Anti-HERV-K102-Env human antibodies and uses thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an anti-HERV-K102-Env human antibody and application thereof. According to the invention, a new antibody of an anti-HERV-K102-Env human monoclonal antibody (hmAb) which shows unexpected properties is developed, and a commercial anti-HERV-K-Env human monoclonal antibody does not exist at present. The applicant has shown that the 18 antibodies aiming at HERV-K102-Env according to the invention are human monoclonal antibodies, some of the antibodies can be selectively combined with a TM antigen of HERV-K102-Env, some of the antibodies can be selectively combined with an SU antigen, and some of the antibodies can be simultaneously combined with the SU and TM antigens of HERV-K102-Env.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to anti-HERV-K102-Env humanized antibodies and uses thereof. Background Art

[0002] Human endogenous retroviruses (HERVs) are remnants of retroviruses that infected humans millions of years ago and integrated into the human genome, inherited in a Mendelian manner, and account for about 8% of the human genome. Most HERVs have lost their coding ability due to mutations, deletions, and the presence of stop codons in the coding sequence, and were previously considered junk DNA. However, in recent years, with the in-depth study of the biological functions of HERV, it has been found that the abnormal expression of some envelope proteins, such as HERV-K102 subtype, may be related to tumors (Shah AH, et al. J Clin Invest. 2023 Jul 3; 133 (13): e167929; Li M, et al. Clin Cancer Res. 2017 Oct 1; 23 (19): 5892-5911; Ng KW, et al. Nature. 2023; 616: 563-573; Chen T, Leukemia. 2013; 27: 1469-78.), autoimmune diseases (Tokuyama M, et al. J Exp Med. 2021; 218 (7): e20191766; Khadjinova AI, et al. Clin Exp Rheumatol. 2022; 40: 1306-1312), neurodegenerative diseases (Simula ER, et al. Viruses. 2021; 13: 2301; Johnson K, et al. Sci Transl Med. 2015; 7: 307ra153; Dembny P, et al. JCI Insight. 2020; 5: e131093; Dubowsky M, et al. Mol Neurobiol. 2023; 60: 6330-6345.) are closely related to the occurrence and development of HERVs. It is known that the C-terminal transmembrane region (TM) of the HERVs envelope protein (envelope, Env) has multiple immunosuppressive domains, such as fusion peptide region (Fusion Peptide, FP), immunosuppressive domains (Immunosuppressive Domains, ISD), and transmembrane domain (Transmembrane Domain, TMD), which has a strong immunosuppressive function and is an important cause of tumor immune tolerance. The N-terminus (SU) of the envelope protein is involved in a variety of inflammatory responses. HERV-K102-Env consists of two major parts: the N-terminal SU antigen and the C-terminal transmembrane region TM antigen. SU is involved in a variety of inflammatory responses, and TM has multiple immunosuppressive domains and is one of the important causes of tumor immune tolerance.

[0003] Neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis (ALS) are associated with the accumulation of misfolded proteins inside and outside neurons and nerve cells in the central nervous system, but their exact causes are unknown. Studies have shown that HERV-K envelope protein is abnormally highly expressed in diseased tissues (Simula ER, et al. Viruses. 2021; 13: 2301; Johnson K, et al. Sci Transl Med. 2015; 7: 307ra153; Dembny P, et al. JCI Insight. 2020; 5: e131093; Dubowsky M, et al. Mol Neurobiol. 2023; 60: 6330-6345.), and HERV-K expression in human neuronal cultures causes neuronal cytotoxicity, such as transfection of the entire HERV-K genome or only the HERV-K-env gene. A dose-dependent decrease in the number of neurons and neurite retraction were observed. This suggests that intracellular HERV-K-Env protein may contribute to neurotoxicity.

[0004] Autoimmune diseases such as systemic lupus erythematosus (SLE) are a major category of diseases that seriously endanger human health. So far, there is no effective drug to cure such diseases, and their exact cause and pathogenesis are unknown. It has been reported in the literature that abnormal expression of HERV-K may be associated with autoimmune diseases such as SLE (Tokuyama M, ET AL. J Exp Med. 2021; 218(7): e20191766; Khadjinova AI, et al. Clin Exp Rheumatol. 2022; 40: 1306-1312).

[0005] In summary, the above research results indicate that different subtypes of HERV-K may be involved in pathological processes such as the onset and progression of diseases such as tumors, neurodegenerative diseases, and autoimmune diseases.

[0006] To date, most malignant tumors, neurodegenerative diseases, and autoimmune diseases lack effective therapeutic drugs and technologies. For patients suffering from these fatal diseases, the discovery of effective treatments remains an urgent need. Therefore, it is of great significance to develop effective drugs to address these unmet clinical needs. Summary of the invention

[0007] In order to solve the above technical problems,

[0008] The present invention relates to an antibody that recognizes human HERV-K102-Env, wherein the antibody-binding antigenic epitopes are TM and SU of HERV-K102-Env. The present invention also relates to the use of an antibody that recognizes HERV-K102-Env for treatment, wherein the antibody-binding antigenic epitopes are TM and SU of HERV-K102-Env. The present invention also relates to a human monoclonal antibody that recognizes HERV-K102-Env for the treatment of HERV-K102-related tumors, neurodegenerative diseases, and autoimmune diseases.

[0009] An isolated antibody or antigen-binding fragment thereof, wherein the CDR region of the antibody or antigen-binding fragment thereof comprises HCDR and LCDR, the HCDR region comprises HCDR1, HCDR2 and HCDR3, and the LCDR region comprises LCDR1, LCDR2 and LCDR3;

[0010] The HCDR1 amino acid sequence is as shown in any one of SEQ ID NOs: 1 to 18, the HCDR2 amino acid sequence is as shown in any one of SEQ ID NOs: 19 to 36, the HCDR3 amino acid sequence is as shown in any one of SEQ ID NOs: 37 to 54, the LCDR1 amino acid sequence is as shown in any one of SEQ ID NOs: 55 to 72, the LCDR2 amino acid sequence is as shown in any one of SEQ ID NOs: 73 to 90, and the LCDR3 amino acid sequence is as shown in any one of SEQ ID NOs: 91 to 108.

[0011] Preferably, the antibody or antigen-binding fragment thereof comprises VH and VL, the VH amino acid sequence is sequentially shown in SEQ ID NOs: 109 to 126, and the VL amino acid sequence is sequentially shown in SEQ ID NOs: 127 to 144.

[0012] Preferably, the antibody or antigen-binding fragment thereof comprises any one of 18 antibodies or antigen-binding fragments thereof 1 to 18;

[0013] The VH amino acid sequences of the antibodies or antigen-binding fragments thereof 1 to 18 are shown in SEQ ID NOs: 109 to 126, respectively, and the VL amino acid sequences of the antibodies or antigen-binding fragments thereof 1 to 18 are shown in SEQ ID NOs: 127 to 144, respectively.

[0014] Preferably, the antibody or antigen-binding fragment specifically binds to HERV-K102-Env, and its binding antigen epitope is HERV-K102-Env-SU and / or HERV-K102-Env-TM, the HERV-K102-Env-SU amino acid sequence is shown in SEQ ID NO: 149, and the HERV-K102-Env-TM amino acid sequence is shown in SEQ ID NO: 150.

[0015] Preferably, the antibody is a human monoclonal antibody.

[0016] An isolated polynucleotide encoding the aforementioned antibody or antigen-binding fragment thereof.

[0017] An isolated vector comprises the aforementioned polynucleotide.

[0018] A host cell comprising the aforementioned polynucleotide or the aforementioned vector.

[0019] A kit for detecting HERV-K102-Env expression, comprising any of the aforementioned antibodies or antigen-binding fragments thereof.

[0020] Use of the antibody or antigen-binding fragment thereof as described above in a product for detecting and / or diagnosing and / or treating HERV-K102-Env expression.

[0021] Use of the aforementioned human monoclonal antibody in products for detecting and / or diagnosing and / or treating tumors, neurodegenerative lesions or autoimmune diseases associated with HERV-K102-Env expression.

[0022] Preferably, the product is any one of CAR-T, CAR-NK, CAR-DC, TIL therapeutic cell products or ADC products.

[0023] The beneficial effects of the present invention are mainly:

[0024] The present invention develops novel human monoclonal antibodies (hmAbs) against HERV-K102-Env that show unexpected properties. Currently, there are no commercial human monoclonal antibodies against HERV-K-Env.

[0025] The applicant has shown that the 18 antibodies against HERV-K102-Env according to the present invention are human monoclonal antibodies, some of which can selectively bind to the TM antigen of HERV-K102-Env, some can selectively bind to the SU antigen, and some can simultaneously bind to the SU and TM antigens of HERV-K102-Env.

[0026] These antibodies have demonstrated their biological activity in ELISA, FCM (flow cytometry) and Western blot immunoassays. These antibodies recognize not only the SU and TM antigens of native and denatured HERV-K102-Env, but also the non-glycosylated and glycosylated forms.

[0027] In addition, and most unexpectedly, the TM target epitope appears to be highly conserved, with a conserved amino acid sequence of the HERV-K-Env gene described in the database (the antigen HERV-K102-Env-TM sequence is shown in SEQ ID NO: 150). This gives these antibodies a unique positioning and can be used for the diagnosis and treatment of diseases associated with different subtypes of HERV-K. These unexpected results show that these human monoclonal antibodies against HERV-K102-Env are original tools for targeting HERV-K102-Env. The present invention also shows that even for technicians, it is not possible to foresee or predict the selection and acquisition of such monoclonal antibodies by a given immunization scheme. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figures 1A-1D The activity diagram of self-produced control antibodies and antigens is measured based on the ELISA method; Figure 1A The binding activity of the control antibody GN_mAb_Env_K01 and the antigen K102-SU-hFc antigen protein is shown; Figure 1B The binding activity of the control antibody HERM-1811-5 to the antigen K102-tTM-G4S-FC(IgG1)-strep is shown; Figure 1C The binding activity of antigens K102-SU-hFc, K102-SU-FC(IgG1), K102-tTM-G4S-FC(IgG1)-strep and control antibody GN_mAb_Env_K01 is shown; Figure 1D The binding activities of the antigens K102-SU-FC(IgG1) and K102-tTM-G4S-FC(IgG1)-strep and the control antibody HERM-1811-5 are shown.

[0029] Figure 2 This is an activity diagram of HERV-K102-Env expression in the self-produced overexpression cell line K102Mtm-SP-trN-mTM-G4S-3Flag-6His measured based on the FACS method.

[0030] Figures 3A-3C The figure is a graph showing the competition effect of the candidate antibody binding to the antigen K102-tTM-G4S-hFc-strep and 1G6-Biotin based on ELISA method; Figure 3AThe competitive effect of A014, A050, A089, A068, and 1G6-Biotin on binding to the antigen K102-tTM-G4S-hFc-strep is shown; Figure 3B The binding of A052, A062, A077, A082, A104, and A131 to the antigen K102-tTM-G4S-hFc-strep and the competitive effect of 1G6-Biotin are shown; Figure 3C The binding of A083, A005, A017, A046, A047, A054, A075, and A049 to the antigen K102-tTM-G4S-hFc-strep and the competitive effect of 1G6-Biotin are shown.

[0031] Figure 3D-3E : Shows the binding activity of candidate antibodies to antigen K102-SU-FC (IgG1) determined by ELISA; Figure 3D The binding activities of A014, A050, A089, A068, A049, A052, A062, A077, A082, A104, and A131 to the antigen K102-SU-FC (IgG1) are shown; Figure 3E The binding activities of A083, A005, A017, A046, A047, A054, A075, A050, and A049 to the antigen K102-SU-FC (IgG1) are shown.

[0032] Figures 4A-4E It is a graph showing the binding activity of candidate antibodies to K102Mtm-SP-trN-mTM-G4S-3Flag-6His cells determined based on the FACS method; Figure 4A-4B The binding activities of A014, A050, A089, and A068 to K102Mtm-SP-trN-mTM-G4S-3Flag-6His cells are shown; Figure 4C-4D The binding activities of A049, A052, A062, A077, A082, A104, and A131 to K102Mtm-SP-trN-mTM-G4S-3Flag-6His cells are shown; Figure 4E The binding activities of A005, A083, A017, A046, A047, A054, A075, A050, and A049 to K102Mtm-SP-trN-mTM-G4S-3Flag-6His cells are shown.

[0033] Figures 5A-5I This is a diagram showing the pairing effect of the antibody on the K102-tTM-G4S-FC(IgG1)-strep antigen protein. Figure 5AThe pairing effect of 1G6 as capture antibody and 1G6-Biotin, A017-Biotin, A050-Biotin, A014-Biotin, A068-Biotin, A052-Biotin, A062-Biotin, and A082-Biotin as detection antibodies on K102-tTM-G4S-FC(IgG1)-strep antigen protein is shown; Figure 5B The pairing effect of A017 as capture antibody and 1G6-Biotin, A017-Biotin, A050-Biotin, A014-Biotin, A068-Biotin, A052-Biotin, A062-Biotin, and A082-Biotin as detection antibodies on K102-tTM-G4S-FC(IgG1)-strep antigen protein is shown; Figure 5C The pairing effect of A014 as capture antibody and 1G6-Biotin, A017-Biotin, A050-Biotin, A014-Biotin, A068-Biotin, A052-Biotin, A062-Biotin, and A082-Biotin as detection antibodies on K102-tTM-G4S-FC(IgG1)-strep antigen protein is shown; Figure 5D The pairing effect of A068 as capture antibody and 1G6-Biotin, A017-Biotin, A050-Biotin, A014-Biotin, A068-Biotin, A052-Biotin, A062-Biotin, and A082-Biotin as detection antibodies on K102-tTM-G4S-FC(IgG1)-strep antigen protein is shown; Figure 5E The pairing effect of A052 as capture antibody and 1G6-Biotin, A017-Biotin, A050-Biotin, A014-Biotin, A068-Biotin, A052-Biotin, A062-Biotin, and A082-Biotin as detection antibodies on K102-tTM-G4S-FC(IgG1)-strep antigen protein is shown; Fig. 5F The pairing effect of A062 as capture antibody and 1G6-Biotin, A017-Biotin, A050-Biotin, A014-Biotin, A068-Biotin, A052-Biotin, A062-Biotin, and A082-Biotin as detection antibodies on K102-tTM-G4S-FC(IgG1)-strep antigen protein is shown; Figure 5GThe pairing effect of A082 as capture antibody and 1G6-Biotin, A017-Biotin, A050-Biotin, A014-Biotin, A068-Biotin, A052-Biotin, A062-Biotin, and A082-Biotin as detection antibodies on K102-tTM-G4S-FC(IgG1)-strep antigen protein is shown; Figure 5H The pairing effect of A050 as capture antibody and 1G6-Biotin, A017-Biotin, A050-Biotin, A014-Biotin, A068-Biotin, A052-Biotin, A062-Biotin, and A082-Biotin as detection antibodies on K102-tTM-G4S-FC(IgG1)-strep antigen protein is shown; Fig.5I The pairing effect of 1G6, A017, A050, A014, A068, A052, A062, and A082 as capture antibodies and A082-Biotin as detection antibody on K102-tTM-G4S-FC(IgG1)-strep antigen protein is shown. DETAILED DESCRIPTION

[0034] 1. Glossary:

[0035] Human endogenous retroviruses (HERVs);

[0036] Envelope protein (Envelope, Env);

[0037] The C-terminal transmembrane region (TM), which is a transmembrane protein formed after Env is hydrolyzed by furin protease;

[0038] Fusion Peptide (FP);

[0039] Immunosuppressive Domains (ISD);

[0040] Transmembrane Domain (TMD);

[0041] N-terminus of envelope protein (SU);

[0042] HERV-K envelope protein (HERV-K-Env);

[0043] HERV-K102 envelope protein (HERV-K102-Env);

[0044] extracellular domain of human HERV-K102-Env (huHERV-K102-Env-ECD);

[0045] Antibody light chain variable region (VL);

[0046] Antibody heavy chain variable region (VH);

[0047] Antibody light chain complementarity determining region (LCDR);

[0048] Antibody heavy chain complementarity determining region (HCDR);

[0049] N-terminus of HERV-K102 envelope protein (HERV-K102-Env-SU);

[0050] the C-terminal transmembrane region of the HERV-K102 envelope protein (HERV-K102-Env-TM);

[0051] Systemic lupus erythematosus (SLE);

[0052] High frequency mutation regions: CDR (complementarity determining regions);

[0053] Antibody-conjugated toxin drug (ADC);

[0054] Human IgG1 Fc: hIgG1 Fc;

[0055] Human IgG1 heavy chain constant region: hIgG1 CH;

[0056] Human IgG1 light chain constant region of antibody KAPPA: hIgG1 CL (KAPPA);

[0057] Human IgG1 light chain constant region of antibody Lambda: hIgG1 CL (Lambda).

[0058] Example 1. Preparation and identification of raw materials

[0059] 1.1 Preparation and construction of fully human phage display library materials

[0060] 1.1.1 Construction of SLE positive patient sample library

[0061] The K-TM eukaryotic protein made by the research group was used to verify the SLE positive patient samples. The basic steps are as follows: collect peripheral blood from SLE patients, obtain the upper plasma after centrifugation, and add the plasma sample to the primary antibody diluent at a dilution ratio of 1:1000 for subsequent primary antibody verification. Use K-TM eukaryotic protein for western blot experiment loading, follow the basic steps of western blot experiment, place the transferred PVDF membrane in the configured primary antibody, and color development after the primary and secondary antibody incubation. Color development verification confirms that the patient has a clear band, which is confirmed as positive plasma, corresponding to the SLE positive patient.

[0062] K-TM eukaryotic protein preparation method

[0063] Using Strep-tag technology, highly pure K-TM eukaryotic protein with Strep tag was purified. The specific experimental steps are as follows:

[0064] The complete K-TM amino acid sequence was fused with a Strep tag at the carboxyl terminus and cloned into the pcDNA3.1(+) vector to construct the pcDNA3.1(+)-K-TM-Strep plasmid. The Strep tag sequence is shown in SEQ ID NO: 151: 5'-TGGAGCCACCCGCAGTTCGAAAAG-3'. The resulting plasmid was stained with ExpiFectamine TM 293 transfection kit (Gibco TM , A14524) were transiently transfected into HEK293 cells ( CRL-1573 TM ), after 72h of transfection, the cell supernatant was collected and the Strep tag antigen was expressed by Tactin XT (IBA) affinity chromatography was used for purification. The purified target protein was replaced with PBS buffer by ultrafiltration concentration tube (Millipore, UFC901096) to obtain K-TM eukaryotic protein.

[0065] 1.1.2 Preparation of cDNA for fully human antibody library

[0066] 1.1.2.1 Isolation of peripheral blood mononuclear cells (PBMC) from positive SLE patients

[0067] Collect peripheral blood samples from patients, transfer the samples to 15mL centrifuge tubes, and add an equal volume of 1×PBS buffer. Take another 15mL centrifuge tube and add 5mL of lymphocyte separation solution. Carefully add the PBS-diluted blood sample to the lymphocyte separation solution slowly along the wall of the centrifuge tube, taking care not to destroy the stratified interface. 2500rpm, speed up 2, speed down 3, horizontal centrifugation at room temperature for 25min. After centrifugation, aspirate the white film layer in the middle of the sample, which is the mononuclear cell, and add 1×PBS to wash once. 1500rpm, centrifuge at room temperature for 5min. The precipitate is PBMC, resuspend it with 1×PBS and keep it for later use.

[0068] 1.1.2.2 Preparation of cDNA from positive SLE patients

[0069] The RNA from PBMC of positive SLE patients was extracted by Trizol method, the obtained RNA precipitate was dissolved in 20-50 μL Rnase-free water, and the RNA quality was measured by spectrophotometer. TM The full-length RNA was reverse transcribed into cDNA using Takara II One-Step cDNA Synthesis Kit (Takara, 6210A).

[0070] 1.1.3 Construction of a fully human phage display library

[0071] Using the #1 to #32 cDNA of the human positive SLE patient prepared above as a template, the light and heavy chain genes were amplified by molecular cloning technology, the antibody gene was recombined by in vitro ligation, the vector and Fab gene sequences were respectively digested, the antibody nucleotide sequence was constructed into a phage display vector, and the constructed vector was further transformed into Escherichia coli by electroporation to obtain a fully human phage display library.

[0072] 1.2 Preparation and identification of HERV-K102-Env control antibody

[0073] Preparation of HERV-K102-Env control antibody: The positive control antibodies used in the present invention are anti-HERV-K102-Env antibody GN_mAb_Env_K01 (patent number: US2019 / 0345232 A1) and commercial antibody HERM-1811-5 (AUSTRAL Biologicals, CAT NO: HERM-1811-5). The target fragment was synthesized by General Biotechnology Co., Ltd. according to the sequence disclosed in the corresponding patent. Then, it was constructed into the eukaryotic expression vector pcDNA3.4-TOPO (Invitrogen) by homologous recombination, and the constructed recombinant protein expression vector was transformed into Escherichia coli DH5α, cultured overnight at 37°C, and then the plasmid was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01), and the plasmid was extracted by ExpiFectamine TM CHO transfection kit (Thermo Fisher, A29129) was used for expression. Seven days after transfection, the cell expression supernatant was centrifuged at 15000g for 10 min. The obtained supernatant was filtered through a 0.22 μm filter membrane and purified by affinity chromatography using a Protein A / G affinity column. After purification, the target protein was eluted with 100 mM glycine (pH 3.0), and the eluted protein was exchanged into PBS buffer through an ultrafiltration concentration tube (Millipore, UFC901096).

[0074] Identification of HERV-K102-Env control antibody: The activity of the prepared positive control antibodies GN_mAb_Env_K01 and HERM-1811-5 was detected using K102-SU-hFc and K102-tTM-G4S-FC(IgG1)-strep antigen proteins. The specific method is as follows: K102-SU-hFc and K102-tTM-G4S-FC(IgG1)-strep (2 μg / mL, 30 μL / well) were coated on a 96-well ELISA plate at 4°C overnight; after washing the plate 3 times, it was blocked with 5% skim milk prepared in PBS at room temperature for 1 hour; the plate was washed After 3 times, add control antibodies GN_mAb_Env_K01 and HERM-1811-5 diluted with PBS gradient and incubate at room temperature for 1 hour; after washing, add secondary antibodies Anti-humanκ+λ-HRP (Millipore; AP502P; AP506P) and Anti-mouse-IgG-Fc-HRP (abcam; ab97265) diluted with PBS (1:6000) and incubate at room temperature for 1 hour, wash the plate 6 times, add TMB (SurModics, TMBS-1000-01) and color for 5-20 minutes, and stop color development. 450Read the plate, process the data and plot using Graphpad prism. Figure 1A , 1B It shows that the produced control antibodies GN_mAb_Env_K01 and HERM-1811-5 can bind normally to HERV-K102-Env antigen protein (see 1.3 Antigen protein preparation), and the control antibodies GN_mAb_Env_K01 and HERM-1811-5 have normal activity and normal anti-HERV-K102-Env activity.

[0075] 1.3 Antigen protein preparation and identification

[0076] Preparation of antigen protein: The nucleotide sequence encoding the human Fc (hFc, SEQ ID NO: 145) tag was added to the 3′ end of the nucleotide sequence encoding the extracellular domain of human HERV-K102-Env (huHERV-K102-Env-ECD, see: Uniprot ID: P15328 AA25-234), and constructed into the pcDNA3.4 vector by homologous recombination. The constructed recombinant protein expression vector was transformed into Escherichia coli DH5α, cultured at 37°C overnight, and then the plasmid was extracted using an endotoxin-free plasmid extraction kit. The obtained plasmid was stained with ExpiFectamine TM 293 transfection kit (Gibco TM , A14524) were transiently transfected into K102-HEK293 cells ( CRL-1573 TM ), after 7 days of expression, the cell supernatant was collected, and the Fc tag antigen was purified by conventional ProteinA / G affinity chromatography column affinity method. After purification, the target protein was eluted with 100mM glycine salt (pH=3.0), concentrated and replaced, and finally the antigen proteins were obtained, namely K102-SU-hFc, K102-tTM-G4S-FC(IgG1)-strep, and K102-SU-FC(IgG1); the eluted proteins were exchanged into PBS buffer by ultrafiltration concentration tubes (Millipore, UFC901096), and finally the antigen proteins (K102-SU-hFc, K102-tTM-G4S-FC(IgG1)-strep, K102-SU-FC(IgG1)) were obtained.

[0077] Antigen identification: The activity of the prepared antigens (K102-SU-hFc, K102-tTM-G4S-FC(IgG1)-strep, K102-SU-FC(IgG1)) was detected using the control antibodies GN_mAb_Env_K01 and HERM-1811-5 that passed the quality inspection in Example 1.1. The specific method is as follows: 2 μg / mL K102-SU-hFc, K102-tTM-G4S-FC(IgG1)-strep, and K102-SU-FC(IgG1) were coated on Elisa plates at 4°C overnight; after washing the plates three times, the plates were blocked with 5% skim milk prepared in PBS at room temperature for 1 hour; after washing the plates three times, control antibodies GN_mAb_Env_K01 and HERM-1811-5 diluted in gradient with PBS were added and incubated at room temperature for 1 hour; after washing the plates, secondary antibodies Goat-Anti-mouse-IgG-Fc-HRP (abcam; ab97265) and add Anti-mouse Fc-HRP (abcam; ab97265) diluted in PBS (1:8000) were added, and after incubation at room temperature for 1 hour, the plates were washed six times, TMB was added for color development for 5-20 minutes, and the OD value of the microplate reader was calculated after the color development was terminated. 450 Read the plate, process the data and plot using Graphpad prism. Figure 1C-1D The results showed that the antigens K102-SU-FC (IgG1) and K102-SU-hFc constructed and expressed by the applicant can bind normally to the antibody GN_mAb_Env_K01, and the antigen K102-tTM-G4S-FC (IgG1)-strep can bind normally to the antibody HERM-1811-5.

[0078] Example 2. Construction and identification of cell lines overexpressing human HERV-K102-Env

[0079] 2.1 Construction of K102MTM-SP-TRN-MTM-G4S-3FLAG-6HIS cell line overexpressing human HERV-K102-Env:

[0080] The nucleic acid sequence encoding the full-length human HERV-K102-Env (Uniprot ID: P15328) was constructed into the pLVX-puro plasmid (Clontech, Cat#632164). Then, the obtained plasmid was electrotransformed into K102MTM-SP-TRN-MTM-G4S-3FLAG-6HIS cells by an electroporator (Invitrogen, NeonTM Transfection System, MP922947). After electrotransformation, the obtained cells were transferred to DMEM medium (Gibco, 11995065) containing 10% FBS (Gibco, 15140-141) by volume and without antibiotics, and then the cells were inoculated into 10×10 cm cell culture dishes and cultured for 48 hours, followed by an average of 0.5×10 4 The cells were dispensed into 96-well cell culture plates at a density of 100 cells / well, and puromycin with a final concentration of 2 μg / mL was added as a screening pressure. The growth of cell line clones was observed for about 2 weeks, and the cell lines that formed clones were picked for identification.

[0081] 2.2 Flow cytometry identification of huHERV-K102-Env-K102MTM-SP-TRN-MTM-G4S-3FLAG-6HIS cells:

[0082] The cells in the logarithmic growth phase were digested and plated into a 96-well plate, washed with FACS buffer (1×PBS buffer containing 2% FBS by volume), and then stained with primary antibodies diluted in PBS gradient (for huHERV-K102-Env-K102MTM-SP-TRN-MTM-G4S-3FLAG-6HIS cells, the primary antibody used Abs dilutions), and incubated at 4°C for 30 minutes; after washing, the prepared fluorescent secondary antibody Anti-Human IgG, FcγPE (Jackson 109-115-0981:200) was added, and incubated at 4°C for 30 minutes; finally, the detection was performed by flow cytometry (Beckman, CytoFLEXAOO-1-1102). The test results are shown in Figure 2. Figure 2 The results showed that human HERV-K102-Env was highly expressed on the surface of huHERV-K102-Env-K102MTM-SP-TRN-MTM-G4S-3FLAG-6HIS cells.

[0083] Example 3. Construction and screening of human phage-displayed recombinant antibody library

[0084] In this example, an antibody gene phage display library was constructed, and the library was screened using the antigen proteins K102-SU-FC (IgG1) and K102-tTM-G4S-FC (IgG1)-strep prepared in Example 1.3 as screening antigens to obtain multiple antibody molecules with specific binding to human HERV-K102-Env.

[0085] 3.1 Construction of human antibody gene library

[0086] Based on the sequence similarity of the heavy chain and light chain germline genes, degenerate primers were designed at the front end of the heavy chain variable region and the rear end of the first heavy chain constant region, as well as at the front end of the light chain variable region and the rear end of the light chain constant region (Li Xiaolin, Construction and Preliminary Screening of a Large-Capacity Non-Immune Human Fab Phage Antibody Library, Master's Thesis, Peking Union Medical College, June 2007), and the heavy chain variable region gene fragment and light chain variable region gene fragment of the antibody were obtained after PCR. The fragment containing the light chain variable region and heavy chain variable region of the antibody was amplified by the fusion PCR method, and the PCR product and the phage display vector were digested, recovered and connected, and the connection product was recovered by a recovery kit (Omega, catalog number: D6492-02). Finally, the cells were transformed into competent E. coli SS320 (Lucigen, MC1061 F) by an electroporator (Bio-Rad, MicroPulser), and the transformed E. coli SS320 bacterial solution was spread on a 2-YT solid plate with ampicillin resistance (the solid plate was prepared by 1.5% tryptone, 1% yeast extract, 0.5% NaCl, and 1.5% agar, according to the mass volume g / mL). The library capacity was determined to be 3.67×10 8 cfu, i.e. 3.67×10 8 The antibody gene library of each antibody gene (the library capacity calculation method refers to Example 2.2 in patent CN112250763B). VSCM13 helper phage (purchased from Stratagene) was used to package it to obtain an antibody gene phage display library (the preparation of the antibody gene phage display library refers to Example 2.3 in patent CN112250763B).

[0087] 3.2 Screening of antibody gene phage display library

[0088] 3.2.1 Screening of antibody gene phage display library by magnetic bead method

[0089] Magnetic bead screening is based on biotin labeling of antigen proteins K102-SU-FC (IgG1) and K102-tTM-G4S-FC (IgG1)-strep, and then binding to magnetic beads coupled with streptavidin, and incubating, washing and eluting the magnetic beads bound to the antigen and the antibody gene phage display library. Usually 3 to 4 rounds of panning are carried out, thereby enriching a large number of specific monoclonal antibodies against the antigen. In this embodiment, biotin-labeled K102-SU-hFc-Biotin and K102-tTM-G4S-FC (IgG1)-strep-Biotin are used for phage display library screening, and after 3 rounds of panning, monoclonal antibodies for K102-SU-FC (IgG1) and K102-tTM-G4S-FC (IgG1)-strep are initially screened. For specific methods, refer to Example 2.4.1 in patent CN112250763B.

[0090] 3.2.2 Screening of antibody gene phage display library by immunotube method

[0091] The principle of the immunotube screening is to coat the antigen protein K102-SU-FC (IgG1) and K102-tTM-G4S-FC (IgG1)-strep on the surface of the immunotube with high adsorption capacity, and then add the phage display antibody library to the immunotube and incubate, wash and elute with the antigen protein adsorbed on the surface of the immunotube. After 3 to 4 rounds of panning, the specific monoclonal antibody for the antigen is finally enriched. In this embodiment, after 3 rounds of panning, the monoclonal antibody for K102-SU-FC (IgG1) and K102-tTM-G4S-FC (IgG1)-strep is initially screened. The specific method is referred to Example 2.4.2 in patent CN112250763B.

[0092] 3.3 Selection of monoclonal

[0093] The phage pool eluted in each round was tested by ELISA to evaluate the enrichment effect, and a total of 512 clones were randomly selected from the phage pool in each round of screening for initial screening, and 135 positive clones were selected for sequence analysis.

[0094] The ELISA monoclonal initial screening used antigen proteins K102-SU-FC (IgG1) and K102-tTM-G4S-FC (IgG1)-strep, and 68 antibody Fab molecules that specifically bind to K102-SU-FC (IgG1) or K102-tTM-G4S-FC (IgG1)-strep were screened. The screened antibody Fab molecules were named by clone number, and 18 molecules were selected for subsequent verification. The CDR region and light and heavy chain amino acid sequences of the relevant antibody Fab molecules are shown in the sequence table. The AbM definition of CDR was used to determine the complementary determining region sequence.

[0095] Example 4 Antibody Construction, Expression and Purification

[0096] 4.1 Plasmid construction

[0097] The coding sequence of VH in the Fab sequence of the screened monoclonal A005, A014, A017, A046, A047, A049, A050, A052, A054, A062, A068, A075, A077, A082, A083, A089, A104, and A131 was connected to the coding sequence of the heavy chain constant region of human IgG1 to construct the heavy chain coding sequence of the fully human antibody, and the coding sequence of VL in the Fab sequence was connected to the Kappa type coding sequence of the human light chain constant region (CL) to construct the light chain coding sequence of the fully human antibody. The coding sequences of the antibody heavy chain and light chain were respectively inserted into the eukaryotic expression vector plasmid pcDNA3.4 (Invitrogen), transformed into Escherichia coli DH5α, and cultured overnight at 37°C. The plasmid was extracted using an endotoxin-free plasmid extraction kit to obtain an endotoxin-free antibody plasmid for eukaryotic expression.

[0098] 4.2 Antibody expression and purification

[0099] The candidate antibodies were expressed using the ExpiCHO transient expression system (Thermo Fisher, A29133) as follows: On the day of transfection, the cell density was confirmed to be 7×10 6 Up to 1×10 7 The cell viability is >98%. At this time, fresh ExpiCHO expression medium pre-warmed at 37°C is used to add ExpiFectamine TM CHO cells were adjusted to a final concentration of 6 × 10 6 cells / mL. Use OptiPRO pre-cooled at 4°C TM Dilute the target plasmid in SFM (add 1 μg of plasmid to 1 mL of the culture medium), mix the two in equal volumes and gently pipette to mix to prepare ExpiFectamine TMThe CHO / plasmid DNA mixture was incubated at room temperature for 1 to 5 minutes, then slowly added to the prepared cell suspension while gently shaking, and finally placed in a cell culture shaker at 37°C and 8% CO. 2 Cultivated under conditions.

[0100] 18-22 hours after transfection, add ExpiCHO TM Enhancer and ExpiCHO TM Feed the flask and place it in a shaker at 32°C and 5% CO 2 On the 5th day after transfection, add the same volume of ExpiCHO TM Feed, slowly add and gently mix the cell suspension. Seven days after transfection, the cell culture supernatant expressing the target protein was centrifuged at 15000g for 10 min, the resulting supernatant was affinity purified using MabSelect SuRe LX (GE, 17547403), and then the target protein was eluted with 100mM sodium acetate (pH3.0), followed by neutralization with 1M Tris-HCl, and finally the resulting protein was exchanged into PBS buffer using an ultrafiltration concentration tube (Millipore, UFC901096).

[0101] Example 5 Identification of the physicochemical properties of antibodies

[0102] In this example, SDS-PAGE and SEC-HPLC were used to detect the relative molecular weight and purity of the candidate antibodies.

[0103] 5.1 Antibody SDS-PAGE Identification

[0104] Preparation of non-reducing solution: 1 μg of each antibody and quality control product IPI (ipilimumab) were added to 5×SDS loading buffer and 40 mM iodoacetamide, heated in a 75°C dry bath for 10 min, cooled to room temperature, and centrifuged at 12000 rpm for 5 min to obtain the supernatant.

[0105] Preparation of reducing solution: 2 μg of each antibody and quality control product IPI were added to 5×SDS loading buffer and 5 mM DTT, heated in a 100°C dry bath for 10 min, cooled to room temperature, centrifuged at 12000 rpm for 5 min, and the supernatant was collected. The supernatant was added to Bis-tris 4-15% gradient gel (GenScript) for gel electrophoresis and the protein bands were visualized by Coomassie Brilliant Blue staining.

[0106] The protein gel with chromogenic protein bands was scanned using EPSON V550 color scanner (decolorization was performed until the gel background was transparent), and the purity of the reduced and non-reduced bands was calculated using ImageJ according to the peak area normalization method.

[0107] The results are shown in Table 2. The bands of each antibody in the non-reducing gel are around 150 kD, and the bands of the reducing gel are around 55 kD and 25 kD, which are in line with the expected size. The purity of all candidate antibodies tested by reducing gel is greater than 90%

[0108] 5.2 SEC-HPLC Identification of Monomeric Purity of Antibodies

[0109] Material preparation: 1. Mobile phase: 150mmol / L phosphate buffer, pH7.4; 2. Sample preparation: dilute each antibody and quality control product IPI to 0.5mg / mL with mobile phase solution. Agilent HPLC 1100 column (XBridge BEH SEC3.5μm, 7.8mm ID×30cm), Waters flow rate is set to 0.8mL / min, injection volume is 20μL, VWD detector wavelength is 280nm and 214nm. Inject blank solution, IPI quality control solution and sample solution in sequence. According to the area normalization method, the percentage of high molecular weight polymers, antibody monomers and low molecular weight substances in the sample is calculated. The results of the candidate antibody physicochemical properties obtained by the application are shown in Table 1.

[0110] Table 1 Physicochemical properties of candidate antibodies obtained in the present invention

[0111]

[0112]

[0113] Note: N / A means no testing was performed

[0114] Example 6 Antigen Binding Activity Detection of Antibodies

[0115] In this example, the affinity of candidate antibodies (A005, A014, A017, A046, A047, A049, A050, A052, A054, A062, A068, A075, A077, A082, A083, A089, A104, A131) to human HERV-K102-Env antigen protein K102-SU-FC (IgG1) was detected based on the ELISA method, and the binding ability of candidate antibodies to human HERV-K102-Env overexpressing cells K102Mtm-SP-trN-mTM-G4S-3Flag-6His was also detected based on the FACS method.

[0116] 6.1 Detection of the binding ability of candidate antibodies to the antigen protein K102-tTM-G4S-hFc-strep based on ELISA double antibody sandwich method

[0117] force

[0118] The control antibody and candidate antibody (2μg / mL, 30μL / well) were coated on a 96-well ELISA plate at 4°C overnight. The next day, the plate was washed 3 times with PBST and blocked with 5% skim milk for 2 hours. After washing the plate 3 times with PBST, a gradient dilution of K102-tTM-G4S-hFc-strep was added and incubated for 1 hour. After washing 3 times with PBST, the detection antibody 1G6-Biotin (2μg / mL, 30μL / well) was added and incubated for 1 hour. After washing 3 times with PBST, the secondary antibody NeutrAvidin-HRP (Thermo, 31001) was added and incubated for 1 hour. After the incubation is completed, the plate was washed 6 times with PBST and TMB was added for color development. According to the color development results, 2MH 2 SO 4 The reaction was terminated and the OD was measured by microplate reader (Molecular Devices, SpecterMax 190). 450 Read the data below.

[0119] The results are as follows Figures 3A-3C As shown, A014, A050, A068, A052, A062, A077, A082, A104, A131, A083, A054, A075, A050, and A049 formed a pair with 1G6 and had good affinity activity to the antigen protein K102-tTM-G4S-hFc-strep.

[0120] 6.2 ELISA-based detection of the binding ability of candidate antibodies to antigen protein K102-SU-FC (IgG1)

[0121] K102-SU-FC (IgG1) (2μg / mL, 30μL / well) was coated on a 96-well ELISA plate at 4°C overnight. The next day, the plate was washed three times with PBST and blocked with 5% skim milk for 2 hours. After washing the plate three times with PBST, the candidate antibodies with gradient dilutions and the positive control antibody GN_mAb_Env_K01 were added and incubated for 1 hour. After washing three times with PBST, the secondary antibody Anti-Human-κ+λ-HRP (Millipore, AP502P, AP506P) was added and incubated for 1 hour. After incubation, the plate was washed 6 times with PBST and TMB was added for color development. According to the color development results, 2M HCl was added to terminate the reaction and the plate was analyzed by a microplate reader (Molecular Devices, SpecterMax 190) at OD 450 Read the data below.

[0122] The results are as follows Figure 3D-3E As shown, A014, A017, A047, A054, A062, A077, A082, A083, A104, and A131 all have good affinity activity for the antigen protein K102-SU-FC (IgG1).

[0123] 6.3 FACS-based detection of the binding ability of candidate antibodies to K102Mtm-SP-trN-mTM-G4S-3Flag-6His cells

[0124] In this example, K102Mtm-SP-trN-mTM-G4S-3Flag-6His cells were used to evaluate the binding activity of the candidate antibodies.

[0125] The specific method is as follows: K102Mtm-SP-trN-mTM-G4S-3Flag-6His cells in the logarithmic growth phase were prepared into a single cell suspension and the density was adjusted to 1×10 6 cells / mL, add 100 μL per well to a 96-well round-bottom plate, centrifuge at 4°C, 300g and remove the supernatant. Add 20 candidate antibodies and positive control antibody A032 (homemade) or serum in gradient dilutions to the corresponding wells, mix well and incubate at 4°C for 30 minutes. Wash the incubated cell mixture twice and add 100 μL of 1:300 diluted secondary antibody Goat F(ab') 2 Anti-Human IgG-Fc was incubated at 4°C in the dark for 30 min, washed twice, and detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

[0126] The results are as follows Figures 4A-4E As shown, on K102Mtm-SP-trN-mTM-G4S-3Flag-6His cells, candidate antibodies A005, A017, A046, A047, A052, A062, A068, and A089 all have good affinity to K102Mtm-SP-trN-mTM-G4S-3Flag-6His cells.

[0127] Example 7 ELISA double antibody sandwich pairing

[0128] 7.1 Pairing of antibodies against K102-tTM-G4S-FC(IgG1)-strep antigen protein based on ELISA double antibody sandwich method:

[0129] Coat 96-well ELISA plates (30 μL / well) with 4 μg / mL antibody at 4°C overnight. The next day, wash the plates three times with PBST and block with 5% skim milk for 2 hours. Wash the plates three times with PBST, add gradient dilutions (10, 2.0, 0.4, 0.08, 0.016, 0.0032 μg / mL) of K102-tTM-G4S-FC(IgG1)-strep and incubate for 1 hour. Wash the plates three times with PBST, add 4 μg / mL of biotinylated detection antibody and incubate for 1 hour. After that, wash the plates three times with PBST, add secondary antibody NeutrAvidin-HRP (Thermo, 31001) and incubate for 1 hour. After incubation, wash the plates six times with PBST and add TMB (SurModics, TMBS-1000-01) for color development. According to the color development results, 2 M stop solution was added to terminate the reaction, and the OD 450 Read the absorbance at

[0130] The results are as follows Figure 5A to Figure 5I As shown: the coating antibodies are A050, A068, A062, and A052, and the detection antibody is 1G6-Biotin. These four pairs of paired antibodies are more effective in detecting K102-tTM-G4S-FC (IgG1)-strep antigen. 50 The eight paired antibodies 1G6 and A062-Biotin, A050 and A062-Biotin, 1G6 and A082-Biotin, 1G6 and A068-Biotin, A082 and 1G6-Biotin, 1G6 and A052-Biotin, A068 and A014-Biotin, A062 and A014-Biotin were excellent in detecting K102-tTM-G4S-FC(IgG1)-strep antigen, and their EC 50 Within 0.1256μg / mL~0.2824μg / mL.

[0131] Relatively short sequences are distributed in the variable regions of antibodies, showing extremely strong diversity, which are called high-frequency mutation regions. There are three such sequences in the variable regions of the light chain and heavy chain, respectively, which are called CDRs (complementarity determining regions). These sequences are related to the antibody's specific recognition of antigens.

[0132] In Table 2, according to the clone number from top to bottom, the HCDR1 sequence is: SEQ ID NO: 1-18, the HCDR2 sequence is: SEQ ID NO: 19-36, the HCDR3 sequence is: SEQ ID NO: 37-54, the LCDR1 sequence is: SEQ ID NO: 55-72, the LCDR2 sequence is: SEQ ID NO: 73-90, and the LCDR3 sequence is: SEQ ID NO: 91-108.

[0133] Table 2: Sequence Listing 1, amino acid sequences of the delivered antibody CDR regions

[0134]

[0135]

[0136] In Table 3, according to the clone number, the VH sequences are as follows: SEQ ID NO: 109-126, and the VL sequences are as follows: SEQ ID NO: 109-126.

[0137] NO:127~144.

[0138] Table 3: Sequence Listing 2, light and heavy chain sequences of delivered antibodies

[0139]

[0140]

[0141]

[0142] Table 4: Antibody pairing table

[0143]

[0144]

[0145]

[0146] Table 5: Antigen binding of 18 antibody molecules

[0147] Protein name Antigen binding A005 Cross-linked SU-TM A083 Single Binding TM A017 Single combined SU A046 Cross-linked SU-TM A047 Cross-linked SU-TM A054 Single Binding TM A075 Single Binding TM A050 Single Binding TM A049 Cross-linked SU-TM A014 Cross-linked SU-TM A068 Single Binding TM A052 Single Binding TM A062 Single Binding TM A077 Single Binding TM A082 Cross-linked SU-TM A104 Single Binding TM A131 Single Binding TM A089 Cross-linked SU-TM

[0148] In Table 5, TM refers to HERV-K102-Env-TM, SU refers to HERV-K102-Env-SU, and SU-TM refers to HERV-K102-Env-SU and HERV-K102-Env-TM together.

[0149] The sequence of the antigen HERV-K102-Env-SU is shown in SEQ ID NO: 149:

[0150] MVTPVTWMDNPIEIYVNDSVWVPGPIDDRCPAKPEEEGMMINISIGYRYPPICLGRAP

[0151] GCLMPAVQNWLVEVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQRSLKFRPKGKP

[0152] CPKEIPKESKNTEVLVWEECVANSAVILQNNEFGTIIDWAPRGQFYHNCSGQTQSCPS

[0153] AQVSPAVDSDLTESLDKHKHKKLQSFYPWEWGEKRISTPRPKIVSPVSGPEHPELWRL

[0154] TVASHHIRIWSGNQTLETRDCKPFYTIDLNSSLTVPLQSCVKPPYMLVVGNIVIKPDSQ

[0155] TITCENCRLLSCIDSTFNWQHRILLVRAREGVWIPVSMDRPWEASPSVHILTEVLKGV

[0156] LNRSKR

[0157] The antigen HERV-K102-Env-TM sequence is shown in SEQ ID NO: 150:

[0158] FIFTLIAVIMGLIAVTATAAVAGVALHSSVQSVNFVNDWQKNSTRLWNSQSSIDQKLA

[0159] NQINDLRQTVIWMGDRLMSLEHRFQLQCDWNTSDFCITPQIYNESEHHWDMVRRHL

[0160] QGREDNLTLDISKLKEQIFEASKAHLNLVPGTEAIAGVADGLANLNPVTWVKTIGSTT

[0161] IINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQI

[0162] VTVSV

[0163] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in the present invention, just as each document is cited as reference separately.

Claims

1. An isolated antibody or antigen-binding fragment thereof, characterized in that: The CDR region of the antibody or antigen-binding fragment thereof includes HCDR and LCDR, the HCDR region includes HCDR1, HCDR2 and HCDR3, and the LCDR region includes LCDR1, LCDR2 and LCDR3; The HCDR1 amino acid sequence is as shown in any one of SEQ ID NOs: 1 to 18, the HCDR2 amino acid sequence is as shown in any one of SEQ ID NOs: 19 to 36, the HCDR3 amino acid sequence is as shown in any one of SEQ ID NOs: 37 to 54, the LCDR1 amino acid sequence is as shown in any one of SEQ ID NOs: 55 to 72, the LCDR2 amino acid sequence is as shown in any one of SEQ ID NOs: 73 to 90, and the LCDR3 amino acid sequence is as shown in any one of SEQ ID NOs: 91 to 108.

2. An isolated antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof comprises VH and VL, wherein the amino acid sequence of VH is shown in sequence as SEQ ID NOs: 109 to 126, and the amino acid sequence of VL is shown in sequence as SEQ ID NOs: 127 to 144.

3. An isolated antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof includes any one of 18 antibodies or antigen-binding fragments thereof 1 to 18; The VH amino acid sequences of the antibodies or antigen-binding fragments thereof 1 to 18 are shown in SEQ ID NOs: 109 to 126, respectively, and the VL amino acid sequences of the antibodies or antigen-binding fragments thereof 1 to 18 are shown in SEQ ID NOs: 127 to 144, respectively.

4. An isolated antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment specifically binds to HERV-K102-Env, and its binding antigen epitope is HERV-K102-Env-SU and / or HERV-K102-Env-TM. The amino acid sequence of HERV-K102-Env-SU is shown in SEQ ID NO: 149, and the amino acid sequence of HERV-K102-Env-TM is shown in SEQ ID NO:

150.

5. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: The antibody is a human monoclonal antibody.

6. An isolated polynucleotide, characterized in that It encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

7. A separated vector, characterized in that It comprises the polynucleotide according to claim 6.

8. A host cell, characterized in that It comprises the polynucleotide according to claim 6 or the vector according to claim 7.

9. A kit for detecting HERV-K102-Env expression, characterized in that: It comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in a product for detecting and / or diagnosing and / or treating HERV-K102-Env expression.

11. Use of the human monoclonal antibody according to claim 5 in a product for detecting and / or diagnosing and / or treating tumors, neurodegenerative diseases or autoimmune diseases associated with HERV-K102-Env expression.

12. The use of the human monoclonal antibody according to claim 11, characterized in that: The product is any one of CAR-T, CAR-NK, CAR-DC, TIL therapeutic cell products or ADC products.

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