An antibody targeting prostate-specific membrane antigen, an antigen-binding fragment thereof, a preparation method and uses thereof

Abstract

The present invention discloses an antibody targeting prostate-specific membrane antigen, an antigen-binding fragment thereof, a preparation method and uses thereof. In the antibody or its antigen-binding fragment, 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 LCDR1, LCDR2 and LCDR3 are respectively as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9. The antibody can specifically bind to the extracellular domain protein of human and monkey PSMA, can effectively bind to C4-2 and 22RV1 cells, and the ADC containing the same 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 thereof, and uses 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 first 19 amino acids at the amino terminus of this protein form a short intracellular region, followed by 22-24 amino acids forming a single transmembrane region, and the remaining most sequences form an extracellular region. The extracellular portion of PSMA folds to form three protein domains, including a protease domain (amino acids 57-116 and amino acids 352-590), an apical domain (amino acids 117-351), and a 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 dimer formation. PSMA has folate hydrolase activity and N-acetylated α-linked acidic dipeptidase activity. In the intestine, PSMA mediates folate uptake. In the brain, PSMA regulates excitatory neurotransmission. In the prostate, PSMA is expressed in normal and malignantly transformed prostatic 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 its expression on cancer tissues is higher than that in normal tissues at different stages of the development of prostate cancer. In addition, PSMA is also expressed in tumor neovessels, such as those of thyroid cancer, liver cancer, renal cell carcinoma, bladder cancer, glioblastoma, pancreatic cancer, non-small cell lung cancer, and breast cancer, and is not expressed on normal blood vessels, which makes PSMA a potential target for the treatment of prostate cancer and various other cancers. Summary of the Invention

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

[0005] In the first aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof that targets prostate-specific membrane antigen. The antibody or the 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 HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 respectively; and / or, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown as 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 murine framework region or a human framework region.

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

[0008] In some technical solutions of the present invention, the antibody or the 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 solutions of the present invention, the antibody or the antigen-binding fragment thereof is a full-length antibody, and its heavy-chain constant region and / or light-chain constant region is derived from a human antibody.

[0010] In some more preferred technical solutions 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 more preferred technical solutions of the present invention, the amino acid sequence of the heavy-chain constant region is shown as SEQ ID NO: 10; and / or, the amino acid sequence of the light-chain constant region is shown as SEQ ID NO: 11.

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

[0013] In the second aspect of the present invention, there is provided an isolated nucleic acid encoding the antibody or the 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, which comprises 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, which comprises 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, and the transformant is a non-animal or non-plant variety;

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

[0018] The fifth aspect of the present invention provides a method for preparing the antibody or its antigen-binding fragment as described in the first aspect of the present invention, and the method comprises 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, which comprises a cytotoxic agent and the antibody or its antigen-binding fragment as described in the first aspect of the present invention;

[0020] In some preferred technical solutions 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 its antigen-binding fragment 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 its antigen-binding fragment 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] The ninth aspect of the present invention provides a set of medicine boxes, which comprises medicine box A and medicine box B, wherein:

[0024] Medicine box A contains one or more of the antibody or its antigen-binding fragment 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] Medicine box B contains other anti-tumor antibodies or pharmaceutical compositions comprising the other anti-tumor antibodies, and / or other anti-tumor drugs.

[0026] The tenth aspect of the present invention provides a drug delivery device, which comprises one or more of the antibody or its antigen-binding fragment 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;

[0027] In some preferred technical solutions of the present invention, the drug delivery device further comprises a component for administering the antibody or its antigen-binding fragment 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 containing the extracellular region of PSMA for non-diagnostic purposes, the method comprising using one or more of the antibody or its antigen-binding fragment 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;

[0029] In some preferred technical solutions of the present invention, the PSMA is monkey-derived or human-derived PSMA; and / or, the protein containing the extracellular region of PSMA 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 its antigen-binding fragment 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 in the preparation of a drug for diagnosing, preventing and / or treating cancers related to PSMA expression;

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

[0032] On the basis of conforming to common general knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

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

[0034] The positive and progressive effects of the present invention are as follows: The antibody can specifically bind to the extracellular domain protein of human and monkey PSMA, can effectively bind to C4-2 and 22RV1 cells, and the ADC prepared therefrom can effectively kill tumor cell lines. 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 of tumor cells C4-2 by PS-17 ADC. Detailed implementation manners

[0037] The following further describes the present disclosure in combination with embodiments, but these embodiments do not limit the scope of the present disclosure. For the experimental methods without specific conditions noted in the embodiments of the present disclosure, they are generally in accordance with conventional conditions, such as the antibody technology experimental manual of Cold Spring Harbor, the molecular cloning manual; or in accordance with the experimental conditions recommended by the raw material or commodity manufacturer. The reagents without specific sources noted are conventional reagents purchased from the market.

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

[0039] Using the extracellular domain (amino acid sequence at positions 44-750) of human PSMA protein (FOLH1, Uniprot number: Q04609-1) as a template, design a PSMA-ECD antigen with a (his) tag at the N-terminus for animal immunization and subsequent detection. The protein antigen sequence is as follows (SEQ ID NO: 1): 6 Label for animal immunization and subsequent detection. The protein antigen sequence is as follows (SEQ ID NO: 1):

[0040] HHHHHHKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA

[0041] Synthesize the DNA sequence encoding the above protein (Sangon Biotech, Shanghai) and insert it into the pcDNA3.1 expression vector to construct a plasmid expressing the recombinant protein. Transfect the target plasmid into Expi293F (Thermo) cells using PEI (polyscience) to express the protein. Four days later, collect the supernatant of the cell culture medium. After high-speed centrifugation, collect the cell culture supernatant and filter it through a 0.22 μm filter membrane to remove residual cell debris.

[0042] Protein purification was carried out using a nickel column (GenScript, #17524802). First, the column was equilibrated with a 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 onto the column at a flow rate of 3 mL / min. After the loading was completed, the column was rinsed with the equilibration buffer until the baseline was balanced. Subsequently, the target protein bound to the column was eluted with an elution buffer (phosphate buffer containing 40 mM imidazole, 0.5 mM Ca 2+ , 1 mM Mg 2+ ). The eluted protein was concentrated and then subjected to SEC purification. During the SEC purification process, it was found that the PSMA-ECD protein existed in monomeric and dimeric forms (PNAS, October 28, 2003 (vol.100) no.22, 12590 - 12595), with the dimeric form accounting for approximately 70%. The dimeric protein was collected. The collected protein was analyzed by SDS-PAGE and its activity was detected. After passing the detection, it was aseptically filtered, aliquoted, and stored at -80 o °C for standby.

[0043] Method for detecting the activity of PSMA-ECD dimeric protein: The purified PSMA-ECD protein hydrolyzes the substrate N-acetylaspartylglutamate (NAAG, Sigma, #A4930) to produce N-acetylaspartic acid and L-glutamic acid. The enzyme activity was characterized by measuring the fluorescence derivative formed by L-glutamic acid and 1,2-phthalaldehyde (excitation light: 330 nm, emission light: 450 nm). Standard curves were plotted by reacting different concentrations of L-glutamic acid standards with an excess of 1,2-phthalaldehyde (Sigma, #G8415) and recording the fluorescence signals. The enzyme activity was quantified according to the standard curves. The enzyme activity of the PSMA-ECD dimeric protein hydrolyzing the substrate was 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 well-known to those skilled in the art. Specifically, freshly collected mouse spleen cells or lymph node cells were prepared into single cell suspensions. Erythrocyte lysate (Beyotime, C3702) was added and incubated at room temperature for 5 minutes to lyse erythrocytes. After centrifugation and counting, the cells were mixed with the Sp2 / 0 myeloma cell line (the ratio of spleen cells to sp2 / 0 cells was 2:1). After thorough washing with 20 mL of electrofusion buffer (BTX cytofusion medium c C), electrofusion was performed (BTX ECM2001) to prepare hybridoma cells. A 9 mL fusion chamber was used, and the electrofusion parameters were set as follows (AC parameters: 75 V / 75V / 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). Approximately 7.2 mL of the well-mixed cell suspension was added to the 9 mL preparation fusion chamber, 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, 1×pyruvate), and seeded in a 96-well cell culture plate (1×10 5 / 100 μL / well), and cultured at 37 °C in 5% CO 2 . The medium was changed every 3 days. On the 14th day after fusion, ELISA detection was performed according to the cell growth situation.

[0051] 3) Screening of hybridoma cells

[0052] 3 μg / mL of antigen protein (100 μL / well) was added to a 96-well high-binding plate (Nunc MaxiSorp™, 44-2404-21) and incubated overnight at 4 o °C. The next day, the 96-well plate was blocked with PBS blocking solution containing 3% skim milk powder (300 μL / well), placed at room temperature for 1 hour, washed 3 times with PBS washing solution containing 0.1% Tween-20, then 100 μL of hybridoma supernatant was added to each well and incubated at room temperature for 1 hour. The 96-well plate was washed 3 times, and 100 μL of HRP-labeled goat anti-mouse antibody diluted 2000-fold (Thermo, A10521) was added to each well and incubated at room temperature for 1 hour. After washing the plate 4 times with the washing solution, 50 μL of TMB chromogenic 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 with an enzyme-linked immunosorbent assay (ELISA) reader. Positive hybridoma cells were sorted by flow cytometry, seeded at 1 cell / well into a new 96-well plate, cultured, amplified, and cryopreserved.

[0053] 4) Amplification and sequencing of antibody sequences

[0054] The positive hybridoma cells are cultured and amplified after single-cell sorting or limited dilution. After amplifying 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). The target genes are cloned into vectors for sequencing.

[0055] Example 4 Construction and Screening of a Phage Display Library of Anti-human PSMA Antibodies

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

[0057] A single-chain antibody (scFv)-phage display library is constructed by synthesizing cDNA from extracted RNA of immunized mouse spleen cells or lymph nodes, and the target antibody is obtained by screening the constructed mouse immune library through phage surface display technology. Specifically, after synthesizing cDNA, degenerate primers, such as the primer combination given by A Krebber et al., Journal of Immunological Methods 201 (1997) 35–55 or degenerate primer combinations designed through the human germline antibody sequences included in IMGT, are used for PCR amplification to obtain the DNA sequences of the heavy and light chains of the target antibody and construct an immune library for single-chain antibody-phage display.

[0058] 2) Screening of the single-chain antibody-phage library

[0059] Antibody molecules that bind to the target antigen are screened through phage panning. The specific operation is as follows: In the first round of screening, the PSMA protein is diluted in PBS, and 100 μL of 5 μg / mL protein is added to each well of a high-binding 96-well plate and incubated overnight at 4 °C. On the second day, the 96-well plate is blocked with PBS containing 3% skim milk powder (PBSM) (room temperature, 2 hours). After washing the plate 3 times with PBS containing 0.1% Tween 20 (PBST), 10 12 phages blocked with PBSM for 1 hour are added to each well and incubated at room temperature for 1 hour. After incubation, the plate is washed 10 times with PBST to remove phage particles that do not bind to the antigen, and the phages that can bind to the antigen are eluted with trypsin. The eluted phages are amplified by infecting TG1. In the second round of screening, a similar screening method is used, increasing the number of washing times to 20 times, and the eluted phages are used to infect TG1 and plated. Monoclonal colonies obtained from the second round of screening are directly picked for ELISA detection. Clones positive in the ELISA detection are sent for sequencing.

[0060] Example 5 Expression and Purification of Anti-PSMA Antibodies

[0061] Through hybridoma screening and phage display screening, antibody clones with different sequences that can specifically bind to the PSMA-ECD dimer protein were obtained, and these clones were constructed into the form of human-mouse chimeric IgG1 for expression and purification. The specific operations are as follows: Primers were designed and the VH and VL gene fragments of the antibody were obtained by PCR amplification, and were respectively subjected to overlap PCR with the constant region gene fragments CH1-Hinge-CH2-CH3 (SEQ ID NO: 10) and CL (SEQ ID NO: 11) of the human IgG1 heavy chain and light chain to obtain a fused DNA sequence, which was inserted into the mammalian expression vector pCDNA3.1 to construct plasmid vectors for expressing the heavy and light chains of the chimeric antibody. After the sequence was verified to be correct by sequencing, the plasmid vectors were extracted using an endotoxin-free plasmid extraction kit and stored at -20 °C for later use. Expi293F cells were diluted to a density of approximately 4×10 6 cells / mL, and the plasmids expressing the antibody light and heavy chains were co-transfected into Expi293F cells using the PEI40000 (polysciences) transfection reagent. Four days later, the cell culture supernatant was collected, and after high-speed centrifugation, the cell culture supernatant was collected and filtered through a 0.22 μm filter membrane 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 miscellaneous proteins. 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 filtration column ENrichTM SEC650 (Bio-red) to remove aggregates, and the monomer peak was collected. After the collected sample was detected by 4-12% SDS-PAGE gradient gel electrophoresis, it was aliquoted and stored at -80 °C for later use.

[0062] Example 6 Species-specific ELISA Detection of Anti-PSMA Antibody

[0063] The recombinant antigen proteins of human and monkey PSMA extracellular domains (the monkey PSMA protein was purchased from Acro Biosystems, and the human PSMA protein was prepared in Example 1) were diluted in PBS buffer (final concentration 1 μg / mL), and the antigen proteins were added to a 96-well high-binding plate (100 μL / well), 4 oIncubate overnight at C. The next day, block a 96-well plate with PBS blocking solution containing 3% non-fat milk powder (300 μL / well) and let it stand at room temperature for 1 hour. After washing the 96-well plate 3 times with PBS washing solution containing 0.1% Tween-20, add 100 μL of the purified antibody to be tested (diluted to 10 μg / mL in PBS) to each well and let it stand at room temperature for 1 hour. Wash the 96-well plate 3 times, and add 100 μL of HRP-labeled goat anti-mouse antibody (Thermo) diluted 2000-fold to each well and incubate at room temperature for 1 hour. After washing the plate 4 times with the washing solution, add 50 μL of TMB chromogenic solution (Invitrogen) for color development for 2 - 5 minutes. Add 1 M sulfuric acid to terminate the reaction, and record the absorbance value at 450 nm with an enzyme-linked immunosorbent assay reader. As shown in Table 1, the positive clone PS-17 has a good monomer ratio after expression and purification (detected by SEC) and can specifically bind to the extracellular domain proteins of human and monkey PSMA.

[0064] Table 1 Detection of the purity and species specificity of anti-PSMA monoclonal antibodies

[0065]

[0066] Example 7 Detection of the EC50 of anti-PSMA antibody binding to PSMA-positive tumor cell lines

[0067] Dilute the purified PSMA antibody to 10 μg / mL with ice-cold flow cytometry staining buffer (biolegend, # 420201), and then perform serial dilutions from this starting concentration to obtain a series of antibody solutions with decreasing concentrations. Incubate the antibody with 1×10 5 The prostate cancer cell line C4-2 with high PSMA expression and the prostate cancer cell line 22Rv1 with low PSMA expression are incubated at 4 °C for 1 hour respectively, washed 2 times with ice-cold flow cytometry staining solution, 1 mL each time, centrifuged at 300 g for 3 minutes, and the washed cells are resuspended in 50 μL containing Alexa Fluor TM 488-labeled goat anti-human IgG secondary antibody (Thermo, # A-11013), incubated at 4 °C for 30 minutes, and then washed twice for flow cytometry analysis. Record the mean fluorescence intensity value (MFI) and plot the dose-response curve of each antibody to calculate the EC50 value and the maximum binding value Max. As Figure 1 shown, the PS-17 antibody can effectively bind to C4-2 and 22RV1 cells, and the EC50 values are 1.3 μg / mL and 5.5 μg / mL respectively.

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

[0069] The ADC sample (AGLink® ADC Conjugation Kit, MMAE, DAR4, AcroBiosystems) was prepared using a glycosylation site-directed conjugation kit for evaluating the internalization of ADC and its killing effect on tumor cells. As Figure 2 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] QVQLQQSGPELVKPGASVKVSCKASGYAFISYNMYWVKQSRGKSLEWIGYIDPFHGGTTYNQKFKGKATLTVDKSSSTAYMHLNSLTSEDSAVYYCAREEYYYGNYLDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

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

[0088] DIVMTQAAFSNPVTLGTSTSISCRSSKSLLHSNGITFLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSCSGSGTDFTLRISRVEAEDVGVYYCAQNLELPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0089] Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that these are only examples. Without departing from the principle and essence of the present invention, various changes or modifications can be made to these embodiments. Therefore, the protection scope of the present invention is defined 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, 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 box B contains other anti-tumor drugs.

21. The kit of claim 20, wherein: The other anti-tumor drugs are other anti-tumor antibodies or pharmaceutical compositions comprising the other anti-tumor antibodies.

22. 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.

23. The drug delivery device according to claim 22, 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.

24. 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.

25. The method of claim 24, 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.

26. 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 and / or treating cancer associated with PSMA expression; the cancer is prostate cancer.

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