Antibody targeting prostate specific membrane antigen, antigen binding fragment thereof, preparation method and application thereof

Abstract

The invention discloses an antibody targeting a prostate specific membrane antigen, an antigen binding fragment thereof, a preparation method and application thereof. In the antibody or the antigen binding fragment thereof, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are respectively as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; and / or the amino acid sequences of the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9. The antibody can be specifically combined with PSMA extracellular domain proteins of human and monkeys and can be effectively combined with C4-2 and 22RV1 cells, and ADC containing the antibody can effectively kill tumor cell lines.

Description

Technical Field

[0001] The present invention relates to the fields of biology and medicine; more specifically, the present invention relates to an antibody targeting prostate-specific membrane antigen, an antigen-binding fragment thereof, a preparation method and use thereof. Background Art

[0002] Prostate specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), is a homodimeric type II membrane protein composed of 750 amino acids with a molecular weight of 110 kDa. The 19 amino acids at the amino terminal of the protein constitute a short intracellular region, the following 22-24 amino acids constitute a single transmembrane region, and the remaining majority of the sequence constitutes the extracellular region. The extracellular part of PSMA folds to form three protein domains, including the protease domain (amino acids 57-116 and amino acids 352-590), the apical domain (amino acids 117-351) and the C-terminal domain (amino acids 591-750). All three protein domains are involved in substrate binding, and the C-terminal domain is also involved in the formation of dimers. PSMA has folate hydrolase activity and N-acetylated α-linked acid dipeptidase activity. In the intestine, PSMA mediates the uptake of folate. In the brain, PSMA regulates excitatory neurotransmission. In the prostate, PSMA is expressed in normal as well as malignantly transformed prostate epithelial cells.

[0003] The high expression of PSMA in prostate cancer cells makes it an ideal target for the diagnosis and treatment of prostate cancer. Compared with other normal tissues, such as salivary glands, kidneys and small intestines, PSMA is highly expressed in prostate cancer tissues, and the expression of PSMA in cancer tissues is higher than that in normal tissues at different stages of prostate cancer development. In addition, PSMA is also expressed in tumor neovascularization, such as neovascularization of thyroid cancer, liver cancer, renal cell carcinoma, bladder cancer, brain glioma, pancreatic cancer, non-small cell lung cancer and breast cancer, but not in normal blood vessels, which makes PSMA a potential target for the treatment of prostate cancer and many other cancers. Summary of the invention

[0004] In order to provide a new antibody for diagnosing and treating prostate cancer, the present invention provides an antibody targeting prostate-specific membrane antigen, an antigen-binding fragment thereof, a preparation method and its use. The antibody can specifically bind to the PSMA extracellular domain protein of humans and monkeys, can effectively bind to C4-2 and 22RV1 cells, and the ADC prepared therefrom can effectively kill tumor cell lines.

[0005] The first aspect of the present invention provides an antibody or an antigen-binding fragment thereof targeting prostate-specific membrane antigen, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3; wherein: the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; and / or, the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

[0006] In some technical solutions of the present invention, the framework region of the heavy chain variable region and / or the light chain variable region is a mouse framework region or a human framework region.

[0007] In some preferred technical embodiments of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2; and / or, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3.

[0008] In some technical solutions of the present invention, the antibody or antigen-binding fragment thereof is a full-length antibody, Fab, Fab', F(ab') 2 Or Fv; the Fv is preferably scFv.

[0009] In some preferred technical embodiments of the present invention, the antibody or antigen-binding fragment thereof is a full-length antibody, and its heavy chain constant region and / or light chain constant region are derived from a human antibody.

[0010] In some better technical embodiments of the present invention, the heavy chain constant region is derived from a human heavy chain IgG1 constant region; and / or, the light chain constant region is derived from a human light chain κ chain constant region.

[0011] In some further preferred technical embodiments of the present invention, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 10; and / or, the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 11.

[0012] In some technical embodiments of the present invention, the amino acid sequence of the heavy chain of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 12; and / or, the amino acid sequence of the light chain of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 13.

[0013] The second aspect of the present invention provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention.

[0014] The third aspect of the present invention provides a recombinant expression vector, the recombinant expression vector comprising the isolated nucleic acid as described in the second aspect of the present invention;

[0015] In some technical solutions of the present invention, preferably, the backbone of the recombinant expression vector is pCDNA3.1.

[0016] The fourth aspect of the present invention provides a transformant, the transformant comprising the nucleic acid as described in the second aspect of the present invention or the recombinant expression vector as described in the third aspect of the present invention, the transformant is a non-animal or plant variety;

[0017] In some technical solutions of the present invention, preferably, the host cell of the transformant is a mammalian cell, such as an Expi293F cell.

[0018] The fifth aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, the method comprising culturing the transformant as described in the fourth aspect of the present invention.

[0019] The sixth aspect of the present invention provides an antibody-drug conjugate, wherein the antibody-drug conjugate comprises a cytotoxic agent and the antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0020] In some preferred technical embodiments of the present invention, the cytotoxic agent is MMAE.

[0021] The seventh aspect of the present invention provides a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention and / or the antibody-drug conjugate as described in the sixth aspect of the present invention, and a pharmaceutically acceptable carrier.

[0022] The eighth aspect of the present invention provides a kit, which comprises one or more of the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, the antibody-drug conjugate as described in the sixth aspect of the present invention, and the pharmaceutical composition as described in the seventh aspect of the present invention.

[0023] A ninth aspect of the present invention provides a set of medicine kits, the set of medicine kits comprising medicine kit A and medicine kit B, wherein:

[0024] The drug kit A contains one or more of the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, the antibody-drug conjugate as described in the sixth aspect of the present invention, and the pharmaceutical composition as described in the seventh aspect of the present invention;

[0025] The medicine kit B contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and / or other anti-tumor drugs.

[0026] The tenth aspect of the present invention provides a drug delivery device, the drug delivery device comprising one or more of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the antibody-drug conjugate according to the sixth aspect of the present invention, and the pharmaceutical composition according to the seventh aspect of the present invention;

[0027] In some preferred technical solutions of the present invention, the drug delivery device further comprises a component for administering the antibody or antigen-binding fragment thereof or the pharmaceutical composition to a subject, such as a syringe or an infusion device.

[0028] The eleventh aspect of the present invention provides a method for detecting a protein comprising the extracellular region of PSMA for non-diagnostic purposes, the method comprising using one or more of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the antibody-drug conjugate according to the sixth aspect of the present invention, and the pharmaceutical composition according to the seventh aspect of the present invention;

[0029] In some preferred technical solutions of the present invention, the PSMA is monkey-derived or human-derived PSMA; and / or, the protein comprising the PSMA extracellular region is a full-length PSMA protein.

[0030] The twelfth aspect of the present invention provides the use of one or more of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the antibody-drug conjugate according to the sixth aspect of the present invention, and the pharmaceutical composition according to the seventh aspect of the present invention in the preparation of a drug for diagnosing, preventing and / or treating cancer associated with PSMA expression;

[0031] In some preferred technical embodiments of the present invention, the cancer is prostate cancer, liver cancer, thyroid cancer, renal cell carcinoma, bladder cancer, glioma, pancreatic cancer, non-small cell lung cancer or breast cancer.

[0032] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0033] The reagents and raw materials used in the present invention are commercially available.

[0034] The positive and progressive effects of the present invention are that the antibody can specifically bind to the PSMA extracellular domain protein of humans and monkeys, can effectively bind to C4-2 and 22RV1 cells, and the ADC prepared therefrom can effectively kill tumor cell lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 EC50 detection of PS-17 binding to PSMA-positive tumor cells C4-2 and 22RV1.

[0036] Figure 2The killing effect of PS-17 ADC on tumor cells C4-2. DETAILED DESCRIPTION

[0037] The present disclosure is further described below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure. Experimental methods in the examples of the present disclosure that do not specify specific conditions are generally performed under conventional conditions, such as the Cold Spring Harbor Antibody Technology Laboratory Manual and the Molecular Cloning Manual; or according to the experimental conditions recommended by the raw material or product manufacturer. Reagents that do not specify specific sources are conventional reagents purchased from the market.

[0038] Example 1 Preparation of PSMA antigen and detection protein

[0039] Using the extracellular domain (amino acid sequence 44-750) of human PSMA protein (FOLH1, Uniprot No.: Q04609-1) as a template, we designed a (his) 6 The labeled PSMA-ECD antigen is used for animal immunization and subsequent detection. The protein antigen sequence is as follows (SEQ ID NO: 1):

[0040] HHHHHHKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVK NAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAA VVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFK YHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA

[0041] The DNA sequence encoding the above protein was synthesized (Shanghai Sangon Biotechnology) and inserted into the pcDNA3.1 expression vector to construct a plasmid expressing the recombinant protein. The target plasmid was transfected into Expi293F (Thermo) cells using PEI (polyscience) to express the protein. The cell culture supernatant was collected four days later, and the cell culture supernatant was collected after high-speed centrifugation and filtered with a 0.22 μm filter to remove residual cell debris.

[0042] A nickel column (GenScript, #17524802) was used for protein purification. First, the column was equilibrated with equilibration buffer (50 mM Tris-HCl, 150 mM NaCl) at a flow rate of 5 mL / min for 10-15 column volumes until the baseline was stable. Then, the culture supernatant containing the target protein was loaded at a flow rate of 3 mL / min. After loading, the column was flushed with equilibration buffer until the baseline was balanced. Then, the column was eluted with elution buffer (containing 40 mM imidazole, 0.5 mM Ca 2+ , 1 mM Mg 2+ The target protein bound to the column was eluted with phosphate buffer (PBS). The eluted protein was concentrated and then purified by SEC. During the SEC purification process, it was found that PSMA-ECD protein existed in monomer and dimer forms (PNAS, October 28, 2003 (vol.100) no.22,12590-12595), of which the dimer form accounted for about 70%. The dimer protein was collected. The collected protein was analyzed by SDS-PAGE and activity detection. If the test was qualified, it was sterile filtered, packaged and frozen at -80 o C is for backup.

[0043] PSMA-ECD dimer protein activity detection method: The purified PSMA-ECD protein hydrolyzes the substrate N-acetylaspartylglutamate (NAAG, Sigma, #A4930) to produce N-acetylaspartate and L-glutamate. The enzyme activity is characterized by measuring the fluorescent derivatives formed by L-glutamate and 1,2-benzenedicaldehyde (excitation light: 330 nm, emission light: 450 nm). Different concentrations of L-glutamate standards are reacted with excess 1,2-benzenedicaldehyde (Sigma, #G8415) and the fluorescence signal is recorded to draw a standard curve. The enzyme activity is quantified according to the standard curve. The enzyme activity of the PSMA-ECD dimer hydrolyzing the substrate is measured to be 800-1000 pmol / min / μg.

[0044] Example 2 Construction of a stable cell line expressing PSMA

[0045] The full-length DNA sequence encoding human PSMA with a flag tag at the N-terminus was synthesized (Shanghai Sangon Biotechnology), and the target gene was cloned into the pcDNA3.1 vector to obtain the plasmid pcDNA3.1-flag-hPSMA. The extracted plasmid was linearized by enzyme digestion and transfected into HEK293, Cos7 or CHO K1 cells using Lipofectamine LTX (Thermo). 24 hours after transfection, 500-1000 µg / mL of G418 was added for screening. After continuous culture in the culture medium containing G418 for 2 weeks, cells with high expression of the flag tag were sorted by flow cytometry. Stable cell lines HEK293-PSMA, CHOK1-PSMA and Cos7-PSMA expressing PSMA were constructed by single cell sorting and amplification culture.

[0046] Example 3 Obtaining and preparing anti-human PSMA hybridoma monoclonal antibodies

[0047] 1) Mouse immunization

[0048] Select 6-8 week old Balb / c female mice (Guangdong Yaokang Biotechnology Co., Ltd.) and transfect the recombinant expressed protein antigen (his) 6 -hPSMA-ECD (protein concentration: 1-2 mg / mL) and an equal volume of adjuvant [optional: Freund's complete adjuvant (Sigma, F5881-10ML), Freund's incomplete adjuvant (344291-10ML), TiterMax Gold adjuvant (Sigma, T2684), Imject® Alum (Thermo, 77161), etc.] are fully mixed for immunization. The antigen emulsion prepared with Freund's complete adjuvant or TiterMax Gold adjuvant is used for the first immunization, and 50-100 μg of antigen is injected subcutaneously per mouse. Booster immunization is performed on the 14th and 28th days after the first immunization. The antigen suspension is prepared by mixing protein with Freund's incomplete adjuvant or Imject® Alum. Each mouse is injected intraperitoneally or subcutaneously with 25-50 μg of antigen. Blood is collected on the 35th day to test the antibody titer, and the antibody titer is determined to determine whether to continue the booster immunization or the last sprint immunization. For sprint immunization, 50-100 µg of pure protein solution (protein dissolved in phosphate buffer or saline) was injected through the tail vein or intraperitoneal cavity of mice. Three days after immunization, mice were euthanized and blood, spleen and lymph node samples were collected for detection and hybridoma preparation.

[0049] 2) Hybridoma preparation

[0050] Hybridoma cells were prepared by methods known to those skilled in the art. Specifically, freshly collected mouse spleen cells or lymph node cells were prepared into a single cell suspension. Red blood cells were lysed by adding red blood cell lysis buffer (Biyuntian, C3702) at room temperature for 5 minutes, centrifuged and counted, and then mixed with Sp2 / 0 myeloma cell line (the ratio of spleen cells: sp2 / 0 cells was 2:1), and then washed thoroughly with 20 mL of electrofusion buffer (BTX cytofusion mediumc C) and electrofused (BTX ECM2001) to prepare hybridoma cells. A 9 mL fusion pool was used, and the electrofusion parameters were set as follows (AC parameters: 75 V / 75 V / 30 s / 1.0 MHz; DC parameters: 800 V / 40 μs / 1 / 0.000 s; AC parameters: 75 V / 75 V / 30 s / 1.0 MHz). About 7.2 mL of the well-mixed cell suspension was added to the 9 mL prepared fusion pool, and fusion was started immediately. The fused hybridoma cells were resuspended in HAT complete medium (RPMI-1640 medium containing 10% FBS, 1× HAT, 1× P / S, 1× Glutamax, and 1× pyruvate) and inoculated into a 96-well cell culture plate (1×10 5 / 100 μL / well), 37°C, 5% CO 2 Culture. Change the medium every 3 days. On the 14th day after fusion, perform ELISA test based on the cell growth.

[0051] 3) Hybridoma cell screening

[0052] 3 µg / mL of antigen protein (100 µL / well) was added to a 96-well high-absorption plate (Nunc MaxiSorp™, 44-2404-21). o C overnight. The next day, the 96-well plate was blocked with PBS blocking solution containing 3% skim milk powder (300 µL / well) and placed at room temperature for 1 hour. After washing the 96-well plate 3 times with PBS washing solution containing 0.1% Tween-20, 100 µL of hybridoma supernatant was added to each well and placed at room temperature for 1 hour. The 96-well plate was washed 3 times, and 100 µL of 2000-fold diluted HRP-labeled goat anti-mouse antibody (Thermo, A10521) was added to each well and incubated at room temperature for 1 hour. After washing the plate 4 times with washing solution, 50 µL of TMB color development solution (Invitrogen, 002023) was added for color development for 2-5 minutes. 1 M sulfuric acid was added to terminate the reaction, and the absorbance at 450 nm was recorded using an enzyme reader. Positive hybridomas were flow sorted, and 1 cell / well was inoculated into a new 96-well plate, cultured, expanded and frozen.

[0053] 4) Antibody sequence amplification and sequencing

[0054] Positive hybridoma cells are cultured and expanded after single cell sorting or limiting dilution. After amplification to a sufficient number of cells, RNA is extracted and the heavy and light chain genes of the target antibody are amplified by 5'RACE (Clontech, #634859), and the target gene is cloned into a vector for sequencing.

[0055] Example 4 Construction and screening of phage display library of anti-human PSMA antibodies

[0056] 1) Construction of single-chain antibody-phage library

[0057] After extracting RNA from spleen cells or lymph nodes of immune mice to synthesize cDNA, a single-chain antibody (scFv)-phage display library is constructed, and the constructed mouse immune library is screened by phage surface display technology to obtain the target antibody. Specifically, after synthesizing cDNA, PCR amplification is performed using degenerate primers, such as the primer combination given by A Krebber et al., Journal of Immunological Methods 201 (1997) 35-55, or a degenerate primer combination designed by using human germline antibody sequences included in IMGT, to obtain the heavy chain and light chain sequence DNA of the target antibody and construct a single-chain antibody-phage display immune library.

[0058] 2) Single-chain antibody-phage library screening

[0059] The antibody molecules that bind to the target antigen were screened by phage panning. The specific operation was as follows: In the first round of screening, PSMA protein was diluted in PBS, and 100 μL of 5 μg / mL protein was added to each well of a high-absorption 96-well plate at 4°C overnight. On the second day, the 96-well plate was blocked with PBS containing 3% skim milk powder (PBSM) (room temperature, 2 hours), and the plate was washed 3 times with PBS containing 0.1% Tween20 (PBST), and 10 12 The phages were blocked with PBSM for 1 hour and incubated at room temperature for 1 hour. After the incubation, the plates were washed 10 times with PBST to remove the phage particles that did not bind to the antigen, and the phages that could bind to the antigen were eluted with trypsin. The eluted phages were amplified by infecting TG1. The second round of screening used a similar screening method, increasing the number of washes to 20 times, and the eluted phages were infected with TG1 coated plates. The single clones obtained in the second round of screening were directly picked for ELISA testing. The clones that were positive in the ELISA test were sent for sequencing.

[0060] Example 5 Expression and purification of anti-PSMA antibodies

[0061] Through hybridoma screening and phage surface display screening, antibody clones with different sequences that can specifically bind to PSMA-ECD dimer proteins were obtained, and these clones were constructed into human-mouse chimeric IgG1 forms for expression and purification. The specific operations are as follows: primers were designed and PCR amplified to obtain the VH and VL gene fragments of the antibody, and overlap PCR was performed with the constant region gene fragments of the human IgG1 heavy chain and light chain CH1-Hinge-CH2-CH3 (SEQ ID NO: 10) and CL (SEQ ID NO: 11) to obtain the fusion DNA sequence, which was inserted into the mammalian expression vector pCDNA3.1 to construct the heavy and light chain plasmid vectors expressing the chimeric antibody. After sequencing to verify the correct sequence, the plasmid vector was extracted using an endotoxin-free plasmid extraction kit and stored at -20°C for future use. Expi293F was diluted to a density of approximately 4×10 6 cells / mL, and the plasmids expressing the light and heavy chains of the antibody were co-transfected into Expi293F cells using PEI40000 (polysciences) transfection reagent. Four days later, the cell culture supernatant was collected, and the cell culture supernatant was collected after high-speed centrifugation, and filtered with a 0.22 μm filter to remove residual cell debris. The filtered cell culture supernatant was purified using a Protein A column, and the Protein A column was rinsed with PBS buffer to remove impurities. After the A280 reading dropped to a stable baseline, the target protein was eluted with a 0.1 M acetic acid-sodium acetate solution at pH 3.2, and the target protein peak was collected and neutralized with a 1 M Tris-HCl, pH 8.0 solution. After the sample was concentrated, it was further purified using a gel chromatography column ENrichTM SEC650 (Bio-red), aggregates were removed, and the monomer peak was collected. The collected samples were detected by 4-12% SDS-PAGE gradient gel electrophoresis, and then aliquoted and stored at -80°C for later use.

[0062] Example 6 Species-specific ELISA detection of anti-PSMA antibodies

[0063] Human and monkey PSMA extracellular domain recombinant antigen proteins (monkey PSMA protein was purchased from Acro Biosystems, and human PSMA protein was prepared in Example 1) were diluted into PBS buffer (final concentration was 1 μg / mL), and the antigen proteins were added to a 96-well high adsorption plate (100 μL / well). oC overnight. The next day, the 96-well plate was blocked with PBS blocking solution containing 3% skim milk powder (300 µL / well) and placed at room temperature for 1 hour. After washing the 96-well plate 3 times with PBS washing solution containing 0.1% Tween-20, 100 µL of purified test antibody (10 µg / mL diluted in PBS) was added to each well and placed at room temperature for 1 hour. The 96-well plate was washed 3 times, and 100 µL of 2000-fold diluted HRP-labeled goat anti-mouse antibody (Thermo) was added to each well and incubated at room temperature for 1 hour. After washing the plate 4 times with washing solution, 50 µL of TMB color development solution (Invitrogen) was added for color development for 2-5 minutes. 1 M sulfuric acid was added to terminate the reaction, and the absorbance at 450 nm was recorded using an enzyme reader. As shown in Table 1, the positive clone PS-17 had a good monomer ratio (SEC detection) after expression and purification, and could specifically bind to the PSMA extracellular domain protein of human and monkey.

[0064] Table 1 Purity and species specificity of anti-PSMA monoclonal antibodies

[0065]

[0066] Example 7 EC50 Detection of Anti-PSMA Antibodies Binding to PSMA-Positive Tumor Cell Lines

[0067] The purified PSMA antibody was diluted to 10 μg / ml with ice-cold flow cytometry staining buffer (BioLegend, #420201), and then graded diluted to obtain a series of antibody solutions with decreasing concentrations. 5 The prostate cancer cell line C4-2 with high expression of PSMA and the prostate cancer cell line 22Rv1 with low expression of PSMA were incubated at 4 degrees Celsius for 1 hour, washed twice with ice-cold flow cytometry staining solution (1 ml / time), centrifuged at 300 g for 3 minutes, and the washed cells were resuspended in 50 μl of Alexa Fluor TM 488-labeled goat anti-human IgG secondary antibody (Thermo, # A-11013), incubated at 4 degrees Celsius for 30 minutes, washed twice, and analyzed by flow cytometry. The mean fluorescence intensity (MFI) was recorded and the dose response curve of each antibody was plotted to calculate the EC50 value and the maximum binding value Max. Figure 1 As shown, PS-17 antibody can effectively bind to C4-2 and 22RV1 cells, with EC50 of 1.3 μg / mL and 5.5 μg / mL, respectively.

[0068] Example 8 ADC prepared by PS-17 can effectively kill tumor cells C4-2

[0069] ADC samples were prepared using a sugar-specific site-specific conjugation kit (AGLink® ADC Conjugation Kit, MMAE, DAR4, AcroBiosystems) to evaluate the internalization of ADC and its ability to kill tumor cells. Figure 2 As shown, PS-17 ADC can effectively kill tumor cell lines.

[0070] Sequence Listing

[0071] PS-17 VH (SEQ ID NO: 2):

[0072] QVQLQQSGPELVKPGASVKVSCKASGYAFISYNMYWVKQSRGKSLEWIGYIDPFHGGTTYNQKFKGKATLTVDKSSSTAYMHLNSLTSEDSAVYYCAREEYYYGNYLDYWGQGTTLTVSS

[0073] PS-17 VL (SEQ ID NO: 3):

[0074] DIVMTQAAFSNPVTLGTSTSISCRSSKSLLHSNGITFLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSCSGSGTDFTLRISRVEAEDVGVYYCAQNLELPWTFGGGTKLEIK

[0075] HCDR1 (SEQ ID NO: 4): GYAFISYN

[0076] HCDR2 (SEQ ID NO: 5): IDPFHGGT

[0077] HCDR3 (SEQ ID NO: 6): AREEYYYGNYLDY

[0078] LCDR1 (SEQ ID NO: 7):KSLLHSNGITF

[0079] LCDR2 (SEQ ID NO: 8): QMS

[0080] LCDR3 (SEQ ID NO: 9): AQNLELPWT

[0081] CH1-Hinge-CH2-CH3 (SEQ ID NO: 10):

[0082] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0083] CL (SEQ ID NO: 11):

[0084] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0085] PS-17 heavy chain (SEQ ID NO: 12):

[0086] QVQLQQSGPELVKPGASVKVSCKASGYAFISYNMYWVKQSRGKSLEWIGYIDPFHGGTTYNQKFKGKATLTVDKSSSTAYMHLNSLTSEDSAVYYCAREEYYYGNYLDYWGQ GTTLTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0087] PS-17 light chain (SEQ ID NO: 13):

[0088] DIVMTQAAFSNPVTLGTSTSISCRSSKSLLHSNGITFLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSCSGSGTDFTLRISRVEAEDVGVYYCAQNLELPWTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0089] Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that these are only examples, and various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the protection scope of the present invention is limited by the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof targeting prostate-specific membrane antigen, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3; wherein: the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; and / or, the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The framework region of the heavy chain variable region and / or the light chain variable region is a murine framework region or a human framework region.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2; and / or, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

3.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein: The antibody or antigen-binding fragment thereof is a full-length antibody, Fab, Fab', F(ab')2 or Fv.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein: The Fv is a scFv.

6. The antibody or antigen-binding fragment thereof according to claim 4, wherein: The antibody or antigen-binding fragment thereof is a full-length antibody, and the heavy chain constant region and / or light chain constant region thereof are derived from a human antibody.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein: The heavy chain constant region is derived from a human heavy chain IgG1 constant region; and / or the light chain constant region is derived from a human light chain κ chain constant region.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein: The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 10; and / or, the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

11.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein The amino acid sequence of the heavy chain of the antibody or its antigen-binding fragment is shown in SEQ ID NO: 12; and / or, the amino acid sequence of the light chain of the antibody or its antigen-binding fragment is shown in SEQ ID NO:

13.

10. An isolated nucleic acid, characterized in that The nucleic acid encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the isolated nucleic acid of claim 10.

12. The recombinant expression vector according to claim 11, characterized in that The backbone of the recombinant expression vector is pCDNA3.

1.

13. A transformant, characterized in that: The transformant comprises any one of the nucleic acid according to claim 10 or the recombinant expression vector according to claim 11, and the transformant is a non-animal or plant species.

14. The transformant according to claim 13, characterized in that The host cell of the transformant is a mammalian cell.

15. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that: The method comprises culturing the transformant according to any one of claims 13 or 14.

16. An antibody-drug conjugate, characterized in that: The antibody-drug conjugate comprises a cytotoxic agent and the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

17. The antibody-drug conjugate according to claim 16, characterized in that: The cytotoxic agent is MMAE.

18. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 or the antibody-drug conjugate according to claim 16 or 17, and a pharmaceutically acceptable carrier.

19. A kit, characterized in that: The kit comprises any one of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the antibody-drug conjugate according to claim 16 or 17, or the pharmaceutical composition according to claim 18.

20. A medicine kit, characterized in that: The kit comprises kit A and kit B, wherein: The drug kit A contains any one of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the antibody-drug conjugate according to claim 16 or 17, or the pharmaceutical composition according to claim 18; The medicine kit B contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and / or other anti-tumor drugs.

21. A drug delivery device, characterized in that: The drug delivery device comprises any one of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the antibody-drug conjugate according to claim 16 or 17, or the pharmaceutical composition according to claim 18.

22. The drug delivery device according to claim 21, characterized in that The drug delivery device also includes a component for administering the antibody or antigen-binding fragment thereof or the pharmaceutical composition to a subject.

23. A method for detecting a protein comprising the extracellular region of PSMA for non-diagnostic purposes, characterized in that: The method comprises using any one of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the antibody-drug conjugate according to claim 16 or 17, or the pharmaceutical composition according to claim 18.

24. The method of claim 23, wherein: The PSMA is monkey-derived or human-derived PSMA; and / or, the protein comprising the PSMA extracellular region is a full-length PSMA protein.

25. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the antibody-drug conjugate according to claim 16 or 17, or the pharmaceutical composition according to claim 18 in the preparation of a medicament for diagnosing, preventing and / or treating cancer associated with PSMA expression.

26. The use according to claim 25, characterized in that The cancer is prostate cancer, liver cancer, thyroid cancer, renal cell carcinoma, bladder cancer, glioma, pancreatic cancer, non-small cell lung cancer or breast cancer.

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