Application of GPRC5D humanized antibody and concatemer in construction of CAR-T cells

Abstract

The invention provides an application of a GPRC5D humanized antibody and a concatemer in construction of a CAR-T cell, particularly relates to a GPRC5D-targeted single-domain antibody and the humanized antibody thereof, and a serial configuration of the antibody, and also provides a chimeric antigen receptor (CAR) molecule constructed by using the antibody, the CAR-T cell and application thereof. Under a standardized condition, the tandem configuration shows remarkably improved tumor cell killing compared with a corresponding monomer configuration so as to realize a high CAR-T maximum effect, and a new treatment choice is provided for GPRC5D positive multiple myeloma patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering and antibody engineering, in particular to the application of GPRC5D humanized antibodies and concatemers in constructing CAR-T cells. BACKGROUND

[0002] Multiple myeloma (MM) is a malignant clonal proliferation of plasma cells, accompanied by abnormal proliferation of plasma cells in the bone marrow and increased monoclonal immunoglobulin or light chain (M protein), which can cause bone destruction, hypercalcemia, anemia, and kidney damage. Although the existing BCMA-targeted therapy has made progress, BCMA low expression / deletion and antigen escape still lead to recurrence.

[0003] GPRC5D (G protein-coupled receptor class C group 5 subtype D) is highly expressed on the surface of MM cells and has limited normal tissue distribution, and is considered as a preferred alternative / complementary target in addition to BCMA. Therefore, obtaining a high-affinity and high-specificity humanized single-domain antibody against GPRC5D and realizing stable and enhanced cytotoxicity in CAR configuration has important clinical and industrial value. SUMMARY

[0004] In order to make up for the shortcomings of the prior art, the purpose of the present application is to provide a family of humanized single-domain antibodies targeting GPRC5D and their two-by-two concatenated configuration, and to construct CAR molecules and CAR-T cells, which exhibit significantly improved tumor cell killing relative to the corresponding monomer configuration under standardized conditions to achieve high CAR-T maximum effect, providing a new treatment option for multiple myeloma patients positive for GPRC5D.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0006] The first aspect of the present application provides a humanized single-domain antibody targeting GPRC5D, wherein the humanized single-domain antibody is obtained by humanizing the residues at key positions in the framework region of the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 28 or SEQ ID NO: 31, with reference to a human germline framework.

[0007] In some embodiments, the humanized single-domain antibody includes any one of B05HS1, B05HS2, B05HS3, F08HS1, F08HS2, F08HS3, C07HS1, C07HS2, and C07HS3.

[0008] In some embodiments, B05HS1, B05HS2, B05HS3, F08HS1, F08HS2, F08HS3, C07HS1, C07HS2, and C07HS3 each have an amino acid sequence as shown in SEQ ID NO:26, SEQ ID NO:1, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:5, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:9, or SEQ ID NO:33.

[0009] In some embodiments, the humanized single-domain antibody has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO:26, SEQ ID NO:1, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:5, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:9, or SEQ ID NO:33.

[0010] A second aspect of the present invention provides a tandem antibody targeting GPRC5D, said tandem antibody being formed by sequentially tandemly connecting the humanized single-domain antibodies described in the first aspect.

[0011] In some embodiments, the tandem configuration antibody further includes a linker protein.

[0012] In some embodiments, the linker protein is a flexible polypeptide with the structure (G4S). n (n = 2-6), with a total length of 10–30 amino acids.

[0013] In some embodiments, the linker protein is (G4S)3, whose amino acid sequence is shown in SEQ ID NO:16.

[0014] In some embodiments, the tandem configuration antibody is formed by sequentially linking a humanized single-domain antibody, a linker protein, and a humanized single-domain antibody.

[0015] In some embodiments, the humanized single-domain antibody is selected from any two of B05HS2, F08HS2, and C07HS2.

[0016] In some embodiments, the humanized single-domain antibody is formed by sequentially linking B05HS2, a linker protein, and F08HS2.

[0017] In some embodiments, the humanized single-domain antibody is formed by sequentially linking C07HS2, a linker protein, and B05HS2.

[0018] In some embodiments, the humanized single-domain antibody is formed by sequentially linking F08HS2, a linker protein, and C07HS2.

[0019] In a specific embodiment of the present invention, the tandem configuration antibody has an amino acid sequence as shown in SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15.

[0020] A third aspect of the present invention provides a chimeric antigen receptor comprising the humanized single-domain antibody described in the first aspect, or comprising the tandem antibody described in the second aspect.

[0021] In some embodiments, the chimeric antigen receptor further includes an SP exomembrane signal, a hinge region, a transmembrane region, a co-stimulatory signaling domain, and an intracellular signal transduction region.

[0022] In some embodiments, the chimeric antigen receptor further includes CD8 SP, the amino acid sequence of which is shown in SEQ ID NO:17.

[0023] In some embodiments, the hinge region is selected from the hinge regions of the following molecules: CD8, 41BB, IgG1, IgG4, PD1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT, and variants thereof.

[0024] In some embodiments, the transmembrane region is selected from the transmembrane regions of the following molecules: CD8, 41BB, IgG1, IgG4, PD1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT, and variants thereof.

[0025] In some embodiments, 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 variants thereof.

[0026] In some embodiments, 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, ZAP70, and variants thereof.

[0027] In a specific embodiment of the present invention, the hinge region is a CD8 hinge region, and the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO:18.

[0028] In a specific embodiment of the present invention, the transmembrane region is the CD8 transmembrane region, and the amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO:19.

[0029] In a specific embodiment of the present invention, the co-stimulatory signaling domain is a 4 1BB co-stimulatory signaling domain, and the amino acid sequence of the 4 1BB co-stimulatory signaling domain is shown in SEQ ID NO:20.

[0030] In a specific embodiment of the present invention, the intracellular signal transduction region is the CD3ζ intracellular signal transduction region, and the amino acid sequence of the CD3ζ intracellular signal transduction region is shown in SEQ ID NO:21.

[0031] In a specific embodiment of the present invention, the chimeric antigen receptor is formed by sequentially connecting the SP exomembrane signal, the humanized single-domain antibody described in the first aspect or the tandem conformation antibody described in the second aspect, CD8 SP, CD8 hinge region, CD8 transmembrane region, 41BB co-stimulatory signal domain, and CD3ζ intracellular signal transduction region.

[0032] A fourth aspect of the present invention provides an immune effector cell that expresses the chimeric antigen receptor described in the third aspect.

[0033] In some embodiments, the immune effector cells are T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, or macrophages.

[0034] In some embodiments, the immune effector cells are T cells.

[0035] A fifth aspect of the present invention provides a pharmaceutical composition comprising the chimeric antigen receptor described in the third aspect or the immune effector cells described in the fourth aspect.

[0036] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier, excipient, or buffer solution.

[0037] In some embodiments, the pharmaceutical composition is an injectable cell preparation.

[0038] The sixth aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the humanized single-domain antibody of the first aspect, the tandem antibody of the second aspect, or the chimeric antigen receptor of the third aspect.

[0039] In some embodiments, the nucleotide sequence is as shown in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or is a nucleotide sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24.

[0040] A seventh aspect of the present invention provides a recombinant expression vector comprising the nucleic acid molecule described in the sixth aspect.

[0041] The eighth aspect of the present invention provides a recombinant host cell expressing the humanized single-domain antibody of the first aspect, the tandem antibody of the second aspect, or the chimeric antigen receptor of the third aspect.

[0042] In some embodiments, the recombinant host cell comprises the recombinant expression vector described in the seventh aspect.

[0043] In some embodiments, the recombinant host cell comprises recombinant immune cells.

[0044] In some embodiments, the recombinant immune cells are T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, or macrophages.

[0045] In a specific embodiment of the present invention, the recombinant immune cell is a T cell.

[0046] The ninth aspect of the present invention provides any of the following applications, the applications including:

[0047] 1) The use of the humanized single-domain antibody described in the first aspect or the tandem antibody described in the second aspect in the construction of receptor constructs or the preparation of immune effector cells.

[0048] In some embodiments, the receptor construct is selected from chimeric antigen receptor (CAR), TCR fusion construct (TRuC-T), or signal transduction and activation receptor (STAR-T).

[0049] 2) The application of the nucleic acid molecule described in the sixth aspect or the recombinant expression vector described in the seventh aspect in the preparation of recombinant host cells, wherein the recombinant host cells are the recombinant host cells described in the eighth aspect.

[0050] 3) The use of the humanized single-domain antibody described in the first aspect, the tandem antibody described in the second aspect, the chimeric antigen receptor described in the third aspect, the immune effector cells described in the fourth aspect, the pharmaceutical composition described in the fifth aspect, the nucleic acid molecule described in the sixth aspect, the recombinant expression vector described in the seventh aspect, or the recombinant host cell described in the eighth aspect in the preparation of a drug for treating GPRC5D positive tumors.

[0051] In some embodiments, the GPRC5D positive tumor is multiple myeloma.

[0052] The tenth aspect of the present invention provides any one of the following methods, the method comprising:

[0053] 1) A method for producing any one of the humanized single-domain antibody of the first aspect, the tandem antibody of the second aspect, or the chimeric antigen receptor of the third aspect, the method comprising: culturing the recombinant host cell of the eighth aspect.

[0054] 2) A method for preparing a recombinant host cell according to the eighth aspect, the method comprising: introducing the recombinant expression vector according to the seventh aspect into a host cell.

[0055] 3) A method for treating a subject with a GPRC5D-positive tumor, the method comprising: administering to the subject the chimeric antigen receptor described in the third aspect, the immune effector cells described in the fourth aspect, or the pharmaceutical composition described in the fifth aspect.

[0056] In some embodiments, the administration method includes an ex vivo-prepared immune cell reinfusion or an in vivo delivery expression system.

[0057] In some embodiments, the subject includes a mammal; in a particular embodiment of the invention, the subject is preferably a human.

[0058] Advantages and benefits of the present invention: The present invention provides a family of humanized single-domain antibodies targeting GPRC5D and their pairwise tandem configurations, which are used to construct CAR molecules and CAR-T cells. Under standardized conditions, they exhibit significantly enhanced tumor cell killing compared to the corresponding monomer configurations to achieve the maximum CAR-T effect, providing a new treatment option for GPRC5D-positive multiple myeloma patients. Attached Figure Description

[0059] Figure 1 This is a schematic diagram of the structure of a humanized antibody CAR.

[0060] Figure 2 The image shows the results of detecting the killing power of CAR-T cells constructed with humanized antibodies.

[0061] Figure 3 This is a schematic diagram of the humanized antibody tandem configuration CAR structure.

[0062] Figure 4 The image shows the results of detecting the killing power of CAR-T cells constructed using a humanized antibody tandem configuration. Detailed Implementation

[0063] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. The embodiments given are merely illustrative of the invention and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation of the invention in any way.

[0064] In the context of this invention, the term "antibody" is used in the broadest sense and explicitly covers single-domain antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, chimeric antibodies, and multispecific antibodies (e.g., bivalent antibodies) formed from at least two intact antibodies, provided they exhibit the desired biological activity.

[0065] In this invention, the modified antibody sequence also falls within the scope of protection of this invention. The term "modification" refers to any form of modification to an 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 a different amino acid. The term "deletion" refers to reducing one or more amino acids in the original amino acid sequence. The terms "insertion" or "addition" refer to changes in the amino acid sequence resulting in the addition of one or more amino acids compared to the original amino acid sequence. In this invention, modification preferably occurs in regions other than variable regions, such as the constant region or frame region of the antibody, and the modified antibody retains the desired functional characteristics of the antibody or its antigen-binding fragment of this invention, or has improved antigen-binding properties. The term "identity," also known as "homology," refers to an amino acid sequence that is at least 80% identical to the sequence provided in this invention. To determine sequence identity, sequence alignment can be performed using various methods known to those skilled in the art, such as using BLAST, BLAST-2, ALIGN, NEEDLE, Megalign (DNASTAR), Snapgene, or DNAMAN software. Those skilled in the art can determine the appropriate parameters for the alignment, including any algorithm required to achieve optimal alignment across the full-length sequences being compared.

[0066] In this invention, the CDR area is defined according to the IMGT numbering system.

[0067] Unless otherwise stated, the antibodies described herein are isolated antibodies. The term "isolated" as used herein means nucleic acids or antibodies or fragments thereof that have been extracted from their natural environment. "Isolated" nucleic acids or antibodies or fragments thereof therefore include nucleic acids or antibodies or fragments thereof purified by standard purification methods. The term also includes nucleic acids or antibodies or fragments thereof prepared through recombinant expression in host cells, as well as chemically synthesized nucleic acids and / or antibodies.

[0068] In some embodiments, those skilled in the art may change the combination of signal peptide, extracellular hinge region and transmembrane domain, co-stimulatory domain and intracellular immune receptor 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 single-domain antibody or humanized CLDN6 single-domain antibody described in this invention, it falls within the protection scope of this invention.

[0069] In this invention, the term "pharmaceutical composition" refers to a composition containing at least one bioactive compound. The pharmaceutical compositions of this invention can be administered orally, non-gastrointestinally, via inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted drug delivery device. The pharmaceutical compositions of this invention may contain any commonly used non-toxic, pharmaceutically acceptable carrier, excipient, or excipient. In some cases, pharmaceutical 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 "non-gastrointestinal" as used in this invention includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions of this invention can be administered to the receptor via any route, provided the target tissue can be reached.

[0070] The pharmaceutical compositions of the present invention can also be used in combination with other drugs for treating solid tumors. These other compounds for treating solid tumors can be administered simultaneously with the main active ingredient (e.g., the antibody described in the first aspect of the invention), or even simultaneously in the same composition. Other therapeutic compounds can also be administered alone as a single composition or in a dosage form different from that of the main active ingredient.

[0071] In this invention, the term "pharmaceutically acceptable carrier" refers to any pharmaceutical carrier that does not 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 skilled in the art. Pharmaceutically acceptable carriers in pharmaceutical compositions may comprise fluids such as water, saline, glycerol, and ethanol. Such carriers may also contain auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc.

[0072] In this invention, the term "nucleic acid" or "nucleic acid molecule" is intended to include polymeric forms of nucleotides of any length containing deoxyribonucleotides, ribonucleotides, and / or their analogues, including DNA, RNA, and DNA / RNA hybrids, and also including DNA or RNA analogues, such as those containing a modified backbone (e.g., peptide nucleic acid (PNA) or phosphate thioester) or modified bases. Therefore, the nucleic acids of this invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc. Once the coding sequence of the antibody described in this invention is isolated, the antibody can be obtained in large quantities using recombinant technology. An exemplary method is to clone its coding gene into a vector, transform it into cells, and then isolate it from the proliferated host cells using conventional methods.

[0073] In this invention, the terms "recombinant expression vector," "vector," and "expression vector" are used interchangeably to refer to an artificial construct capable of delivering and preferentially expressing one or more target genes or sequences in a host cell. The vector can be a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenovirus vectors, AAV viral vectors, herpesvirus vectors, and other viral vectors. Other viral vectors may include bacteriophage vectors, baculovirus vectors, animal viral vectors, plant viral vectors, and may include papillomavirus vectors, herpesvirus vectors, poxvirus vectors, RNA virus vectors, bovine papillomavirus vectors, EB virus vectors, retroviral vectors, etc.

[0074] The recombinant host cell of this invention refers to any cell type suitable for transformation, transfection, transduction, etc., using a nucleic acid construct or expression vector containing the nucleic acid molecules of this invention. The host cell includes any progeny of the parent cell that differs from the parent cell due to mutations occurring during replication. Preferably, the recombinant host cell includes prokaryotic cells and eukaryotic cells; more preferably, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsiae; even more preferably, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells; most preferably, the recombinant host cell is an immune cell; most preferably, the immune cell includes T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages, or any combination thereof; most preferably, the immune cell is a T cell.

[0075] In some embodiments, the antibodies of the present invention are obtained through artificial synthesis. Methods for artificially synthesizing antibodies are known in the art, for example, the antibodies of the present invention are obtained through direct amino acid synthesis. In some embodiments, the antibodies of the present invention are obtained through genetic engineering expression. Genetic engineering expression systems include prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Prokaryotic cell expression systems include *Escherichia coli* expression systems. Eukaryotic cell expression systems include enzyme expression systems, insect cell expression systems, and mammalian cell expression systems.

[0076] In this invention, the term "T cell" refers to a major type of leukocyte that matures in the thymus and plays various roles in the immune system, including identifying specific foreign antigens in the body and activating or inactivating other immune cells. T cells can be any type of T cell, such as cultured T cells, for example, primary T cells, or T cells derived from cultured T cell lines (e.g., Jurkat, SupT1, etc.) or derived from mammals. T cells can be CD3+ cells. T cells can be any type of T cell and can be at any developmental stage, including but not limited to: CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), memory T cells, primary T cells, regulatory T cells, γδ T cells, etc. Other types of helper T cells include cells such as Th3, Th17, Th9, or Tfh cells. Other types of memory T cells include cells such as central memory T cells (Tcm cells) and effector memory T cells (Tern cells and TEMRA cells). T cells can also refer to genetically engineered T cells, such as proprietary T cell fusion constructs (TRuCs) modified to express T cell receptor (TCR), chimeric antigen receptor (CAR), synthetic signal transduction and activation receptor (STAR), or TCR2.

[0077] Example

[0078] 1. Humanization design and sequence acquisition of VHH antibodies

[0079] This invention uses non-human VHH antibodies as templates and employs the Abysis online analysis tool (http: / / www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi) to perform domain segmentation and key site annotation of their amino acid sequences, identifying the variable regions FR1-FR4 and CDR1-CDR3, and selecting the human antibody template with the highest homology to its framework region as the humanized backbone. Based on this, mutation design is performed according to humanization principles: while maintaining antigen-binding ability, non-human amino acid residues are replaced with human template residues as much as possible; conserved structural sites in the FR region are preferentially replaced with assimilated amino acids to avoid secondary structure perturbation; in the FR2-FR3 region, if the original amino acid residue is hydrophilic, it is not mutated to a hydrophobic residue to maintain solubility. The sequence humanization results of the three VHH antibodies B05 / C07 / F08 are shown in Table 1, where the T20 score is in parentheses; HS2 adopts a "conservative strategy" for the FR2 segment, that is, no mutation is performed on this segment to maintain the stability of the antibody backbone.

[0080] Table 1. Humanized antibody sequences

[0081]

[0082]

[0083] 2. Construction of humanized antibody CAR-T and detection of its killing power

[0084] Based on the above sequences, lentiviral system plasmids (pCDH-EF1α lentiviral expression plasmids) were constructed respectively. The CAR structures are as follows: Figure 1As shown. The obtained plasmids were co-transfected into logarithmically growing adherent 293T cells using a four-plasmid lentiviral system (pCDH-EF1α expression vector, PsPAX2, pMD2.G, and helper plasmid pRev, mixed in a mass ratio of 4:3:2:1). Cell supernatants were collected after 48–72 hours, concentrated, filtered, and lentivirus was obtained and stored at -80°C for later use. Peripheral blood was collected from patients or healthy volunteers via leukoablation, and PBMCs were obtained by ficoll density gradient centrifugation. T cells were then isolated using the EasySep™ Human T Cell Isolation Kit (STEMCELL, #17951). X-vivo (lonza) medium containing 10 ng / mL IL-7 (nearshore protein, GMP-C086), 5 ng / mL IL-15 (nearshore protein, GMP-C016), and ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Activator (STEMCELL, #10970) antibody was used to activate T cells. After activation for 2-3 days, lentiviral transduction was performed. The medium without activation antibody was then replaced, and the cells were amplified for 9-11 days. The CAR-T cell positivity rate was measured, and the positivity rate of each group was adjusted to a consistent level by mixing in T cells from the same batch. The killing effect was verified by co-incubating K562 target cells overexpressing GPRC5D with the antibody at an effector-to-target ratio of 1:1 and 1:10 for 16 hours. The results showed that the humanized CAR-T cells all exhibited good killing ability. However, when co-incubated with K562 target cells not expressing GPRC5D at an effector-to-target ratio of 1:1, no non-specific killing was observed, indicating that the humanized sequence still retains the antibody's specificity. Figure 2 ).

[0085] 3. Construction of CAR-T antibodies with tandem configuration and detection of cytotoxicity

[0086] For the HS2 humanized clone sequence using a "conservative strategy" for the FR2 segment, new sequences were obtained by tandemly connecting pairs of sequences (Table 2), and lentiviral system plasmids (pCDH-EF1α lentiviral expression plasmids) were constructed accordingly. The CAR structures are shown below. Figure 3As shown. The obtained plasmids were co-transfected into logarithmically growing adherent 293T cells using a four-plasmid lentiviral system (pCDH-EF1α expression vector, PsPAX2, pMD2.G, and helper plasmid pRev, mixed in a mass ratio of 4:3:2:1). Cell supernatants were collected after 48-72 hours, concentrated, filtered, and lentivirus was obtained and stored at -80°C for later use. Peripheral blood was collected from patients or healthy volunteers via leukoablation, and PBMCs were obtained by ficoll density gradient centrifugation. T cells were then isolated using the EasySep™ Human TCell Isolation Kit (STEMCELL, #17951). X-vivo (lonza) medium containing 10 ng / mL IL-7 (nearshore protein, GMP-C086), 5 ng / mL IL-15 (nearshore protein, GMP-C016), and ImmunoCult™ Human CD3 / CD28 / CD2 TCell Activator (STEMCELL, #10970) antibody was used to activate T cells. After 2-3 days of normal activation, lentiviral transduction was performed. The medium without activation antibody was then replaced, and the cells were amplified to 9-11 days. The CAR-T cell positivity rate was measured, and the positivity rate of each group was adjusted to a consistent level by mixing in T cells from the same batch. Using 109 clone (reference patent: US10633426) and F08HS2 clone alone as positive controls, CAR-T cells overexpressing GPRC5D were co-incubated with K562 target cells at effector-to-target ratios of 1:1 and 1:5 for 16 hours to verify cytotoxicity. The results showed that CAR-T cells constructed with tandem antibody configurations had higher targeted cytotoxicity than those constructed with monomeric clones. Culturing K562 target cells without GPRC5D expression at an effector-to-target ratio of 1:1 for cytotoxicity verification showed no non-specific cytotoxicity. Figure 4 ).

[0087] Table 2. Tandem configuration antibodies and CAR sequences

[0088]

[0089]

[0090]

[0091] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A humanized single-domain antibody targeting GPRC5D, characterized in that, the humanized single-domain antibody is obtained by humanizing the residues in the framework region key positions of the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 28 or SEQ ID NO: 31 with reference to a human germline framework; Preferably, the humanized single-domain antibody comprises any one of B05HS1, B05HS2, B05HS3, F08HS1, F08HS2, F08HS3, C07HS1, C07HS2, C07HS3; Preferably, the B05HS1, B05HS2, B05HS3, F08HS1, F08HS2, F08HS3, C07HS1, C07HS2, C07HS3 respectively has an amino acid sequence as shown in SEQ ID NO: 26, SEQ ID NO: 1, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 5, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 9 or SEQ ID NO: 33; Preferably, the humanized single-domain antibody has an amino acid sequence identical to SEQ ID NO: 26, SEQ ID NO: 1, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 5, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 9 or SEQ ID NO: 33 by at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.

2. A tandem configuration antibody targeting GPRC5D, characterized in that, The tandem configuration antibody is sequentially formed by the humanized single-domain antibodies of claim 1; Preferably, the tandem configuration antibody further comprises a linker protein; Preferably, the connexin is a flexible polypeptide having the structure (G4S) n (n = 2-6), having a total length of 10 - 30 amino acids; Preferably, the linker protein is (G4S)3, and the amino acid sequence thereof is shown in SEQ ID NO: 16; Preferably, the tandem configuration antibody is sequentially formed by a humanized single-domain antibody, a linker protein and a humanized single-domain antibody; Preferably, the humanized single-domain antibody is selected from any two of B05HS2, F08HS2, C07HS2; More preferably, the humanized single-domain antibody is sequentially formed by B05HS2, a linker protein and F08HS2; More preferably, the humanized single-domain antibody is sequentially formed by C07HS2, a linker protein and B05HS2; More preferably, the humanized single-domain antibody is sequentially formed by F08HS2, a linker protein and C07HS2. Most preferably, the tandem configuration antibody has an amino acid sequence as set forth in SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:

15.

3. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises the humanized single domain antibody of claim 1, or the chimeric antigen receptor comprises the tandem configuration antibody of claim 2; Preferably, the chimeric antigen receptor further comprises a SP membrane exit signal, a hinge region, a transmembrane region, a costimulatory signaling domain, an intracellular signal transduction region; Preferably, the chimeric antigen receptor further comprises a CD8 SP, and the amino acid sequence of the CD8 SP is as set forth in SEQ ID NO: 17; More preferably, the hinge region is selected from the hinge region of CD8, 4 1BB, IgG1, IgG4, PD 1, CD28, CD34, OX40, CD3 epsilon, NKG2D, NKG2C, Fc epsilon RI gamma, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT, and variants thereof; More preferably, the transmembrane region is selected from the transmembrane region of CD8, 4 1BB, IgG1, IgG4, PD 1, CD28, CD34, OX40, CD3 epsilon, NKG2D, NKG2C, Fc epsilon RI gamma, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT, and variants thereof; More preferably, the costimulatory signaling domain is selected from the costimulatory signaling domain of 4 1BB, HVEM, CD27, CD19, CD28, ICOS, CD4, CD8 alpha, CD8 beta, CD40, OX40, DR3, CD2, GITR, CD30, TIM1, CD226, CD278, and variants thereof; More preferably, the intracellular signal transduction region is selected from the intracellular signal transduction region of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD278, CD21, CD22, Fc epsilon RI, FcR gamma, FcR beta, CD4, CD5, CD8, CD79a, CD79b, DAP10, DAP12, CD66d, ZAP70, and variants thereof; Most preferably, the hinge region is a CD8 hinge region, and the amino acid sequence of the CD8 hinge region is as set forth in SEQ ID NO: 18; Most preferably, the transmembrane region is a CD8 transmembrane region, and the amino acid sequence of the CD8 transmembrane region is as set forth in SEQ ID NO: 19; Most preferably, the co-stimulatory signaling domain is a 4 1BB co-stimulatory signaling domain, and the amino acid sequence of the 4 1BB co-stimulatory signaling domain is as set forth in SEQ ID NO: 20; Most preferably, the intracellular signaling region is a CD3ζ intracellular signaling region, and the amino acid sequence of the CD3ζ intracellular signaling region is as set forth in SEQ ID NO: 21; Most preferably, the chimeric antigen receptor is obtained in sequence from a SP membrane-out signal, the humanized single-domain antibody of claim 1, the tandem configuration antibody of claim 2, a CD8 SP, a CD8 hinge region, a CD8 transmembrane region, a 4 1BB co-stimulatory signaling domain, and a CD3ζ intracellular signaling region.

4. An immune effector cell, characterized in that, The immune effector cell expresses the chimeric antigen receptor of claim 3; Preferably, the immune effector cell is a T cell, an NK cell, an iNKT cell, a B cell, a CTL cell, a monocyte, a myeloid cell, a dendritic cell, or a macrophage cell; More preferably, the immune effector cell is a T cell.

5. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the chimeric antigen receptor of claim 3 or the immune effector cell of claim 4; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, or buffer; More preferably, the pharmaceutical composition is a cell preparation for injection.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a nucleotide sequence encoding the humanized single-domain antibody of claim 1, the tandem configuration antibody of claim 2, or the chimeric antigen receptor of claim 3; Preferably, the nucleotide sequence is as set forth in SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO:

24.

7. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule of claim 6.

8. A recombinant host cell, characterized in that, The recombinant host cell expresses the humanized single-domain antibody of claim 1, the tandem configuration antibody of claim 2, or the chimeric antigen receptor of claim 3; Preferably, the recombinant host cell comprises the recombinant expression vector of claim 7; Preferably, the recombinant host cell comprises a recombinant immune cell; More preferably, the recombinant immune cell is a T cell, an NK cell, an iNKT cell, a B cell, a CTL cell, a monocyte, a myeloid cell, a dendritic cell, or a macrophage cell; Most preferably, the recombinant immune cell is a T cell.

9. An application characterized by any one of The use comprises: 1) use of the humanized single-domain antibody of claim 1 or the tandem configuration antibody of claim 2 in constructing a receptor construct or preparing an immune effector cell; 2) Use of the nucleic acid molecule of claim 6 or the recombinant expression vector of claim 7 for the manufacture of a recombinant host cell, which is the recombinant host cell of claim 8; 3) Use of the humanized single domain antibody of claim 1, the tandem configuration antibody of claim 2, the chimeric antigen receptor of claim 3, the immune effector cell of claim 4, the pharmaceutical composition of claim 5, the nucleic acid molecule of claim 6, the recombinant expression vector of claim 7 or the recombinant host cell of claim 8 for the manufacture of a medicament for treating a GPRC5D-positive tumor; Preferably, the receptor construct is selected from a chimeric antigen receptor, a TCR fusion construct or a signal transducing and activating receptor; Preferably, the GPRC5D-positive tumor is multiple myeloma.

10. Any of the following methods, characterized in that, The method comprises: 1) A method for producing any one of the humanized single domain antibody of claim 1, the tandem configuration antibody of claim 2 or the chimeric antigen receptor of claim 3, the method comprising: culturing the recombinant host cell of claim 8; 2) A method for preparing the recombinant host cell of claim 8, the method comprising: introducing the recombinant expression vector of claim 7 into a host cell.

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