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CN115386006A - anti-GPRC 5D antibody, preparation method and application thereof - Google Patents

anti-GPRC 5D antibody, preparation method and application thereof Download PDF

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CN115386006A
CN115386006A CN202110561819.5A CN202110561819A CN115386006A CN 115386006 A CN115386006 A CN 115386006A CN 202110561819 A CN202110561819 A CN 202110561819A CN 115386006 A CN115386006 A CN 115386006A
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魏海涛
褚敏
尚玉栓
李亚男
王平
董国良
李秀兰
罗雪琴
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Shanghai Xiangyao Biotechnology Co ltd
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Abstract

The invention discloses an anti-GPRC 5D antibody, a preparation method and application thereof, wherein the antibody comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprises amino acid sequences shown as SEQ ID NO:29 (HCDR 1), SEQ ID NO:30 (HCDR 2) and SEQ ID NO:31 (HCDR 3), and the light chain variable region comprises amino acid sequences shown as SEQ ID NO:47 (LCDR 1), YAS (LCDR 2) and SEQ ID NO:48 (LCDR 3). The anti-GPRC 5D antibody has stronger binding activity, reaction specificity, species cross-reactivity and endocytosis activity, and is suitable for treatment and product development of related diseases such as subsequent cancers.

Description

anti-GPRC 5D antibody, preparation method and application thereof
Technical Field
The invention relates to the technical field of antibodies, in particular to an anti-GPRC 5D antibody, a preparation method and application thereof.
Background
Multiple Myeloma (MM) is the second most common hematological tumor worldwide after non-hodgkin's lymphoma, accounting for approximately 10% of hematological malignancies. According to the global cancer statistical report issued by the WHO, 20,066 cases of new-onset multiple myeloma patients and 14,665 cases of death patients in the Chinese of 2018; the number of patients with the disease is 44,643 cases in 5 years. And the morbidity and mortality of patients escalate with age. Therefore, with the increasing aging degree of the population in China, the total number of patients will gradually increase, and the clinical demand is huge. In the last decade, the application of immunomodulators represented by thalidomide and derivatives lenalidomide thereof and small-molecule proteasome inhibitors represented by bortezomib greatly improves the remission rate and survival time of MM patients. At present, the treatment scheme aiming at multiple myeloma in China can be divided into three categories: immunomodulators, proteasome inhibitors and anti-CD 38 monoclonal antibodies. The immunomodulator and the proteasome inhibitor are mainly used for the first-line combination treatment and the maintenance treatment after transplantation, which are received by patients meeting the transplantation condition of stem cells before transplantation. First line treatment second line treatment regimen for relapsed or refractory multiple myeloma a 2019 conditional approval by the national drug administration (NMPA) of the anti-CD 38 mab Daratumumab marketed by import registration is considered. After a short period of treatment, however, the patient may be in an embarrassment situation where no medicine is available after a relapse. Thus, the relapse and refractory nature of MM continues to plague people, driving the search for new therapeutic targets and methods for use in the case of MM relapse/refractory.
The development of targeting therapies for MM is accelerated by the rapid development of related technologies of small-molecule inhibitors for treating relapse and biological drugs. Currently FDA approved biomacromolecule targeted therapies include targeting three major targets, plasma Cell surface protein CD38, signaling lymphocyte Activating Molecule family member 7 (slamf 7), and B Cell Maturation Antigen (BCMA), and the types of approved drugs mainly include: monoclonal antibodies and Antibody-conjugated drugs (ADC). The expression of BCMA protein on MM cells was more specific compared to CD38, SLAMF7 target. The FDA accelerated approval of BMCA-targeted ADC drug, blenrep, for the treatment of patients with median 7-line recurrence in the united states 8 months of 2020, with an overall remission rate still reaching 31% and median duration of remission (DoR) greater than 6 months. Furthermore, the anti-tumor efficacy of BCMA-targeting CAR-T or CD3 bispecific antibodies by mediating T cells is also significant. The BCAM medicament development racetrack is participated by a plurality of international famous biological medicine companies including blue birds, BMS, advance, producer, renaugh, nanjing legend and the like, and the competition is very violent. Under the vigorous guidance of the government of China in recent years, the Chinese biomedical industry rises rapidly, and a plurality of MM-layout targeted new drug research and development companies are developed, but most of MM-layout targeted new drug research and development companies focus on the double resistance and CAR-T direction of CD38 and BCMA targets, and the MM-layout targeted new drug research and development companies face great homogenization competition pressure at home and abroad. In addition, although BCMA-targeted ADC, dual antibody and CAR-T therapies have shown positive clinical efficacy, negative (or low expression) of BCMA and associated post-treatment relapse cases have been reported, highlighting the need for more targets for multiple myeloma treatment.
The G protein-coupled receptor (GPCR) is a protein receptor with 7 transmembrane helices, which is divided into 5 subfamilies according to sequence similarity and ligand binding, is the largest protein family in human body, and is also an important drug target. G protein-coupled receptor subtype C5D (GPRC 5D) is the earliest orphan atypical C-class GPCR identified in 2001 (Brauner-Osborne, h., et al. Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D. Biochim biophysis Acta,2001.1518 (3): p.237-48). GPCR family group C5 receptors (GPRC 5 receptors) share 4 subtypes, namely GPRC5A, GPRC5B, GPRC5C and GPRC5D, which are expressed induced by retinoic acid, and are therefore also known as retinoic acid-induced orphan G protein-coupled receptors (Inoue, s., t.nambu, and t.shimomura, the RAIG family member, GPRC5D, associated with hard-keyed structures. Journal of Investigative Dermatology,2004.122 (3): p.565-573).
GPRC5D has been previously identified in cells from patients with multiple myeloma, however has not been applied to clinical development because of the lack of protein expression profiling studies. Studies until 2019 reported that GPRC5D was highly expressed in plasma cells of multiple myeloma, much not expressed in normal tissues, and expressed only in the region of the hair follicle with immune privilege (Smith, e.l., et al, GPRC5D is a target for the immunization of multiple myelomas with a rating assigned CAR T cells, science relative Medicine,2019.11 (485)). It has been reported that high expression of GPRC5D is associated with an adverse prognosis in multiple myeloma (Atamaniuk, J., et al., overexpression of G protein-coupled receptor 5D in the bone marrow associated with bone cancer with multiple myeloma. European Journal of Clinical investigation,2012.42 (9): p.953-960). Furthermore, another important finding is that the expression profile of GPRC5D does not overlap with BCMA and therefore would likely be a novel therapeutic target for low/no BCMA expression or following recurrence of BCMA therapy. The inventor group deeply ploughs the GPCR and tumor immunity field for a long time, and then starts a new medicine development project for the GPRC5D specific antibody when the research result of the project is concerned. However, since GPCR proteins belong to 7 transmembrane proteins, the structure is complex and the antigen is difficult to obtain, conventional antibody development schemes are not basically feasible. Based on the fact that an inventor group has long dedicated to the innovation, research and development and accumulation of key technologies in the field of antibody new drug development, the method breaks through multiple key technical difficulties of target immunization, monoclonal antibody generation, screening and the like, successfully screens multiple specific and sensitive GPRC5D antibodies, and verifies the pharmaceutical activity of the antibodies.
GPRC5D is a surface antigen expected to be used for MM immunotherapy, but at present, most GPRC5D drugs are still in clinical trial or development stage, and no drug targeting GPRC5D is yet on the market, so that there is a need to further develop GPRC5D drugs with higher activity, higher affinity, higher specificity, and better therapeutic effect for therapeutic research and application of related diseases.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides an antibody which can be combined with a G protein coupled receptor C5 family subtype D (GPRC 5D) with high specificity.
The invention also provides a recombinant protein, a pharmaceutical composition, a polynucleotide, a recombinant plasmid and an isolated cell related to the antibody.
The invention also provides a preparation method of the antibody.
The invention also provides the application of the antibody in preparing anti-cancer drugs.
The invention also provides application of the antibody in preparation of an antibody detection kit.
An antibody according to an embodiment of the first aspect of the invention, which antibody comprises a heavy chain variable region and/or a light chain variable region:
the heavy chain variable region comprising: a heavy chain complementarity determining region HCDR1 consisting of the amino acid sequence shown by SEQ ID NO. 29, a heavy chain complementarity determining region HCDR2 consisting of the amino acid sequence shown by SEQ ID NO. 30, and a heavy chain complementarity determining region HCDR3 consisting of the amino acid sequence shown by SEQ ID NO. 31;
the light chain variable region comprising: a light chain complementarity determining region LCDR1 consisting of the amino acid sequence shown in SEQ ID NO:47, a light chain complementarity determining region LCDR2 consisting of the YAS amino acid sequence, and a light chain complementarity determining region LCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 48.
The antibody according to the embodiment of the present invention has at least the following advantageous effects: the antibody of the invention has (1) good binding activity and stronger binding capacity with GPRC5D protein; (2) The specificity is strong, the antibody is not combined with antibodies such as CD19, CD3, CD11b and CD14, and the GPRC5D can be targeted more specifically, so that the toxicity caused by off-target is reduced, and the safety is better; (3) Species cross-reactivity, namely, the compound has cross-reactivity with monkey GPRC5D, is favorable for developing verification experiments and subsequent product development with therapeutic application; (4) The antibody has endocytosis effect, has endocytosis activity in cells, and is suitable for the development of ADC drugs.
According to some embodiments of the invention, the antibody is a murine antibody, a chimeric antibody or a human antibody.
In the invention, the base sequence of CDS region of human GPRC5D gene is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO. 4.
In the present invention, the antibody comprises, in addition to the sequences of the heavy chain variable region and the light chain variable region, a heavy chain constant region amino acid sequence comprising lgG1 and a light chain constant region amino acid sequence comprising Kappa.
According to some embodiments of the invention, the heavy chain variable region is selected from the group consisting of the amino acid sequence shown as SEQ ID NO 59, SEQ ID NO 61, SEQ ID NO 63, SEQ ID NO 65, and SEQ ID NO 67; the light chain variable region is selected from the amino acid sequence shown in SEQ ID NO 69, 71, 73, 75 or 77.
According to some embodiments of the 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 has an amino acid sequence shown as SEQ ID NO. 59, and the light chain variable region has an amino acid sequence shown as SEQ ID NO. 73;
or the sequence of the heavy chain variable region is the amino acid sequence shown by SEQ ID NO. 59, and the sequence of the light chain variable region is the amino acid sequence shown by SEQ ID NO. 75;
or the sequence of the heavy chain variable region is the amino acid sequence shown by SEQ ID NO. 65, and the sequence of the light chain variable region is the amino acid sequence shown by 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 invention, the antibody comprises any one of the following properties i to v:
i. the antibody is capable of specifically binding to subtype D of the C5 family of G protein-coupled receptors;
ii. The anti-GPRC 5D antibody does not bind to any one of CD19, CD3, CD11b, or CD 14;
iii, the anti-GPRC 5D antibody specifically binds to human GPRC5D and cross-reacts with monkey GPRC 5D;
iv, the anti-GPRC 5D antibody has endocytic activity;
v, said antibody has reduced glycosylation or is aglycosylated or is low fucosylated.
In the present invention, the above antibody sequences have optimal binding activity, reaction specificity, species cross-reactivity and endocytic activity. The experiments of the examples can prove that the protein binding level of the anti-GPRC 5D antibody is obviously increased compared with that of the antibody in the prior art; the polypeptide has binding specificity, is not non-specifically bound with CD19, CD3, CD11b and CD14 positive cells, and is more specifically targeted to GPRC5D, so that the toxicity risk caused by off-target is reduced; the method has the characteristic of species cross reaction, is beneficial to the toxicity analysis of the antibody in the monkey, is beneficial to the development of verification tests in related animals, and is further beneficial to the application and development of subsequent therapeutic application; has obvious endocytic activity and is more suitable for the subsequent development of the drugs of antibody-drug conjugates (ADC).
In the present invention, the glycosylation of the Fc region is modified to enhance Fc γ R binding compared to an unmodified Fc region. In some embodiments, the Fc region lacks fucose content or has reduced fucose content.
A recombinant protein, pharmaceutical composition, polynucleotide, vector or isolated cell according to an embodiment of the second aspect of the invention: the recombinant protein comprises the antibody; the pharmaceutical composition comprises the above antibody or the above recombinant protein; the polynucleotide comprises a nucleotide sequence encoding the antibody or recombinant protein; the vector comprises the polynucleotide; the isolated cells produce the above antibodies.
According to some embodiments of the invention, the recombinant protein further comprises a tag sequence that aids in expression and/or purification.
According to some embodiments of the invention, the recombinant protein is a diabody, i.e. also comprises an antibody capable of binding to other target proteins. Further, the double antibodies also include antibodies that specifically bind to CD3, antibodies that specifically bind to different epitopes of GPRC5D.
According to some embodiments of the invention, the pharmaceutical composition further comprises the above-described diabody.
According to some embodiments of the invention, the pharmaceutical composition further comprises an ADC medicament comprising the above antibody. Further, the ADC drug further comprises a linker arm and a toxic molecule.
According to some embodiments of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
The production method according to an embodiment of the third aspect of the present invention includes the steps of: culturing the isolated cell, and recovering the antibody from the culture.
In the invention, the specific preparation method comprises the following steps: the heavy chain variable region sequence encoding the anti-GPRC 5D antibody is cloned into a recombinant plasmid 1 containing an IgG1 heavy chain constant region amino acid sequence, the light chain variable region sequence is cloned into a recombinant plasmid 2 containing a Kappa light chain constant region amino acid sequence, the recombinant plasmid 1 and the recombinant plasmid 2 are transfected into cells at the same time and cultured, and the anti-GPRC 5D antibody is recovered from the culture.
According to the use of the embodiment of the fourth aspect of the present invention, the antibody may be used in the preparation of an anticancer drug and/or an antibody detection kit.
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, renal cancer, liver cancer, pancreatic cancer, colorectal cancer, esophageal cancer, bladder cancer, cervical cancer, hematological cancer, lymphoma or malignant melanoma expressing GPRC5D protein. Further wherein the cancer is multiple myeloma that expresses GPRC5D protein.
In the present invention, the anti-cancer drug also comprises antibodies that target other targets, such as: CD3, BCMA, CD38, etc. are constructed as bispecific antibodies and developed into various tumor cell regulating methods.
In the present invention, the antibodies of the invention may also be prepared by coupling other types of molecules, such as: toxins, nucleic acid molecules, etc., which specifically bring the conjugated molecule into the tumor cell body via the antibody, thereby modulating the action of the tumor cell. For example, the antibodies may be used to treat cancer by interfering with GPRC 5D-receptor interactions or wherein the antibodies are conjugated to toxins, thereby targeting the toxins to GPRC 5D-expressing cancers.
In the present invention, the GPRC 5D-specific antibody or antigen-binding fragment may be labeled for use in the methods described 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 labeled with a radiolabel, a fluorescent label, an epitope tag, biotin, a chromophore label, an ECL label, an enzyme, ruthenium, 111In-DOTA, 111 In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase, and β -galactosidase, or polyhistidine or similar such labels known In the art.
In the present invention, the antibody can be developed as a method for detecting the expression level of GPRC5D on the surface of a tissue cell by an immunological means. For example, the antibodies may 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 GPRC 5D-expressing cancer, to determine a method of treating a subject having cancer, or to monitor the progression of GPRC 5D-expressing cancer in a subject, among others.
In the present invention, the term "antibody" specifically includes antibodies in the narrow sense, as well as "chimeric" antibodies and fragments of such antibodies, in which a portion of the heavy and/or light chain is identical to or homologous to the corresponding sequence derived from an antibody of a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to the corresponding sequence derived from an antibody of another species or belonging to another antibody class or subclass, so long as it specifically binds to the target antigen and/or exhibits the desired biological activity (U.S. Pat. No. 4,816,567, and Morrison et al, proc. Natl. Acad. Sci. Usa 81. The term "narrow-sense antibody" refers to a kind of globulin containing glycosyl which can specifically bind to the corresponding antigen and generate immune effect, and is secreted by the antigen entering the body to stimulate the differentiation and proliferation of B cells into plasma cells. The world health organization conferred in 1964 that globulins having antibody activity and chemical structure similar to antibodies were collectively called immunoglobulins. Modern immunology considers antibodies and immunoglobulins to be equivalent concepts, with the emphasis on the description of their biological activities being placed on antibodies, and the emphasis on their chemical structures being placed on immunoglobulins.
The basic structure of immunoglobulins comprises four peptide chains: two Heavy chains (H chain) and two Light chains (L chain), wherein the Light chains and the Heavy chains are connected by disulfide bonds to form a symmetrical tetrapeptide chain molecule which becomes an immunoglobulin molecule monomer, and the monomer is the basic structure of all immunoglobulins. Each heavy and light chain is divided into an amino terminus (N-terminus) and a carboxy terminus (C-terminus). Through the comparative analysis of the amino acid sequences of the H chain or the L chain, the following results are found: the N-terminal sequence varies greatly, and this region is called Variable region (V region); the C-terminal amino acid is relatively stable and has little variation, and this region is called the Constant region (C region). The variable region can be divided into a Hypervariable region (HVR) and a Framework Region (FR). The variable regions of the heavy and light chains have 3 HVRs, from N-terminus to C-terminus, referred to as HVR1, HVR2 and HVR3 of the heavy or light chain, respectively. The hypervariable regions are the binding sites of antibodies to antigens, called Complementarity-determining regions (CDRs), and thus HVR1, HVR2 and HVR3 of the heavy or light chain are also referred to as CDR1, CDR2 and CDR3.
In the present invention, the term "diabody", i.e. "bispecific" antibody, refers to an antibody, typically a monoclonal antibody, having the binding properties of at least two different antigenic epitopes. In one embodiment, the epitopes are from the same antigen. In another embodiment, the epitope is from two different antigens. Methods for making bispecific antibodies are known in the art. For example, bispecific antibodies can be recombinantly produced by co-expression of two immunoglobulin heavy/light chain pairs. See, e.g., milstein et al, nature305:537-39 (1983). Alternatively, bispecific antibodies can be prepared using chemical ligation. See, e.g., brennan et al, science229:81 (1985). Bispecific antibodies include bispecific antibody fragments (e.g., 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 containing sequences derived from a non-human (e.g., murine) antibody as well as a human antibody. The antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. Typically, the humanized antibody will comprise substantially all of at least one and typically two variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody also optionally comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., cabilly U.S. patent nos. 4,816,567; queen et al (1989) Proc.Natl.Acad.Sci.USA86:10029-10033; and Antibody Engineering: A Practical Approach (Oxford university Press) 1996).
The three letter and one letter codes for 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 part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 shows binding of panned monoclonal scFv to HEK293-GPRC5D-ZSGreen1 stably transfected cells;
FIG. 2 shows binding of panned monoclonal scFv to NCI-H929 cells;
FIG. 3 shows the binding of different chimeric antibodies to NCI-H929 cells;
FIG. 4 shows the binding of humanized HTS0370 antibodies to HEK293-GPRC5D-ZSGreen1 cells;
FIG. 5 shows the binding of humanized HTS0370 antibody to NCI-H929 cells;
FIG. 6 shows the binding of humanized HTS0375 antibody to HEK293-GPRC5D-ZSGreen1 cells;
FIG. 7 shows the binding of humanized HTS0375 antibody to NCI-H929 cells;
figure 8 shows the cross-reactivity of humanized HTS0370, HTS0375 antibody with CD19 positive PBMC cells;
figure 9 shows the cross-reactivity of humanized HTS0370, HTS0375 antibody to CD3 positive PBMC cells;
figure 10 shows the cross-reactivity of humanized HTS0370, HTS0375 antibody to CD11b positive PBMC cells;
figure 11 shows the cross-reactivity of humanized HTS0370, HTS0375 antibody to CD14 positive PBMC cells;
figure 12 shows the activity of humanized HTS0370, HTS0375 antibody binding to GPRC5D protein;
figure 13 shows the endocytic activity of humanized HTS0370, HTS0375 antibody;
figure 14 shows the species cross-reactivity of humanized HTS0370, HTS0375 antibody.
Detailed Description
In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.
In the following examples of the invention, the carrier information used is as follows:
the pTT5-hIgG1.CH vector is capable of synthesizing human IgG1 constant region sequences (SEQ ID NO: 97) obtained by construction of EcoRI/HindIII cleavage sites into the pTT5 vector (purchased from < v > 28156ling >;
the pTT5-hKappa.CL vector is capable of synthesizing the constant region sequence of the human Kappa chain (SEQ ID NO: 98), obtained by construction of EcoRI/BamHI cleavage sites into the pTT5 vector (purchased from a. Organism, \ 28156ling.
Example 1 preparation of human GPRC5D expression vector and Stable transfected cell line
1-1 preparation of human GPRC5D expression vectors pLVX-huGPRC 5D-IRES-ZSGGreen 1 and pTT5-huGPRC5D
Obtaining a base sequence (NM-018654.1) encoding CDS region of human GPRC5D gene from NCBI database, designing PCR primers 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2), selecting cDNA of MM.1S (purchased from Nanjing model animal research institute) and NCI-H929 (ATCC) two Multiple Myeloma (MM) cell lines as templates, carrying out PCR amplification, digesting PCR products with BamHI and EcoRI, connecting the digested products to pLVX-IRES-ZSGreen1 (purchased from Clonetech) and pTT5 vector (purchased from < v > 28156Ling organism), transfecting DH5a, and selecting a single clone for sequencing. The sequencing verifies that the sequence is correct, namely strains pLVX-huGPRC5D-IRES-ZSGreen1 and pTT5-huGPRC5D for successfully constructing expression target plasmids. The strain containing the objective plasmid was cultured in liquid LB medium, and plasmid DNA was extracted using Tiangen Biochemical technology (Beijing) Ltd. Endotoxin-free plasmid Large extraction kit (DP 117) according to the conventional procedures described in the specification.
The base sequence of CDS region of human GPRC5D gene is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO. 4.
1-2 preparation of HEK293 stable transgenic cell strain over-expressing human GPRC5D antigen
HEK293 cells (purchased from ATCC) with good growth state and long log growth period were selected, 8E6 cells were inoculated into a culture dish (10 cm), 10% fetal bovine serum was added to DMEM, and the concentration of CO was 5% at 37 ℃% 2 Culturing in an incubator, and transfecting when the cell fusion rate (cell density observed under a microscope) reaches about 70-80%. The three plasmids psPAX2 (from Clonetech), pMD2.G (from Clonetech) and pLVX-huGPRC5D-IRES-ZSGreen1 were co-transfected with the transfection reagent PEI. Virus fluid was collected 48-72 hours after transfection, virus was filtered through a 0.45 μ M syringe filter to remove residual cells, and cells of interest were infected at 10 MOI in HEK293 (purchased from ATCC) in DMEM plus 10% fetal bovine serum. Viral infection for 24 hoursAnd then changing the solution to obtain a stable cell pool for stably and highly expressing the hGPRC 5D. The constructed cell detects the positive rate of human GPRC5D expression by using a positive antibody of ET150 in a flow mode, the positive rate is over 98 percent and is completely consistent with the positive rate of GFP, the co-high expression of ZSGreen and antigen is detected, and the HEK293-GPRC5D-ZSGreen1 stable transgenic cell strain is successfully constructed.
EXAMPLE 2 preparation of monoclonal antibodies and screening of antibodies
2-1. Immunization and serum titer detection
8 SPF-grade Balb/C female healthy mice (purchased from Shanghai Jihui laboratory animal Breeding Co., ltd.) aged 6 weeks were selected, divided into two groups A and B, and immunized on days 0, 7, 14, 21, 28, 35, 42 and 49, respectively. Group A was immunized with HEK293-GPRC5D-ZSGreen1 stable transgenic cell line by intraperitoneal injection of 2X 10 per mouse 7 A cell; group B was immunized with GPRC5D expression plasmid pTT5-hGPRC5D, and each mouse was subcutaneously inoculated with 20. Mu.g of the plasmid in the back by a gene gun. The mice were subjected to orbital bleeds on days 34, 41, 48 and 55, and the mouse immune serum titers were measured by a conventional method of flow cytometry (FACS). Briefly, 96-well V-plate format plates were loaded with 5 × 10 wells per well 5 An HEK293-GPRC5D-ZSGreen1 stably transfected cell or endogenously expressed cell NCI-H929 (ATCC), was centrifuged at 1500r/min for 1min and the supernatant was discarded. After the immune serum is diluted by PBS according to the ratio of 1 to 50, 8 concentrations are diluted by 4 times of gradient, 50 mu L/hole is added into a micropore plate, after incubation for 30min on ice, 150 mu L PBS is added into each hole, centrifugation is carried out for 1min at 1500r/min, and the plate washing is repeated for 2 times. APC-labeled goat anti-mouse IgG (Jackson, cat # 115-605-164, PBS 1 diluted 800) was added at 50. Mu.L per well and incubated on ice for 30min. After washing the plate 2 times with PBS finally, 150. Mu.L of PBS per well was added to resuspend the cells and examined with CytoFLEX (Beckman). Selecting mice A1 and B1 with highest titer and stable titer of immune serum for two consecutive times, and using 2 × 10 antibody library 3 days before constructing antibody library 7 One-time impact immunization was performed by intraperitoneal injection of HEK293-GPRC 5D-ZSGGreen 1 cells.
2-2 construction of phage antibody library
Shock-immunized Balb/C mice A1 and B1, spleen cells, and literature references (Krebber, A., bornhauser, S., burmester, J., hone)A library of mouse immunophages was constructed by the method of gger, A., willuda, J., bosshard, H.R., and Pl ü ckthun, A. (1997) Reliable cloning of functional antibody variable domains from hybrid and spacer cell recombinant expression system, journal of immunological methods 201, 35-55), briefly after lysis with Trizol lysate, total RNA of the cells was extracted, cDNA was inverted, variable region genes of the antibody were amplified using specific antibody heavy and light chain primers, cloned onto a phage display vector, and the host E.coli cell TG1 was transformed. And (3) evaluating the diversity and effectiveness of the antibody library by NGS sequencing and utilizing quality control indexes such as library capacity, clone positive rate, heavy and light chain pairing, CDR3 distribution, phage display rate and the like. Library of antibody library at 1X10 8 Above, the positive rate of the clone insertion is above 90%, the scFv display rate is above 60%, and NGS sequencing shows that the antibody library has rich diversity and can be used for subsequent screening.
2-3 screening and identification of phage libraries
The phages were first pretreated with HEK293 cells at a phage input of 1 × 10 per antibody pool 10 And then performing enrichment panning on the pretreated phage supernatant by using HEK293-GPRC5D-ZSGreen1 stable cells, washing away unbound phage by using PBS, eluting phage bound on the cells by using 0.1M HCl-Glycine, neutralizing the eluent by using Tris-HCl, infecting escherichia coli TG 1in a logarithmic growth phase with the phage, and preparing the phage for the next panning. Gradually increasing the screening and washing times of each round, and stopping the elutriation when the enrichment degree reaches more than 10 times of the previous round.
The phage-infected TGl monoclonals after termination of panning were selected and inoculated into 96-well plates in a medium of 2YT/2% glucose/(100. Mu.g/ml Ampicilline). After 6 hours at 37 ℃ and 220rpm, the scFv were centrifuged at 4000rpm for 10min and induced by overnight culture at 220rpm in 2 YT/(100. Mu.g/ml Ampicilline)/(1. Mu.M IPTG) medium at 30 ℃. And centrifuging at 4000rpm for 10min, taking an induced supernatant, and detecting the binding activity of the scFv and the HEK293-GPRC5D-ZSGreen1 cells by a conventional method of a flow cytometer. Briefly, 96-well V-plate wells were loaded with 2.5 × 10 wells per well 5 HEK293-GPRC5D-ZSGreen1 stably transfected cells,at the same time, 50% HEK293 cells are mixed, centrifuged at 1500r/min for 1min, and the supernatant is discarded. Add 35. Mu.L PBS to 15. Mu.L scFv supernatant, add 50. Mu.L/well to the plate, incubate for 30min on ice, add 150. Mu.L PBS to each well, centrifuge for 1min at 1500r/min, repeat plate washing 2 times. APC-labeled mouse anti-6 × His antibody (kasumi, cat # a01802,1, 800 dilution) was added, 50 μ L per well, and incubated on ice for 30min. And washing the plate for 2 times by using PBS, adding 100 mu L of PBS into each hole to resuspend cells, detecting by using CytoFLEX (Beckman), carrying out binding verification on endogenous cells H929 with positive clones selected as the positive clones, wherein the MFI value of the cells HEK293-GPRC5D-ZSGreen1 is more than 2 times higher than that of a HEK293 control group, and selecting double-binding positive clones for sequencing. The positive scFv control BMK-ET150-8 was constructed according to the ET150-8 scFv sequence in patent US 20180118803. The results of cell binding are shown in FIGS. 1 and 2. Through the analysis of binding activity, 6 single-chain antibodies (scFv) are preferred, which are named as HTS0370, HTS0371, HTS0372, HTS0373, HTS0374, and HTS0375, respectively, and the 6 scFv single-chain antibodies are subsequently studied. After sequencing, the VH and VL sequences and CDR sequences of the 6 antibodies are shown in tables 1-4 below.
TABLE 1
Amino acid sequence table 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
scFv single chain antibody nucleotide sequence table 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
Figure BDA0003079321320000111
Figure BDA0003079321320000121
TABLE 4
Antibody light chain CDR sequence Listing 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 antibodies 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 by Biotechnology (Shanghai) Inc., and cloned by homologous recombination into pTT5-hIgG1.CH vector containing IgG1 heavy chain constant region amino acid sequence to obtain 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-hIgG1 and pTT5-HTS0375.VH-hIgG1.
3-2 construction of chimeric antibody light chain expression vector pTT5-VL
Light chain variable region sequences of HTS0370, HTS0371, HTS0372, HTS0373, HTS0374 and HTS0375 were routinely synthesized by Biotechnology (Shanghai) Inc., and cloned into pTT5-hKappa.CL vector containing antibody kappa light chain constant region amino acid sequence CL by homologous recombination to obtain 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 purification of chimeric antibodies
Good log-extended 293F cells (purchased from ThermoFisher) were harvested and seeded into 250mL cell culture flasks and cultured in 50mL media, 25. Mu.g each of PEI co-transfected light and heavy chain expression plasmids. Cell supernatants from post-transfection culture day 7 were collected, centrifuged and filtered using a 0.45 μ M filter, protein a media purified the antibody and replaced by dialysis into PBS ph7.2 buffer. Antibody concentration and purity were determined by Nanodrop determination of absorbance and purity 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 was named "xw.HTS0370", the antibody obtained by the combination of pTT5-HTS0371.VH-hIgG1 and pTT5-HTS0371.VL-hKappa was named "xw.HTS0371", the antibody obtained by the combination of pTT5-HTS0372.VH-hIgG1 and pTT5-HTS0372.VL-hKappa was named "xw.HTS0372", the antibody obtained by the combination of pTT5-HTS0373.VH-hIgG1 and pTT5-HTS0373.VL-hKappa was named "xw.HTS0373", the antibody obtained by the combination of pTT5-HTS0374.VH-hIgG1 and pTT5-HTS0374. VL-h0373. Vl-hKappa was named "xw.0375", and the antibody obtained by the combination of pTT5-HTS0374.VH-hIgG1 and pTT5-HTS0374. 0375".
3-4 detection of the binding Activity of chimeric antibodies
The binding activity of the chimeric antibody obtained in example 3-3 to HEK293-GPRC 5D-ZSGGreen 1 stably transfected cells overexpressing human GPRC5D and endogenously expressed cells NCI-H929 (ATCC) was examined by the method of FACS, the antibody sequence of the positive reference antibody was from ET150-8 in 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, 5X 10 of which is added per well 5 HEK293-GPRC5D-ZSGreen1 cells or NCI-H929 are placed in a 96-hole V-shaped microplate, centrifuged at 1500r/min for 1min, and the supernatant is discarded. Chimeric antibody was diluted in gradient, 50 μ L per well, and incubated on ice for 30min. Then 150. Mu.L PBS was added to each well, centrifuged at 1500r/min for 1min, the supernatant was discarded, and the plate washing was repeated 4 times. APC-labeled goat anti-human IgG (Jackson, cat # 109-605-098, PBS 1 diluted 800) was added, 50. Mu.L per well, and incubated on ice for 30min. After washing the plate 4 times with PBS, 100. Mu.L of PBS per well was added to resuspend the cells and examined with CytoFLEX (Beckman). The binding activity of xw.HTS0370, xw.HTS0371, xw.HTS0372, xw.HTS0373, xw.HTS0374 and xw.HTS0375 to the endogenously expressed cell line NCI-H929 was significantly higher than that of the positive control antibody xw.ET150-8 as analyzed by GraphPad 8.0.2, and the results are shown in FIG. 3. The half maximal effector concentration EC50 of 6 resulting chimeric antibodies and the control antibody are shown in table 5.
TABLE 5
Antibodies 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, and it can be seen from the above table that the binding activity of the remaining 5 chimeric antibodies except xw. Hts0371 was better than that of the control.
Through light and heavy chain Germline analysis and sequence alignment, the sequences of HTS 0372-375 antibodies are similar and possibly differentiated from the same B cell clone, and the sequences Germline between the group of clones and HTS0370 and HTS0371 and between HTS0370 and HTS0371 are completely different, so that the difference of CDR regions is larger. The sequences of the two clones that bound best xw.hts0370 and xw.hts0375 were selected for subsequent humanization in conjunction with the data on antibody binding to cellular EC 50.
Example 4 humanization of murine GRPC5D antibody
4-1. Humanization design protocol
Murine anti-human HTS0370 and HTS0375 antibodies were humanized engineered according to a commonly-called CDR-grafting approach. Briefly, the sequences of the VH and VK base sequences of HTS0370, HTS0375 antibodies were analyzed using the IMGT/V-QUEST tool (http:// www.imgt. Org/IMGT _ vquest/input) to determine the CDR regions of the light and heavy chains of the antibodies. The amino acid sequences of HTS0370 and HTS0375 antibodies were analyzed using the Igblast tool (https:// www.ncbi.nlm.nih.gov/IgBlast /) to obtain the human germline VH and VK sequences that are closest to HTS0370 and HTS0375 antibodies. The CDRs of HTS0370 and HTS0375 antibodies were grafted into the framework regions of selected VH and VK human germline sequences, respectively, i.e. humanized antibody sequences. Human germline VH of HTS0370 and HTS0375 antibodies VH were analyzed, 5 different sequences were selected, and CDRs of HTS0370 and HTS0375 antibodies VH were grafted into framework regions of these 5 sequences, respectively, to yield 5 heavy chain sequence variable regions. In the same manner, CDR grafting was performed on the light chains of HTS0370 and HTS0375, respectively, to obtain 5 light chain variable region sequences.
The humanized sequence of the light and heavy chains is shown in a sequence table 6-7:
TABLE 6
HTS0370 humanized sequences 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
Figure BDA0003079321320000141
Figure BDA0003079321320000151
4-2 preparation of humanized antibody
HTS0370 and HTS0375 heavy chain humanized sequences are conventionally synthesized by biological engineering (Shanghai) GmbH, and are respectively cloned into pTT5-hIgG1.CH vectors containing IgG1 heavy chain constant region amino acid sequences in a homologous recombination mode to obtain chimeric antibody heavy chain expression plasmids; HTS0370 and HTS0375 light chain humanized sequences are conventionally synthesized by Biotechnology engineering (Shanghai) GmbH and cloned into pTT5-hKappa.CL vectors respectively by means of homologous recombination. 293F cells with good log growth status were inoculated into 250mL cell culture flasks and cultured in 50mL medium, 25. Mu.g each of PEI co-transfected light and heavy chain expression plasmids. Cell supernatants from post-transfection day 7 cultures were collected, centrifuged and filtered using a 0.45 μ M filter, protein A media purified the antibody and replaced by dialysis methods into PBS pH7.2 buffer. Antibody concentration and purity were determined by Nanodrop determination of absorbance and purity checked by sodium dodecyl sulfate gel electrophoresis and coomassie staining. The names of antibodies for each heavy and light chain combination are shown in tables 8-9:
TABLE 8
Figure BDA0003079321320000152
Figure BDA0003079321320000161
TABLE 9
Figure BDA0003079321320000163
4-3 detection of binding Activity of humanized antibody
The humanized antibody obtained in example 4-2 was examined with GPRC5D by FACS methodBinding activity of the cell line and the endogenously expressed cell NCI-H929 (ATCC). The detection method is briefly described as follows, 5X 10 of which is added per well 5 The NCI-H929 cells are placed in a 96-hole V-shaped microplate, centrifuged at 1500r/min for 1min, and the supernatant is discarded. Humanized antibody was diluted in a gradient, 50. Mu.L per well, and incubated on ice for 30min. Then 150. Mu.L PBS per well was added, centrifuged at 1500 rpm for 1min, the supernatant was discarded, and the plate washing was repeated 4 times. APC-labeled goat anti-human IgG (Jackson, cat # 109-605-098, PBS 1 diluted 800) was added at 50. Mu.L per well and incubated on ice for 30min. After washing the plate 4 times with PBS, 100. Mu.L of PBS per well was added to resuspend the cells and examined with CytoFLEX (Beckman). By GraphPad 8.0.2 analysis, multiple sequences after HTS0370 humanization showed better binding activity with GPRC5D overexpressed 293 cells and endogenous cell NCI-H929 (results are shown in FIG. 4 and FIG. 5). The EC50 values of 293 cells with over-expressed GPRC5D and endogenous NCI-H929 of HTS0370 humanized sequences and controls are shown in tables 10-11.
Watch 10
Figure BDA0003079321320000162
Figure BDA0003079321320000171
TABLE 11
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
From the results of the above two tables, humanized antibodies humanized by HTS0370 were selected for superior binding activity in view of superior binding activity with GPRC5D over-expressed 293 cells as well as endogenous cell NCI-H929: zw. Htsz0370z02, zw. Htsz0370z03, zw. Htsz0370z04, zw. Htsz0370z16, zw. Htsz0370z18, zw. Htsz0370z22, zw. Htsz0370z23. Wherein, two results are combined, and zw.HTS0370Z22 is selected as the best and is subjected to subsequent research. The EC50 values of the HTS0375 humanized sequences and control and GPRC5D overexpressed 293 cells and endogenous cell NCI-H929 are shown in tables 12-13.
TABLE 12
EC50
293T_GPRC5D zw.HTS0375Z56 1.936
293T_GPRC5D xw.HT0375 1.936
293T_GPRC5D_zw.BMK.ET150-8λ 1.719
Watch 13
EC50
H929zw.H0375Z56 0.9438
H929xw.H0375 0.2424
H929zw.BMK.ET150-8λ 10.59
Similarly, the comprehensive binding activity of zw. HTS0375Z56 molecules obtained by humanizing HTS0375 by the same method is optimized, and the results of subsequent studies are shown in FIGS. 6 and 7.
Example 5: detection of tissue cross-reactivity of antibodies with various blood cells
Populations of T cells, monocytes, B cells, NK cells, granulocytes were represented by the flow method using FITC-CD3, FITC-CD14, FITC-CD19, percpCy5.5-CD11B, respectively, and the humanized antibody obtained in section 4-2 of example 4 biotinylated was used for diabody staining, and secondary antibody labeling of the GPRC5D antibody to be detected was performed using an antibody to PE-SA. Briefly, 1X10 additions per well 6 Individual PBMC cells (purchased from Shanghai Miaoshun biosciences) were centrifuged at 1500 rpm for 1min in a 96-well V-type microplate, and the supernatant was discarded. Add 1. Mu.g/well of the humanized antibody for GPRC5D to be detected, 50. Mu.L/well, and incubate on ice for 30min. Then 150. Mu.L PBS was added to each well, centrifuged at 1500r/min for 1min, the supernatant was discarded, and the plate washing was repeated 4 times. PE-SA (BD, cat # 554061, PBS 1 dilution) was added at 25. Mu.L per well, along with antibodies to various PBMC blood cells at a final concentration of 25. Mu.L in volume according to the instructions for antibodies, and after mixing all, the cells were incubated on ice for 30min. After washing the plate 4 times with PBS, 100. Mu.L of PBS per well was added to resuspend the cells and examined with CytoFLEX (Beckman). The humanized antibody was not bound to these 5 types of blood cells as analyzed by the software of the instrument itself, but the xw.et150-8 antibody from the prior art was non-specifically bound to both granulocytes represented by CD11b and monocytes represented by CD14, suggesting that there may be serious side effects with this antibody. The results of non-specific binding of the humanized antibody and the control to CD19, CD3, CD11b, and CD14 positive cells are shown in fig. 8 to 11, respectively. From the results, it was found that the humanized molecules zw. Hts0370z22 and zw. Hts0375z56 did not bind non-specifically to various cell populations positive for CD19, CD3, CD11b, and CD14, compared to ETC150 which binds non-specifically to cell populations of CD11b and CD 14. Since HTS0370, HTS0375 molecules and chimeric and humanized antibodies derived therefrom have identical CDRs, i.e., identical surface antigen binding sites, the results show HTS0370, HTS0375 and the sameOther antibody formats, CDR-derived, will target GPRC5D more specifically, thereby reducing the risk of toxicity due to off-target.
Example 6: binding capacity of antibodies to GPRC5D protein
The binding activity of the humanized antibody obtained in section 4-2 of test example 4 to the VLP-like protein of GPRC5D (Kactus biosystems) was examined by means of ELISA. The detection method is summarized as follows, the concentration of VLP protein of diluted GPRC5D is 1 mug/ml, the VLP protein is laid on a 384-hole enzyme label plate overnight, the protein is discarded the next day, 80 mul of 3% milk (dissolved in PBS) is used for blocking for 2 hours, 80 mul of PBST (1 thousandth Tween 20) is used for washing 3 times, then the humanized antibody obtained in 4-2 is subjected to gradient dilution, 25 mul of the humanized antibody is added into the enzyme label plate which is blocked, the reaction is carried out at room temperature for 1 hour, then the antibody is discarded, 80 mul of PBST (1 thousandth Tween 20) is used for washing 5 times, 25 mul of HRP-labeled goat anti-human secondary antibody (Sino biological, product number SSA002, 1. After the reaction was stopped by adding 2M HCl, the reading was completed within 30 minutes after the color development was carried out for 10 minutes by adding 25. Mu.L of TMB developing solution. The antibody sequence of the positive reference antibody GC5B596 is from SEQ90 and SEQ96 in patent US10562968, and the analysis by GraphPad 8.0.2 shows that the humanized antibody binds to the VLP-like protein of GPRC5D with better binding activity than GC5B596, and the results are shown in fig. 12. Comparing the cell binding activity results of the humanized antibody, the protein binding level difference between the HTS0375 humanized antibody zw.hts0375z56 and the HTS0370 humanized antibody zw.hts0370z22 is obviously increased compared with the cell binding difference.
Example 7: species cross-reactivity of antibodies binding to human, monkey GPRC5D cell lines
The binding activity of the humanized antibody obtained in section 4-2 of example 4 to HEK293-GPRC 5D-overexpressing monkey GPRC 5D-transfected cells was examined by FACS method using the constructed monkey GPRC5D cells, and the antibody sequences of the positive reference antibody GC5B596 were derived from SEQ90 and SEQ96 of patent US 10562968. The detection method is briefly described as follows, 5X 10 is added to each well 5 HEK293-GPRC5D cells are put in a 96-hole V-shaped micro-perforated plate,centrifuging at 1500r/min for 1min, and discarding the supernatant. Humanized antibody was diluted in a gradient, 50. Mu.L per well, and incubated on ice for 30min. Then 150. Mu.L PBS was added to each well, centrifuged at 1500r/min for 1min, the supernatant was discarded, and the plate washing was repeated 4 times. APC-labeled goat anti-human IgG (Jackson, cat # 109-605-098, PBS 1 diluted 800) was added, 50. Mu.L per well, and incubated on ice for 30min. After washing the plate 4 times with PBS, 100. Mu.L of PBS per well were added to resuspend the cells and detected with CytoFLEX (Beckman). The binding activity of zw.HTS0370Z22 and zw.HTS0375Z56 with monkey expressing cells is obviously higher than that of a positive control antibody by GraphPad 8.0.2 analysis, and the result is shown in FIG. 13. The humanized antibody has the characteristic of species cross reaction, is favorable for toxicity analysis of the antibody in monkeys, is favorable for carrying out verification tests in related animals, and is further favorable for subsequent application development of therapeutic purposes.
Example 8: endocytosis of antibodies
The endocytic activity of the humanized antibody obtained in section 4-2 of example 4 was tested using the method of FACS using cells of NCI-H929, the antibody sequence of the positive reference antibody GC5B596 being derived from SEQ90 and SEQ96 in patent US10562968, and another positive reference antibody being constructed according to the ET150-8 scFv sequence in patent US 20180118803. The detection method is briefly described as follows, and 4 portions of 5X 10 are subpackaged 5 Each NCI-H929 cell was centrifuged at 1500 rpm for 1min in a 96-well V-type microplate, and the supernatant was discarded. Saturated 1. Mu.g/ml of the antibody to be detected, 200. Mu.l per well, are added and incubated on ice for 30min. Then 150. Mu.L PBS was added to each well, centrifuged at 1500 rpm for 1min, the supernatant was discarded, and the plate washing was repeated 4 times. Resuspend with 200. Mu.L PBS, take out 1 part and put into 37 ℃ cell culture box, stand for 2 hours, another 2 parts are respectively placed under the same conditions for 1 hour, 0.5 hour, the rest last part is placed on ice, after the time is over, all are placed at 4 ℃ and centrifuged at 1500r/min, APC labeled goat anti-human IgG (Jackson, cat # 109-605-098, PBS 1 diluted 800) is added, 50. Mu.L per well, and incubated on ice for 30min. After washing the plate 4 times with PBS, 100. Mu.L of PBS per well was added to resuspend the cells and examined with CytoFLEX (Beckman). By GraphPad 8.0.2 analysis, zw.HTS0370Z22 and zw.HTS0375Z56 have obvious endocytosis activity, wherein the endocytosis activity of zw.HTS0370Z22 is obvious, and the positive control antibody GC5B596 has no endocytosis activityThe results are shown in FIG. 14. The results suggest that zw.hts0370z22 and zw.hts0375z56 are more suitable for subsequent development of ADC drugs than positive controls, and zw.hts0370z22 has better potential.
The above experimental results show that compared with the existing published antibodies, the antibodies disclosed by the invention have at least the following advantages: 1) The CDR sequence has large difference and is different from the GPRC5D antigen binding epitope; 2) The binding activity is good; 3) The specificity is stronger, and the safety is good; 4) Has species cross-reactivity, and is convenient for the development of products for therapeutic use.
The antibodies of the invention may also be used with antibodies that target other targets, such as: CD3, BCMA, CD38, etc. are constructed into bispecific antibody and developed into various tumor cell regulating methods. In addition, the antibodies of the present invention may also be prepared by coupling other types of molecules, such as: toxins, nucleic acid molecules, etc., which specifically bring the conjugated molecule into the tumor cell body via the antibody, thereby modulating the action of the tumor cell. Furthermore, the antibody of the present invention can be developed as a method for detecting the expression level of GPRC5D on the surface of a tissue cell by an immunological method.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.
Sequence listing
<110> Shanghai Xiang Yao Biotechnology Limited liability company
<120> anti-GPRC 5D antibody, preparation method and application thereof
<160> 98
<170> SIPOSequenceListing 1.0
<210> 1
<211> 52
<212> DNA
<213> Artificial sequence ()
<400> 1
agcgaattct ctagacaatt gcttgccgcc accatgtaca aggactgcat cg 52
<210> 2
<211> 40
<212> DNA
<213> Artificial sequence ()
<400> 2
cgggggatcc ctcgagtgat catactcctc ctgcatcttg 40
<210> 3
<211> 1035
<212> DNA
<213> person ()
<400> 3
atgtacaagg actgcatcga gtccactgga gactattttc ttctctgtga cgccgagggg 60
ccatggggca tcattctgga gtccctggcc atacttggca tcgtggtcac aattctgcta 120
ctcttagcat ttctcttcct catgcgaaag atccaagact gcagccagtg gaatgtcctc 180
cccacccagc tcctcttcct cctgagtgtc ctggggctct tcggactcgc ttttgccttc 240
atcatcgagc tcaatcaaca aactgccccc gtacgctact ttctctttgg ggttctcttt 300
gctctctgtt tctcatgcct cttagctcat gcctccaatc tagtgaagct ggttcggggt 360
tgtgtctcct tctcctggac gacaattctg tgcattgcta ttggttgcag tctgttgcaa 420
atcattattg ccactgagta tgtgactctc atcatgacca gaggtatgat gtttgtgaat 480
atgacaccct gccagctcaa tgtggacttt gttgtactcc tggtctatgt cctcttcctg 540
atggccctca cattcttcgt ctccaaagcc accttctgtg gcccgtgtga gaactggaag 600
cagcatggaa ggctcatctt tatcactgtg ctcttctcca tcatcatctg ggtggtgtgg 660
atctccatgc tcctgagagg caacccgcag ttccagcgac agccccagtg ggacgacccg 720
gtcgtctgca ttgctctggt caccaacgca tgggttttcc tgctgctgta catcgtccct 780
gagctctgca ttctctacag atcgtgtaga caggagtgcc ctttacaagg caatgcctgc 840
cccgtcacag cctaccaaca cagcttccaa gtggagaacc aggagctctc cagagcccga 900
gacagtgatg gagctgagga ggatgtagca ttaacttcat atggtactcc cattcagccg 960
cagactgttg atcccacaca agagtgtttc atcccacagg ctaaactaag cccccagcaa 1020
gatgcaggag gagta 1035
<210> 4
<211> 345
<212> PRT
<213> person ()
<400> 4
Met Tyr Lys Asp Cys Ile Glu Ser Thr Gly Asp Tyr Phe Leu Leu Cys
1 5 10 15
Asp Ala Glu Gly Pro Trp Gly Ile Ile Leu Glu Ser Leu Ala Ile Leu
20 25 30
Gly Ile Val Val Thr Ile Leu Leu Leu Leu Ala Phe Leu Phe Leu Met
35 40 45
Arg Lys Ile Gln Asp Cys Ser Gln Trp Asn Val Leu Pro Thr Gln Leu
50 55 60
Leu Phe Leu Leu Ser Val Leu Gly Leu Phe Gly Leu Ala Phe Ala Phe
65 70 75 80
Ile Ile Glu Leu Asn Gln Gln Thr Ala Pro Val Arg Tyr Phe Leu Phe
85 90 95
Gly Val Leu Phe Ala Leu Cys Phe Ser Cys Leu Leu Ala His Ala Ser
100 105 110
Asn Leu Val Lys Leu Val Arg Gly Cys Val Ser Phe Ser Trp Thr Thr
115 120 125
Ile Leu Cys Ile Ala Ile Gly Cys Ser Leu Leu Gln Ile Ile Ile Ala
130 135 140
Thr Glu Tyr Val Thr Leu Ile Met Thr Arg Gly Met Met Phe Val Asn
145 150 155 160
Met Thr Pro Cys Gln Leu Asn Val Asp Phe Val Val Leu Leu Val Tyr
165 170 175
Val Leu Phe Leu Met Ala Leu Thr Phe Phe Val Ser Lys Ala Thr Phe
180 185 190
Cys Gly Pro Cys Glu Asn Trp Lys Gln His Gly Arg Leu Ile Phe Ile
195 200 205
Thr Val Leu Phe Ser Ile Ile Ile Trp Val Val Trp Ile Ser Met Leu
210 215 220
Leu Arg Gly Asn Pro Gln Phe Gln Arg Gln Pro Gln Trp Asp Asp Pro
225 230 235 240
Val Val Cys Ile Ala Leu Val Thr Asn Ala Trp Val Phe Leu Leu Leu
245 250 255
Tyr Ile Val Pro Glu Leu Cys Ile Leu Tyr Arg Ser Cys Arg Gln Glu
260 265 270
Cys Pro Leu Gln Gly Asn Ala Cys Pro Val Thr Ala Tyr Gln His Ser
275 280 285
Phe Gln Val Glu Asn Gln Glu Leu Ser Arg Ala Arg Asp Ser Asp Gly
290 295 300
Ala Glu Glu Asp Val Ala Leu Thr Ser Tyr Gly Thr Pro Ile Gln Pro
305 310 315 320
Gln Thr Val Asp Pro Thr Gln Glu Cys Phe Ile Pro Gln Ala Lys Leu
325 330 335
Ser Pro Gln Gln Asp Ala Gly Gly Val
340 345
<210> 5
<211> 127
<212> PRT
<213> Artificial sequence ()
<400> 5
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr
20 25 30
Trp Met Glu Trp Val Lys Gln Arg Pro Gly Leu Gly Leu Glu Trp Ile
35 40 45
Gly Gln Ile Leu Pro Gly Ser Ser Tyr Thr Asn Tyr Asn Asp Lys Phe
50 55 60
Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Lys Gly Gly Pro Ile Tyr Tyr Gly Asn Arg Pro Phe Tyr Tyr
100 105 110
Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120 125
<210> 6
<211> 107
<212> PRT
<213> Artificial sequence ()
<400> 6
Asn Ile Met Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Gln Ile
35 40 45
Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 7
<211> 119
<212> PRT
<213> Artificial sequence ()
<400> 7
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Lys Leu Asn Trp Tyr Phe Asp Val Trp Gly Ala Gly
100 105 110
Thr Thr Val Thr Val Ser Ser
115
<210> 8
<211> 111
<212> PRT
<213> Artificial sequence ()
<400> 8
Asn Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr
20 25 30
Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp
65 70 75 80
Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn
85 90 95
Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Asn
100 105 110
<210> 9
<211> 118
<212> PRT
<213> Artificial sequence ()
<400> 9
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Tyr Asn Gly Arg Thr Ile Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Ala Leu Arg Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Leu Thr Val Ser Ser
115
<210> 10
<211> 107
<212> PRT
<213> Artificial sequence ()
<400> 10
Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Asn
100 105
<210> 11
<211> 119
<212> PRT
<213> Artificial sequence ()
<400> 11
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ala Gly Tyr
20 25 30
Thr Val Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Thr Tyr Thr Pro Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Gly Gly Phe Tyr Arg Tyr Asp Phe Asp Phe Trp Gly Gln Gly
100 105 110
Thr Ser Val Thr Val Ser Ser
115
<210> 12
<211> 107
<212> PRT
<213> Artificial sequence ()
<400> 12
Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr
20 25 30
Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser
65 70 75 80
Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Met Lys
100 105
<210> 13
<211> 119
<212> PRT
<213> Artificial sequence ()
<400> 13
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr
1 5 10 15
Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Thr Val Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Thr Tyr Thr Pro Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Gly Gly Phe Tyr Arg Tyr Asp Phe Asp Phe Trp Gly Gln Gly
100 105 110
Thr Ser Leu Thr Val Ser Ser
115
<210> 14
<211> 107
<212> PRT
<213> Artificial sequence ()
<400> 14
Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser
20 25 30
Leu Asn Trp Leu Gln Gln Glu Pro Asp Gly Thr Ile Lys Arg Leu Ile
35 40 45
Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser
65 70 75 80
Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys
100 105
<210> 15
<211> 118
<212> PRT
<213> Artificial sequence ()
<400> 15
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Tyr Asn Gly Arg Thr Ile Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Ala Leu Arg Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Leu Thr Val Ser Ser
115
<210> 16
<211> 107
<212> PRT
<213> Artificial sequence ()
<400> 16
Ser Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Lys Val Ser Val Thr Cys Lys Ala Ser Gln Ser Val Tyr Thr Asn
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ser Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Asn Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Val
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 17
<211> 381
<212> DNA
<213> Artificial sequence ()
<400> 17
gaggttcagc tgcagcagtc tggagctgag ctgatgaagc ctggggcctc agtgaagata 60
tcctgcaagg ctactggcta cacattcagt agctactgga tggagtgggt aaagcagagg 120
cctggacttg gccttgagtg gattggacag attttacctg gaagtagtta tactaactac 180
aatgacaaat tcaagggcaa ggccacattc actgcagata catcctccaa cacagcctac 240
atgcaactca gcagcctgac atctgaggac tctgccgtct attactgtgc aaaaaagggg 300
ggcccgatat actatggtaa ccgtcctttt tactatgcta tggactactg gggtcaagga 360
acctcagtca ccgtctcgag t 381
<210> 18
<211> 321
<212> DNA
<213> Artificial sequence ()
<400> 18
aacattatga tgacacagac tcccaaattc ctgcttgtat cagcaggaga cagggttacc 60
ataacctgca aggccagtca gagtgtgagt aatgatgtag cttggtacca acagaagcca 120
gggcagtctc ctaaactgca gatatactat gcatccaatc gctacactgg agtccctgat 180
cgcttcactg gcagtggata tgggacggat ttcactttca ccatcagcac tgtgcaggct 240
gaagacctgg cagtttattt ctgtcagcag gattatagct ctccgctcac gttcggtgct 300
gggacaaagt tggaaataaa a 321
<210> 19
<211> 357
<212> DNA
<213> Artificial sequence ()
<400> 19
gaggttcagc tgcagcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60
tcctgcaagg cttctggata cacattcact agctatgtta tgcactgggt gaagcagaag 120
cctgggcagg gccttgagtg gattggatat attaatcctt acaatgatgg tactaagtac 180
aatgagaagt tcaaaggcaa ggccacactg acttcagaca aatcctccag cacagcctac 240
atggagctca gcagcctgac ctctgaggac tctgcggtct attactgtgc aagagggggg 300
aagctaaact ggtacttcga tgtctggggc gcagggacca cggtcaccgt ctcgagt 357
<210> 20
<211> 333
<212> DNA
<213> Artificial sequence ()
<400> 20
aacattgtaa tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60
atatcctgca gagccagtga aagtgttgat agttacggca atagttttat gcactggtac 120
cagcagaaac caggacagcc acccaaactc ctcatctatc ttgcatccaa cctagaatct 180
ggggtccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccattgat 240
cctgtggagg ctgatgatgc tgcaacctat tactgtcagc aaaataatga ggatccgtac 300
acgttcggag ggggcaccaa gctggaaatc aat 333
<210> 21
<211> 354
<212> DNA
<213> Artificial sequence ()
<400> 21
gaggtccagc tgcaacagtc tggacctgag ctggtgaagc ctggagcttc aatgaagata 60
tcctgcaagg cttctggtta ctcattcact ggctacacca tgaactgggt gaaacagagc 120
catggaaaga accttgagtg gattggactt attaatcctt ataatggtcg tactatatac 180
aaccagaagt tcaagggcaa ggccacatta actgtagaca agtcatccag cacggcctac 240
atggagctcc tcagtctgac gtccgaggac tctgcagtct attactgtgc aagagtggca 300
ttacggtatg ctatggacta ctggggtcaa ggcacctctc tcacagtctc gagt 354
<210> 22
<211> 321
<212> DNA
<213> Artificial sequence ()
<400> 22
gacattgtga tgactcagtc tcaaaaattc atgtccacat cagtaggaga cagggtcagc 60
gtcacctgca aggccagtca gaatgtgggt actaatgtag cctggtatca acagaaacca 120
gggcaatctc ctaaagcact gatttactcg gcatcctacc ggtacagtgg agtccctgat 180
cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct 240
gaagacttgg cagagtattt ctgtcagcaa tataacagct atcctctcac gttcggtgct 300
ggcaccaagc tggaaatcaa t 321
<210> 23
<211> 357
<212> DNA
<213> Artificial sequence ()
<400> 23
gaggtccagc ttcagcagtc tggacctgag ctggtgaagc ctggagcttc aatgaagatt 60
tcctgcaagg cttctggtta ctcattcgct ggctacaccg tgaactgggt gaagcagagc 120
catggcaaga accttgagtg gattggactt attaatcctt acaatggtgg tactacctac 180
accccgaagt tcaaggacaa ggccacatta actgtagaca agtcatccag cacagcctat 240
atggagctcc tcagtctgac atctgaggac tctgcagtct actactgtac aagagggggt 300
ttctataggt acgactttga cttctggggc caaggaacct cagtcaccgt ctcgagt 357
<210> 24
<211> 321
<212> DNA
<213> Artificial sequence ()
<400> 24
gacatcctga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60
ctcacttgtc gggcaagtca ggaaattagt ggttacttaa gttggcttca gcagaaacca 120
gatggaacta ttaaacgcct gatctacgcc gcatccactt tagattctgg tgtcccaaaa 180
aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240
gaagattttg cagactatta ctgtctacaa tatgctagtt atccattcac gttcggctcg 300
gggaccaagc tggaaatgaa a 321
<210> 25
<211> 357
<212> DNA
<213> Artificial sequence ()
<400> 25
caggttcagc tgcagcagtc tggacctgag ctggtgaagc ctggaacttc aatgaagatt 60
tcctgcaagg cttctggtta ctcattcact ggctacaccg tgaactgggt gaagcagagc 120
catggcaaga accttgagtg gattggactt attaatcctt acaatggtgg tactacctac 180
accccgaagt tcaaggacaa ggccacatta actgtagaca agtcatccag cacagcctat 240
atggaactcc tcagtctgac atctgaggac tctgcagtct actactgtac aagagggggt 300
ttctataggt acgactttga cttttggggc caaggcacct ctctcacagt ctcgagt 357
<210> 26
<211> 321
<212> DNA
<213> Artificial sequence ()
<400> 26
gaaatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60
ctcacttgtc gggcaagtca ggacattggt agtagcttaa actggcttca gcaggaacca 120
gatggaacta ttaaacgcct gatctacgcc acatccagtt tagattctgg tgtccccaaa 180
aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240
gaagattttg tagactatta ctgtctacaa tatgctagtt ctccgtacac gttcggaggg 300
gggaccaagc tggaaatgaa a 321
<210> 27
<211> 354
<212> DNA
<213> Artificial sequence ()
<400> 27
gaggttcagc tgcagcagtc tggacctgag ctggtgaagc ctggagcttc aatgaagata 60
tcctgcaagg cttctggtta ctcattcact ggctacacca tgaactgggt gaaacagagc 120
catggaaaga accttgagtg gattggactt attaatcctt ataatggtcg tactatatac 180
aaccagaagt tcaagggcaa ggccacatta actgtagaca agtcatccag cacggcctac 240
atggagctcc tcagtctgac gtctgaggac tctgcagtct attactgtgc aagagtggca 300
ttacggtatg ctatggacta ctggggtcaa ggcacctctc tcacagtctc gagt 354
<210> 28
<211> 321
<212> DNA
<213> Artificial sequence ()
<400> 28
agcattgtga tgacccagtc tcaaaaattc atgtccacat cagtaggaga caaagtcagc 60
gtcacctgca aggccagtca gagtgtgtat actaatgttg cctggtatca acagaaacca 120
gggcaatctc ctaaatcact gatttactcg gcatcctacc gatacaatgg ggtccctgat 180
cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct 240
gaagacttgg cagagtattt ctgtcagcaa tataacagct atccggtcac gttcggtgct 300
gggacaaaat tggaaataaa a 321
<210> 29
<211> 8
<212> PRT
<213> Artificial sequence ()
<400> 29
Gly Tyr Thr Phe Ser Ser Tyr Trp
1 5
<210> 30
<211> 8
<212> PRT
<213> Artificial sequence ()
<400> 30
Ile Leu Pro Gly Ser Ser Tyr Thr
1 5
<210> 31
<211> 20
<212> PRT
<213> Artificial sequence ()
<400> 31
Ala Lys Lys Gly Gly Pro Ile Tyr Tyr Gly Asn Arg Pro Phe Tyr Tyr
1 5 10 15
Ala Met Asp Tyr
20
<210> 32
<211> 8
<212> PRT
<213> Artificial sequence ()
<400> 32
Gly Tyr Thr Phe Thr Ser Tyr Val
1 5
<210> 33
<211> 8
<212> PRT
<213> Artificial sequence ()
<400> 33
Ile Asn Pro Tyr Asn Asp Gly Thr
1 5
<210> 34
<211> 12
<212> PRT
<213> Artificial sequence ()
<400> 34
Ala Arg Gly Gly Lys Leu Asn Trp Tyr Phe Asp Val
1 5 10
<210> 35
<211> 8
<212> PRT
<213> Artificial sequence ()
<400> 35
Gly Tyr Ser Phe Thr Gly Tyr Thr
1 5
<210> 36
<211> 8
<212> PRT
<213> Artificial sequence ()
<400> 36
Ile Asn Pro Tyr Asn Gly Arg Thr
1 5
<210> 37
<211> 11
<212> PRT
<213> Artificial sequence ()
<400> 37
Ala Arg Val Ala Leu Arg Tyr Ala Met Asp Tyr
1 5 10
<210> 38
<211> 8
<212> PRT
<213> Artificial sequence ()
<400> 38
Gly Tyr Ser Phe Ala Gly Tyr Thr
1 5
<210> 39
<211> 8
<212> PRT
<213> Artificial sequence ()
<400> 39
Ile Asn Pro Tyr Asn Gly Gly Thr
1 5
<210> 40
<211> 12
<212> PRT
<213> Artificial sequence ()
<400> 40
Thr Arg Gly Gly Phe Tyr Arg Tyr Asp Phe Asp Phe
1 5 10
<210> 41
<211> 8
<212> PRT
<213> Artificial sequence ()
<400> 41
Gly Tyr Ser Phe Thr Gly Tyr Thr
1 5
<210> 42
<211> 8
<212> PRT
<213> Artificial sequence ()
<400> 42
Ile Asn Pro Tyr Asn Gly Gly Thr
1 5
<210> 43
<211> 12
<212> PRT
<213> Artificial sequence ()
<400> 43
Thr Arg Gly Gly Phe Tyr Arg Tyr Asp Phe Asp Phe
1 5 10
<210> 44
<211> 8
<212> PRT
<213> Artificial sequence ()
<400> 44
Gly Tyr Ser Phe Thr Gly Tyr Thr
1 5
<210> 45
<211> 8
<212> PRT
<213> Artificial sequence ()
<400> 45
Ile Asn Pro Tyr Asn Gly Arg Thr
1 5
<210> 46
<211> 11
<212> PRT
<213> Artificial sequence ()
<400> 46
Ala Arg Val Ala Leu Arg Tyr Ala Met Asp Tyr
1 5 10
<210> 47
<211> 6
<212> PRT
<213> Artificial sequence ()
<400> 47
Gln Ser Val Ser Asn Asp
1 5
<210> 48
<211> 9
<212> PRT
<213> Artificial sequence ()
<400> 48
Gln Gln Asp Tyr Ser Ser Pro Leu Thr
1 5
<210> 49
<211> 10
<212> PRT
<213> Artificial sequence ()
<400> 49
Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe
1 5 10
<210> 50
<211> 9
<212> PRT
<213> Artificial sequence ()
<400> 50
Gln Gln Asn Asn Glu Asp Pro Tyr Thr
1 5
<210> 51
<211> 6
<212> PRT
<213> Artificial sequence ()
<400> 51
Gln Asn Val Gly Thr Asn
1 5
<210> 52
<211> 9
<212> PRT
<213> Artificial sequence ()
<400> 52
Gln Gln Tyr Asn Ser Tyr Pro Leu Thr
1 5
<210> 53
<211> 6
<212> PRT
<213> Artificial sequence ()
<400> 53
Gln Glu Ile Ser Gly Tyr
1 5
<210> 54
<211> 9
<212> PRT
<213> Artificial sequence ()
<400> 54
Leu Gln Tyr Ala Ser Tyr Pro Phe Thr
1 5
<210> 55
<211> 6
<212> PRT
<213> Artificial sequence ()
<400> 55
Gln Asp Ile Gly Ser Ser
1 5
<210> 56
<211> 9
<212> PRT
<213> Artificial sequence ()
<400> 56
Leu Gln Tyr Ala Ser Ser Pro Tyr Thr
1 5
<210> 57
<211> 6
<212> PRT
<213> Artificial sequence ()
<400> 57
Gln Ser Val Tyr Thr Asn
1 5
<210> 58
<211> 9
<212> PRT
<213> Artificial sequence ()
<400> 58
Gln Gln Tyr Asn Ser Tyr Pro Val Thr
1 5
<210> 59
<211> 127
<212> PRT
<213> Artificial sequence ()
<400> 59
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr
20 25 30
Trp Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Leu Pro Gly Ser Ser Tyr Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Lys Gly Gly Pro Ile Tyr Tyr Gly Asn Arg Pro Phe Tyr Tyr
100 105 110
Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 60
<211> 381
<212> DNA
<213> Artificial sequence ()
<400> 60
caagtccagc ttgttcagag tggtgcagag gttaagaagc cggggagctc cgtaaaggtg 60
tcctgcaagg caacaggcta caccttcagt agctattgga ttagctgggt gagacaggcg 120
ccgggacaag ggcttgagtg gatggggcgt attctaccgg gctcatcata cacaaactat 180
gcccaaaaat tccaaggccg agtcacgatc actgcggaca agagtactag tacagcgtac 240
atggaacttt catcattacg gagcgaggat accgcggtgt attactgtgc gaaaaaggga 300
ggcccaatat attatgggaa tcgacctttc tattatgcga tggattactg gggacaaggt 360
accaccgtta cagtctcgag t 381
<210> 61
<211> 127
<212> PRT
<213> Artificial sequence ()
<400> 61
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr
20 25 30
Trp Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Leu Ile Leu Pro Gly Ser Ser Tyr Thr Ile Tyr Ala Glu Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Lys Gly Gly Pro Ile Tyr Tyr Gly Asn Arg Pro Phe Tyr Tyr
100 105 110
Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 62
<211> 381
<212> DNA
<213> Artificial sequence ()
<400> 62
gaggtgcaac tcgtacagtc gggcgcggaa gttaagaaac ccggcgccac ggtcaagatc 60
tcatgtaaag ccacaggata tacgttttcg tcttactgga tgcattgggt tcaacaagca 120
cccgggaaag gcctggagtg gatgggctta atcctccccg gatcttccta caccatttac 180
gccgagaaat tccagggaag ggtgacgatc accgctgaca cgagcaccga tacagcctac 240
atggagctaa gctccctgcg gtctgaggac accgcggtat attattgcgc caaaaaggga 300
ggacccatat attacggcaa ccgaccgttt tactacgcaa tggactattg gggacaaggc 360
accaccgtaa cagtctcgag t 381
<210> 63
<211> 127
<212> PRT
<213> Artificial sequence ()
<400> 63
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Leu Pro Gly Ser Ser Tyr Thr Thr Tyr Ala Gln Gly Phe
50 55 60
Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Lys Gly Gly Pro Ile Tyr Tyr Gly Asn Arg Pro Phe Tyr Tyr
100 105 110
Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 64
<211> 381
<212> DNA
<213> Artificial sequence ()
<400> 64
caagtacaac tggtgcagag cggctctgag ctgaagaagc ccggagccag cgtgaaggtc 60
tcatgtaagg ctactggcta tactttcagt agttactgga tgaactgggt ccgacaagcg 120
ccagggcaag gactagagtg gatgggctgg attttaccag gcagttcata cacgacatac 180
gcccagggct tcaccggcag attcgtgttc agcctggaca ccagcgtgtc caccgcctac 240
ctgcagatca gcagcctgaa ggccgaagat actgccgtgt actactgcgc gaagaaaggg 300
ggacctatat attatggcaa ccgcccattt tattatgcga tggattattg gggccagggt 360
accacggtga cagtctcgag t 381
<210> 65
<211> 127
<212> PRT
<213> Artificial sequence ()
<400> 65
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Leu Pro Gly Ser Ser Tyr Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Lys Gly Gly Pro Ile Tyr Tyr Gly Asn Arg Pro Phe Tyr Tyr
100 105 110
Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 66
<211> 381
<212> DNA
<213> Artificial sequence ()
<400> 66
caagttcaac ttgtgcaaag tggggctgaa gttaaaaaac ccggagcgag tgtaaaggtc 60
agctgcaaag ctacgggata taccttttcc tcatactgga tgcactgggt acgccaggcc 120
cccgggcagg gcctagaatg gatgggttgg attctcccgg gctcatctta tactaattac 180
gcacagaagt tccaaggtag agtaacaatg actagagata ccagcatcag tacagcatac 240
atggaactga gccgcttaag aagcgacgac accgccgtct attattgtgc taaaaaagga 300
ggtccaattt attacgggaa cagacccttc tactatgcga tggactactg gggacagggg 360
actaccgtta cagtctcgag t 381
<210> 67
<211> 127
<212> PRT
<213> Artificial sequence ()
<400> 67
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Leu Pro Gly Ser Ser Tyr Thr Ser Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Lys Gly Gly Pro Ile Tyr Tyr Gly Asn Arg Pro Phe Tyr Tyr
100 105 110
Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 68
<211> 381
<212> DNA
<213> Artificial sequence ()
<400> 68
caagtccagt tggtccagag tggggctgaa gtaaaaaagc caggcgcaag tgtcaaagtt 60
tcctgtaaag ccactggtta tacattttct agctactgga tgcattgggt acggcaagcg 120
ccgggacaag gtttagagtg gatggggata atccttcctg gctccagcta tacctcgtat 180
gcgcaaaaat tccaaggacg ggtgaccatg actagggata caagcacctc cacagtgtac 240
atggaactct ctagcctgcg tagcgaggat acagccgtgt attattgtgc aaaaaaaggc 300
ggccccattt actatgggaa tagacccttt tattacgcaa tggattattg gggccaggga 360
accactgtga cagtctcgag t 381
<210> 69
<211> 107
<212> PRT
<213> Artificial sequence ()
<400> 69
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 70
<211> 321
<212> DNA
<213> Artificial sequence ()
<400> 70
gacatccaga tgacccagtc tcctagcagc ctgtctgcat cggtgggcga tagggtgacg 60
attacctgta aagccagcca gagcgtcagc aacgacctca actggtacca acaaaagcca 120
ggcaaagccc caaagctcct catttactac gcttctaatc tggagaccgg ggtaccgagc 180
cggttctccg gatcgggctc aggcacagat ttcacattca ctataagcag cttacaacca 240
gaggatatcg ctacttatta ttgtcaacag gactattcca gcccccttac ctttgggggc 300
ggcaccaagg ttgagattaa g 321
<210> 71
<211> 107
<212> PRT
<213> Artificial sequence ()
<400> 71
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Ser Asn Asp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala
65 70 75 80
Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 72
<211> 321
<212> DNA
<213> Artificial sequence ()
<400> 72
gatatcgtga tgacgcaatc acctgacagt ctggccgtta gcctgggcga acgcgccaca 60
attaattgta aggccagcca aagcgtatca aacgacttgg cctggtatca gcaaaaaccg 120
ggccagccgc cgaaattgct tatttattat gcatcaacac gtgaaagcgg agttcccgac 180
cggttcagcg gctctggatc gggaactgat ttcacgctga cgatatcttc tctccaggca 240
gaggacgtcg ccgtttatta ttgccaacag gattactcct cacccctgac ttttggcggc 300
ggcaccaaag tcgagatcaa g 321
<210> 73
<211> 107
<212> PRT
<213> Artificial sequence ()
<400> 73
Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 74
<211> 321
<212> DNA
<213> Artificial sequence ()
<400> 74
gatatacagt tgacccagtc accgagcttc ctgagcgcca gcgttggcga ccgagtgacc 60
attacgtgta aagcgagtca atcggtgtcc aacgatttgg cgtggtatca acagaagcct 120
ggaaaggccc ctaaactcct gatatactac gcaagcacat tacaaagcgg cgtcccatcc 180
cgtttctctg ggagcggcag tggcactgag ttcactctta ccatcagcag tctccaaccg 240
gaagacttcg ccacctacta ctgtcagcaa gactactcta gtccgctgac atttggcggc 300
ggtacgaagg tggaaattaa g 321
<210> 75
<211> 107
<212> PRT
<213> Artificial sequence ()
<400> 75
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ser Val Ser Asn Asp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Tyr Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 76
<211> 321
<212> DNA
<213> Artificial sequence ()
<400> 76
gagatagtga tgacccaaag tcccgctact ttgtcggtaa gtcccggcga aagagctaca 60
ttatcatgca aagcatccca aagtgtaagc aatgatcttg catggtacca gcagaaacct 120
ggccaggccc ctcgattatt gatttattac gccagcacca gagccacggg catccccgcc 180
agatttagtg ggagtggttc tggtacggaa tttaccctga cgataagtag ccttcagagc 240
gaggacttcg cggtatatta ttgccagcag gattacagtt ctcccctaac ttttggagga 300
ggcaccaaag tggaaattaa a 321
<210> 77
<211> 107
<212> PRT
<213> Artificial sequence ()
<400> 77
Glu Ile Val Met Thr Gln Ser Pro Pro Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Val Thr Leu Ser Cys Lys Ala Ser Gln Ser Val Ser Asn Asp
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Tyr Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 78
<211> 321
<212> DNA
<213> Artificial sequence ()
<400> 78
gaaatagtga tgactcaatc accccccacc ctgtccctgt cgcctggcga aagagtgacg 60
ctctcgtgta aggcgtccca aagtgtgtct aatgacctgt cctggtatca acagaagcca 120
gggcaggccc cccgattgct gatttattac gcatcgacta gagctaccgg tatccctgcc 180
cgtttcagcg gttccggtag tggtaccgat ttcaccctga ctattagctc cctacagcct 240
gaagactttg cagtgtatta ctgccaacag gactatagca gccccctgac gttcgggggc 300
ggcactaagg tagagataaa a 321
<210> 79
<211> 118
<212> PRT
<213> Artificial sequence ()
<400> 79
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Pro Tyr Asn Gly Arg Thr Lys Tyr Ser Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Ala Leu Arg Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 80
<211> 354
<212> DNA
<213> Artificial sequence ()
<400> 80
caggtacaat tagtgcagtc cggcgccgag gtgaagaagc ctggcgccag cgtgaaagtt 60
agctgtaaag cgtctggtta cagtttcaca gggtatacca tgcattgggt aagacaggcc 120
ccggggcaac gtcttgagtg gatgggctgg ataaacccct acaacggccg taccaaatac 180
agccaaaaat ttcagggccg ggtaaccatt acacgcgaca cttccgccag cacggcatac 240
atggaattgt cgagtctccg gtctgaggat acggcagtat attattgtgc acgggtggca 300
ctgcgctatg ctatggacta ttggggacaa gggacgttag tcacagtctc gagt 354
<210> 81
<211> 118
<212> PRT
<213> Artificial sequence ()
<400> 81
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Thr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Asn Pro Tyr Asn Gly Arg Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Ala Leu Arg Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 82
<211> 354
<212> DNA
<213> Artificial sequence ()
<400> 82
caagtgcaat tggtgcaaag tggcgcagaa gttaaaaagc caggtagtag cgttaaagtg 60
tcatgcaagg cgagcggcta ctcatttact ggttacacga tcagctgggt gagacaagca 120
ccgggtcaag gactcgaatg gatggggaga atcaaccctt acaatggcag gacaaattac 180
gcccaaaagt tccaaggaag agtgacaatc accgcggaca aatcaaccag tacggcgtac 240
atggaacttt caagtctgcg atctgaagat acggctgtat actactgcgc ccgtgtcgcc 300
ttaagatatg ctatggatta ctgggggcaa gggactttag tgacagtctc gagt 354
<210> 83
<211> 118
<212> PRT
<213> Artificial sequence ()
<400> 83
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Asn Pro Tyr Asn Gly Arg Thr Ser Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Ala Leu Arg Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 84
<211> 354
<212> DNA
<213> Artificial sequence ()
<400> 84
caggttcagt tggtgcaaag cggggctgag gtcaaaaagc ctggtgcatc tgtcaaggta 60
agttgtaaag caagtgggta cagcttcacc ggatatacta tgcactgggt cagacaagcc 120
cctgggcaag gccttgaatg gatggggata attaatcctt acaacggcag aacttcctat 180
gcccagaaat ttcaagggcg ggtgacaatg acgagagaca ccagcacttc cactgtgtat 240
atggagttat caagcctgag atctgaagac accgccgttt actattgcgc aagagtcgcc 300
ctgcgttacg caatggatta ttggggccaa gggaccctgg ttacagtctc gagt 354
<210> 85
<211> 118
<212> PRT
<213> Artificial sequence ()
<400> 85
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Thr Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Leu Ile Asn Pro Tyr Asn Gly Arg Thr Ile Tyr Ala Glu Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Ala Leu Arg Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 86
<211> 354
<212> DNA
<213> Artificial sequence ()
<400> 86
gaggtacagc ttgtgcagag tggagctgaa gtgaaaaagc ccggcgcaac cgtaaagatt 60
agctgcaaag cgtcgggata ctcctttacg ggatatacca tgcactgggt acagcaagcc 120
ccaggcaaag ggctggaatg gatgggcttg atcaatccat ataacggcag aacaatctat 180
gccgaaaagt tccagggtcg tgtaacaatt actgcagata ccagcaccga caccgcgtac 240
atggaactgt cctctctcag aagcgaggac acggcagtgt actactgtgc cagggtagct 300
ctaaggtatg caatggacta ttggggtcaa ggaacgctgg ttacagtctc gagt 354
<210> 87
<211> 118
<212> PRT
<213> Artificial sequence ()
<400> 87
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Thr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Leu Ile Asn Pro Tyr Asn Gly Arg Thr Ile Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Ala Leu Arg Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Val Thr Val Ser Ser
115
<210> 88
<211> 354
<212> DNA
<213> Artificial sequence ()
<400> 88
caagtgcaat tggtgcaaag tggcgcagaa gttaaaaagc caggtagtag cgttaaagtg 60
tcatgcaagg cgagcggcta ctcatttact ggttacacga tcagctgggt gagacaagca 120
ccgggtcaag gactcgaatg gatggggctg atcaaccctt acaatggcag gacaatctac 180
aaccaaaagt tcaaaggaag agtgacaatc accgcggaca aatcaaccag tacggcgtac 240
atggaacttt caagtctgcg atctgaagat acggctgtat actactgcgc ccgtgtcgcc 300
ttaagatatg ctatggatta ctgggggcaa gggactaccg tgacagtctc gagt 354
<210> 89
<211> 107
<212> PRT
<213> Artificial sequence ()
<400> 89
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Tyr Thr Asn
20 25 30
Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile
35 40 45
Tyr Ser Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Val
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 90
<211> 321
<212> DNA
<213> Artificial sequence ()
<400> 90
gatatccaaa tgactcagtc accgtcgagc ctaagcgcct ccgtgggcga ccgggttacc 60
ataacatgca aagcctctca aagcgtatac acgaatttgg cttggttcca acagaaaccc 120
ggtaaggctc cgaaatctct gatctacagc gccagctcac tgcaatctgg tgtaccaagt 180
cgcttctccg ggtccgggtc cgggacagac tttacgctga cgatttcgtc attgcagccc 240
gaggactttg ccacgtacta ttgccaacag tataacagct acccggtaac tttcgggcaa 300
ggaaccaaag ttgaaatcaa g 321
<210> 91
<211> 107
<212> PRT
<213> Artificial sequence ()
<400> 91
Glu Ile Val Met Thr Gln Ser Pro Pro Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Val Thr Leu Ser Cys Lys Ala Ser Gln Ser Val Tyr Thr Asn
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Val
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 92
<211> 321
<212> DNA
<213> Artificial sequence ()
<400> 92
gaaatagtta tgacccaatc accgccaacc ctgagcttaa gccccggtga aagagtgacc 60
ctgagttgca aggcatcgca atcagtctat acgaacctga gttggtatca acagaaaccc 120
ggccaggctc caagactcct gatctactct gcctcaaccc gcgccaccgg cattcccgcc 180
cggtttagcg gttcaggatc gggtacggac ttcacactga ccatatcttc tctacagccg 240
gaagactttg ccgtctacta ctgtcagcag tataacagct atcccgtcac cttcggccag 300
ggtactaagg tagaaataaa a 321
<210> 93
<211> 107
<212> PRT
<213> Artificial sequence ()
<400> 93
Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Tyr Thr Asn
20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile
35 40 45
Lys Ser Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala
65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Val
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 94
<211> 321
<212> DNA
<213> Artificial sequence ()
<400> 94
gagatcgtgc ttacccaatc tcctgatttt caaagcgtga cccccaagga gaaggttaca 60
ataacctgca aggcgtcaca gtctgtgtat accaacttgc actggtacca gcaaaagcct 120
gaccaaagcc ccaagctact gattaagagc gcctcgcaat ccttcagcgg tgttccgtca 180
cggttttccg ggtccggctc agggaccgat tttaccctga ctataaattc gctggaggca 240
gaggatgccg ccacttacta ctgccagcag tacaatagct atcccgttac ctttggccaa 300
ggaacgaagg ttgagatcaa g 321
<210> 95
<211> 107
<212> PRT
<213> Artificial sequence ()
<400> 95
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Tyr Thr Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala
65 70 75 80
Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Val
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 96
<211> 321
<212> DNA
<213> Artificial sequence ()
<400> 96
gatatagtaa tgacccaaag cccagactcc ctggcggtca gcctaggaga gagagcaacc 60
ataaactgca aggccagcca gagtgtctac acaaacctag cctggtacca gcaaaaaccc 120
ggacaacctc ccaagctcct gatctattcc gccagcaccc gtgagtctgg ggtacctgac 180
agattctctg gatcaggatc cggcacggac ttcaccttaa ccatctcctc cctgcaggca 240
gaagacgtgg ctgtatacta ttgccaacaa tacaatagct atcctgtgac ttttgggcag 300
ggcacaaagg tggagattaa a 321
<210> 97
<211> 1101
<212> DNA
<213> Artificial sequence ()
<400> 97
aattcccgcc gccaccatgg gctggtcctg catcatcctg tttctggtgg ccacagccac 60
cggcgtgcac tctggtacca cgcgtgtcga ccaattgacc gtgtcctccg cctccacaaa 120
ggggccttct gtgtttccac tggccccctc ctctaagagc accagcggtg ggacagccgc 180
cctgggatgc ctggtgaagg actatttccc tgagcccgtg accgtgtcct ggaactccgg 240
cgccctgact tccggcgtgc acaccttccc cgccgtgctg cagagctctg gcctgtactc 300
tctgtcctcc gtggtgaccg tgccctcttc ctctctgggc acccagacct acatctgcaa 360
tgtgaaccac aagccttcca acaccaaggt ggataaaaag gtggagccca agtcctgcga 420
caagacccat acctgccccc cctgccccgc ccctgaactg ctgggaggac cctccgtgtt 480
cctgttcccc cccaagccca aggacactct gatgatctcc cgcacccccg aggtcacctg 540
cgtggtcgtg gacgtctctc acgaagaccc cgaggtgaaa ttcaactggt acgtggacgg 600
cgtcgaggtg cacaacgcca agaccaaacc ccgcgaggaa cagtataaca gcacctacag 660
agtggtgtcc gtgctcaccg tgctgcacca ggactggctc aacggcaagg aatacaagtg 720
caaggtgtcc aacaaggccc tccccgcccc cattgaaaag accatctcca aagccaaagg 780
ccagcccaga gaaccccagg tctacaccct ccccccctcc agagaagaaa tgaccaagaa 840
ccaggtgagc ctgacctgcc tcgtcaaagg gttctacccc tccgacatcg ccgtggagtg 900
ggagagcaac ggccagcccg agaacaacta caaaaccacc ccccccgtgc tcgactccga 960
cggatccttc ttcctgtaca gcaaactgac cgtggacaaa tcccgctggc agcagggcaa 1020
cgtgttctcc tgctccgtga tgcacgaggc cctgcacaac cattacaccc agaagtccct 1080
gtccctgtcc cccggcaagt a 1101
<210> 98
<211> 455
<212> DNA
<213> Artificial sequence ()
<400> 98
gaattcccgc cgccaccatg ggctggtcct gcatcatcct gtttctggtg gccacagcca 60
ccggcgtgca ctctggtacc acgcgtgtcg accaattgag aaccgtggcc gccccctccg 120
tgtttatctt ccccccctct gacgagcagc tcaagtccgg caccgcctcc gtcgtctgcc 180
tcctgaacaa tttctacccc cgcgaagcta aagtccagtg gaaggtggat aacgccctgc 240
agtccggcaa ctcccaggag agcgtcaccg agcaggactc caaggactcc acctactccc 300
tctcctccac cctgaccctc tccaaagccg attacgagaa acacaaagtg tacgcctgcg 360
aggtgacaca ccagggcctg tccagccccg tcaccaaaag cttcaacaga ggcgagtgct 420
gataagcttg ctagcggccg ctcgaggccg gatcc 455

Claims (13)

1. An antibody comprising a heavy chain variable region and/or a light chain variable region:
the heavy chain variable region comprising:
a heavy chain complementarity determining region HCDR1 consisting of the amino acid sequence shown in SEQ ID NO. 29,
a heavy chain complementarity determining region HCDR2 consisting of an amino acid sequence shown in SEQ ID NO. 30,
a heavy chain complementarity determining region HCDR3 consisting of an amino acid sequence represented by SEQ ID NO. 31;
the light chain variable region comprising:
a light chain complementarity determining region LCDR1 consisting of the amino acid sequence shown in SEQ ID NO:47,
a light chain complementarity determining region LCDR2 consisting of a YAS amino acid sequence,
and a light chain complementarity determining region LCDR3 consisting of the amino acid sequence shown in SEQ ID NO. 48.
2. The antibody of claim 1, wherein the antibody is a murine antibody, a chimeric antibody, or a human antibody.
3. The antibody of claim 1 or 2, wherein said heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO 59, SEQ ID NO 61, SEQ ID NO 63, SEQ ID NO 65 and SEQ ID NO 67; the light chain variable region is selected from the amino acid sequence shown in SEQ ID NO 69, 71, 73, 75 or 77.
4. The antibody of claim 3, wherein the heavy chain variable region is the amino acid sequence of SEQ ID NO 59 and the light chain variable region is the amino acid sequence of SEQ ID NO 71;
or the heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 59, and the light chain variable region has an amino acid sequence shown as SEQ ID NO. 73;
or the sequence of the heavy chain variable region is the amino acid sequence shown by SEQ ID NO. 59, and the sequence of the light chain variable region is the amino acid sequence shown by SEQ ID NO. 75;
or the sequence of the heavy chain variable region is the amino acid sequence shown by SEQ ID NO. 65, and the sequence of the light chain variable region is the amino acid sequence shown by 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.
5. The antibody according to claim 1 or 2, characterized in that it comprises any one of the following properties i to v:
i. the antibody is capable of specifically binding to subtype D of the C5 family of G protein-coupled receptors;
ii. The antibody does not bind to any of CD19, CD3, CD11b, or CD 14;
iii, the antibody specifically binds to human GPRC5D and cross-reacts with monkey GPRC 5D;
iv, the antibody has endocytic activity;
v, said antibody has reduced glycosylation or is aglycosylated or is hypofucosylated.
6. A recombinant protein comprising the antibody of claim 1 or 2.
7. A pharmaceutical composition comprising the antibody of claim 1 or 2 or the recombinant protein of claim 6.
8. A polynucleotide comprising a nucleotide sequence encoding the antibody of claim 1 or 2 or the recombinant protein of claim 6.
9. A vector comprising the polynucleotide of claim 8.
10. An isolated cell that produces the antibody of claim 1 or 2 or the recombinant protein of claim 6.
11. A method for producing an antibody, comprising the steps of: culturing the isolated cell of claim 10 and recovering the antibody from the culture.
12. Use of an antibody according to claim 1 or 2 or a recombinant protein according to claim 6 for the manufacture of a medicament or pharmaceutical composition for the treatment of cancer; preferably, the cancer is multiple myeloma.
13. Use of the antibody of claim 1 or 2 or the recombinant protein of claim 6 for the preparation of an antibody detection kit.
CN202110561819.5A 2021-05-23 2021-05-23 anti-GPRC 5D antibody, preparation method and application thereof Pending CN115386006A (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202110561819.5A CN115386006A (en) 2021-05-23 2021-05-23 anti-GPRC 5D antibody, preparation method and application thereof
PCT/CN2022/094551 WO2022247804A1 (en) 2021-05-23 2022-05-23 Anti-gprc5d antibody, preparation method therefor, and use thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110561819.5A CN115386006A (en) 2021-05-23 2021-05-23 anti-GPRC 5D antibody, preparation method and application thereof

Publications (1)

Publication Number Publication Date
CN115386006A true CN115386006A (en) 2022-11-25

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Country Link
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117700558A (en) * 2024-02-06 2024-03-15 苏州艾凯利元生物科技有限公司 Monoclonal anti-GPRC 5D antibody and anti-GPRC 5D-CAR-NK cell
WO2024087731A1 (en) * 2022-10-25 2024-05-02 上海祥耀生物科技有限责任公司 Anti-gprc5d multi-specific antibody
WO2024131962A1 (en) * 2022-12-23 2024-06-27 成都恩沐生物科技有限公司 Novel anti-gprc5d antibody
WO2025002251A1 (en) * 2023-06-28 2025-01-02 浙江博锐生物制药有限公司 Anti-gprc5d antibody and medical use thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024087731A1 (en) * 2022-10-25 2024-05-02 上海祥耀生物科技有限责任公司 Anti-gprc5d multi-specific antibody
WO2024131962A1 (en) * 2022-12-23 2024-06-27 成都恩沐生物科技有限公司 Novel anti-gprc5d antibody
WO2025002251A1 (en) * 2023-06-28 2025-01-02 浙江博锐生物制药有限公司 Anti-gprc5d antibody and medical use thereof
CN117700558A (en) * 2024-02-06 2024-03-15 苏州艾凯利元生物科技有限公司 Monoclonal anti-GPRC 5D antibody and anti-GPRC 5D-CAR-NK cell
CN117700558B (en) * 2024-02-06 2024-05-03 苏州艾凯利元生物科技有限公司 A monoclonal anti-GPRC5D antibody and anti-GPRC5D-CAR-NK cell

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