A FZD7 mouse monoclonal antibody and its preparation method and application

Abstract

The present invention discloses a FZD7 mouse monoclonal antibody, its preparation method, and application. The FZD7 mouse monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO: 1, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO: 2. The FZD7 mouse monoclonal antibody obtained by the present invention can bind to the FZD7 protein both in vivo and in vitro, and has clinical development value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a FZD7 mouse monoclonal antibody and its preparation method and application; in particular, a high-titer FZD7 mouse monoclonal antibody, its preparation method and application; the present invention also provides the antibody encoding nucleic acid molecule, expression vector, host cell, and method for expressing the antibody. Background Art

[0002] The Frizzleds (FZDs) receptor family is a member of the G protein-coupled receptor (GPCR) F family, with 10 subtypes widely expressed in the cardiovascular system. Reports have confirmed the targeting effect of FZDs in tumors.

[0003] The Frizzled7 (FZD7) gene is located on chromosome 2q33 and consists of 574 amino acids. It is highly expressed in melanoma, lung cancer, esophageal cancer, gastric cancer, colon cancer, liver cancer, and lymphocytic leukemia (Khan NI et al., Br J Haematol, 2007, 138: 338-348). Studies have confirmed that FZD7 is an essential biomarker in the development of various cancers (Yang L et al., Oncogene, 2011, 30(43): 4437-4446) and is one of the potential cancer treatment targets in vivo.

[0004] There are currently no FZD-related drugs on the market. The most advanced FZD-related drug under development globally is in Phase I clinical trials, with the multi-targeted drug vantictumab targeting Wnt, FZDs, and β-catenin signaling. There are no drugs specifically targeting FZD7 in clinical development. Therefore, the development of monoclonal antibodies targeting the FZD7 subtype is highly necessary and holds significant medical value. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defect of the lack of effective FZD7 antibody drugs in the prior art and to provide a FZD7 mouse monoclonal antibody and its preparation method and application.

[0006] The technical solution of the present invention to solve the above technical problems is as follows.

[0007] One of the technical solutions of the present invention is: a FZD7 murine monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO: 1, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO: 2.

[0008] The antibody of the present invention further comprises a heavy chain constant region and a light chain constant region; preferably, the subclass of the heavy chain constant region is IgG1, and the light chain constant region is a kappa chain.

[0009] In a preferred embodiment of the present invention, the target protein targeted by the FZD7 murine monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:3.

[0010] The FZD7 murine monoclonal antibody of the present invention may exist in the form of a full-length antibody, Fab, Fab', F(ab')2, Fv, a bispecific antibody or a multispecific antibody, that is, it may be in the form of a full-length antibody, Fab, Fab', F(ab')2, Fv, a bispecific antibody or a multispecific antibody.

[0011] In the present invention, the term "full-length antibody" is used interchangeably to refer to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in the present invention) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in the present invention) and a light chain constant region (abbreviated as CL in the present invention). The light chain constant region consists of one domain, CL. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulins (Ig) A, IgD, IgE, IgG, and IgM. Complete antibodies form a "Y" shape. The stem of the Y is composed of the second and third constant regions of the two heavy chains (and the fourth constant region for IgE and IgM), and disulfide bonds (between chains) are formed in the hinge. The heavy chains γ, α, and δ have a constant region consisting of three tandem (in a row) Ig domains and a hinge region for increasing flexibility; the heavy chains μ and ε have a constant region consisting of four immunoglobulin domains. The second and third constant regions are called "CH2 domains" and "CH3 domains," respectively. Each arm of the Y includes the variable region and the first constant region of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0012] As used herein, a "Fab fragment" consists of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions within the CH3 domain. A "Fab' fragment" contains one light chain and a portion of one heavy chain comprising the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains. This allows for interchain disulfide bonds to form between the two heavy chains of the two Fab' fragments, forming a F(ab')2 molecule. A "F(ab')2 fragment" contains two light chains and two heavy chains comprising a portion of the constant region between the CH1 and CH2 domains. This allows for interchain disulfide bonds to form between the two heavy chains. Thus, a F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains. The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody, but lacking the constant region.

[0013] In the present invention, the scFv (single chain antibody fragment) may be a conventional single chain antibody in the art, comprising a heavy chain variable region, a light chain variable region and a short peptide of 15 to 20 amino acids. The VL and VH domains are paired to form a monovalent molecule by enabling them to be produced as a linker for a single polypeptide chain [see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers are composed of repeated G4S amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (G4S)4 or (G4S)3 may be used, but variants thereof may also be used.

[0014] The term "multispecific antibody" is used in its broadest sense to encompass antibodies with multiple epitope specificities. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has multiple epitope specificities; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable domains, each single variable domain binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, bispecific antibodies (diabodies), and triabodies, antibody fragments linked together covalently or non-covalently, etc.

[0015] In a preferred embodiment of the present invention, the method for preparing the FZD7 murine monoclonal antibody comprises the following steps:

[0016] (1) constructing an expression vector to express a partial DNA sequence of the FZD7 gene as shown in SEQ ID NO: 4 to obtain the FZD7 linker protein;

[0017] (2) immunizing mice with the FZD7 linker protein obtained in step (1);

[0018] (3) Prepare a spleen cell suspension from immunized mice and fuse it with SP2 / 0 myeloma cells to produce hybridoma cells;

[0019] (4) Preparation of antibodies from ascites.

[0020] The second technical solution of the present invention is: an isolated nucleic acid encoding the FZD7 murine monoclonal antibody as described in the first technical solution.

[0021] The third technical solution of the present invention is: a recombinant expression vector comprising the isolated nucleic acid described in the second technical solution; preferably, the recombinant expression vector is a plasmid, a cosmid, a phage or a viral vector, and the viral vector is preferably a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector; the backbone of the recombinant expression vector is preferably plasmid pET28a-Sumo.

[0022] In the present invention, the term "host cell" includes cells into which exogenous nucleic acids have been introduced, including the progeny of these cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its derived progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. The present invention includes mutant progeny that have the same function or biological activity as cells screened or selected in the initial transformed cell.

[0023] The fourth technical solution of the present invention is: a transformant, which contains the recombinant expression vector as described in the third technical solution in a host cell.

[0024] As used herein, "vector" refers to a construct that is capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0025] The fifth technical solution of the present invention is: a chimeric antigen receptor, which comprises the FZD7 murine monoclonal antibody as described in the first technical solution.

[0026] The sixth technical solution of the present invention is: a genetically modified cell comprising the chimeric antigen receptor as described in the fifth technical solution; preferably, the genetically modified cell is a eukaryotic cell, preferably an isolated human cell; more preferably, an immune cell such as a T cell or a NK cell.

[0027] The seventh technical solution of the present invention is: a method for preparing FZD7 mouse monoclonal antibody, which comprises culturing the transformant as described in the fourth technical solution, and obtaining FZD7 mouse monoclonal antibody from the culture.

[0028] The eighth technical solution of the present invention is: an antibody-drug conjugate, comprising a cytotoxic agent and the FZD7 murine monoclonal antibody as described in one of the technical solutions; preferably, the cytotoxic agent is MMAF or MMAE.

[0029] A ninth technical solution of the present invention is: a pharmaceutical composition comprising the FZD7 murine monoclonal antibody according to the first technical solution and / or the antibody-drug conjugate according to the eighth technical solution, and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is preferably a cancer-related therapeutic drug that specifically targets FZD7;

[0030] Preferably, the pharmaceutical composition further contains one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.

[0031] In some embodiments, the pharmaceutical composition or pharmaceutical preparation of the present invention comprises a suitable pharmaceutically acceptable carrier such as a pharmaceutical excipient, such as a pharmaceutical carrier or pharmaceutical excipient known in the art, including a buffer. As used in the present invention, "pharmaceutically acceptable carrier" or "pharmaceutical carrier" includes any and all solvents, dispersion media, isotonic agents, and absorption delaying agents that are physiologically compatible. Pharmaceutical carriers suitable for the present invention can be sterile liquids such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. For the use and purposes of excipients, see also "Handbook of Pharmaceutical Excipients", Fifth Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago. If desired, the composition may also contain a small amount of a wetting agent or emulsifier, or a pH buffer. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may include standard pharmaceutical carriers and / or excipients, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin. Pharmaceutical preparations or pharmaceutical compositions comprising the present invention may be prepared by mixing an antibody or antigen-binding fragment thereof of the present invention with the desired purity and one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. ed. (1980)), preferably in the form of a lyophilized formulation or aqueous solution. Pharmaceutical composition of the present invention or preparation can also comprise more than a kind of active ingredient, and described active ingredient is required for the specific indication being treated, preferably has those active ingredients of complementary activity that can not adversely affect each other.For example, it is desirable to also provide other anti-infective active ingredients, for example other antibodies, anti-infective active agents, small molecule drugs or immunomodulators etc..Described active ingredient is suitably combined with the effective amount for purpose use.Sustained release preparation can be prepared.Sustained release preparation suitable example comprises the semi-permeable matrix of the solid hydrophobic polymer containing antibody of the present invention or its Fab, and described matrix is ​​shaped article, for example film or microcapsule form.

[0032] The tenth technical solution of the present invention is: use of the FZD7 murine monoclonal antibody according to the first technical solution, the antibody-drug conjugate according to the eighth technical solution, and / or the pharmaceutical composition according to the ninth technical solution in the preparation of a drug for diagnosing, preventing and / or treating a cancer specifically targeting FZD7; preferably, the tumor is a FZD7-positive tumor.

[0033] The present invention adopts the following technical solutions:

[0034] A mouse antibody that can specifically bind to FZD7, wherein the amino acid sequence of the antibody is derived from mouse preparation.

[0035] Method for preparing anti-FZD7 monoclonal antibodies: Mice are immunized with the constructed pET28a-Sumo expression vector, transformed, and induced to express. After the mouse serum titer exceeds 1:10,000, spleens are harvested to prepare a splenocyte suspension. Hybridoma cells are then fused with SP2 / 0 myeloma cells to generate hybridomas. Hybridoma cell lines that stably secrete FZDT antibodies are screened by ELISA and subcloned. Selected hybridoma cells are injected into the peritoneal cavity of paraffin oil-sensitized mice. Ascites fluid is collected before the mice die, and the antibodies in the ascites fluid are purified to obtain the FZD7 mouse monoclonal antibody.

[0036] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0037] The reagents and raw materials used in the present invention are commercially available.

[0038] The positive progress effect of the present invention is:

[0039] The immunogen used to prepare the FZD7 murine monoclonal antibody of the present invention is synthesized in vitro, constructed into a pET28a-Sumo expression vector, and then recombinantly expressed. The target protein was confirmed by ELISA. The antibody preparation method is based on hybridoma cell technology to produce monoclonal antibodies. In vitro SEC binding experiments and Western blot validation confirmed that the FZD7 murine monoclonal antibody obtained in the present invention can bind to the FZD7 protein. Live cell binding experiments also confirmed that the FZD7 murine monoclonal antibody obtained in the present invention can bind to the FZD7 protein. It is speculated that the FZD7 murine monoclonal antibody obtained in the present invention has clinical development value. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The plasmid map of the FZD7 DNA template.

[0041] Figure 2 This is the peak diagram of analytical molecular sieve experiment.

[0042] Figure 3 Binding was verified by western blotting.

[0043] Figure 4 Flow cytometry (FACS) binding experiments are shown. DETAILED DESCRIPTION

[0044] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0045] Example 1 Construction of FZD7 DNA template

[0046] According to the expression gene of the target protein (SEQ ID NO: 4), the pET28a-Sumo expression vector ( Figure 1 ), expressing FZD7 linker region protein.

[0047] Example 2 Preparation of hybridoma cells

[0048] 1. Animal immunization

[0049] In order to obtain a monoclonal antibody targeting the FZD7 linker protein, the present invention selected the expressed protein obtained in Example 1 as an antigen to immunize mice (6-8 week old BALB / c mice, Shanghai Xipul-Bikai Experimental Animal Co., Ltd.). Each mouse was immunized with 2 ml of the first immunization antigen, and the antigen was mixed with an equal amount of complete Freund's adjuvant to prepare an emulsion, which was injected intraperitoneally. At intervals of two weeks, 1 ml of the immunization antigen was mixed with 1 ml of incomplete Freund's adjuvant to prepare an emulsion, which was injected intraperitoneally for a total of four immunizations. One week after the third immunization, blood was collected from the mouse's orbital vein, and the serum was collected. The mouse serum titer was detected by Elisa. Qualified mice that had been immunized four times were selected, and 40 μg of antigen was injected intraperitoneally. Cell fusion was performed three days later.

[0050] 2. Elisa test

[0051] 1) Coating: Dilute the antigen to 1 μg / ml with CBS and add 100 μl per well of the ELISA plate at 4°C overnight.

[0052] 2) Blocking: Discard the coating solution, add 200 μl of blocking solution (5% skim milk powder) to each well, and incubate at 37°C for 1.5 h;

[0053] 3) Sample addition: Discard the blocking solution and add 100 μl of sample to each well. Incubate at 37°C for 1 hour.

[0054] 4) Washing: Wash 3 times with detergent and pat dry;

[0055] 5) Secondary antibody: Dilute enzyme-labeled goat anti-mouse antibody to the working concentration in blocking buffer, add 100 μl to each well, and incubate at 37°C for 30 min.

[0056] 6) Washing: Wash 3 times with detergent and pat dry;

[0057] 7) Color development: Add 100 μl of TMB color development substrate to each well and incubate at 37°C for 15 min.

[0058] 8) Stop: Add 50 μl of stop solution to each well;

[0059] 9) Reading: Read the OD at 450 nm using a microplate reader.

[0060] 3. Cell fusion: Perform according to standard procedures in this field.

[0061] 3.1 SP2 / 0 cell collection:

[0062] 3.1.1 Collect 50 ml of SP2 / 0 cells in logarithmic growth phase and in good condition and centrifuge at 1200 rpm for 5 minutes;

[0063] 3.1.2 Discard the supernatant, pipette 1 ml of preheated fusion agent (PEG1450) and add it dropwise to the cells. Incubate in a 37°C water bath for 1 min, then add stop solution (25 ml of DMEM medium) and centrifuge at 1200 rpm for 5 min.

[0064] 3.2 Fusion board detection:

[0065] 3.2.1 After fusion, observe the growth status of the clones in the 96-well plate every day to check for contamination and ensure normal cell growth;

[0066] 3.2.2 When the number of cloned cells reaches hundreds or more, change the medium and add 200 μl of fresh HT medium to each well. Incubate the cells in an incubator (37°C, 5% CO2).

[0067] 3.2.3 After 2 days, change the medium and transfer 100 μl of supernatant from each well to a coated 96-well ELISA plate for ELISA detection.

[0068] 3.2.4 For cells detected by Elisa, discard the culture medium in the wells and add 200 μl of fresh HT medium. Incubate in an incubator (37°C, 5% CO2).

[0069] 3.2.5 After 2 days, change the medium and retest by ELISA. Prepare the wells that show positive results for subcloning.

[0070] Example 3 Preparation of FZD7 Mouse Monoclonal Antibody

[0071] 1. Preparation of antibodies from ascites

[0072] 1.1 Intraperitoneal injection of paraffin oil into mice 7 days in advance;

[0073] 1.2 When the hybridoma cells have basically covered the bottom of the bottle and are in good condition, the cells are collected, centrifuged at 1200 rpm for 5 minutes, and injected intraperitoneally into paraffin mice (2×10 6 cell);

[0074] 1.3 Observe the condition of the mice every day. Kill the mice by cervical dislocation before they become moribund. Cut the skin with surgical scissors to expose the peritoneum. Then make a small cut in the peritoneum and aspirate the ascites with a dropper.

[0075] 1.4 Centrifuge the ascites at 3000 rpm for 30 min, take the supernatant, aliquot and freeze at -80°C, and take a small amount of ELISA to detect the titer of the ascites.

[0076] 2. Antibody purity identification

[0077] 2.1 Take 1 ml of ascites, centrifuge at 12000 rpm for 4 min, take the supernatant and dilute with PBS;

[0078] 2.2 Equilibrate a 1 ml column volume of Protein G column with 10 ml PBS, load the ascites fluid onto the column, and collect the flow-through;

[0079] 2.3 After washing with PBS, elute with 2.5 ml of 0.1 M glycine solution (pH 2.5). Neutralize the eluate by adding 250 μl of 1 M Tris solution (pH 8.8).

[0080] 2.4 The antibody eluate was dialyzed against PBS overnight, and the affinity was detected by ELISA.

[0081] The reagents used in Elisa are as follows:

[0082] 1) PBS (Phosphate Buffered Saline) Solution:

[0083] ① PBS storage solution (10×) preparation method:

[0084]

[0085] Dissolve in 100 ml of deionized water and adjust the pH to 7.2.

[0086] ②PBS solution preparation method (1×):

[0087] Take 50ml of storage solution and add 450ml of deionized water, and store at room temperature or 4℃ for later use.

[0088] 2) Antigen coating solution (CBS, 1×):

[0089] Weigh 1.59 g of Na2CO3, 2.93 g of NaHCO3, and 950 ml of deionized water, adjust the pH to 9.6, add deionized water to make up to 1000 ml, and store at 4°C.

[0090] 3) Blocking solution

[0091] Weigh 5g skim milk powder, dissolve it in 100ml PBS, mix well, and store at 4℃ for later use (storage at 4℃ should not exceed 3 days)

[0092] 4) TMB colorimetric solution

[0093] ① TMB stock solution: Weigh TMB powder and prepare 1.5 mg / ml stock solution with DMSO. Store in aliquots at -20℃.

[0094] ②NaAc buffer: Prepare 200mM NaAc solution and adjust the pH to 5.3 with HAc

[0095] ③H2O2 solution: prepare 0.03% H2O2 solution

[0096] ④ When using, prepare the color developing solution according to the ratio of TMB storage solution: NaAc buffer solution: H2O2 solution = 1:4:5, and use it immediately after preparation.

[0097] 5) Stop solution (2M sulfuric acid)

[0098] Slowly add 100ml of concentrated sulfuric acid into 900ml of water (note: concentrated sulfuric acid must be added dropwise to the water), and store at room temperature for later use.

[0099] The specific operations are as follows:

[0100] 1) Coating: Dilute the antigen with CBS, usually 1 μg / ml, add 100 μl to each well of the ELISA plate, and incubate at 4°C overnight.

[0101] 2) Blocking: Discard the coating solution, add 200 μl of blocking solution to each well, and place at 37°C for 1.5 hours

[0102] 3) Add sample: discard the blocking solution, add sample (diluted serum or primary antibody), add 100 μl to each well of the ELISA plate, and place at 37°C for 1 hour

[0103] 4) Washing: Rinse 10 times with tap water and pat dry

[0104] 5) Add secondary antibody: Dilute the secondary antibody to the working concentration with blocking solution, add 100 μl to each well of the ELISA plate, and place at 37°C for 30 minutes

[0105] 6) Washing: Rinse 10 times with tap water and pat dry

[0106] 7) Add colorimetric substrate: add 100 μl per well to the ELISA plate and place at 37°C for 15 minutes

[0107] 8) Add stop solution: Add 50 μl of 2M H2SO4 to each well

[0108] 9) Reading: Microplate reader OD450 nm reading

[0109] Antibody ELISA test data report

[0110] (1) Fusion mouse ELISA test data (mouse triple immune test results)

[0111] Table 1

[0112]

[0113] ELISA tests showed that antibodies 3301-M1 and 3301-M3 had the highest detection values. Therefore, spleen cells from these two groups of mice were fused with SP2 / 0 cells to prepare hybridoma cells.

[0114] The following 7 monoclonal antibodies were prepared.

[0115] (2) Ascites antibody ELISA test results

[0116] Table 2

[0117]

[0118] Example 4 In vitro binding assay of antibodies

[0119] 1. Analytical molecular sieve experiment

[0120] 1) Membrane washing: In a cell homogenizer, add low-salt and high-salt solutions containing protease inhibitors (cocktail, bimake) to wash the membrane, respectively, and centrifuge at 35,000 rpm to retain the precipitate.

[0121] 10× low salt solution (pH 7.4): 1 L of solution contains 100 ml of 1 M HEPES, 20.3 g of MgCl2·6H2O, and 14.9 g of KCl.

[0122] 1× high salt solution (pH 7.4): 5 L of solution contains 50 ml of 1 M HEPES, 10.165 g of MgCl2·6H2O, 7.45 g of KCl, and 292.5 g of NaCl.

[0123] 2) Dissolution: The precipitate was resuspended in a low-salt homogenate solution, and then incubated with 20 mg / ml iodoacetamide. Then, dissolution solution was added and incubated at 4°C for 2 h.

[0124] 2x membrane dissolution solution: 1 M HEPES, 5 M NaCl, 2% DDM, 0.4% CHS.

[0125] 3) Affinity chromatography: centrifuge at 35,000 rpm, take the supernatant, add cobalt medium (Co 2+resin) and 20 mM imidazole and incubated at 4°C overnight.

[0126] 4) Purification: Transfer the cobalt medium containing the membrane protein to a purification column. Add W1 and W2 solutions sequentially by gravity to remove contaminants nonspecifically bound to the cobalt medium. Finally, elute the membrane protein from the cobalt medium with Elute solution and collect it in a centrifuge tube for later use.

[0127] W1 solution: 50 mM HEPES pH 7.4, 500 mM NaCl, 10% glycerol, 0.1% DDM, 0.02% CHS, 20 mM imidazole

[0128] W2 solution: 50 mM HEPES pH 7.4, 500 mM NaCl, 10% glycerol, 0.005% DDM, 0.001% CHS, 50 mM imidazole

[0129] Elute solution: 50 mM HEPES pH 7.4, 500 mM NaCl, 10% glycerol, 0.025% DDM, 0.005% CHS, 50 mM imidazole

[0130] FZD7 membrane protein was expressed in an insect cell system and obtained through membrane washing, membrane dissolution, affinity chromatography, and purification. 30 μg of FZD7 membrane protein was incubated with 120 μg of antibody #5 (3301-05 monoclonal antibody) overnight at 4°C. A nanofilm SEC-250 column (Sepax) was used to analyze the protein, utilizing the characteristic absorption peak of aromatic amino acids at 280 nm. Binding experiments were performed using an Agilent Technology 1260 Infinity instrument to detect the binding of FZD7 membrane protein and antibody #5 (3301-05 monoclonal antibody).

[0131] Figure 2 Analytical size-exclusive chromatography (aSEC) revealed that, when using the same amount of FZD7 membrane protein, the curve of FZD7 membrane protein bound to Antibody #5 (3301-05 mAb) showed a significant peak shift compared to the curve of FZD7 membrane protein alone, demonstrating that Antibody #5 (3301-05 mAb) can bind to FZD7 membrane protein in vitro.

[0132] 2. Western blot (WB)

[0133] Primary antibody: Antibody #5 (3301-05 monoclonal antibody)

[0134] Secondary antibody: Monoclonal ANTI-FLAG(R)M2 antibody produced in mouse 1mg / mL,clone M2,affinity isolated antibody,buffered aqueous solution(Sigma)

[0135] The FZD7 membrane protein was extracted and subjected to SDS-PAGE electrophoresis (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE), electrotransferred to a PVDF membrane, blocked with TBST solution containing 5% milk, incubated with the primary antibody, washed with TBST solution, incubated with the secondary antibody, washed with TBST solution, and developed with HRP to verify the binding of the antibody to the FZD7 membrane protein ( Figure 3 ), it can be seen that antibody #5 (3301-05 monoclonal antibody) can effectively bind to FZD7 membrane protein.

[0136] 3. Flow cytometry (FACS) experiment

[0137] Collect 10×10 6 293F / 293T cells transiently transfected with FZD7 receptor (purchased from ATCC) were incubated with 5# antibody (3301-05 monoclonal antibody) as primary antibody for 30 min, washed and then added with secondary antibody (A31571 Alexa The cells were incubated with 647 Donkey Anti-Mouse IgG (H+L), 1:2000, for 30 min, washed, and then detected using a Millipore Guava assay. The negative control consisted of the addition of secondary antibody alone.

[0138] Figure 4 Figures a, b, and c were transiently transfected with FZD7 plasmid into 293F cells using Polyethylenimine and Linear (PEI, Polysciences). Figure a shows that the transfection efficiency reached 63.6%. Compared with the control b where only the secondary antibody was added, after adding the 5# antibody (3301-05 monoclonal antibody) and the secondary antibody, cells were visible at the location of stronger red fluorescence (647). This suggests that the 293F living cells transiently transfected and expressing FZD7 can be bound by the 5# antibody (3301-05 monoclonal antibody) ( Figure 4 (c)

[0139] Figure 4In d and e, FZD7 plasmid was transiently transfected into 293T cells using Lipofectamine 2000 (ThermoFisher). Compared with control d, the 293T living cells transiently transfected and expressing FZD7 could be bound by antibody 5# (3301-05 monoclonal antibody) ( Figure 4 e), the binding efficiency is 6.5% ( Figure 4 f, 594 cells / 9132 cells). SEQUENCE LISTING <110> Shanghai University of Science and Technology <120> A FZD7 mouse monoclonal antibody and its preparation method and application <130> P21011411C <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 120 <212> PRT <213> Mus musculus <400> 1 Gln Val Gln Leu Leu Gln Ser Gly Pro Glu Leu Met Lys Pro Gly Thr 1 5 10 15 Ser Val Arg Met Ser Cys Lys Thr Ser Ser Gly Tyr Thr Phe Ser Gly Tyr 20 25 30 Trp Met Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Thr Asp Lys Thr Asn Tyr Asn Lys Asn Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Leu Gln Val Thr Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Val Arg Gly Tyr Gly Asp Ser Gly Ser Trp Phe Leu Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Thr 115 120 <210> 2 <211> 112 <212> PRT <213> Mus musculus <400> 2 Asn Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Cys Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Val Tyr Tyr Cys His Gln 85 90 95 Tyr Leu Ser Ser Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 3 <211> 74 <212> PRT <213> Mus musculus <400> 3 Asp Gly Ser Gly Gly Pro Gly Gly Gly Pro Thr Ala Tyr Pro Thr Ala 1 5 10 15 Pro Tyr Leu Pro Asp Leu Pro Phe Thr Ala Leu Pro Pro Gly Ala Ser 20 25 30 Asp Gly Arg Gly Arg Pro Ala Phe Pro Phe Ser Cys Pro Arg Gln Leu 35 40 45 Lys Val Pro Pro Tyr Leu Gly Tyr Arg Phe Leu Gly Glu Arg Asp Cys 50 55 60 Gly Ala Pro Cys Glu Pro Gly Arg Ala Asn 65 70 <210> 4 <211> 222 <212> DNA <213> Mus musculus <400> 4 gacggctccg gcggccccgg cggcggcccc accgcctacc ccacagcccc ttacctccct 60 gatctgcctt tcaccgccct gcctcccgga gctagcgatg gcagaggcag acctgccttc 120 cccttcagct gccccagaca gctgaaggtg ccccctatc tgggctacag gttcctgggc 180 gagagagatt gcggcgctcc ctgcgaacct ggaagagcca ac 222 <210> 5 <211> 1035 <212> DNA <213> Mus musculus <220> <221> misc_feature <222> (1)..(3) <223> n is a, c, g, or t <400> 5 nnnagtggtg gctacgtata ctccggaata ttaatagatc atggagataa ttaaaatgat 60 aaccatctcg caaataaata agtattttac tgttttcgta acagttttgt aataaaaaaa 120 cctataaata ttccggatta ttcataccgc cccaccatcg ggcgcgcgga attcgccaaa 180 atgaagacaa tcatcgccct gagctatatc ttctgcctgg tgttcgccga ctacaaggac 240 gacgacgaca aggaaaaggg cattagcgtc cccgaccacg gcttctgtca gcccattagc 300 atccccctgt gcaccgacat cgcctacaat caaaccatcc tgcccaacct cctgggccat 360 accaaccagg aagacgctgg actcgaagtc caccagttct accccctggt gaaggtccag 420 tgcagccccg agctgaggtt cttctctctgc tccatgtatg cccccgtgtg caccgtgctg 480 gatcaggcca tccccccctg tagaagcctc tgtgaaaggg ccaggcaggg atgcgaggct 540 ctgatgaaca agttcggctt ccagtggccc gagaggctga ggtgtgagaa cttccccgtg 600 cacggcgccg gcgaaatttg cgtgggacag aacacatccg acggctccgg cggccccggc 660 ggcggcccca ccgcctaccc cacagcccct tacctccctg atctgccttt caccgccctg 720 cctcccggag ctagcgatgg cagaggcaga cctgccttcc ccttcagctg ccccagacag 780 ctgaaggtgc ccccctatct gggctacagg ttcctgggcg agagagattg cggcgctccc 840 tgcgaacctg gaagagccaa cggcctcatg tacttcaagg aggagagag gaggttcgcc 900 aggctctggg tgggagtgtg gtccgtgctg tgctgcgcct ccacactctt caccgtgctg 960 acctacctcg tggacatgag gagattcagc taccccgaaa gacccatcat cttcctgtcc 1020 ggctgctatt tcatg 1035

Claims

1. A FZD7 murine monoclonal antibody, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2.

2. The FZD7 murine monoclonal antibody according to claim 1, wherein The FZD7 mouse monoclonal antibody further comprises a heavy chain constant region and a light chain constant region; the subclass of the heavy chain constant region is IgG1, and the light chain constant region is a kappa chain.

3. The FZD7 murine monoclonal antibody according to claim 1, wherein It is present in a full-length antibody, Fab, Fab', F(ab')2, Fv, bispecific antibody or multispecific antibody.

4. An isolated nucleic acid encoding the FZD7 murine monoclonal antibody according to any one of claims 1 to 3. A recombinant expression vector comprising the isolated nucleic acid according to claim 4 .

6. The recombinant expression vector according to claim 5, wherein The recombinant expression vector is a plasmid or a phage.

7. The recombinant expression vector according to claim 5, wherein The recombinant expression vector is a cosmid.

8. The recombinant expression vector according to claim 5, wherein The recombinant expression vector is a viral vector.

9. The recombinant expression vector according to claim 8, wherein The viral vector is selected from a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

10. The recombinant expression vector according to claim 5, wherein The skeleton of the recombinant expression vector is plasmid pET28a-Sumo.

11. A transformant comprising the recombinant expression vector according to any one of claims 5 to 10 in a host cell. 12 . A method for preparing a FZD7 murine monoclonal antibody, comprising culturing the transformant according to claim 11 and obtaining the FZD7 murine monoclonal antibody from the culture. 13 . A pharmaceutical composition comprising the FZD7 murine monoclonal antibody according to claim 1 , and a pharmaceutically acceptable carrier.

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