WO2022247804A1 - Anti-gprc5d antibody, preparation method therefor, and use thereof - Google Patents

Abstract

Provided are an anti-GPRC5D antibody, a preparation method therefor, and a use thereof. The antibody comprises a heavy chain variable region and/or a light chain variable region. The heavy chain variable region comprises amino acid sequences represented by SEQ ID NO: 44(HCDR1), SEQ ID NO: 99(HCDR2), and SEQ ID NO: 100(HCDR3), and the light chain variable region comprises amino acid sequences represented by SEQ ID NO: 101(LCDR1), SAS(LCDR2), and SEQ ID NO: 102(LCDR3). Or, the heavy chain variable region comprises amino acid sequences represented by SEQ ID NO: 29(HCDR1), SEQ ID NO: 30(HCDR2), and SEQ ID NO: 31(HCDR3), and the light chain variable region comprises amino acid sequences represented by SEQ ID NO: 47(LCDR1), YAS(LCDR2), and SEQ ID NO: 48(LCDR3). The anti-GPRC5D antibody can be used in the treatment of related diseases such as cancer and product development.

Description

Anti-GPRC5D antibody, its preparation method and use

The present invention claims the priority of the Chinese patent application submitted to the China Patent Office on May 23, 2021, with the application number 202110561818.0, and the application name is "Anti-GPRC5D antibody, its preparation method and use", and requires that it be filed on May 23, 2021. The priority of the Chinese patent application with the application number 202110561819.5 and the application name "Anti-GPRC5D antibody, its preparation method and use" filed with the China Patent Office on 12th, the entire content of which is incorporated by reference in this application.

technical field

The present invention relates to the technical field of antibodies, in particular to an anti-GPRC5D antibody, its preparation method and application.

Background technique

Multiple myeloma (Multiple Myeloma, MM) is the second most common hematological tumor after non-Hodgkin's lymphoma in the world, accounting for about 10% of hematological malignancies. According to the global cancer statistics report released by WHO, in 2018, there were 20,066 new cases of multiple myeloma in China, and 14,665 cases of death; the cumulative number of cases in 5 years was 44,643. And the morbidity and mortality of patients gradually increase with age. Therefore, as the aging population in my country continues to increase, the total number of patients will gradually increase, and the clinical demand is huge. In the past ten years, the application of immunomodulators represented by thalidomide and its derivative lenalidomide and small molecule proteasome inhibitors represented by bortezomib has greatly improved the remission rate and lifetime. At present, the treatment options for multiple myeloma in my country can be divided into three categories: immunomodulators, proteasome inhibitors and targeted therapy of anti-CD38 monoclonal antibody. Immunomodulators and proteasome inhibitors are mainly used for first-line combination therapy and maintenance therapy after transplantation for patients eligible for stem cell transplantation. For the second-line treatment of relapsed or refractory multiple myeloma, the anti-CD38 monoclonal antibody Daratumumab, which was conditionally approved by the National Medical Products Administration (NMPA) in 2019 and marketed through import registration, is considered. However, after a short period of treatment, patients who relapse again are in an embarrassing situation where no medicine is available. Therefore, the relapse and refractory nature of MM still plague people, prompting people to constantly seek new therapeutic targets and methods to be applied to recurrent/refractory MM.

The rapid development of small molecule inhibitors in the treatment of relapse and biopharmaceutical-related technologies has accelerated the development of targeted therapies for MM. Biomacromolecular targeted therapies currently approved by the FDA include targeting plasma cell surface protein CD38, signaling lymphocyte activating molecule family member 7 (Signaling Lymphocytes Activating Molecule Factor 7, SLAMF7) and B Cell Maturation Antigen (BCMA). ) and other three major targets, the types of approved drugs mainly include: monoclonal antibodies and antibody-drug conjugates (Antibody-Drug Conjugate, ADC). Compared with CD38 and SLAMF7 targets, the expression of BCMA protein on MM cells is more specific. In August 2020, the U.S. FDA accelerated the approval of the BMCA-targeting ADC drug Blenrep for the treatment of patients with median 7-line recurrence. The overall response rate can still reach 31%, and the median duration of response (DoR) is greater than 6 months. In addition, the anti-tumor effect of CAR-T or CD3 bispecific antibody targeting BCMA by mediating T cells is also significant. The BCMA drug research and development track has many internationally renowned biopharmaceutical companies including Bluebird, BMS, Amgen, Johnson & Johnson, Regeneron, and Nanjing Legend, and the competition is fierce. In recent years, under the vigorous guidance of the Chinese government, my country's biopharmaceutical industry has also risen rapidly, and a number of targeted new drug R&D companies have emerged, but most of them focus on CD38, BCMA target double antibodies and CAR- In the T direction, it is facing great pressure from domestic and foreign homogenization competition. In addition, although BCMA-targeted ADCs, dual antibodies, and CAR-T therapy have shown positive clinical effects, BCMA-negative (or low expression) and related post-treatment recurrence cases have been reported, highlighting the multiple myeloma More targets are urgently needed for treatment.

G protein-coupled receptor (GPCR) is a protein receptor with 7 transmembrane helices, which can be divided into 5 subfamilies according to their sequence similarity and ligand binding. The largest protein family in the human body and an important drug target. G protein-coupled receptor C5 family subtype D (GPRC5D) is the first orphan SARS class C GPCR identified in 2001 (Brauner-Osborne, H., et al. Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D. Biochim Biophys Acta, 2001.1518(3):p.237-48). There are four subtypes of GPCR family C group 5 receptors (GPRC5 receptors), namely GPRC5A, GPRC5B, GPRC5C and GPRC5D, which are induced by retinoic acid, so they are also called retinoic acid-induced orphan G protein-coupled receptors. Body (Inoue, S., T. Nambu, and T. Shimomura, The RAIG family member, GPRC5D, is associated with hard-keratinized structures. Journal of Investigative Dermatology, 2004.122(3): p.565-573).

GPRC5D has previously been identified in cells from patients with multiple myeloma, but has not been used in clinical development due to lack of protein expression profiling studies. Research reports until 2019 have shown that GPRC5D is highly expressed in plasma cells of multiple myeloma, mostly not expressed in normal tissues, and only expressed in hair follicles with immune pardon (Smith, E.L., et al., GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells. Science Translational Medicine, 2019.11(485)). Studies have reported that high expression of GPRC5D is associated with poor prognosis in multiple myeloma (Atamaniuk, J., et al., Overexpression of G protein-coupled receptor 5D in the bone marrow is associated with poor prognosis in patients with multiple myeloma. European Journal of Clinical Investigation, 2012.42(9):p.953-960). In addition, another important finding is that the expression profile of GPRC5D does not overlap with BCMA, so it may become a new therapeutic target for low/no expression of BCMA or relapse after BCMA treatment. However, because the GPCR protein is a 7-transmembrane protein with a complex structure, antigens and specific antibodies are difficult to obtain, only a few drugs targeting GPRC5D are still in the clinical trial stage or research and development stage. In view of this, the present invention develops GPRC5D drugs with higher activity, higher affinity, higher specificity and better therapeutic effect by combining immunization method and antibody library technology, so as to carry out diagnosis, treatment research and application of related diseases.

Contents of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes an antibody that can bind to G protein-coupled receptor C5 family subtype D (GPRC5D) with high specificity.

The present invention also proposes recombinant proteins, pharmaceutical compositions, polynucleotides, recombinant plasmids and isolated cells related to the above antibodies.

The present invention also proposes a preparation method of the above-mentioned antibody.

The present invention also proposes the application of the above antibody in the preparation of anticancer drugs.

The present invention also proposes the application of the above-mentioned antibody in the preparation of an antibody detection kit.

According to the antibody according to the first aspect of the present invention, the antibody comprises a heavy chain variable region and/or a light chain variable region:

The heavy chain variable region comprises: the heavy chain complementarity determining region HCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 44, the heavy chain complementarity determining region consisting of the amino acid sequence of INPX ₁ NGX ₂ T (SEQ ID NO: 99) Chain complementarity determining region HCDR2, heavy chain complementarity determining region HCDR3 consisting of the amino acid sequence of ARX ₃ ALRYAMDY (SEQ ID NO: 100);

The light chain variable region comprises: the light chain complementarity determining region LCDR1 consisting of the amino acid sequence of QX ₄ X ₅ X ₆ TX ₇ (SEQ ID NO: 101), the light chain complementarity determining region consisting of the amino acid sequence of SAS Region LCDR2, light chain complementarity determining region LCDR3 consisting of the amino acid sequence of QQX ₈ X ₉ X ₁₀ X ₁₁ PVT (SEQ ID NO: 102);

in,

X ₁ : represent any amino acid of Y, P, R, L, D, S, G, K, V, T, M and Q,

X ₂ : represent any amino acid of R and A,

X ₃ : represent any amino acid of V and A,

X ₄ : represent any amino acid of S and A,

X ₅ : represent any amino acid of V and A,

X ₆ : represent any amino acid of S, V, L, R, H, E, G, Q, M and Y,

X ₇ : represent any amino acid of R, P, H, S, W, I, G, V and N,

X ₈ : represent any amino acid of Y and A,

X ₉ : represent any amino acid of N and A,

X ₁₀ : represent any amino acid of S and A,

X ₁₁ : represents any amino acid of Y and A.

According to the antibody according to the first aspect of the present invention, the antibody comprises a heavy chain variable region and/or a light chain variable region:

The heavy chain variable region comprises: the heavy chain complementarity determining region HCDR1 consisting of the amino acid sequence shown in SEQ ID NO:29, the heavy chain complementarity determining region HCDR2 consisting of the amino acid sequence shown in SEQ ID NO:30 , the heavy chain complementarity determining region HCDR3 consisting of the amino acid sequence shown in SEQ ID NO:31;

The light chain variable region comprises: a light chain complementarity determining region LCDR1 composed of the amino acid sequence shown in SEQ ID NO: 47, a light chain complementarity determining region LCDR2 composed of a YAS amino acid sequence, composed of SEQ ID NO: 48 The indicated amino acid sequence consists of the light chain complementarity determining region LCDR3.

The antibody according to the embodiment of the present invention has at least the following beneficial effects: the antibody of the present invention has (1) good binding activity and strong binding ability to GPRC5D protein; (2) strong specificity and strong binding ability to CD19, CD3, CD11b and CD14 Antibodies such as these are non-binding, and can more specifically target GPRC5D to reduce off-target toxicity, so the safety is better; (3) Species cross-reactivity, which has cross-reactivity with monkey GPRC5D, is conducive to carrying out verification experiments and facilitating Subsequent product development for therapeutic use; (4) Antibody endocytosis, which has endocytic activity in cells and is suitable for the development of ADC drugs.

According to some embodiments of the present invention, the antibody is a murine antibody, a chimeric antibody or a human antibody.

In the present invention, the base sequence of the human GPRC5D gene CDS region is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.

In the present invention, in addition to the sequences of the heavy chain variable region and the light chain variable region, the antibody also includes the amino acid sequence of the heavy chain constant region containing IgG1 and the amino acid sequence of the light chain constant region containing Kappa.

According to some embodiments of the present invention, the heavy chain variable region is selected from the amino acids shown in SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85 or SEQ ID NO:87 Sequence; The light chain variable region is selected from the amino acid sequence shown in SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95 or SEQ ID NO:97.

According to some preferred embodiments of the present invention, the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:87, and the light chain variable region is the amino acid sequence shown in SEQ ID NO:89.

According to some embodiments of the present invention, the heavy chain variable region is selected from the amino acids shown in SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 or SEQ ID NO:67 Sequence; The light chain variable region is selected from the amino acid sequence shown in SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75 or SEQ ID NO:77.

According to some preferred embodiments of the present invention, the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:59, and the light chain variable region is the amino acid sequence shown in SEQ ID NO:71;

Or the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:59, and the sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:73;

Or the sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:59, and the sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:75;

Or the sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:65, and the sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:69;

Or the sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:65, and the sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:73;

Or the sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:67, and the sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:71;

Or the sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:67, and/or the sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:73.

According to some embodiments of the present invention, the antibody comprises any one of the following properties i to iv:

i. The antibody can specifically bind to G protein-coupled receptor C5 family subtype D;

ii. The anti-GPRC5D antibody does not bind to any one of CD19, CD3, CD11b or CD14;

iii. The anti-GPRC5D antibody specifically binds to human GPRC5D and cross-reacts with monkey GPRC5D;

iv. The anti-GPRC5D antibody has endocytic activity.

In the present invention, the above-mentioned antibody sequences have the best binding activity, reaction specificity, species cross-reactivity and endocytosis activity. Experiments in the examples can prove that the protein binding level of the anti-GPRC5D antibody of the present invention is significantly higher than that of prior art antibodies; it has binding specificity, does not bind non-specifically to CD19, CD3, CD11b, and CD14 positive cells, and targets more specifically GPRC5D, thereby reducing the risk of toxicity caused by off-target; it has the characteristics of species cross-reactivity, which is helpful for the toxicity analysis of antibodies in monkeys, and is conducive to carrying out verification tests in related animals, which in turn facilitates the development of subsequent therapeutic uses Application development; with obvious endocytic activity, it is more suitable for subsequent development of drugs of antibody drug conjugate (antibody-drug conjugate, ADC).

In the present invention, the glycosylation of the Fc region is modified to enhance FcγR binding compared to an unmodified Fc region. In some embodiments, the Fc region lacks or has reduced fucose content.

The recombinant protein, pharmaceutical composition, polynucleotide, vector or isolated cell according to the embodiment of the second aspect of the present invention: the recombinant protein comprises the above-mentioned antibody; the pharmaceutical composition comprises the above-mentioned antibody or the above-mentioned recombinant protein; the multinuclear The nucleotide contains the nucleotide sequence encoding the above-mentioned antibody or recombinant protein; the vector contains the above-mentioned polynucleotide; and the isolated cell produces the above-mentioned antibody.

According to some embodiments of the present invention, the recombinant protein further comprises a tag sequence to assist expression and/or purification.

According to some embodiments of the present invention, the recombinant protein is a double antibody, which also includes antibodies capable of binding to other target proteins. Further, the double antibody also includes antibodies specifically binding to CD3 and antibodies specifically binding to different epitopes of GPRC5D.

According to some embodiments of the present invention, the pharmaceutical composition further includes the above-mentioned double antibody.

According to some embodiments of the present invention, the pharmaceutical composition further includes an ADC drug containing the above-mentioned antibody. Further, the ADC drug also includes a linker and a toxic molecule.

According to some embodiments of the present invention, the pharmaceutical composition further includes pharmaceutically available excipients.

The preparation method according to the embodiment of the third aspect of the present invention includes the following steps: culturing the above-mentioned isolated cells, and recovering the antibody from the culture.

In the present invention, the specific preparation method is: clone the heavy chain variable region sequence encoding the above-mentioned anti-GPRC5D antibody into recombinant plasmid 1 containing the IgG1 heavy chain constant region amino acid sequence, and clone the light chain variable region sequence into recombinant plasmid 1 containing the Kappa In the recombinant plasmid 2 of the amino acid sequence of the light chain constant region, the recombinant plasmid 1 and the recombinant plasmid 2 were simultaneously transfected into cells and cultured, and the anti-GPRC5D antibody was recovered from the culture.

According to the application of the embodiment of the fourth aspect of the present invention, the antibody can be applied to the preparation of anticancer drugs and/or antibody detection kits.

In the present invention, the anticancer drug is mainly used for preventing or treating cancer, wherein the cancer is breast cancer, endometrial cancer, ovarian cancer, lung cancer, gastric cancer, prostate cancer, kidney cancer, liver cancer, Cancer of the pancreas, colorectum, esophagus, bladder, cervix, blood, lymphoma, or malignant melanoma. Further, the cancer is multiple myeloma expressing GPRC5D protein.

In the present invention, anti-cancer drugs also include antibodies targeting other targets, such as: CD3, BCMA, CD38, etc. to construct bispecific antibodies, and develop various methods for regulating tumor cells.

In the present invention, the antibody of the present invention can also be coupled to other types of molecules, such as toxins, nucleic acid molecules, etc., and the antibody can specifically bring the coupled molecules into the body of tumor cells, thereby regulating the effect of tumor cells. For example, the antibody can be used to treat cancer by interfering with GPRC5D-receptor interactions or wherein the antibody is conjugated to the toxin, thereby targeting a toxin to a GPRC5D expressing cancer.

In the present invention, the GPRC5D-specific antibody or antigen-binding fragment can be labeled for the method or other methods known to those skilled in the art. For example, the antibodies or antigen-binding fragments thereof of the present invention can be radiolabeled, fluorescently labeled, epitope tags, biotin, chromophore labels, ECL labels, enzymes, ruthenium, 111In-DOTA, 111In-di Ethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, or polyhistidine or similar such labels known in the art.

In the present invention, the antibody can also be developed into a method for detecting the expression level of GPRC5D on the surface of tissue cells by means of immunization. For example, the antibody can also be used to detect the presence of GPRC5D in a biological sample such as blood or serum, to quantitatively analyze the amount of GPRC5D in a biological sample such as blood or serum, to diagnose GPRC5D expressing cancers, to determine treatment for cancer patients A method in a subject, or for monitoring the progression of a GPRC5D expressing cancer in a subject, etc.

In the present invention, the term "antibody" specifically includes antibodies in a narrow sense, and also includes "chimeric" antibodies and antibody fragments, wherein part of the heavy chain and/or light chain corresponds to an antibody derived from a specific species or belonging to a specific antibody type or subclass. The sequence is identical or homologous, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody type or subclass, so long as it specifically binds the target antigen and/or exhibits the desired Biological activity (US Patent No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Among them, "antibody in a narrow sense" refers to a type of glycosyl-containing globulin that is secreted by antigens entering the body to stimulate B cells to differentiate and proliferate into plasma cells, which can specifically bind to corresponding antigens and produce immune effects. In 1964, the World Health Organization held a meeting and collectively referred to globulins with antibody activity and chemical structure similar to antibodies as immunoglobulins. Modern immunology believes that antibodies and immunoglobulins are equivalent concepts, but antibodies focus on the description of their biological activities, while immunoglobulins focus on their chemical structures.

The basic structure of immunoglobulin consists of four peptide chains: two heavy chains (Heavy chain, H chain) and two light chains (Light chain, L chain), the light chain and the heavy chain are connected by disulfide bonds to form a symmetrical tetrapeptide Chain molecules become monomers of immunoglobulin molecules, which are the basic structure of all immunoglobulins. Each heavy and light chain is divided into amino-terminus (N-terminus) and carboxy-terminus (C-terminus). Through the comparison and analysis of the amino acid sequences of the H chain or L chain, it is found that the N-terminal sequence changes greatly, and this region is called the variable region (Variable region, V region); the C-terminal amino acid is relatively stable, and the change is small, called this region. It is a constant region (Constant region, C region). The variable region can be divided into hypervariable region (HVR) and framework region (Framework region, FR). There are three HVRs in the heavy chain variable region and the light chain variable region, respectively, from the N-terminal to the C-terminal, called HVR1, HVR2 and HVR3 of the heavy chain or light chain, respectively. The hypervariable region is the binding site of the antibody and the antigen, and is called the complementarity-determining region (CDR). Therefore, HVR1, HVR2, and HVR3 of the heavy chain or light chain are also called CDR1, CDR2, and CDR3.

In the present invention, the term "diabodies", that is, "bispecific" antibodies, refers to an antibody, generally a monoclonal antibody, that has the binding properties of at least two different antigenic epitopes. In one embodiment, the epitopes are from the same antigen. In another embodiment, the epitopes are from two different antigens. Methods of making bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly by co-expressing two immunoglobulin heavy/light chain pairs. See, eg, Milstein et al., Nature 305:537-39 (1983). Alternatively, bispecific antibodies can be prepared using chemical linkage. See, eg, Brennan et al., Science 229:81 (1985). Bispecific antibodies include bispecific antibody fragments (eg, Hollinger et al., Proc. Natl. Acad. Sci. U.S.A. 90:6444-48 (1993), Gruber et al., J. Immunol. 152:5368 (1994)).

In the present invention, the term "humanized antibody" refers to a form of antibody that contains sequences derived from non-human (eg, murine) antibodies as well as human antibodies. The antibody is a chimeric antibody containing minimal sequence derived from non-human immunoglobulin. Typically, such humanized antibodies will comprise substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, all or substantially all of which The FR regions are those of human immunoglobulin sequences. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., Cabilly U.S. Patent No. 4,816,567; Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033; Oxford University Press) 1996).

The three-letter codes and one-letter codes of amino acids used in the present invention are as described in J. Biol. Chem, 243, P3558 (1968).

Additional aspects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Description of drawings

Figure 1 shows the binding of panned monoclonal scFv to HEK293-GPRC5D-ZSGreen1 stably transfected cells;

Figure 2 shows the binding of panned monoclonal scFv to NCI-H929 cells;

Figure 3 shows the binding ability of different chimeric antibodies to NCI-H929 cells;

Figure 4 shows the binding force of humanized HTS0370 antibody to HEK293-GPRC5D-ZSGreen1 cells;

Figure 5 shows the binding force of humanized HTS0370 antibody to NCI-H929 cells;

Figure 6 shows the binding force of humanized HTS0375 antibody to HEK293-GPRC5D-ZSGreen1 cells;

Figure 7 shows the binding force of humanized HTS0375 antibody to NCI-H929 cells;

Figure 8 shows the cross-reactivity of humanized HTS0370 and HTS0375 antibodies with CD19-positive PBMC cells;

Figure 9 shows the cross-reactivity of humanized HTS0370 and HTS0375 antibodies with CD3-positive PBMC cells;

Figure 10 shows the cross-reactivity of humanized HTS0370, HTS0375 antibody and CD11b positive PBMC cells;

Figure 11 shows the cross-reactivity of humanized HTS0370, HTS0375 antibodies and CD14-positive PBMC cells;

Figure 12 shows the binding activity of humanized HTS0370 and HTS0375 antibodies to GPRC5D protein;

Figure 13 shows the endocytic activity of humanized HTS0370 and HTS0375 antibodies;

Figure 14 shows the species cross-reactivity of humanized HTS0370, HTS0375 antibodies;

Figure 15 shows the FACS verification results of the mutant whose mutation site in the CDR region of the antibody is _X1 ;

Figure 16 shows the FACS verification results of the mutant whose mutation site in the CDR region of the antibody is _X2 ;

Figure 17 shows the FACS verification results of the mutant whose mutation site in the CDR region of the antibody is _X3 ;

Figure 18 shows the FACS verification results of the mutant whose mutation site in the CDR region of the antibody is _X4 ;

Figure 19 shows the FACS verification results of the mutant whose mutation site in the CDR region of the antibody is _X5 ;

Figure 20 shows the FACS verification results of the mutant whose mutation site in the CDR region of the antibody is _X6 ;

Figure 21 shows the FACS verification results of the mutant whose mutation site in the CDR region of the antibody is _X7 ;

Figure 22 shows the FACS verification results of the mutant whose mutation site in the CDR region of the antibody is _X8 ;

Figure 23 shows the FACS verification results of the mutant whose mutation site in the CDR region of the antibody is _X9 ;

Figure 24 shows the FACS verification results of the mutant whose mutation site in the CDR region of the antibody is _X10 ;

Figure 25 shows the FACS verification results of the mutant whose mutation site in the CDR region of the antibody is _X11 ;

Figure 26 shows the overexpression effect of the constructed GPRC5A, GPRC5B, and GPRC5C three overexpression cell lines;

Figure 27 shows the binding reactivity of the antibody to three overexpression cell lines of GPRC5A, GPRC5B, and GPRC5C;

Figure 28 shows the schematic structure of four chimeric molecules of GPRC5D-A1-A4;

Figure 29 shows the overexpression effect of GPRC5D-A1～A4 four kinds of chimeric molecular overexpression cell lines;

Figure 30 shows the binding reactivity of antibodies to cell lines overexpressing four chimeric molecules GPRC5D-A1-A4.

Detailed ways

In order to describe the technical content, achieved goals and effects of the present invention in detail, the following descriptions will be made in conjunction with the embodiments and accompanying drawings.

In the following embodiments of the present invention, the carrier information used is as follows:

The pTT5-hIgG1.CH vector can synthesize the constant region sequence of human IgG1 (SEQ ID NO:97), which is obtained by constructing the pTT5 vector (purchased from Miaoling Biology) through EcoRI/HindIII restriction sites;

The pTT5-hKappa.CL vector can synthesize the constant region sequence of the human Kappa chain (SEQ ID NO:98), which is obtained by constructing the pTT5 vector (purchased from Miaoling Biology) through the EcoRI/BamHI restriction site.

Example 1. Preparation of human GPRC5D expression vector and stable transfection cell line

1-1. Preparation of human GPRC5D expression vectors pLVX-huGPRC5D-IRES-ZSGreen1 and pTT5-huGPRC5D

Obtain the nucleotide sequence of the CDS region of the human GPRC5D gene (NM_018654.1) from the NCBI database, design PCR primers 1 (SEQ ID NO:1) and 2 (SEQ ID NO:2), and select MM.1S with high expression of GPRC5D (purchased from Nanjing Institute of Model Animals), NCI-H929 (ATCC) cDNA of two multiple myeloma (MM) cell lines were used as templates for PCR amplification, and the PCR products were digested with BamHI/EcoRI and ligated to pLVX-IRES - ZSGreen1 (purchased from Clonetech) and pTT5 vectors (purchased from Miaoling Biotech) were transfected with DH5a, and single clones were picked for sequencing. The sequences were verified to be correct by sequencing, that is, the strains pLVX-huGPRC5D-IRES-ZSGreen1 and pTT5-huGPRC5D expressing the target plasmids were successfully constructed. Cultivate the strains containing the target plasmid in liquid LB medium, and use Tiangen Biochemical Technology (Beijing) Co., Ltd. Endotoxin-Free Plasmid Large-scale Extraction Kit (DP117) to extract plasmid DNA according to the routine steps in the manual.

The base sequence of the CDS region of the human GPRC5D gene is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.

1-2. Preparation of HEK293 stably transfected cell line overexpressing human GPRC5D antigen

HEK293 cells in logarithmic growth phase (purchased from ATCC) in good growth state were selected, 8E6 cells were inoculated in a culture dish (10 cm), 10% fetal bovine serum was added to DMEM, and cultured in a 37°C, 5% _CO2 incubator. Transfect when the cell fusion rate (cell density observed under a microscope) reaches about 70% to 80%. The three plasmids psPAX2 (purchased from Clonetech), pMD2.G (purchased from Clonetech) and pLVX-huGPRC5D-IRES-ZSGreen1 were co-transfected using transfection reagent PEI. The virus liquid was collected from 48 hours to 72 hours after transfection, filtered through a 0.45 μM syringe filter to remove residual cells, and infected with the target cell HEK293 (purchased from ATCC) at an MOI of 10, cultured in DMEM plus 10% fetal bovine serum. After 24 hours of virus infection, the medium was changed to obtain a stably transfected cell pool with a stable and high expression of hGPRC5D. The positive rate of human GPRC5D expression was detected by flow cytometry using the positive antibody of ET150, and the positive rate was over 98%, which was completely consistent with the positive rate of GFP. At the same time, the co-high expression of ZSGreen and antigen was detected, and HEK293-GPRC5D-ZSGreen1 was stable. The transgenic cell line was successfully constructed.

Example 2. Preparation of Monoclonal Antibodies and Screening of Antibodies

2-1. Immunization and serum titer detection

Eight 6-week-old SPF grade Balb/C female healthy mice (purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.) were selected and randomly divided into two groups, A and B, at the 0th, 7th, 14th, 21st, 28th, and Immunization was carried out on days 35, 42, and 49. Group A was immunized with HEK293-GPRC5D-ZSGreen1 stably transfected cell line, and each mouse was injected intraperitoneally with 2×10 ⁷ cells; Group B was immunized with GPRC5D expression plasmid pTT5-hGPRC5D, and each mouse was subcutaneously inoculated on the back by gene gun 20 μg of plasmid. On the 34th day, 41st day, 48th day and 55th day, blood was collected from the orbit of the mice, and the immune serum titer of the mice was detected by conventional methods of flow cytometry (FACS). Briefly, 5×10 ⁵ HEK293-GPRC5D-ZSGreen1 stably transfected cells or endogenously expressed cells NCI-H929 (ATCC) were added to each well of a 96-well V-shaped microplate, centrifuged at 1500 r/min for 1 min, and the supernatant was discarded. After diluting the immune serum with PBS at 1:50, dilute 8 concentrations in a 4-fold gradient, add 50 μL/well to a microwell plate, incubate on ice for 30 minutes, add 150 μL PBS to each well, centrifuge at 1500 r/min for 1 min, and wash repeatedly. Plate 2 times. Add APC-labeled goat anti-mouse IgG (Jackson, Cat. No. 115-605-164, diluted in PBS 1:800), 50 μL per well, and incubate on ice for 30 min. Finally, the plate was washed twice with PBS, and 150 μL of PBS was added to each well to resuspend the cells, and CytoFLEX was used for detection (Beckman). Select mice A1 and B1 with the highest titer and two consecutive immunization serum titers that tend to be stable, and perform a shock immunization with 2×10 ⁷ HEK293-GPRC5D-ZSGreen1 cells by intraperitoneal injection 3 days before the construction of the antibody library.

2-2. Construction of phage antibody library

Take immunized Balb/C mice A1 and B1, and take spleen cells, refer to the literature (Krebber, A., Bornhauser, S., Burmester, J., Honegger, A., Willuda, J., Bosshard, HR, and Plückthun, A. (1997). Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. Journal of immunological methods 201, 35-55.) method to construct mouse immune phage library, briefly After lysing with Trizol lysate, extract the total RNA of the cells, reverse the cDNA, use specific antibody heavy chain primers and light chain primers, amplify the variable region gene of the antibody, clone it into the phage display vector, and transfer it into the host E. coli cells TG1. Through NGS sequencing, the diversity and effectiveness of the antibody library were evaluated by quality control indicators such as library capacity, clone positive rate, heavy and light chain pairing, CDR3 distribution, and phage display rate. The capacity of the antibody library is above ^1x108 , the positive rate of clone insertion is above 90%, and the display rate of scFv is above 60%. NGS sequencing shows that the antibody library is rich in diversity and can be used for subsequent screening.

2-3. Screening and identification of phage library

Firstly, HEK293 cells were used to pretreat phages, and the input amount of phages for each antibody library was 1×10 ¹⁰ , and then HEK293-GPRC5D-ZSGreen1 stably transfected cells were used to enrich and pan the pretreated phage supernatant, and wash with PBS first. Remove unbound phages, then use 0.1M HCl-Glycine to elute the phages bound to the cells, then use Tris-HCl to neutralize the eluate, and take the phages to infect Escherichia coli TG1 in the logarithmic growth phase to prepare phages in the next round of panning. Gradually increase the number of screening washes in each round, and stop panning when the enrichment degree reaches more than 10 times in the previous round.

The TG1 single clones infected by the phages after the termination of panning were selected and inoculated in a 96-well plate, and the culture medium was 2YT/2% glucose/(100 μg/ml Ampicilline). After culturing at 37°C and 220rpm for 6 hours, centrifuge at 4000rpm for 10min, and use 2YT/(100μg/ml Ampicilline)/(1μM IPTG) culture medium at 30°C and 220rpm overnight to induce scFv. The induced supernatant was obtained by centrifugation at 4000 rpm for 10 min, and the binding activity of the scFv to HEK293-GPRC5D-ZSGreen1 cells was detected by conventional methods of flow cytometry. In short, 2.5×10 ⁵ HEK293-GPRC5D-ZSGreen1 stably transfected cells were added to each well of a 96-well V-shaped microplate, and 50% HEK293 cells were mixed at the same time, centrifuged at 1500 r/min for 1 min, and the supernatant was discarded. Add 35 μL PBS to 15 μL scFv supernatant, add 50 μL/well to the microwell plate, incubate on ice for 30 min, add 150 μL PBS to each well, centrifuge at 1500 r/min for 1 min, and wash the plate twice. Add APC-labeled mouse anti-6×His antibody (GenScript, Cat. No. A01802, diluted 1:800), 50 μL per well, and incubate on ice for 30 min. After washing the plate twice with PBS, add 100 μL of PBS to each well to resuspend the cells, and use CytoFLEX to detect (Beckman). The MFI value of HEK293-GPRC5D-ZSGreen1 cells is more than 2 times higher than that of the HEK293 control group. For H929 binding verification, double binding positive clones were selected for sequencing. The positive scFv control BMK-ET150-8 was constructed according to the ET150-8 scFv sequence in the patent US20180118803. The results of cell binding are shown in Figures 1 and 2. Through the analysis of binding activity, 6 single-chain variable fragments (single-chain variable fragments, scFv) were selected, respectively named as HTS0370, HTS0371, HTS0372, HTS0373, HTS0374, HTS0375, and these 6 scFv single-chain antibodies were followed up. Research. After sequence determination, the VH, VL sequences and CDR sequences of the six antibodies are shown in Tables 1-4 below.

Table 1

Amino acid sequence list of scFv single chain antibody VH VL HTS0370 SEQ ID NO:5 SEQ ID NO:6 HTS0371 SEQ ID NO:7 SEQ ID NO:8 HTS0372 SEQ ID NO:9 SEQ ID NO:10 HTS0373 SEQ ID NO:11 SEQ ID NO:12 HTS0374 SEQ ID NO:13 SEQ ID NO:14 HTS0375 SEQ ID NO:15 SEQ ID NO:16

Table 2

Nucleotide sequence list of scFv single chain antibody VH VL HTS0370 SEQ ID NO:17 SEQ ID NO:18 HTS0371 SEQ ID NO:19 SEQ ID NO:20 HTS0372 SEQ ID NO:21 SEQ ID NO:22 HTS0373 SEQ ID NO:23 SEQ ID NO:24 HTS0374 SEQ ID NO:25 SEQ ID NO:26 HTS0375 SEQ ID NO:27 SEQ ID NO:28

table 3

Antibody heavy chain CDR sequence list HCDR1 HCDR2 HCDR3 HTS0370 SEQ ID NO:29 SEQ ID NO: 30 SEQ ID NO: 31 HTS0371 SEQ ID NO:32 SEQ ID NO:33 SEQ ID NO:34 HTS0372 SEQ ID NO:35 SEQ ID NO:36 SEQ ID NO:37 HTS0373 SEQ ID NO:38 SEQ ID NO:39 SEQ ID NO:40 HTS0374 SEQ ID NO:41 SEQ ID NO:42 SEQ ID NO:43 HTS0375 SEQ ID NO:44 SEQ ID NO:45 SEQ ID NO:46

Table 4

Antibody light chain CDR sequence list LCDR1 LCDR2 LCDR3 HTS0370 SEQ ID NO:47 YAS SEQ ID NO:48 HTS0371 SEQ ID NO:49 LAS SEQ ID NO:50 HTS0372 SEQ ID NO:51 SAS SEQ ID NO:52 HTS0373 SEQ ID NO:53 AAS SEQ ID NO:54 HTS0374 SEQ ID NO:55 ATS SEQ ID NO:56 HTS0375 SEQ ID NO:57 SAS SEQ ID NO:58

Example 3. Preparation of chimeric antibody and detection of binding activity

3-1. Construction of Chimeric Antibody Heavy Chain Expression Vector pTT5-hIgG1

The heavy chain variable region sequences of HTS0370, HTS0371, HTS0372, HTS0373, HTS0374 and HTS0375 were routinely synthesized at Sangon Bioengineering (Shanghai) Co., Ltd., and cloned into IgG1 heavy chain constant region amino acid sequences by homologous recombination. In the pTT5-hIgG1.CH vector, the chimeric antibody heavy chain expression vectors pTT5-HTS0370.VH-hIgG1, pTT5-HTS0371.VH-hIgG1, pTT5-HTS0372.VH-hIgG1, pTT5-HTS0373.VH-hIgG1, pTT5 - HTS0374.VH-hlgG1 and pTT5-HTS0375.VH-hlgG1.

3-2. Construction of chimeric antibody light chain expression vector pTT5-VL

The light chain variable region sequences of HTS0370, HTS0371, HTS0372, HTS0373, HTS0374, and HTS0375 were routinely synthesized at Sangon Bioengineering (Shanghai) Co., Ltd., and cloned into antibody-containing κ light chain constant regions by homologous recombination. In the pTT5-hKappa.CL vector of amino acid sequence CL, chimeric antibody light chain expression vectors pTT5-HTS0370.VL-hKappa, pTT5-HTS0371.VL-hKappa, pTT5-HTS0372.VL-hKappa, pTT5-HTS0373.VL- hKappa, pTT5-HTS0374.VL-hKappa and pTT5-HTS0375.VL-hKappa.

3-3. Expression and purification of chimeric antibodies

293F cells (purchased from ThermoFisher) in the logarithmic growth phase in good growth state were collected and inoculated into 250 mL cell culture flasks and cultured in 50 mL medium, and 25 μg each of light and heavy chain expression plasmids were co-transfected with PEI. The cell supernatant on the 7th day of culture after transfection was collected, centrifuged and filtered using a 0.45 μM filter, the protein A medium was used to purify the antibody, and the antibody was replaced by dialysis into PBS pH7.2 buffer. Absorbance was measured by Nanodrop to determine antibody concentration and purity, and purity was checked by sodium dodecyl sulfate gel electrophoresis and Coomassie staining. The antibody obtained by the combination of pTT5-HTS0370.VH-hIgG1 and pTT5-HTS0370.VL-hKappa is named "xw.HTS0370", and will be obtained by the combination of pTT5-HTS0371.VH-hIgG1 and pTT5-HTS0371.VL-hKappa The antibody obtained through the combination of pTT5-HTS0372.VH-hIgG1 and pTT5-HTS0372.VL-hKappa was named "xw.HTS0371", and the antibody obtained through the combination of pTT5-HTS0372.VH-hIgG1 and pTT5-HTS0372.VL-hKappa was named "xw.HTS0372", and the antibody obtained through the combination of pTT5-HTS0373.VH-hIgG1 and pTT5-HTS0373.VH-hIgG1 and The antibody obtained by combining pTT5-HTS0373.VL-hKappa was named "xw.HTS0373", and the antibody obtained by combining pTT5-HTS0374.VH-hIgG1 and pTT5-HTS0374.VL-hKappa was named "xw.HTS0374", The antibody obtained by combining pTT5-HTS0375.VH-hIgG1 and pTT5-HTS0375.VL-hKappa was named "xw.HTS0375".

3-4. Detection of chimeric antibody binding activity

By means of FACS, the binding activity of the chimeric antibody obtained in Example 3-3 to HEK293-GPRC5D-ZSGreen1 stably transfected cells and endogenous expression cells NCI-H929 (ATCC) overexpressing human GPRC5D was tested, and the positive reference antibody The antibody sequence is from ET150-8 in the patent US20180118803, a chimeric expression vector was constructed and a chimeric antibody xw.ET150-8 was prepared. The detection method is briefly described as follows. Add 5×10 ⁵ HEK293-GPRC5D-ZSGreen1 cells or NCI-H929 to each well in a 96-well V-shaped microplate, centrifuge at 1500r/min for 1min, and discard the supernatant. The chimeric antibody was serially diluted, 50 μL per well, and incubated on ice for 30 min. Then add 150 μL of PBS to each well, centrifuge at 1500 r/min for 1 min, discard the supernatant, and wash the plate 4 times repeatedly. Add APC-labeled goat anti-human IgG (Jackson, Cat. No. 109-605-098, diluted in PBS 1:800), 50 μL per well, and incubate on ice for 30 min. After washing the plate 4 times with PBS, 100 μL of PBS was added to each well to resuspend the cells, and the CytoFLEX was used for detection (Beckman). By GraphPad 8.0.2 analysis, the binding activity of xw.HTS0370, xw.HTS0371, xw.HTS0372, xw.HTS0373, xw.HTS0374 and xw.HTS0375 to the endogenous expression cell line NCI-H929 was significantly higher than that of the positive control antibody xw.ET150 -8, the result is shown in Figure 3. Table 5 shows the half-maximal effect concentration EC50 of the 6 obtained chimeric antibodies and the control antibody.

table 5

Antibody EC50 xw.HTS0370 6.782 xw.HTS0371 148.5 xw.HTS0372 4.817 xw.HTS0373 9.167 xw.HTS0374 4.625 xw.HTS0375 4.307 xw.ET150-8 14.55

The lower the EC50 value, the better the antibody binding activity. According to the above table, except for xw.HTS0371, the binding activity of the other five chimeric antibodies is better than that of the control.

After light and heavy chain germline analysis and sequence comparison, it was found that the antibody sequences of HTS0372-375 were similar, which may be differentiated from the same B cell clone, not only between this group of clones and HTS0370 and HTS0371, but also between HTS0370 and HTS0371 The sequence germlines are completely different, and the differences in the CDR regions are large. Combined with the data of antibody binding cell EC50, the sequences of the two clones with the best binding, xw.HTS0370 and xw.HTS0375, were screened for subsequent humanization.

Example 4. Humanization of murine GRPC5D antibody

4-1. Humanized design scheme

The murine anti-human HTS0370 and HTS0375 antibodies were humanized according to the so-called CDR grafting method. In short, the VH and VK base sequences of HTS0370 and HTS0375 antibodies were analyzed using the IMGT/V-QUEST tool (http://www.imgt.org/IMGT_vquest/input), and the CDR region sequences of the antibody light and heavy chains were determined. The amino acid sequences of the HTS0370 and HTS0375 antibodies were analyzed using the IgBlast tool (https://www.ncbi.nlm.nih.gov/igblast/), and the closest human germline VH and VK sequences of the HTS0370 and HTS0375 antibodies were obtained. The CDRs of the HTS0370 and HTS0375 antibodies were grafted into the framework regions of the selected VH and VK human germline sequences, respectively, and the sequences were humanized antibody sequences. The human germline VH of the HTS0370 and HTS0375 antibody VH was analyzed, and 5 different sequences were selected, and the CDRs of the HTS0370 and HTS0375 antibody VH were grafted into the framework regions of these 5 sequences, and 5 variable heavy chain sequences were obtained for each Area. The light chains of HTS0370 and HTS0375 were CDR-grafted in the same way, and five light chain variable region sequences were obtained respectively.

The humanized sequences of the light and heavy chains are shown in Sequence Listing 6-7:

Table 6

HTS0370 humanized sequence amino acid sequence Nucleotide sequence 370-H1 SEQ ID NO:59 SEQ ID NO:60 370-H2 SEQ ID NO:61 SEQ ID NO:62 370-H3 SEQ ID NO:63 SEQ ID NO:64 370-H4 SEQ ID NO:65 SEQ ID NO:66 370-H5 SEQ ID NO:67 SEQ ID NO:68 370-L1 SEQ ID NO:69 SEQ ID NO:70 370-L2 SEQ ID NO:71 SEQ ID NO:72 370-L3 SEQ ID NO:73 SEQ ID NO:74 370-L4 SEQ ID NO:75 SEQ ID NO:76 370-L5 SEQ ID NO:77 SEQ ID NO:78

Table 7

HTS0375 humanized sequence amino acid sequence Nucleotide sequence 375-H1 SEQ ID NO:79 SEQ ID NO:80 375-H2 SEQ ID NO:81 SEQ ID NO:82 375-H3 SEQ ID NO:83 SEQ ID NO:84 375-H4 SEQ ID NO:85 SEQ ID NO:86 375-H2B SEQ ID NO:87 SEQ ID NO:88 375-L1 SEQ ID NO:89 SEQ ID NO:90 375-L2 SEQ ID NO:91 SEQ ID NO:92 375-L3 SEQ ID NO:93 SEQ ID NO:94 375-L4 SEQ ID NO:95 SEQ ID NO:96

4-2. Preparation of humanized antibody

The heavy chain humanized sequences of HTS0370 and HTS0375 were routinely synthesized at Sangon Bioengineering (Shanghai) Co., Ltd., and cloned into the pTT5-hIgG1.CH vector containing the amino acid sequence of IgG1 heavy chain constant region by means of homologous recombination , to obtain chimeric antibody heavy chain expression plasmids; HTS0370 and HTS0375 light chain humanized sequences were routinely synthesized at Sangon Bioengineering (Shanghai) Co., Ltd., and cloned into pTT5-hKappa.CL vectors by homologous recombination middle. The 293F cells in the logarithmic growth phase in good growth state were inoculated into 250 mL cell culture flasks and cultured in 50 mL medium, and 25 μg of each light and heavy chain expression plasmids were co-transfected with PEI. The cell supernatant on the 7th day of culture after transfection was collected, centrifuged and filtered using a 0.45 μM filter, the protein A medium was used to purify the antibody, and the antibody was replaced by dialysis into PBS pH7.2 buffer. Absorbance was measured by Nanodrop to determine antibody concentration and purity, and purity was checked by sodium dodecyl sulfate gel electrophoresis and Coomassie staining. The names of antibodies corresponding to various combinations of heavy and light chains are listed in List 8-9:

Table 8

Table 9

4-3. Detection of humanized antibody binding activity

By means of FACS, the binding activity of the humanized antibody obtained in Example 4-2 to the GPRC5D overexpression cell line and the endogenous expression cell NCI-H929 (ATCC) was examined. The detection method is briefly described as follows. Add 5×10 ⁵ NCI-H929 cells per well into a 96-well V-shaped microplate, centrifuge at 1500 r/min for 1 min, and discard the supernatant. The humanized antibody was serially diluted, 50 μL per well, and incubated on ice for 30 min. Then add 150 μL of PBS to each well, centrifuge at 1500 r/min for 1 min, discard the supernatant, and wash the plate 4 times repeatedly. Add APC-labeled goat anti-human IgG (Jackson, Cat. No. 109-605-098, diluted in PBS 1:800), 50 μL per well, and incubate on ice for 30 min. After washing the plate 4 times with PBS, 100 μL of PBS was added to each well to resuspend the cells, and the CytoFLEX was used for detection (Beckman). According to GraphPad 8.0.2 analysis, multiple humanized sequences of HTS0370 showed good binding activity to 293 cells with GPRC5D overexpression and endogenous cells NCI-H929 (results are shown in Figure 4 and Figure 5). Table 10-11 shows the EC50 values of various sequences after humanization of HTS0370 and controls, 293 cells with GPRC5D overexpression and endogenous cells NCI-H929.

Table 10

the EC50 293T_GPRC5D_zw.HTS0370Z02 3.513 293T_GPRC5D_zw.HTS0370Z03 2.829 293T_GPRC5D_zw.HTS0370Z04 1.594 293T_GPRC5D_zw.HTS0370Z16 3.812 293T_GPRC5D_zw.HTS0370Z18 1.802 293T_GPRC5D_zw.HTS0370Z22 1.094 293T_GPRC5D_zw.HTS0370Z23 3.887 293T_GPRC5D_xw.HTS0370 2.930 293T_GPRC5D_zw.BMK.ET150-8 4.102

Table 11

the EC50 H929zw.HTS0370Z02 0.8621 H929zw.HTS0370Z03 0.3915 H929zw.HTS0370Z04 0.2475 H929zw.HTS0370Z16 0.4676 H929zw.HTS0370Z18 0.2726 H929zw.HTS0370Z22 0.2536 H929zw.HTS0370Z23 0.4940 H929xw.HTS0370 1.231 H929zw.BMK.ET150-8 19.85

According to the results of the above two tables, from the perspective of showing good binding activity with GPRC5D overexpressed 293 cells and endogenous cell NCI-H929, two humanized HTS0370 with good binding activity were selected. Humanized antibodies: zw.HTS0370Z02, zw.HTS0370Z03, zw.HTS0370Z04, zw.HTS0370Z16, zw.HTS0370Z18, zw.HTS0370Z22, zw.HTS0370Z23. Among them, combining the two results, we selected zw.HTS0370Z22 as the best, and carried out follow-up research on it. Table 12-13 shows the humanized sequence of HTS0375 and the EC50 values of the control, 293 cells with GPRC5D overexpression and endogenous cell NCI-H929.

Table 12

the EC50 293T_GPRC5D zw.HTS0375Z56 1.936 293T_GPRC5D xw.HT0375 1.936 293T_GPRC5D_zw.BMK.ET150-8λ 1.719

Table 13

the EC50 H929zw.H0375Z56 0.9438 H929xw.H0375 0.2424 H929zw.BMK.ET150-8λ 10.59

Similarly, the zw.HTS0375Z56 molecule obtained after humanizing HTS0375 by the same method was selected to have the best comprehensive binding activity, and entered into follow-up research. The results are shown in Figure 6 and Figure 7 .

Example 5: Detection of tissue cross-reactivity between antibodies and various blood cells

By flow cytometry, FITC-CD3, FITC-CD14, FITC-CD19, and PercpCy5.5-CD11b represent T cells, monocytes, B cells, NK cells, and granulocytes, respectively, using biotinylated The humanized antibody obtained in part 4-2 of Example 4 was stained with double antibodies, and the antibody of PE-SA was used for the secondary antibody labeling of the GPRC5D antibody to be detected. Briefly, 1×10 ⁶ human PBMC cells (purchased from Shanghai Miaoshun Biotechnology Co., Ltd.) were added to each well in a 96-well V-shaped microplate, centrifuged at 1500 r/min for 1 min, and the supernatant was discarded. Add 1 μg/well of GPRC5D humanized antibody to be detected, 50 μL per well, and incubate on ice for 30 minutes. Then add 150 μL of PBS to each well, centrifuge at 1500 r/min for 1 min, discard the supernatant, and wash the plate 4 times repeatedly. Add PE-SA (BD, Cat. No. 554061, PBS 1:20 dilution), 25 μL per well, and add various PBMC blood cell antibodies at the same time, the final concentration refers to the antibody manual, the volume is 25 μL, mix everything, and incubate on ice for 30 minutes. After washing the plate 4 times with PBS, 100 μL of PBS was added to each well to resuspend the cells, and the CytoFLEX was used for detection (Beckman). According to the analysis of the instrument’s built-in software, the humanized antibody does not bind to these five types of blood cells, but the xw.ET150-8 antibody from the prior art binds to granulocytes represented by CD11b and monocytes represented by CD14 , all have non-specific binding, suggesting that this antibody may have serious side effects. The results of non-specific binding of humanized antibody and control to CD19, CD3, CD11b, CD14 positive cells respectively are shown in Figure 8 to Figure 11. According to the results, the humanized molecules zw.HTS0370Z22 and zw.HTS0375Z56 had no non-specific binding to CD19, CD3, CD11b, and CD14-positive cell populations. In contrast, ETC150 had non-specific binding to CD11b and CD14 cell populations. binding effect. Since HTS0370, HTS0375 molecules and their derived chimeric antibodies and humanized antibodies have the same CDR, that is, the same surface antigen binding site, therefore, the results show that HTS0370, HTS0375 and other antibody forms derived from the same CDR, will Target GPRC5D more specifically, thereby reducing the risk of off-target toxicity.

Example 6: The binding ability of antibodies to bind to GPRC5D protein

The binding activity of the humanized antibody obtained in part 4-2 of Example 4 to the VLP-like protein of GPRC5D (Kactus Biosystems) was tested by ELISA. The detection method is briefly described as follows. Dilute the VLP protein of GPRC5D to a concentration of 1 μg/ml, spread it on a 384-well microtiter plate overnight, discard the protein the next day, and use 80 μl of 3% milk (dissolved in PBS) to block for 2 hours , wash 3 times with 80 μl of PBST (1‰ Tween20), then serially dilute the humanized antibody obtained in 4-2, add 25 μL per well to the blocked microtiter plate, and react at room temperature for 1 hour , then discarded the antibody, washed 5 times with 80 μL of PBST (1‰ Tween20), added 25 μL of HRP-labeled goat anti-human secondary antibody (Sino biological, Cat. No. SSA002, 1:10000), incubated at room temperature for 1 hour, and then Discard the antibody and wash 7 times with 80 μL of PBST (1‰ Tween20). Add 25 μL of TMB chromogenic solution to develop color for 10 minutes, add 2M HCl to terminate the reaction, and complete the reading within 30 minutes. The antibody sequence of the positive reference antibody GC5B596 comes from SEQ90 and SEQ96 in the patent US10562968. According to GraphPad 8.0.2 analysis, the humanized antibody binds to the VLP protein of GPRC5D, and its binding activity is better than that of GC5B596. The results are shown in Figure 12. Comparing the results of the cell binding activity of the humanized antibodies above, it can be seen that the difference in protein binding level between HTS0375 humanized antibody zw.HTS0375Z56 and HTS0370 humanized antibody zw.HTS0370Z22 is significantly larger than the difference in cell binding.

Example 7: Species cross-reactivity of antibody binding to human and monkey GPRC5D cell lines

The constructed monkey GPRC5D cells were tested for binding activity between the humanized antibody obtained in part 4-2 of Example 4 and HEK293-GPRC5D stably transfected cells overexpressing monkey GPRC5D by FACS method, positive reference antibody The antibody sequence of GC5B596 comes from SEQ90 and SEQ96 in the patent US10562968. The detection method is briefly described as follows. Add 5×10 ⁵ HEK293-GPRC5D cells to each well in a 96-well V-shaped microplate, centrifuge at 1500 r/min for 1 min, and discard the supernatant. The humanized antibody was serially diluted, 50 μL per well, and incubated on ice for 30 min. Then add 150 μL of PBS to each well, centrifuge at 1500 r/min for 1 min, discard the supernatant, and wash the plate 4 times repeatedly. Add APC-labeled goat anti-human IgG (Jackson, Cat. No. 109-605-098, diluted in PBS 1:800), 50 μL per well, and incubate on ice for 30 min. After washing the plate 4 times with PBS, 100 μL of PBS was added to each well to resuspend the cells, and the CytoFLEX was used for detection (Beckman). According to GraphPad 8.0.2 analysis, the binding activity of zw.HTS0370Z22 and zw.HTS0375Z56 to monkey expression cell lines was significantly higher than that of the positive control antibody. The results are shown in Figure 13. Humanized antibodies have the characteristic of cross-species reactivity, which will facilitate the toxicity analysis of antibodies in monkeys, facilitate verification tests in related animals, and facilitate the subsequent development of therapeutic applications.

Example 8: Endocytosis of antibodies

Using the FACS method, using NCI-H929 cells, to test the endocytic activity of the humanized antibody obtained in part 4-2 of Example 4, the antibody sequence of the positive reference antibody GC5B596 comes from SEQ90 and SEQ96 in the patent US10562968, and A positive reference antibody was constructed based on the ET150-8scFv sequence in the patent US20180118803. The detection method is briefly described as follows. 4 parts of 5×10 ⁵ NCI-H929 cells were divided into 96-well V-shaped microplates, centrifuged at 1500 r/min for 1 min, and the supernatant was discarded. Add saturated 1 μg/ml antibody to be detected, 200 μl per well, and incubate on ice for 30 minutes. Then add 150 μL of PBS to each well, centrifuge at 1500 r/min for 1 min, discard the supernatant, and wash the plate 4 times repeatedly. Resuspend with 200 μL of PBS, take out 1 portion and place it in a 37°C cell culture incubator for 2 hours, and divide the other 2 portions under the same conditions and place it for 1 hour and 0.5 hour respectively, and place the last portion on ice for time After the end, put them all at 4°C and centrifuge together at 1500r/min, then add APC-labeled goat anti-human IgG (Jackson, product number 109-605-098, diluted in PBS 1:800), 50μL per well, and incubate on ice for 30min. After washing the plate 4 times with PBS, 100 μL of PBS was added to each well to resuspend the cells, and the CytoFLEX was used for detection (Beckman). According to GraphPad 8.0.2 analysis, zw.HTS0370Z22 and zw.HTS0375Z56 had obvious endocytic activity, among which zw.HTS0370Z22 had obvious endocytic activity, while the positive control antibody GC5B596 had no endocytic activity. The results are shown in Figure 14. It is suggested that zw.HTS0370Z22 and zw.HTS0375Z56 are more suitable for the subsequent development of ADC drugs than the positive control, and zw.HTS0370Z22 has better potential.

Example 9: Mutation of the CDR region of an antibody

375H2B and 375-L1 were constructed together on the phage vector to construct the wild-type template of the single-chain antibody. The CDR region of the single-chain antibody is mutated based on this template. After analyzing amino acid conservation and mutation by using the Kabat database for HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 regions, point mutations were performed at defined sites for HCDR2, HCDR3, LCDR1, and LCDR3, respectively. The primers used for the mutation of the CDR region are shown in Table 14, wherein S=G/C, N=A/G/C/T.

Table 14. Primers used for CDR region mutations

X1-F GGATGGGGCTGATCAACCCTNNSAATGGCAGGACAATCTACAA X1-R TTGTAGATTGTCCTGCCATTSNNAGGGTTGATCAGCCCCATCC X2-F ATCAACCCCTTACAATGGCNNSACAATCTACAAACCAAAAG X2-R CTTTTGGTTGTAGATTGTSNNNGCCATTGTAAGGGTTGAT X3-F GTATACTACTGCGCCCGTNNSGCCTTAAGATATGCTATG X3-R CATAGCATATCTTAAGGCSNNACGGGCGCAGTAGTATAC X4-F ACATGCAAAGCCTCTCAANNSGTATACACGAATTTGGCT X4-R AGCCAAATTCGTGTATACSNNTTGAGAGGCTTTGCATGT X5-F TGCAAAGCCTCTCAAAGCNNSTACACGAATTTGGCTTGG X5-R CCAAGCCAAATTCGTGTASNNGCTTTGAGAGGCTTTGCA X6-F AAAGCCTCTCAAAAGCGTANNSACGAATTTGGCTTGGTTC

X6-R GAACCAAGCCAAATTCGTSNNTACGCTTTGAGAGGCTTT X7-F TCTCAAAGCGTATACACGNNSTTGGCTTGGTTCCAACAG X7-R CTGTTGGAACCAAGCCAASNNCGTGTATACGCTTTGAGA X8-F ACGTACTATTGCCAACAGNNSAACAGCTACCCGGTAACT X8-R AGTTACCGGGTAGCTGTTSNNCTGTTGGCAATAGTACGT X9-R TACTATTGCCAACAGTATNNSAGCTACCCGGTAACTTTC X9-R GAAAGTTACCGGGTAGCTSNNATACTGTTGGCAATAGTA X10-F TATTGCCAACAGTATAACNNSTACCCGGTAACTTTCGGG X10-R CCCGAAAGTTACCGGGTASNNGTTATACTGTTGGCAATA X11-F TGCCAACAGTATAACAGCNNSCCGGTAACTTTCGGGCAA X11-R TTGCCCGAAAGTTACCGGSNNGCTGTTATACTGTTGGCA

Using fast high-fidelity enzyme PrimeSTAR Max DNA Polymerase (Takara Company, cat#R045A) and primers for each CDR region, the four CDR regions were subjected to full-plasmid PCR amplification, and the PCR amplification products were recovered from the gel and digested with Dpn I and precipitated with ethanol. After that, it was transformed into the host Escherichia coli cell TG1 to obtain the mutant. The mutants were verified by FACS using the induction method and detection method of Example 2.3, and the verification results of the mutants are shown in Figures 15-25. Confirm the final amino acid position by selecting mutants with the same binding ability as wild type. Confirmed sequences are shown in SEQ ID NO:99-102.

Among them, the heavy chain complementarity determining region HCDR2 is the amino acid sequence of INPX ₁ NGX ₂ T (SEQ ID NO: 99), as shown by the results in Figure 15 and Figure 16, X ₁ can be selected from Y, P, R, L, D, Any amino acid of S, G, K, V, T, M and Q, _X2 can be selected from any amino acid of R and A. The heavy chain complementarity determining region HCDR3 is the amino acid sequence of ARX ₃ ALRYAMDY (SEQ ID NO: 100). The results in Figure 17 show that X ₃ can be selected from any amino acid of V and A. The light chain complementary determining region LCDR1 is the amino acid sequence of QX ₄ X ₅ X ₆ TX ₇ (SEQ ID NO: 101), as shown by the results of Figures 18-21, X ₄ can be selected from any amino acid of S and A, X ₅ Can be selected from any amino acid of V and A, _X6 can be selected from any amino acid of S, V, L, R, H, E, G, Q, M and Y, _X7 can be selected from R, P, H , any amino acid of S, W, I, G, V and N. The light chain complementary determining region LCDR3 is the amino acid sequence of QQX ₈ X ₉ X ₁₀ X ₁₁ PVT (SEQ ID NO: 102), as shown by the results in Figures 22-25, X ₈ can be selected from any amino acid of Y and A, X ₉ can be selected from any amino acid of N and A, X ₁₀ can be selected from any amino acid of S and A, and X ₁₁ can be selected from any amino acid of Y and A.

Example 10: Antibody cross-binding reaction with paralogous proteins

In order to verify the binding specificity of the antibody, by comparing the homology relationship between GPRC5D and paralogous proteins, it was found that it has the closest homology relationship with the three proteins of the same family, GPRC5A, GPRC5B, and GPRC5C, about 20-40% similarity. In order to detect the binding specificity of the antibody of the present invention, the present invention constructed three gene overexpression cell lines, and the construction method of the overexpression cells is as described in Examples 1-2. The overexpression effect of the cells was verified by tag antibody FACS experiments. According to the FACS results of antibody binding to these types of cells, it can be seen that the humanized HTS0375Z56 and HTS0370Z22 molecules do not cross-link related proteins, indicating that the antibody has no cross-reactivity with the collateral genes of the same family and has good binding specificity. The results are shown in Figure 26 and Figure 27.

Example 11: Identification of antibody binding to GPRC5D region

Since GC5B596, ET150.8 and candidate HTS0375Z56 and HTS0370Z22 molecules do not bind to GPRC5A molecules (the closest homologous molecules of GPRC5D), we respectively constructed GPRC5D overexpression cell lines chimerized with four different GPRC5A extracellular domains: GPRC5D-A1, GPRC5D-A2, GPRC5D-A3, GPRC5D-A4 are used to verify the binding region of the antibody. The schematic diagram of the four chimeric molecules is shown in Figure 28, and the construction method of overexpression cells is as described in Example 1-2. The overexpression effect of the constructed cell line was detected by tag antibody FACS, and the results are shown in Figure 29, all molecules had a good overexpression effect. On this basis, we tested the binding ability of humanized and control antibodies to these four kinds of cells respectively. The specific experimental method is as follows: 5×10 ⁵ overexpressed cells of GPRC5D-A1-A4 were placed in a 96-well V-shaped microwell plate, centrifuged at 1500 r/min for 1 min, and the supernatant was discarded. Add saturated 1 μg/ml antibody to be detected, 200 μL per well, and incubate on ice for 30 min. Then add 150 μL of PBS to each well, centrifuge at 1500 r/min for 1 min, discard the supernatant, and wash the plate 4 times repeatedly. Add APC-labeled goat anti-human IgG (Jackson, diluted in PBS 1:800), 50 μL per well, and incubate on ice for 30 min. After washing the plate 4 times with PBS, 100 μL of PBS was added to each well to resuspend the cells, and CytoFLEX was used for detection (Beckman). The results are shown in Figure 30. The results showed that: 1) ET150.8 mainly binds to the N-terminal extracellular region of GRPC5D, while the other three molecules do not bind to the N-terminal extracellular region; 2) GC5B596 antibody binds to three extracellular regions except the N-terminal; 3) The HTS0375Z56 and HTS0370Z22 molecules mainly bind through the second extracellular region near the C-terminus, which is different from the binding regions of other molecules.

The nucleotide sequence of GPRC5D-A1 is:

The nucleic acid amino acid sequence of GPRC5D-A1 is:

The nucleotide sequence of GPRC5D-A2 is:

The amino acid sequence of GPRC5D-A2 is:

The nucleotide sequence of GPRC5D-A3 is:

The amino acid sequence of GPRC5D-A3 is:

The nucleotide sequence of GPRC5D-A4 is:

The amino acid sequence of GPRC5D-A4 is:

The above experimental results show that compared with the existing published antibodies, the antibodies disclosed in the present invention have at least the following advantages: 1) The CDR sequence has a large difference, which is different from the GPRC5D antigen-binding epitope; 2) The binding activity is good; 3) The specificity is stronger , good safety; 4) has species cross-reactivity, which is convenient for the development of therapeutic products.

At the same time, the antibody of the present invention can also be combined with antibodies targeting other targets, such as: CD3, BCMA, CD38, etc. to construct bispecific antibodies, and developed into various methods that can regulate tumor cells. In addition, the antibodies invented by this technology can also be coupled to other types of molecules, such as toxins, nucleic acid molecules, etc., and the antibodies can specifically bring the coupled molecules into the tumor cells, thereby regulating the effect of tumor cells. In addition, the antibody invented by the present technology can also be developed into a method for detecting the expression level of GPRC5D on the surface of tissue cells by means of immunization.

The above description is only an embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent transformations made by using the description of the present invention and the contents of the accompanying drawings, or directly or indirectly used in related technical fields, are all included in the same principle. Within the scope of patent protection of the present invention.

Claims (17)

An antibody, characterized in that the antibody comprises a heavy chain variable region and/or a light chain variable region: The heavy chain variable region, which comprises: The heavy chain complementarity determining region HCDR1 consisting of the amino acid sequence shown in SEQ ID NO:44, The heavy chain complementarity determining region HCDR2 consisting of the amino acid sequence of INPX ₁ NGX ₂ T, Heavy chain complementarity determining region HCDR3 consisting of ARX ₃ ALRYAMDY amino acid sequence; The light chain variable region, which comprises: The light chain complementarity determining region LCDR1 consisting of the amino acid sequence of QX ₄ X ₅ X ₆ TX ₇ , The light chain complementarity determining region LCDR2 consisting of the amino acid sequence of SAS, Light chain complementarity determining region LCDR3 composed of the amino acid sequence of QQX ₈ X ₉ X ₁₀ X ₁₁ PVT; in, X ₁ : represent any amino acid of Y, P, R, L, D, S, G, K, V, T, M and Q, X ₂ : represent any amino acid of R and A, X ₃ : represent any amino acid of V and A, X ₄ : represent any amino acid of S and A, X ₅ : represent any amino acid of V and A, X ₆ : represent any amino acid of S, V, L, R, H, E, G, Q, M and Y, X ₇ : represent any amino acid of R, P, H, S, W, I, G, V and N, X ₈ : represent any amino acid of Y and A, X ₉ : represent any amino acid of N and A, X ₁₀ : represent any amino acid of S and A, X ₁₁ : represents any amino acid of Y and A. The antibody according to claim 1, wherein the antibody is a murine antibody, a chimeric antibody or a human antibody. The antibody according to claim 1 or 2, wherein the heavy chain variable region is selected from the group consisting of SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85 or SEQ ID The amino acid sequence shown in NO:87; the light chain variable region is selected from the amino acid sequence such as SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95 or SEQ ID NO:97 . The antibody according to claim 3, wherein the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:87, and the light chain variable region is the amino acid sequence shown in SEQ ID NO:89 sequence. An antibody, characterized in that the antibody comprises a heavy chain variable region and/or a light chain variable region: The heavy chain variable region, which comprises: The heavy chain complementarity determining region HCDR1 consisting of the amino acid sequence shown in SEQ ID NO:29, The heavy chain complementarity determining region HCDR2 consisting of the amino acid sequence shown in SEQ ID NO:30, The heavy chain complementarity determining region HCDR3 consisting of the amino acid sequence shown in SEQ ID NO:31; The light chain variable region, which comprises: The light chain complementarity determining region LCDR1 consisting of the amino acid sequence shown in SEQ ID NO:47, Light chain complementarity determining region LCDR2 consisting of YAS amino acid sequence, A light chain complementarity determining region LCDR3 consisting of the amino acid sequence shown in SEQ ID NO:48. The antibody according to claim 5, wherein the antibody is a murine antibody, a chimeric antibody or a human antibody. The antibody according to claim 5 or 6, wherein the heavy chain variable region is selected from the group consisting of SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 or SEQ ID The amino acid sequence shown in NO:67; The light chain variable region is selected from those shown in SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75 or SEQ ID NO:77 amino acid sequence. The antibody according to claim 7, wherein the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:59, and the light chain variable region is the amino acid sequence shown in SEQ ID NO:71 sequence; Or the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:59, and the sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:73; Or the sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:59, and the sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:75; Or the sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:65, and the sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:69; Or the sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:65, and the sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:73; Or the sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:67, and the sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:71; Or the sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:67, and/or the sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:73. The antibody according to any one of claims 1 to 8, characterized in that it comprises any of the following properties i to iv: i. The antibody can specifically bind to G protein-coupled receptor C5 family subtype D; ii. the antibody does not bind any of CD19, CD3, CD11b or CD14; iii. The antibody specifically binds to human GPRC5D and cross-reacts with monkey GPRC5D; iv. The antibody has endocytic activity. A recombinant protein, characterized in that the recombinant protein comprises the antibody according to any one of claims 1-8. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the antibody according to any one of claims 1 to 8 or the recombinant protein according to claim 10. A polynucleotide, characterized in that the polynucleotide comprises a nucleotide sequence encoding the antibody according to claim 1 or 2 or the recombinant protein according to claim 10. A vector, characterized in that the vector comprises the polynucleotide according to claim 12. An isolated cell, characterized in that the cell produces the antibody according to any one of claims 1 to 8 or the recombinant protein according to claim 10. A method for preparing an antibody, characterized by comprising the following steps: cultivating the isolated cell according to claim 14, and recovering the antibody from the culture. Use of the antibody according to any one of claims 1 to 8 or the recombinant protein according to claim 10 in the preparation of a drug or pharmaceutical composition for treating cancer; preferably, the cancer is multiple myeloma. Application of the antibody described in any one of claims 1 to 8 or the recombinant protein described in claim 10 in the preparation of an antibody detection kit.

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