GPRC5D cells, with C07, F08, B05, A12, and B11 showing the strongest binding.

[0103] 1) GPRC5D antibody cell binding verification: Flow cytometry specific parameters: using 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). The results are shown in Figure 2. Figure 3 shown.

[0104] 2) Validation of affinity of purified GPRC5D antibody: Flow cytometry 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 serial 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. 5GPRC5D candidate antibody + positive antibody (10 μg / ml, 100 μl / well) was used as primary antibody, PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution) was used as secondary antibody, and flow cytometry was performed. 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 plasmids (pCDH-EF1α lentiviral expression plasmid, PsPAX2, and pMD2.G plasmid system, mixed at a mass ratio of 3:2:1) were transfected into adherent 293T cells in the logarithmic growth phase. The cell culture supernatant was harvested 48-72 hours after transfection, concentrated and filtered, and the CAR lentivirus was obtained and stored at -80°C for later use.

[0110] 2. CAR-T cell construction: Peripheral blood mononuclear cells (PBMC) were isolated from human peripheral blood, and T cells were isolated using human CD3 / 28 magnetic beads. Viral transduction was performed within 72 hours of activation. The medium was changed 24 hours after transduction and cultured until the 8th day. 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. Subsequent killing experiments were carried out according to the CAR-T positive ratio and mixed with target cells (effective target ratio 1:1 and 5:1) 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 A range of framework amino acids is required, as these amino acid positions often affect the conformation or antigen-binding activity of the CDRs. Humanized germlines are obtained through IMGT analysis. After splicing the selected humanized germline framework with the antibody's CDRs, the framework regions of the designed humanized antibody are aligned with those of the original antibody. By analyzing homology modeling results of the parental antibody, amino acid substitutions similar to those on the surface of human antibodies are selected to design the humanized antibody sequence, while maintaining antibody activity and minimizing heterogeneity. The humanized antibodies designed above are synthesized and subcloned into the pcDNA3.4-IgG1Fc expression vector. After the vectors are verified by sequencing, endotoxin-free plasmids are prepared and expressed in 293F cells. Affinity is then measured after incubation with CHO-S and CHO-S-GPRC5D cells.

[0112] 1) Affinity test results such as Figure 8 As shown, humanized B05 antibody was verified 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 serial 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) B05 humanized antibody FACS combined with CHO-S cells (negative cells) detection, using CHO-S cells (3×10 5 The primary antibody was GPRC5D target candidate antibody and positive antibody (starting from 30 μg / ml, 3-fold serial dilution 10 points, 100 μl / well); the secondary antibody was PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution). Figure 9 shown.

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

[0115] 4. The candidate B05HM2 and B05HM3 sequences were synthesized to construct the pCDH-EF1α lentiviral expression plasmid. The lentiviral system plasmid (pCDH-EF1α lentiviral expression plasmid, PsPAX2, and pMD2.G plasmid system, mixed at a mass ratio of 3:2:1) was transfected into adherent 293T cells in the logarithmic growth phase. 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. Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood, and T cells were isolated using human CD3 / 28 magnetic beads. Viral transduction was performed within 72 hours of activation. After 24 hours of transduction, the medium was changed and cultured until day 8. The cells were collected by centrifugation and resuspended in 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 cells were mixed with target cells (effect-target ratio 1:1 and 5:1) to conduct subsequent killing experiments and detect the killing rate. The results are as follows Figure 11 shown.

[0116] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention can be implemented over a wide range under equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides embodiments, it will be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any variations, uses, or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the disclosed scope of this application.

Claims

1. A single domain antibody that binds to GPRC5D, characterized in that The single-domain antibody comprises heavy chain complementary determining regions HCDR1-HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 13; the amino acid sequence of HCDR2 is shown in SEQ ID NO: 14; and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

15.

2. The single domain antibody according to claim 1, characterized in that The antibody comprises a heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO:

16.

3. The single domain antibody according to claim 2, characterized in that The sequence of the heavy chain variable region is obtained by replacing some amino acids in the framework region of SEQ ID NO: 16 with humanized amino acids. The amino acid sequence of the heavy chain variable region obtained by replacing the humanized amino acids is shown in SEQ ID NO:

17.

4. The single domain antibody according to claim 2, characterized in that The sequence of the heavy chain variable region is obtained by replacing some amino acids in the framework region of SEQ ID NO: 16 with humanized amino acids. The amino acid sequence of the heavy chain variable region obtained by replacing the humanized amino acids is shown in SEQ ID NO:

18.

5. The single domain antibody according to claim 2, characterized in that The sequence of the heavy chain variable region is obtained by replacing some amino acids in the framework region of SEQ ID NO: 16 with humanized amino acids. The amino acid sequence of the heavy chain variable region obtained by replacing the humanized amino acids is shown in SEQ ID NO:

19.

6. The single domain antibody according to claim 2, characterized in that The single domain antibody further comprises an Fc sequence.

7. The single domain antibody according to claim 6, characterized in that The Fc sequence is shown in SEQ ID NO:

35.

8. The single domain antibody according to claim 2, characterized in that The single domain antibody further comprises an SP membrane export signal.

9. The single domain antibody according to claim 8, characterized in that The sequence of the SP membrane-exiting signal is shown in SEQ ID NO:

34.

10. An antibody derivative, characterized in that The antibody derivative comprises any one of the following: 1) The single domain antibody according to any one of claims 1 to 9 and a detectable label conjugated thereto; 2) A chimeric antigen receptor comprising the heavy chain variable region of the single domain antibody according to any one of claims 1 to 9.

11. The antibody derivative according to claim 10, characterized in that The detectable label comprises at least one of a radioisotope, a metal nanomaterial, fluorescein, biotin, avidin, a biotin / avidin complex, a biotin / avidin complex, an electron-dense substance, and an enzyme.

12. The antibody derivative according to claim 10, characterized in that The chimeric antigen receptor further comprises a hinge region, a transmembrane region, a co-stimulatory signaling domain, an intracellular signal transduction region, a 2A peptide, and a suicide gene.

13. The antibody derivative according to claim 12, characterized in that The hinge region is CD8 Hinge.

14. The antibody derivative according to claim 13, characterized in that The amino acid sequence of the CD8 Hinge is shown in SEQ ID NO:

37.

15. The antibody derivative according to claim 12, characterized in that The transmembrane region is the CD8 TM.

16. The antibody derivative according to claim 15, characterized in that The amino acid sequence of the CD8 TM is shown in SEQ ID NO:

38.

17. The antibody derivative according to claim 12, characterized in that The intracellular signal transduction domain is CD3ζ.

18. The antibody derivative according to claim 17, characterized in that The amino acid sequence of CD3ζ is shown in SEQ ID NO:

40.

19. The antibody derivative according to claim 12, characterized in that The co-stimulatory signaling domain is 4-1BB.

20. The antibody derivative according to claim 19, characterized in that The amino acid sequence of 4-1BB is shown in SEQ ID NO:

39.

21. The antibody derivative according to claim 12, characterized in that The suicide gene includes EGFRt.

22. The antibody derivative according to claim 21, characterized in that The amino acid sequence of EGFRt is shown in SEQ ID NO:

43.

23. The antibody derivative according to claim 21, characterized in that The suicide gene also includes a signal peptide of EGFRt.

24. The antibody derivative according to claim 23, characterized in that The amino acid sequence of the EGFRt signal peptide is shown in SEQ ID NO:

42.

25. The antibody derivative according to claim 12, characterized in that The 2A peptide is located between the intracellular signal transduction region and the suicide gene, and the 2A peptide is T2A.

26. The antibody derivative according to claim 25, characterized in that The amino acid sequence of T2A is shown in SEQ ID NO:

41.

27. The antibody derivative according to claim 12, characterized in that The chimeric antigen receptor also includes a signal peptide.

28. The antibody derivative according to claim 27, characterized in that The amino acid sequence of the signal peptide is shown in SEQ ID NO:

36.

29. A nucleic acid molecule, characterized in that The nucleic acid molecule can encode the single domain antibody according to any one of claims 1 to 9 and / or the chimeric antigen receptor according to any one of claims 10 to 28.

30. The nucleic acid molecule according to claim 29, characterized in that The nucleic acid molecule comprises the base sequence encoding HCDR1-3 in the heavy chain variable region of the single-domain antibody according to any one of claims 1 to 9 as shown in SEQ ID NOs: 48-51; or, the nucleic acid molecule comprises the base sequence of an antibody derivative as shown in SEQ ID NO:

58.

31. The nucleic acid molecule according to claim 30, characterized in that The base sequences encoding HCDR1-3 are shown in SEQ ID NOs: 70-72.

32. The nucleic acid molecule according to claim 30, characterized in that The nucleic acid molecule further comprises a base sequence encoding an Fc sequence as shown in SEQ ID NO:

84.

33. The nucleic acid molecule according to claim 30, characterized in that The nucleic acid molecule further comprises a base sequence encoding an SP membrane exit signal as shown in SEQ ID NO:

83.

34. The nucleic acid molecule according to claim 30, characterized in that The nucleic acid molecule further comprises a base sequence encoding CD8 Hinge as shown in SEQ ID NO:

86.

35. The nucleic acid molecule according to claim 30, characterized in that The nucleic acid molecule also comprises a base sequence encoding CD8 TM as shown in SEQ ID NO:

87.

36. The nucleic acid molecule according to claim 30, characterized in that The nucleic acid molecule also comprises a base sequence encoding CD3ζ as shown in SEQ ID NO:

89.

37. The nucleic acid molecule according to claim 30, characterized in that The nucleic acid molecule further comprises a base sequence encoding 4-1BB as shown in SEQ ID NO:

88.

38. The nucleic acid molecule according to claim 30, characterized in that The nucleic acid molecule further comprises a base sequence encoding EGFRt as shown in SEQ ID NO:

92.

39. The nucleic acid molecule according to claim 30, characterized in that The nucleic acid molecule further comprises a base sequence encoding a signal peptide of EGFRt as shown in SEQ ID NO:

91.

40. The nucleic acid molecule according to claim 30, characterized in that The nucleic acid molecule further comprises a base sequence encoding T2A as shown in SEQ ID NO:

90.

41. The nucleic acid molecule according to claim 30, characterized in that The nucleic acid molecule further comprises a base sequence encoding a signal peptide as shown in SEQ ID NO:

85.

42. A carrier, characterized in that The vector comprises the nucleic acid molecule according to any one of claims 29 to 41.

43. A recombinant host cell, characterized in that The recombinant host cell comprises the nucleic acid molecule according to any one of claims 29 to 41 or the vector according to claim 42.

44. Use of the single domain antibody according to any one of claims 1 to 9, the antibody derivative according to any one of claims 10 to 28, and / or the nucleic acid molecule according to any one of claims 29 to 41, characterized in that: The application includes any of the following: 1) Application in the construction of CAR-T cells targeting GPRC5D; 2) Application in the preparation of products for detecting GPRC5D protein.

45. The use according to claim 44, characterized in that The products include test kits, nucleic acid chips, and nucleic acid membrane strips.

46. ​​A method for preparing the single domain antibody according to any one of claims 1 to 9 or the chimeric antigen receptor according to any one of claims 10 to 28, characterized in that: The method includes artificial synthesis or preparation by genetic engineering technology; The preparation using genetic engineering technology refers to culturing the recombinant host cell according to claim 43 under conditions suitable for expressing antibodies.

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