CN117700558B

CN117700558B - Monoclonal anti-GPRC 5D antibody and anti-GPRC 5D-CAR-NK cell

Info

Publication number: CN117700558B
Application number: CN202410166101.XA
Authority: CN
Inventors: 刘晓东; 贺智勇; 张晓芳; 赵彩芝; 孙莎
Original assignee: Suzhou Aikailiyuan Biotechnology Co ltd
Current assignee: Suzhou Aikailiyuan Biotechnology Co ltd
Priority date: 2024-02-06
Filing date: 2024-02-06
Publication date: 2024-05-03
Anticipated expiration: 2044-02-06
Also published as: CN117700558A

Abstract

The invention relates to a monoclonal anti-GPRC 5D antibody and an anti-GPRC 5D-CAR-NK cell, wherein the amino acid sequence of a heavy chain variable region of the antibody is shown as any one of SEQ ID NO.1-3, and the amino acid sequence of a light chain variable region of the antibody is shown as any one of SEQ ID NO. 8-10. The GPRC5D sequence with higher affinity is obtained through screening, and the CAR-NK structure constructed by combining the GPRC5D sequence with NK can show higher in-vitro killing effect.

Description

Monoclonal anti-GPRC 5D antibody and anti-GPRC 5D-CAR-NK cell

Technical Field

The invention relates to the field of cellular immunotherapy, in particular to a monoclonal anti-GPRC 5D antibody and an anti-GPRC 5D-CAR-NK cell.

Background

Multiple Myeloma (MM) is the second most common hematological malignancy next to non-hodgkin's lymphoma. Traditional methods of treating MM include the use of immunomodulators, proteasome inhibitors, and anti-cd 38 antibodies. However, these treatments have limited efficacy in MM patients and generally suffer from poor overall prognosis. In recent years, despite great progress in chemotherapy, proteasome inhibitors and the immunomodulator thalidomide derivatives, the vast majority of patients eventually relapse. Thus, there is an urgent need for new therapeutic regimens.

Compared with BCMA, GPRC5D has better specificity, the expression of the GPRC5D is not reduced along with the time, and the expression of the GPRC5D are independent, so that the single-targeting and double-targeting development of therapeutic drugs can be realized, the overexpression of the GPRC5D is related to the poor prognosis and tumor burden of patients with multiple myeloma, and the association makes the GPRC5D a potential candidate target for treating the multiple myeloma. Enterprises at home and abroad use the leading technology to develop GPRC5D antibodies and cell therapy medicines.

Tacrolimus is a GPRC5D x CD3 bispecific antibody developed by robusta, leading far among many GPRC5D targeted drugs. GPRC5D is a target expressed on multiple myeloma cells, whose expression does not decrease over time, whereas CD3 is a T cell receptor, associated with activated T cells.

OriC-321 and OriCAR-017 are two GPRC5D CAR-T products of the original organism for the indication multiple myeloma. The original organism utilizes a proprietary CAR-T technology platform to construct a unique signal activation domain element Ori. After the original is inserted into a new generation of CAR structure, the amplification efficiency of the memory immune cells can be improved by times, the physical barrier of extracellular matrixes in TME is effectively broken through, the anti-tumor activity and durability of CAR-T in vivo are obviously enhanced, and the anti-tumor effect of CAR-T in vivo have better relapse prevention potential.

MCARH109 is another GPRC5D CAR-T product developed by the commemorative Style Josepioline center (MSKCC) in combination with Yoareke (Eureka Therapeutics). In vitro studies MCARH109,109 showed good efficacy, while it also showed better activity in BCMA drug resistance model. The MM patients (including those who relapse after BCMA CAR T cell therapy) who failed the group multi-line therapy of the phase i dose expansion study were given MCARH treatments at 4 dose levels.

RG6234 (GPRC 5 DxCD) is a GPRC5D T cell engagement bispecific antibody developed by Roche, comprising two protein domains that bind to a target and one protein domain that binds to CD 3. Rogowski indicates that this bispecific antibody is "the most potent". In early clinical trials in patients with multiple myeloma, ORR exceeded 71% and CR exceeded 35%.

LM-305 is a novel targeted GPRC5D antibody conjugated drug (ADC) consisting of an anti-GPRC 5D monoclonal antibody, a degradable protease linker and a cytotoxin-loaded monomethyl auristatin E (MMAE). The novel medicine is a second product independently developed based on an exclusive ADC platform. LM-305 is the GPRC5D-ADC of the first clause worldwide into clinical phase.

At present, for the GPRC5D medicines, neither CAR-T nor ADC, or bispecific antibodies target MM tumors with the specificity of GPRC5D so as to bring killing to achieve clinical treatment effects. The low affinity makes the treatment effect poor and increases the risk of tumor escape, so that the screening of the GPRC5D sequence with high affinity is an effective method for improving the treatment effect.

Disclosure of Invention

Problems to be solved by the invention

The invention aims to solve the current situation that the therapeutic effect is poor and the recurrence rate is high due to low affinity of target protein of multiple myeloma.

Solution for solving the problem

The present invention provides a monoclonal anti-GPRC 5D antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises one or more of the following sequences:

(1) An amino acid sequence as set forth in any one of SEQ ID NO. 1-3;

(2) An amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in any one of SEQ ID nos. 1 to 3, and which retains the activity of the amino acid sequence set forth in any one of SEQ ID nos. 1 to 3;

(3) An amino acid sequence in which 1 or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence shown in any one of SEQ ID NO.1 to 3, and which retains the activity of the amino acid sequence shown in any one of SEQ ID NO.1 to 3;

The amino acid sequence of the light chain variable region comprises one or more of the following sequences:

(1) An amino acid sequence as set forth in any one of SEQ ID NO. 8-10;

(2) An amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in any one of SEQ ID nos. 8 to 10, and which retains the activity of the amino acid sequence set forth in any one of SEQ ID nos. 8 to 10;

(3) An amino acid sequence in which 1 or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence shown in any one of SEQ ID NO.8-10, and which retains the activity of the amino acid sequence shown in any one of SEQ ID NO. 8-10;

wherein, SEQ ID NO.1 is paired with SEQ ID NO.8, SEQ ID NO.2 is paired with SEQ ID NO.9, and SEQ ID NO.3 is paired with SEQ ID NO. 10.

Preferably, the antibody is a single chain antibody and/or a double chain antibody that binds to a cell surface GPRC5D protein.

The invention also provides a single chain variable region fragment scFv, said scFv comprising a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises one or more of the following sequences:

(1) An amino acid sequence as set forth in any one of SEQ ID NO. 1-3;

(1) An amino acid sequence as set forth in any one of SEQ ID NO. 8-10;

Preferably, the scFv further comprises a connecting peptide; wherein the sequence of the connecting peptide is shown as SEQ ID NO.15, and the scFv comprises one or more of the following sequences:

(1) An amino acid sequence as set forth in any one of SEQ ID NO. 16-18;

(2) An amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in any one of SEQ ID nos 16 to 18, and which retains the activity of the amino acid sequence set forth in any one of SEQ ID nos 16 to 18;

(3) An amino acid sequence of 1 or more amino acid residues added, substituted, deleted or inserted in the amino acid sequence shown in any one of SEQ ID NO. 16-18, and which retains the activity of the amino acid sequence shown in any one of SEQ ID NO. 16-18.

The invention also provides a chimeric antigen receptor CAR comprising:

(1) The above-mentioned scFv is used for the preparation of a medicine,

(2) A transmembrane domain comprising a transmembrane domain,

(3) A costimulatory domain, and

(4) The domain is activated.

Preferably, the CAR has the structure of formula I:

L-scFv-H-TM-C-CD3ζ（I），

Wherein,

Each "-" is independently a connecting peptide or peptide bond;

l is an optional signal peptide sequence;

The scFv is the scFv;

H is an optional hinge region;

TM is a transmembrane domain;

C is a costimulatory signaling molecule;

cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ.

The present invention also provides a drug conjugate comprising:

(1) The antibody, or the scFv; and

(2) A coupling moiety;

wherein the coupling moiety is selected from one or more of a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof.

The invention also provides a polynucleotide encoding the antibody, or the scFv, or the CAR.

The invention also provides a vector comprising the polynucleotide; wherein the vector is selected from one or more of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, or a combination thereof.

The invention also provides a genetically engineered cell comprising the vector, the polynucleotide integrated in the chromosome, or expressing the antibody, or expressing the scFv, or expressing the CAR.

Preferably, the cells are NK cells.

The invention also provides a pharmaceutical composition comprising one or more of the antibody, or the scFv, or the CAR, or the drug conjugate, or the cell; wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent and/or excipient.

The invention also provides an application of the antibody, the scFv, the CAR, the drug conjugate, the cell or the pharmaceutical composition in preparing medicines for preventing and/or treating cancers or tumor related diseases.

Preferably, the tumor-associated disease is multiple myeloma.

ADVANTAGEOUS EFFECTS OF INVENTION

The GPRC5D sequence with higher affinity is obtained through screening, and the in vitro killing effect on the CAR-NK can be higher.

Drawings

Figure 1 shows the binding capacity of GPRC5D mab to mm.1 s.

FIG. 2 shows the binding capacity of GPRC5D mab to CHOK1-GPRC 5D.

FIG. 3 shows the structure of CAR-NK.

Figure 4 shows the electrotransformation and positive rate of GPRC5D CAR.

Figure 5 shows killing of mm.1s by GPRC 5D-CAR-NK.

Figure 6 shows killing of NCI-H929 by GPRC 5D-CAR-NK.

Detailed Description

In order to make the technical scheme and the beneficial effects of the application more obvious and understandable, the following detailed description is given by way of example. Wherein the drawings are not necessarily to scale, and wherein local features may be exaggerated or reduced to more clearly show details of the local features; unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts.

As used herein, the term "about" may refer to a value or composition that is within an acceptable error of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or measured.

As used herein, the term "antibody" refers to an immunoglobulin that is a tetrapeptide chain structure formed from two identical heavy chains and two identical light chains joined by an interchain disulfide bond. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be assigned to five classes, or different types of immunoglobulins, i.e., igM, igD, igG, igA and IgE, and the heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. IgG represents the most important class of immunoglobulins, which can be divided into 4 subclasses again due to differences in chemical structure and biological function: igG1, igG2, igG3 and IgG4. Light chains are classified as either kappa or lambda chains by the difference in constant regions. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

The sequences of the heavy and light chains of the antibody near the N-terminus vary widely, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable region includes 3 hypervariable regions (HVRs) and 4 FR Regions (FR) that are relatively conserved in sequence. The amino acid sequences of the 4 FRs are relatively conserved and do not directly participate in the binding reaction. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) consists of 3 CDR regions and 4 FR regions, arranged in sequence from amino-to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain, namely the light chain hypervariable region (LCDR), refer to LCDR1, LCDR2 and LCDR3; the 3 CDR regions of the heavy chain, namely heavy chain hypervariable regions (HCDR), refer to HCDR1, HCDR2 and HCDR3. The CDR amino acid residues of the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the invention are in numbers and positions that meet the known Kabat numbering convention (LCDR 1-3, HCDR 2-3), or that meet the numbering convention of Kabat and chothia (HDR 1). The four FR regions in the natural heavy and light chain variable regions are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form a partially folded structure. The CDRs in each chain are held closely together by the FR regions and form together with the CDRs of the other chain an antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody. The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.

The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.

In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using DNA recombination techniques well known in the art.

As used herein, the term "monoclonal antibody" refers to an antibody secreted from a clone derived from a single cell source. Monoclonal antibodies are highly specific, being directed against a single epitope. The cells may be eukaryotic, prokaryotic or phage clonal cell lines.

As used herein, the term "domain" refers to a region in a polypeptide that folds into a particular structure independently of the other regions.

As used herein, the term "single chain variable region fragment" or "scFv" refers to a single chain polypeptide derived from an antibody that retains the ability to bind an antigen. Examples of ScFv include antibody polypeptides formed by recombinant DNA techniques, and wherein Fv regions of immunoglobulin heavy (H chain) and light (L chain) chain fragments are linked via a spacer sequence. Various methods of engineering scfvs are known to those skilled in the art.

As used herein, the term "tumor antigen" refers to a biomolecule that is antigenic, the expression of which results in cancer.

As used herein, the term "chimeric antigen receptor" or "CAR" refers to a fusion protein comprising an extracellular domain capable of binding an antigen, a transmembrane domain derived from a polypeptide different from the polypeptide from which the extracellular domain is derived, and at least one intracellular domain. "chimeric antigen receptor" is sometimes also referred to as "chimeric receptor", "T-body" or "Chimeric Immune Receptor (CIR)". An "extracellular domain capable of binding an antigen" refers to any oligopeptide or polypeptide that can bind to a particular antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known to function in a cell as a domain that transmits a signal to cause activation or inhibition of a biological process.

As used herein, the term "hinge region" generally refers to the region between the CH1 and CH2 functional regions of an immunoglobulin heavy chain. The hinge region is a region located between the extracellular antigen binding domain (e.g., scFv) and the NK cell membrane. The hinge region is typically derived from the IgG family, e.g., can be derived from IgG1 and IgG4, and can also be derived from IgD and CD8.

As used herein, the term "transmembrane region" generally refers to the transmembrane segment that connects the extracellular antigen binding domain and the intracellular signaling domain, typically from a dimeric membrane protein, including predominantly cd3ζ, CD4, CD8, CD28, and the like, capable of anchoring the CAR structure to the NK cell membrane. Different designs of the transmembrane region can affect the expression of the introduced CAR gene.

As used herein, the term "signaling domain" generally refers to functional signaling domains from cd3ζ, cd3γ, cd3δ, cd3ε, fcrγ (FCER 1G), fcrβ (Fc Epsilon R1 b), CD79a, CD79b, fcγriia, DAP10, and DAP12 proteins.

As used herein, the term "co-stimulatory domain" generally refers to a functional signaling domain of a protein from one or more of the following: CD27, CD28, 41BB (CD 137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, NKG2C, B-H3, ligand 、CDS、ICAM-1、GITR、BAFFR、HVEM（LIGHTR）、SLAMF7、NKp80（KLRF1）、CD160、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、TRANCE/RANKL、DNAM1（CD226）、SLAMF4（CD244、2B4）、CD84、CD96、CEACAM1、CRTAM、Ly9（CD229）、CD160（BY55）、PSGL1、CD100（SEMA4D）、CD69、SLAMF6（NTB-A、Ly108）、SLAM（SLAMF1、CD150、IPO-3）、BLAME（SLAMF8）、SELPLG（CD162）、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46 that specifically binds CD83, and NKG2D.

As used herein, the term "CAR-NK cell" refers to a CAR-expressing NK cell, typically obtained by transducing a NK cell with an expression vector encoding the CAR. Commonly used expression vectors are viral vectors, such as lentiviral expression vectors. NK cells modified by chimeric antigen receptor (CAR-NK) are not limited by major histocompatibility complex, and have specific targeting killing activity and lasting amplification capability. In addition to NK cells, other lymphocytes, such as T cells, can also be transformed with an expression vector encoding the CAR to obtain a targeted killer cell expressing the CAR.

As used herein, the terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably to refer to a polymer of nucleotides. Such nucleotide polymers may contain natural and/or unnatural nucleotides and include, but are not limited to, DNA, RNA, and PNA. "nucleic acid sequence" or "nucleotide sequence" refers to a linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

As used herein, the terms "sequence identity", "sequence identity" and "sequence identity" are used interchangeably to refer to the amount of degree of identity between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), typically expressed as a percentage. Typically, sequence alignment (alignment) is performed and gaps (gaps), if any, introduced prior to calculating the percent identity between two amino acid or nucleotide sequences. If at a certain alignment the amino acid residues or bases in the two sequences are identical, then the two sequences are considered to be identical or matched at that position; amino acid residues or bases in the two sequences differ, and are considered to be inconsistent or mismatched at that position. In some algorithms, the number of matching positions is divided by the total number of positions in the alignment window to obtain sequence identity. In other algorithms, the number of gaps and/or the gap length are also considered. For the purposes of the present invention, the disclosed alignment software BLAST can be used to obtain optimal sequence alignments by using default settings and to calculate sequence identity between two amino acid or nucleotide sequences.

As used herein, the term "vector" refers to a nucleic acid molecule (e.g., a nucleic acid, plasmid, virus, etc.) that can be engineered to contain a polynucleotide of interest (e.g., a coding sequence for a polypeptide of interest) or that can replicate in a host cell. The carrier may include one or more of the following components: an origin of replication, one or more regulatory sequences (such as promoters and/or enhancers) that regulate the expression of the polynucleotide of interest, and/or one or more selectable marker genes (such as an antibiotic resistance gene and a gene useful in colorimetric assays, e.g., β -galactose). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

As used herein, the term "host cell" refers to a mammalian immune effector cell, particularly a human cell, such as a T cell or NK cell, that can express a CAR provided herein. Host cells include the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells also include cells transfected in vivo with the nucleic acid molecules or expression vectors provided herein.

As used herein, the term "pharmaceutically acceptable carrier" refers to a solid or liquid diluent, filler, antioxidant, stabilizer, etc., which may be safely administered, and which is suitable for administration to humans and/or animals without undue adverse side effects, while maintaining the viability of the drug or active agent located therein. Depending on the route of administration, a variety of different carriers well known in the art may be used, including, but not limited to, sugars, starches, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffers, emulsifying agents, isotonic saline, and/or pyrogen-free water and the like. The pharmaceutical composition provided herein can be prepared into clinically acceptable dosage forms such as powder, injection and the like. The pharmaceutical compositions of the invention may be administered to a subject using any suitable route, for example, by oral, intravenous infusion, intramuscular injection, subcutaneous injection, intraperitoneal, rectal, sublingual, or via inhalation, transdermal, etc.

(1) An amino acid sequence as set forth in any one of SEQ ID NO. 1-3;

(3) An amino acid sequence of 1 or more amino acid residues is added, substituted, deleted or inserted in the amino acid sequence shown in any one of SEQ ID NO.1 to 3, and retains the activity of the amino acid sequence shown in any one of SEQ ID NO.1 to 3.

In certain embodiments, the heavy chain variable region has an amino acid sequence as set forth in SEQ ID No. 1:

EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTPDKRLEWVATISNGGSYTYYPDNVKGRFTISRDNAKNTLYLQMSSLKSEDTATYYCARHAWDYWGQGTTLTVSS.

In certain embodiments, the heavy chain variable region has an amino acid sequence as set forth in SEQ ID No. 2:

EVQLQQSGPELVKPGASVKISCKASGFSFTGYTMNWVKQTHGKNLEWIGLINPYTGGTTYKQKFKGKATLTVDKSSNTAYMELLSLTSEDSAVYYCTRGGFYRYDGDYWGQGTTLTVSS.

In certain embodiments, the heavy chain variable region has an amino acid sequence as set forth in SEQ ID No. 3:

EVQLQQSGPELVKPGASVKISCKASGFSFTGYTMNWVKQSHGKNLEWIGLINPYNGGTTYKQKFKGKATLTVDKSSNTAYMELLSLTSEDSAVYYCTRGGFYRYDGDYWGQGTTLTVSS.

(1) An amino acid sequence as set forth in any one of SEQ ID NO. 8-10;

(3) An amino acid sequence of 1 or more amino acid residues added, substituted, deleted or inserted in the amino acid sequence shown in any one of SEQ ID NO. 8-10, and which retains the activity of the amino acid sequence shown in any one of SEQ ID NO. 8-10.

In certain embodiments, the amino acid sequence of the light chain variable region is as set forth in SEQ ID No. 8:

QIVLTQSPAIMSAFPGEKVTMTCNASSSVSYMYWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISIMEAEDAATYYCQHWSTNPLTFGAGTKLELK.

in certain embodiments, the amino acid sequence of the light chain variable region is as set forth in SEQ ID No. 9:

DIQMTQSPSSLSASLGERVSLTCRASQDIGNYLTWLQQEPDGTIKRLIYATSSLHSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYSSSPWTFGGGTKLEIR.

in certain embodiments, the amino acid sequence of the light chain variable region is as set forth in SEQ ID No. 10:

VIQLTQSPSSLSASLGERVSLTCRASQDIGNYLTWLQKEPNGTIKRLIYATFSLHSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYSSSPWTFGGGTKLEIR.

In certain embodiments, the SEQ ID NO.1 is paired with SEQ ID NO. 8.

In certain embodiments, the SEQ ID NO.2 is paired with SEQ ID NO. 9.

In certain embodiments, the SEQ ID NO.3 is paired with SEQ ID NO. 10.

In certain embodiments, the antibody is a single chain antibody and/or a double chain antibody that binds to a cell surface GPRC5D protein.

In certain embodiments, the antibody is a single chain antibody that binds to a cell surface GPRC5D protein.

In certain embodiments, the antibody is a diabody that binds to a cell surface GPRC5D protein.

In certain embodiments, the antibody is selected from one or more of an animal-derived antibody, a chimeric antibody, and/or a humanized antibody.

In certain embodiments, the antibody is an animal-derived antibody.

(1) An amino acid sequence as set forth in any one of SEQ ID NO. 1-3;

(1) An amino acid sequence as set forth in any one of SEQ ID NO. 8-10;

In certain embodiments, the SEQ ID NO.1 is paired with SEQ ID NO. 8.

In certain embodiments, the SEQ ID NO.2 is paired with SEQ ID NO. 9.

In certain embodiments, the SEQ ID NO.3 is paired with SEQ ID NO. 10.

In certain embodiments, the scFv further comprises a connecting peptide; wherein the sequence of the connecting peptide is shown as SEQ ID NO.15, and the scFv comprises one or more of the following sequences.

In certain embodiments, the sequence of the connecting peptide is as shown in SEQ ID No. 15:

GGGGSGGGGSGGGGS。

(1) An amino acid sequence as set forth in any one of SEQ ID NO. 16-18;

In certain embodiments, the scFv has an amino acid sequence as set forth in SEQ ID No. 16:

EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTPDKRLEWVATISNGGSYTYYPDNVKGRFTISRDNAKNTLYLQMSSLKSEDTATYYCARHAWDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSAFPGEKVTMTCNASSSVSYMYWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISIMEAEDAATYYCQHWSTNPLTFGAGTKLELK.

in certain embodiments, the scFv has an amino acid sequence as set forth in SEQ ID No. 17:

EVQLQQSGPELVKPGASVKISCKASGFSFTGYTMNWVKQTHGKNLEWIGLINPYTGGTTYKQKFKGKATLTVDKSSNTAYMELLSLTSEDSAVYYCTRGGFYRYDGDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASLGERVSLTCRASQDIGNYLTWLQQEPDGTIKRLIYATSSLHSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYSSSPWTFGGGTKLEIR.

In certain embodiments, the scFv has an amino acid sequence set forth in SEQ ID No. 18:

EVQLQQSGPELVKPGASVKISCKASGFSFTGYTMNWVKQSHGKNLEWIGLINPYNGGTTYKQKFKGKATLTVDKSSNTAYMELLSLTSEDSAVYYCTRGGFYRYDGDYWGQGTTLTVSSGGGGSGGGGSGGGGSVIQLTQSPSSLSASLGERVSLTCRASQDIGNYLTWLQKEPNGTIKRLIYATFSLHSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYSSSPWTFGGGTKLEIR.

the invention also provides a chimeric antigen receptor CAR comprising:

(1) The above-mentioned scFv is used for the preparation of a medicine,

(2) A transmembrane domain comprising a transmembrane domain,

(3) A costimulatory domain, and

(4) The domain is activated.

In certain embodiments, the CAR has the structure of formula I:

L-scFv-H-TM-C-CD3ζ（I），

Wherein,

Each "-" is independently a connecting peptide or peptide bond;

l is an optional signal peptide sequence;

The scFv is the scFv;

H is an optional hinge region;

TM is a transmembrane domain;

C is a costimulatory signaling molecule;

cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ.

The present invention also provides a drug conjugate comprising:

(1) The antibody, or the scFv; and

(2) A coupling moiety;

In certain embodiments, the cell is an NK cell.

In certain embodiments, the tumor-associated disease is multiple myeloma.

Example 1: anti-GPRC 5D antibody sequence obtained by DNA and cell immunization of 8BALBC mice

Monoclonal anti-GPRC 5D antibody sequences were obtained by DNA immunization of 8BALBC mice with cells, by affinity screening. The specific implementation steps are as follows: coating an anti-human FC antibody, blocking for 1 hour at 4 ℃, adding a primary antibody to be detected after 2% BSA, incubating for 2 hours at room temperature, adding a GPRC 5D-goat anti-human secondary antibody after DPBS cleaning, incubating for 1 hour at room temperature, adding TMB for color development for 5 minutes after DPBS cleaning, adding 2M hydrochloric acid to terminate the reaction, and reading an OD450 value.

The amino acid sequence of the heavy chain variable region of ICA2002-18D8C7-1-hIgG1 is shown in SEQ ID NO. 1:

EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTPDKRLEWVATISNGGSYTYYPDNVKGRFTISRDNAKNTLYLQMSSLKSEDTATYYCARHAWDYWGQGTTLTVSS

the amino acid sequence of the light chain variable region of ICA2002-18D8C7-1-hIgG1 is shown in SEQ ID NO. 8:

QIVLTQSPAIMSAFPGEKVTMTCNASSSVSYMYWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISIMEAEDAATYYCQHWSTNPLTFGAGTKLELK

the amino acid sequence of the heavy chain variable region of ICA2002-50A5-1-hIgG1 is shown in SEQ ID NO. 2:

EVQLQQSGPELVKPGASVKISCKASGFSFTGYTMNWVKQTHGKNLEWIGLINPYTGGTTYKQKFKGKATLTVDKSSNTAYMELLSLTSEDSAVYYCTRGGFYRYDGDYWGQGTTLTVSS

the amino acid sequence of the light chain variable region of ICA2002-50A5-1-hIgG1 is shown in SEQ ID NO. 9:

DIQMTQSPSSLSASLGERVSLTCRASQDIGNYLTWLQQEPDGTIKRLIYATSSLHSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYSSSPWTFGGGTKLEIR

the amino acid sequence of the heavy chain variable region of ICA2002-50C1-1-hIgG1 is shown in SEQ ID NO. 3:

EVQLQQSGPELVKPGASVKISCKASGFSFTGYTMNWVKQSHGKNLEWIGLINPYNGGTTYKQKFKGKATLTVDKSSNTAYMELLSLTSEDSAVYYCTRGGFYRYDGDYWGQGTTLTVSS

the amino acid sequence of the light chain variable region of ICA2002-50C1-1-hIgG1 is shown in SEQ ID NO. 10:

VIQLTQSPSSLSASLGERVSLTCRASQDIGNYLTWLQKEPNGTIKRLIYATFSLHSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYSSSPWTFGGGTKLEIR

the amino acid sequence of the heavy chain variable region of ICA2002-12B6-1-hIgG1 is shown in SEQ ID NO. 4:

EVQLQQSGPELVKPSTSMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLFNPYNGGTTYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGGIRRPPGDYWGQGTSVTVSS

The amino acid sequence of the light chain variable region of ICA2002-12B6-1-hIgG1 is shown in SEQ ID NO. 11:

DIVMTQSHKIMSTSVGDRVSITCKASQDVGPSVAWYQQKAGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPFTFGAGTKLELK

the amino acid sequence of the heavy chain variable region of ICA2002-22E11A8-1-hIgG1 is shown in SEQ ID NO. 5:

EVQLVETGGGLVQPKGSLKLSCATSGFTFNTNAMNWVRQAPGKGLEWVARIRSKSYNYATYYADSVKDRFTISRDDSQSMVYLQMNNLKTEDTAMYYCVRTGAGTWGQGTLVTVSA

The amino acid sequence of the light chain variable region of ICA2002-22E11A8-1-hIgG1 is shown in SEQ ID NO. 12:

DVVMTQSPLTLSFTVGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGRGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKLEIK

The amino acid sequence of the heavy chain variable region of ICA2002-69H5-1-hIgG1 is shown in SEQ ID NO. 6:

EVQLVESGGGLVRPGGSLKLSCAASGFTFNTYGMSWVRQTPDKRLELVATINSNGGNTYYPDSVKGRFTISRDNAKNTLYLQLSSLKSEDTSMYYCARAYTYAMDYWGQGTSVTVSS

the amino acid sequence of the light chain variable region of ICA2002-69H5-1-hIgG1 is shown in SEQ ID NO. 13:

QIVLTQSPTIMSASPGERVTMTCSASSSVSSSYMFWFQQKSGSSPKLWICSISNLTSGVPARFSGSGSGTSYSLTINSMAADDAATYYGQQWSGNTPTFGAGTTLELK

The amino acid sequence of the heavy chain variable region of ICA2002-195B7-2A11-1E4-hIgG1 is shown in SEQ ID NO. 7:

EVQLQQSGPELVKPGASVKMSCKASGYTFTTYVIHWVKRKPGQGLEWIGFFNPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARRQLRYSMDYWGQGTSVTVSS

the amino acid sequence of the light chain variable region of ICA2002-195B7-2A11-1E4-hIgG1 is shown in SEQ ID NO. 14:

DIQMTQSPSSLSASLGERVSLTCRASQDIGSSLNWLQQEPDGTIKRLIYATSSLDSGVPKRFSGSRSGSDYSLTISGLESEDFVDYYCLQYASSPYTFGGGTKLEIK

The amino acid sequence of the connecting peptide is shown in SEQ ID NO. 15:

GGGGSGGGGSGGGGS

Example 2: expression and purification results of anti-GPRC 5D monoclonal antibody

The screened monoclonal antibody is constructed on a eukaryotic expression vector of HIgG1 (Fc). And carrying out transient expression and purifying by a Protein A affinity column (Protein A column), wherein due to the special antibody sequence, the purification process is complicated, partial antibodies are difficult to obtain, and finally the 12B6, 18D8C7, 22E11A8, 50A5 and 50C1 antibodies are obtained. The purification results are shown in the following table:

TABLE 1 purification results of GPRC5D monoclonal antibodies

The single-chain variable region fragment of ICA2002-18D8C7-1-hIgG1 is shown in SEQ ID NO. 16:

EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTPDKRLEWVATISNGGSYTYYPDNVKGRFTISRDNAKNTLYLQMSSLKSEDTATYYCARHAWDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSAFPGEKVTMTCNASSSVSYMYWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISIMEAEDAATYYCQHWSTNPLTFGAGTKLELK

the single-chain variable region fragment of ICA2002-50A5-1-hIgG1 is shown in SEQ ID NO. 17:

EVQLQQSGPELVKPGASVKISCKASGFSFTGYTMNWVKQTHGKNLEWIGLINPYTGGTTYKQKFKGKATLTVDKSSNTAYMELLSLTSEDSAVYYCTRGGFYRYDGDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASLGERVSLTCRASQDIGNYLTWLQQEPDGTIKRLIYATSSLHSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYSSSPWTFGGGTKLEIR

The single-chain variable region fragment of ICA2002-50C1-1-hIgG1 is shown in SEQ ID NO. 18:

EVQLQQSGPELVKPGASVKISCKASGFSFTGYTMNWVKQSHGKNLEWIGLINPYNGGTTYKQKFKGKATLTVDKSSNTAYMELLSLTSEDSAVYYCTRGGFYRYDGDYWGQGTTLTVSSGGGGSGGGGSGGGGSVIQLTQSPSSLSASLGERVSLTCRASQDIGNYLTWLQKEPNGTIKRLIYATFSLHSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYSSSPWTFGGGTKLEIR

The single-chain variable region fragment of ICA2002-12B6-1-hIgG1 is shown in SEQ ID NO. 19:

EVQLQQSGPELVKPSTSMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLFNPYNGGTTYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGGIRRPPGDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSHKIMSTSVGDRVSITCKASQDVGPSVAWYQQKAGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPFTFGAGTKLELK

The single-chain variable region fragment of ICA2002-22E11A8-1-hIgG1 is shown in SEQ ID NO. 20:

EVQLVETGGGLVQPKGSLKLSCATSGFTFNTNAMNWVRQAPGKGLEWVARIRSKSYNYATYYADSVKDRFTISRDDSQSMVYLQMNNLKTEDTAMYYCVRTGAGTWGQGTLVTVSAGGGGSGGGGSGGGGSDVVMTQSPLTLSFTVGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGRGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKLEIK

example 3: binding force detection of anti-GPRC 5D antibodies and cell surface GPRC5D proteins

The detection of the binding capacity of cell surface GPRC5D antigen to antibody was performed by flow cytometry using DPBS containing 1% FBS as buffer. That is, 1E 5/well of cells (MM.1S, CHOK1-GPRC 5D) were prepared, 100 μl/well of 96-well U-shaped bottom plate (Corning 3799) was added, the antibody was prepared to 100nM using the buffer, and then diluted according to 5-fold dilution of the antibody for 8 concentration gradients. The prepared antibodies with different concentration gradients are added into the paved target cells according to 100 mu l/hole, and the mixture is uniformly mixed, and incubated for 1 hour at 4 ℃. Adding the prepared buffer solution according to 100 μl/hole, 1500rpm,4 ℃,5 min, discarding the supernatant, adding the prepared buffer solution according to 200 μl/hole, 1500rpm,4 ℃,5 min, discarding the supernatant. According to 1:200 secondary antibodies were added, 100 μl/well and incubated for 1 hour at 4deg.C. Adding the prepared buffer solution according to 100 μl/hole, 1500rpm,4 ℃,5 min, discarding the supernatant, adding the prepared buffer solution according to 200 μl/hole, 1500rpm,4 ℃,5 min, discarding the supernatant. And adding the prepared buffer solution according to 100 mu l/hole, and detecting the binding force of the GPRC5D antibody and the cell surface antigen by using a BD flow cell sorter.

The binding capacity of the flow assay to tumor cells and GPRC 5D-overexpressing cells is shown in figures 1,2, in mm.is cells and CHOK1-GPRC5D cells, 3 of the 5 sequences of the invention, 3 of which had much higher affinities than the GPRC5D x CD3 bispecific antibody BMK group developed by robusta.

Example 4: electrotransport plasmid construction

Electrotransformation of empty plasmid and GPRC5D core plasmid (12B 6, 18D8C7, 22E11A8, 50A5, 50C 1) the synthesis of the gene from Biotechnology Co., ltd was designated PL-TCAR (G, X) (X is 1,2,3,4, 5), and the CAR structure in the present application is shown in FIG. 3.

Example 5: detection of electrical transitions and positive rates of GPRC5D CARs

First, the plasmid was transferred into NK92 cells by using a cytoelectrotransport. 5 mug of electrotransport plasmids are uniformly mixed in an electrotransport buffer, transferred into NK92 cells of 5E6 through a cell electrotransport device under 480V voltage, and the cells after electrotransport are placed into a pre-heated NK92 cell culture medium (without PS). Incubated at 37℃with 5% CO ₂ overnight. Second, detection of NK92 cell positive rate after electrotransformation. Counting cells of the previous day, using the corresponding cells of each NK92 cell zone 1E5 for flow detection, namely adding the cells prepared by 1E 5/hole into a 96-hole U-shaped bottom plate (Corning 3799), supplementing each hole to 200 mu l/hole by using prepared flow buffer, 1500 turns/min, 4 ℃ for 5 minutes, discarding the supernatant, adding the prepared buffer again according to 200 mu l/hole for 1500 turns/min, 4 ℃ for 5 minutes, and discarding the supernatant. The antibody for detection was prepared according to 1:100 was mixed with DPBS in 1% FBS and incubated at 4℃for 1 hour at 100 μl/well. Adding the prepared buffer solution according to 100 μl/hole, 1500rpm,4 ℃,5 min, discarding the supernatant, adding the prepared buffer solution according to 200 μl/hole, 1500rpm,4 ℃,5 min, discarding the supernatant. And adding the prepared buffer solution according to 100 μl/hole, and detecting the GPRC5D CAR positive rate by using a BD flow cell sorter.

The results are shown in FIG. 4, and positive rates are analyzed by a flow cytometer after the electric transfer, wherein the positive rate of positive control NK92-TCAR (BMK 1) is 28.6%, and the positive rates of experimental group NK92-TCAR (G, 1), NK92-TCAR (G, 2), NK92-TCAR (G, 3) and NK92-TCAR (G, 4) are 37.4% respectively; 30.3%;24%;23.9% of the above groups represent successful transduction of the CAR structure, with a positive rate of 1.06% for the negative control NT-NK 92.

Example 6: killing of NK92 CAR (expression of CAR and killing on mm.1s-luc)

The ability of GPRC5D to kill target cells MM.1S-Luc cells and NCI-H929 cells was examined by a luciferase luminescence method. Positive rates of NK92 cells of electrotransferred GPRC5D were adjusted to be consistent, target cells mm.1s-Luc were seeded to 96-well U-shaped bottom plates (corning 3799) according to 1E4/50 μl/well, and effector cells with consistent positive rates were seeded to 4:1,2:1,1:1,1:2 into target cells, followed by 100g,2 minutes. Incubated at 37℃with 5% CO ₂ overnight. After 24 hours, 50 μl/hole of luciferase reagent is added into each hole, and the luciferase reagent is transferred to a 96-hole black wall plate in a dark place for 5 minutes and put into a multifunctional microplate reader for reading data.

As shown in fig. 5 and 6, GPRC5D has a killing effect equivalent to BMK1 in mm.1s and stronger than NT-NK 92; on NCI-H929-LUC cells, the killing effect is slightly higher than BMK1 and stronger than NT-NK92

It should be understood that the above examples are illustrative and are not intended to encompass all possible implementations encompassed by the claims. Various modifications and changes may be made in the above embodiments without departing from the scope of the disclosure. Likewise, the individual features of the above embodiments can also be combined arbitrarily to form further embodiments of the invention which may not be explicitly described. Therefore, the above examples merely represent several embodiments of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. A monoclonal anti-GPRC 5D antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence as set forth in any one of SEQ ID nos. 1-2;

the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO. 8-9;

Wherein, SEQ ID NO.1 is paired with SEQ ID NO.8, and SEQ ID NO.2 is paired with SEQ ID NO. 9.

2. The antibody of claim 1, wherein the antibody is a single chain antibody and/or a double chain antibody that binds to a cell surface GPRC5D protein.

3. A single chain variable fragment scFv, wherein said scFv comprises a heavy chain variable region and a light chain variable region; wherein the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO. 1-2;

4. The scFv according to claim 3, wherein the scFv further comprises a linker peptide; wherein the sequence of the connecting peptide is shown as SEQ ID NO.15, and the amino acid sequence of the scFv is shown as any one of SEQ ID NO. 16-17.

5. A chimeric antigen receptor CAR, the CAR comprising:

(1) The scFv of claim 4,

(2) A transmembrane domain comprising a transmembrane domain,

(3) A costimulatory domain, and

(4) The domain is activated.

6. The CAR of claim 5, wherein the CAR has the structure of formula I:

L-scFv-H-TM-C-CD3ζ（I），

Wherein,

Each "-" is independently a connecting peptide or peptide bond;

l is an optional signal peptide sequence;

an scFv is the scFv of claim 4;

H is an optional hinge region;

TM is a transmembrane domain;

C is a costimulatory signaling molecule;

cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ.

7. A drug conjugate, the drug conjugate comprising:

(1) The antibody of any one of claims 1-2, or the scFv of any one of claims 3-4; and

(2) A coupling moiety;

8. A polynucleotide encoding the antibody of any one of claims 1-2, or the scFv of any one of claims 3-4, or the CAR of any one of claims 5-6.

9. A vector comprising the polynucleotide of claim 8; wherein the vector is selected from one or more of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, or a combination thereof.

10. Genetically engineered cell, characterized in that it comprises a vector according to claim 9, a chromosome into which the polynucleotide according to claim 8 is integrated, or an antibody according to any one of claims 1-2 is expressed, or an scFv according to any one of claims 3-4 is expressed, or a CAR according to any one of claims 5-6 is expressed.

11. The genetically engineered cell of claim 10, wherein the cell is an NK cell.

12. A pharmaceutical composition comprising one or more of the antibody of any one of claims 1-2, or the scFv of any one of claims 3-4, or the CAR of any one of claims 5-6, or the drug conjugate of claim 7, or the cell of any one of claims 10-11; wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent and/or excipient.

13. Use of an antibody according to any one of claims 1-2, or an scFv according to any one of claims 3-4, or a CAR according to any one of claims 5-6, or a drug conjugate according to claim 7, or a cell according to any one of claims 10-11, or a pharmaceutical composition according to claim 12, in the manufacture of a medicament for the prevention and/or treatment of multiple myeloma.