Anti-HERV-K102-SU murine antibody and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an anti-HERV-K102-SU mouse antibody and application thereof. The present invention relates to novel antibodies directed against the human endogenous retrovirus HERV-K102 subtype envelope protein SU, which target conserved regions that are not affected by glycosylation or native conformation, and their use in diagnosis and / or therapy. These antibodies have demonstrated their biological activity in ELISA, FCM (flow cytometry) and western blot immunoassays. These antibodies recognize not only native and denatured HERV-K102-Env-SU antigen proteins, but also non-glycosylated and glycosylated forms as well.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an anti-HERV-K102-SU mouse-derived antibody and a use thereof. Background Art

[0002] Human endogenous retroviruses (HERVs) are products of retrovirus infection and integration into the human genome during the evolution of the human genome. In the Human Genome Project, it was found that at least about 50% of the human genome is composed of transposon-derived sequences, which include a considerable portion of endogenous retroviruses, accounting for about 8% of the human genome. However, due to the presence of a large number of mutations, deletions and termination codons in HERVs sequences, they have been considered "parasites" or "junk DNA" in the genome. However, in recent years, more and more studies have provided sufficient evidence that HERVs are involved in most biological processes, and that HERVs transcriptional dysregulation and their reactivated expression products play an important role in the occurrence and development of various tumors.

[0003] HERVs, as the product of ancient retroviruses infecting the human genome, have a typical retrovirus structure. From the 5' end to the 3' end, the complete sequence is the 5'-long term repeat (5'-LTR), gag, pol, env and the 3'-long terminal repeat. The env gene encodes the viral envelope glycoprotein (Env). The Env protein is hydrolyzed by furin protease to form surface protein (SU) and transmembrane protein (TM). The SU protein is involved in a variety of inflammatory reactions, and the TM has an immunosuppressive function.

[0004] 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 the HERV-K102 subtype, may be closely related to the occurrence and development of tumors, autoimmune diseases, neurodegenerative diseases, etc. This suggests that SU, as a new superantigen protein, may be involved in the pathological processes such as the onset and progression of diseases such as tumors, neurodegenerative lesions, and autoimmune diseases. At present, most highly malignant tumors, neurodegenerative lesions, and autoimmune diseases still lack effective therapeutic drugs and technologies. For patients with these fatal diseases, the discovery of new treatments remains an urgent need. Therefore, the development of effective antibody drugs may also be an effective treatment strategy for these unmet clinical needs. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes the following technical solutions:

[0006] 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;

[0007] 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 105.

[0008] Preferably, the antibody or antigen-binding fragment thereof comprises VH and VL, the VH amino acid sequence is sequentially shown in SEQ ID NOs: 106-123, and the VL amino acid sequence is sequentially shown in SEQ ID NOs: 124-141.

[0009] Preferably, the antibody or antigen-binding fragment thereof comprises any one of 18 antibodies or antigen-binding fragments thereof 1 to 18;

[0010] The VH amino acid sequences of the antibodies or antigen-binding fragments thereof 1 to 18 are shown in SEQ ID NOs: 106 to 123, respectively, and the VL amino acid sequences of the antibodies or antigen-binding fragments thereof 1 to 18 are shown in SEQ ID NOs: 124 to 141, respectively.

[0011] Preferably, the antibody or antigen-binding fragment specifically binds to HERV-K102-Env, and its binding antigen epitope is HERV-K102-SU. The amino acid sequence of HERV-K102-SU is shown in SEQ ID NO: 146.

[0012] Preferably, the antibody is a mouse monoclonal antibody.

[0013] An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof described in any one of the preceding items.

[0014] An isolated vector comprises the aforementioned polynucleotide.

[0015] A host cell comprising the aforementioned polynucleotide or the aforementioned vector.

[0016] A kit for detecting HERV-K102-Env expression, comprising any of the aforementioned antibodies or antigen-binding fragments thereof.

[0017] Preferably, the antibodies or antigen-binding fragments thereof include 18, whose monoclonal numbers are A020, A049, A089, A092, A093, A098, A107, A108, A114, A115, A119, A124, A128, A159, A164, A167, A179, and A181, and the ELISA double antibody sandwich pairing includes any one of the 8 pairs, specifically: A115 and A108-Biotin, A089 and A108-Biotin, A114 and A108-Biotin, A115 and A181-Biotin, A115 and A164-biotin, A089 and A115-Biotin, A114 and A115-Biotin, A089 and A093-Biotin.

[0018] Use of the antibody or antigen-binding fragment thereof according to any of the preceding items in a product for detecting and / or diagnosing and / or treating HERV-K102-Env expression.

[0019] Use of the aforementioned murine monoclonal antibody in products for detecting and / or diagnosing and / or treating tumors, neurodegenerative diseases or autoimmune diseases associated with HERV-K102-Env expression.

[0020] Preferably, the product is any one of CAR-T, CAR-NK, CAR-DC, TIL therapeutic cell products or ADC products.

[0021] The beneficial effects of the present invention are mainly:

[0022] The present invention relates to novel antibodies against human endogenous retrovirus HERV-K102 subtype envelope protein SU and use thereof in diagnosis and / or treatment, wherein the antibodies target a conserved region that is not affected by glycosylation or native conformation.

[0023] The present inventors have developed novel mouse monoclonal antibodies (mAbs) against HERV-K102-Env-SU envelope protein that exhibit unexpected properties, and our antibodies have higher antigen binding activity than the currently available HERV-K102-SU antibody (GN_mAb_Env_K01).

[0024] The inventors have shown that the 18 antibodies against HERV-K102-Env-SU according to the present invention are murine monoclonal antibodies that specifically bind to the SU antigen of the HERV-K102 subtype.

[0025] These antibodies have demonstrated their biological activity in ELISA, FCM (flow cytometry) and Western blot immunoassays. These antibodies recognize not only native and denatured HERV-K102-Env-SU antigenic proteins, but also non-glycosylated and glycosylated forms. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A-1B :Shows the activity of self-produced control antibodies and antigens measured based on 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 antigen K102-SU-hFc and control antibody GN_mAb_Env_K01 is shown.

[0027] Figure 2 : Representative images showing the relative molecular weight and purity of candidate antibodies in reduced and non-reduced states based on SDS-PAGE.

[0028] Figure 3A-3B :Shows the binding effect of candidate antibodies to antigen K102-SU-hFc determined by ELISA method; Figure 3A The binding effects of A020, A049, A089, A092, A093, A098, A107, A108, and A114 with antigen K102-SU-hFc are shown, and GN-mAb-Env-K01 was used as a positive control antibody; Figure 3B The binding effects of A115, A119, A159, A164, A167, A179, A181, and A128 with the antigen K102-SU-hFc are shown, and GN-mAb-Env-K01 serves as a positive control antibody.

[0029] Figures 4A-4L :Shows the biotin labeling effect of candidate antibodies based on ELISA. Figure AB is a summary of the binding activity of biotin-labeled candidate antibodies to antigen K102-SU-hFc. Figure CL shows the comparison of the binding activity of candidate antibodies to antigen K102-SU-hFc before and after biotin labeling.

[0030] Figures 5A-5L : Shows the pairing effect of candidate antibodies on K102-SU-hFc antigen protein. Figure 5A The pairing effects of A115, A128, A164, A093, A108, and A181 as capture antibodies and A115-Biotin as detection antibody on K102-SU-hFc antigen protein are shown; Figure 5B The pairing effects of A115, A128, A164, A093, A108, and A181 as capture antibodies and A128-Biotin as detection antibody on K102-SU-hFc antigen protein are shown; Figure 5C The pairing effects of A115, A128, A164, A093, A108, and A181 as capture antibodies and A164-Biotin as detection antibody on K102-SU-hFc antigen protein are shown; Figure 5D The pairing effects of A115, A128, A164, A093, A108, and A181 as capture antibodies and A093-Biotin as detection antibody on K102-SU-hFc antigen protein are shown; Figure 5E The pairing effects of A115, A128, A164, A093, A108, and A181 as capture antibodies and A108-Biotin as detection antibody on K102-SU-hFc antigen protein are shown; Fig. 5F The pairing effects of A115, A128, A164, A093, A108, and A181 as capture antibodies and A181-Biotin as detection antibody on K102-SU-hFc antigen protein are shown; Figure 5G The pairing effects of A115, A128, A093, and A108 as capture antibodies and A089-Biotin as detection antibody on K102-SU-hFc antigen protein are shown; Figure 5H The pairing effects of A115, A128, A093, and A108 as capture antibodies and A114-Biotin as detection antibody on K102-SU-hFc antigen protein are shown; Fig.5I The pairing effect of A089 and A114 as capture antibodies and A115-Biotin as detection antibody on K102-SU-hFc antigen protein is shown; Figure 5J The pairing effect of A089 and A114 as capture antibodies and A128-Biotin as detection antibody on K102-SU-hFc antigen protein is shown; Figure 5K The pairing effect of A089 and A114 as capture antibodies and A093-Biotin as detection antibody on K102-SU-hFc antigen protein is shown; Figure 5LThe pairing effect of A089 and A114 as capture antibodies and A108-Biotin as detection antibody on K102-SU-hFc antigen protein is shown. DETAILED DESCRIPTION

[0031] 1. Glossary:

[0032] Human endogenous retroviruses (HERVs);

[0033] Envelope protein (Envelope, Env);

[0034] The C-terminal transmembrane region (TM), which is a transmembrane protein formed after Env is hydrolyzed by furin protease;

[0035] N-terminus of envelope protein (SU);

[0036] HERV-K102 envelope protein (HERV-K102-Env);

[0037] Antibody light chain variable region (VL);

[0038] Antibody heavy chain variable region (VH);

[0039] Antibody light chain complementarity determining region (LCDR);

[0040] Antibody heavy chain complementarity determining region (HCDR);

[0041] The N-terminus of the HERV-K102 envelope protein (HERV-K102-Env-SU, abbreviated as HERV-K102-SU or K102-SU);

[0042] High frequency mutation regions: CDR (complementarity determining regions);

[0043] Antibody-conjugated toxin drug (ADC);

[0044] Human IgG1 Fc: hIgG1 Fc;

[0045] Human IgG1 heavy chain constant region: hIgG1 CH;

[0046] Human IgG1 light chain constant region of antibody KAPPA: hIgG1 CL (KAPPA);

[0047] Human IgG1 light chain constant region of antibody Lambda: hIgG1 CL (Lambda).

[0048] Example 1 Preparation and identification of raw materials

[0049] 1.1 Preparation and identification of HERV-K102-SU control antibody

[0050] Preparation of HERV-K102-SU control antibody: The positive control antibody used in this patent application is GN_mAb_Env_K01 (patent number: US2019 / 0345232 A1). The target fragment was synthesized by General Biotech 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. 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 (OMEGA, D6950-01). 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).

[0051] Identification of HERV-K102-SU control antibody: K102-SU-hFc antigen protein was used to detect the activity of the positive control antibody GN_mAb_Env_K01 prepared above. The specific detection method is as follows: K102-SU-hFc (coating concentration: 2 μg / mL, volume: 30 μL / well) was coated on a 96-well ELISA plate and coated overnight at 4°C; after washing the plate 3 times, it was blocked with 5% skim milk prepared in PBS at room temperature for 1 hour; after washing the plate 3 times, the control antibody GN_mAb_Env_K01 diluted in gradient PBS was added and incubated at room temperature for 1 hour; after washing the plate, the secondary antibody Anti-humanκ+λ-HRP (Millipore; AP502P; AP506P) diluted in PBS (1:6000) was added and incubated at room temperature for 1 hour, and then the plate was washed 6 times and TMB (SurModics, TMBS-1000-01) was added for color development for 20 minutes, the color development was stopped, and the OD value of the microplate reader was 0. 450 The plate was read at 100 nm and the data were processed and plotted using Graphpad prism. Figure 1AIt shows that the produced control antibody GN_mAb_Env_K01 can bind normally to the HERV-K102-SU antigen protein, the control antibody GN_mAb_Env_K01 has normal activity, and has normal anti-HERV-K102-SU protein activity.

[0052] 1.2 Antigen protein preparation and identification

[0053] Preparation of antigen protein: The antigen protein used in this patent application is K102-SU-hFc. The specific preparation direction is as follows: the nucleotide sequence encoding human Fc (hFc, SEQ ID NO: 142) is added to the 3′ end of the nucleotide sequence encoding the extracellular domain of human HERV-K102-SU protein (huHERV-K102-SU-ECD, Uniprot ID: P61567AA1-354), and constructed into the pcDNA3.4 vector by homologous recombination. The constructed recombinant protein expression vector is transformed into Escherichia coli DH5α, cultured at 37°C overnight, and then the plasmid is extracted using an endotoxin-free plasmid extraction kit. The obtained plasmid is stained with ExpiFectamine TM 293 transfection kit (Gibco TM , A14524) were transiently transfected into 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 protein K102-SU-hFc was obtained. The eluted protein was exchanged into 1x PBS buffer using an ultrafiltration concentration tube (Millipore, UFC901096) to obtain the purified antigen protein K102-SU-hFc.

[0054] Antigen protein identification: Use the control antibody GN_mAb_Env_K01 that passed the quality inspection in Example 1.1 to detect the activity of the prepared antigen K102-SU-hFc. The specific experimental method is as follows: Coat the Elisa plate with 2μg / mL K102-SU-hFc antigen protein at 4°C overnight; wash the plate 3 times, seal the plate with 5% skim milk prepared in PBS at room temperature for 1 hour; after washing the plate 3 times, add the control antibody GN_mAb_Env_K01 diluted in gradient with PBS, and incubate at room temperature for 1 hour; after washing the plate, add the secondary antibody Goat-Anti-mouse-IgG-Fc-HRP (abcam; ab97265) diluted with PBS (1:8000), and incubate at room temperature for 1 hour; wash the plate 6 times, add TMB for color development for 20 minutes; stop color development, and the OD value of the microplate reader is 0.450 The plate was read at 100 nm and the data were processed and plotted using Graphpad prism. Figure 1B It shows that the antigen K102-SU-hFc constructed and expressed by the inventors of the present application can bind normally to the antibody GN_mAb_Env_K01.

[0055] 1.3 Preparation and identification of immune plasmids

[0056] The main steps for preparing the GSV0-K102-SU-hFc immune plasmid are as follows. The full-length K102-SU amino acid sequence is derived from the extracellular domain 1-354aa of the human retrovirus K family 6 Env protein (ENK-7, UniProt, P61567), and the amino acid sequence of the human Fc (hFc, SEQ ID NO: 142) tag is added to the 3′ end. It is constructed into the GSV0 vector by homologous recombination, and after PCR amplification of the target gene, vector linearization, DNA fragment recovery, homologous recombination and transformation, positive clone sequencing and other steps, the sequencing results of the obtained positive clones are sequenced and compared. If the expected sequence in the credible region of the sequencing result is consistent, it is a successful construction, and the GSV0-K102-SU-hFc immune plasmid is obtained.

[0057] Example 2 Construction and screening of mouse immune library

[0058] In this example, a mouse immune library was constructed, and the library was screened using the antigen protein K102-SU-hFc prepared in Example 1.2 as a screening antigen to obtain multiple antibody molecules that specifically bind to human HERV-K102-SU.

[0059] 2.1 Construction of mouse immune antibody library

[0060] The antigen protein K102-SU-hFc antigen prepared in Example 1.2 was used to immunize 3 Balb / C mice, and the GSV0-K102-SU-hFc immune plasmid prepared in Example 1.3 was used to immunize 4 Balb / C mice. After 4 immunizations, antibodies against specific immunogens were produced in the mice, and mouse spleens for constructing mouse immune libraries were obtained. The K102-SU-hFc antigen was used to test the titer of the immunized mice, showing that the immune titers of 7 mice all reached 1.09 million, reaching the library construction standard. The RNA of mouse spleen tissue was extracted by chloroform method, and the antibody gene was amplified, identified, tapped and recovered by the method of grouped primer amplification after reverse transcription, and the vector was connected and transformed to construct 8 mouse immune antibody libraries. The storage capacity of the 8 antibody libraries was determined by the dilution point plate method, and the correct insertion rate of the antibody gene was verified by monoclonal sequencing analysis. The results showed that the storage capacity of the 8 antibody libraries was greater than the designed storage capacity and the correct insertion rate was greater than 80%, which met the expected requirements. At the same time, the antibody gene integrity of the Fab antibody library and the source analysis of the inserted antibody genes were performed to confirm that the inserted antibody sequences were all mouse-derived antibodies.

[0061] 2.2 Screening of mouse immune library

[0062] 2.2.1 Screening of mouse immune library by immunotube method

[0063] The immunotube screening is based on coating the antigen protein K102-SU-hFc on the surface of an immunotube with high adsorption capacity, adding the phage display antibody library to the immunotube and incubating, washing and eluting the antigen protein adsorbed on the surface of the immunotube, and finally enriching the specific monoclonal antibody against the antigen after three rounds of screening. In this embodiment, the mouse immune library phage suspension is first prepared, and then the immunotube coated with the remaining proteins and sealed is incubated. After three rounds of panning, the monoclonal antibody against K102-SU-hFc is initially screened. The specific method refers to Example 2.4.2 in patent CN112250763B.

[0064] 2.2.2 Screening of mouse immune library by magnetic bead method

[0065] The magnetic bead screening is based on the process of incubating, washing and eluting the antigen protein K102-SU-hFc with magnetic beads coupled with streptavidin after biotin labeling, and then the magnetic beads bound to the antigen and the mouse antibody gene phage display library are incubated. After 3-4 rounds of panning, a large number of specific monoclonal antibodies for the antigen are enriched. In this embodiment, a mouse antibody library phage suspension is first prepared, and then the biotin-labeled K102-SU-hFc-Biotin is used for phage display library screening. After 3 rounds of panning, the monoclonal antibody for the K102-SU-hFc antigen is initially screened. The specific operation method can refer to Example 2.4.1 in patent CN112250763B.

[0066] 2.3 Selection of monoclones

[0067] The mouse immune library constructed above was used to enrich the specific Fab antibodies against K102-SU-hFc by trypsin elution. The enrichment of different output sets was then detected by ELISA. Most of the output sets with good enrichment were obtained at the ELISA level. After the initial ELISA screening, a total of 534 clones were selected, and 291 positive clones binding to human antigens were obtained. 183 positive clones binding to human antigens with OD values ​​greater than 1.68 were sent for sequence analysis.

[0068] The ELISA monoclonal screening used the antigen protein K102-SU-hFc, and 19 Fab molecules with unique sequences were screened, which specifically bound to K102-SU-hFc. After sequence diversity analysis, 18 were selected for subsequent verification and full-length construction. The unconstructed molecules had low sequence diversity or contained post-translational modification sites, and no full-length construction was arranged.

[0069] Example 3 Antibody Construction, Expression and Purification

[0070] 3.1 Plasmid construction

[0071] The coding sequence of VH in the Fab sequence of the screened monoclonal A020, A049, A089, A092, A093, A098, A107, A108, A114, A115, A119, A124, A128, A159, A164, A167, A179, and A181 was connected to the coding sequence of the heavy chain constant region of human IgG1 to construct the heavy chain coding sequence of the antibody, and the coding sequence of VL in the Fab sequence was connected to the coding sequence of the Kappa type of the human light chain constant region (CL) to construct the light chain coding sequence of the antibody. The coding sequences of the heavy chain and light chain of the antibody 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.

[0072] 3.2 Antibody expression and purification

[0073] 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 was >98%. At this time, the ExpiFectamineTM CHO cells were adjusted to a final concentration of 6×10 6 Cells / mL. Dilute the target plasmid with OptiPROTM SFM precooled at 4°C (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 ExpiFectamineTM CHO / plasmid DNA mixture, incubate at room temperature for 1-5 minutes, slowly add to the prepared cell suspension and gently shake at the same time, and finally place in a cell culture shaker at 37°C, 8% CO 2 Cultivated under conditions.

[0074] 24 h after transfection, ExpiCHOTM Enhancer and ExpiCHOTM Feed were added to the culture medium and the shake flask was placed in a shaker at 32°C and 5% CO 2Continue to culture under the same conditions. On the 5th day after transfection, add the same volume of ExpiCHOTM Feed and gently mix the cell suspension while slowly adding it. 7 days after transfection, the cell culture supernatant expressing the target protein was centrifuged at 15000g for 10 minutes. 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).

[0075] Example 4 Identification of the physicochemical properties of antibodies

[0076] The relative molecular weight and purity of the candidate antibodies were detected by SDS-PAGE and SEC-HPLC.

[0077] 4.1 Antibody SDS-PAGE identification

[0078] Reducing and non-reducing solutions of each antibody were prepared separately, and IPI (Ipilimumab) was used as a quality control. The samples were loaded onto Bis-tris 4-15% gradient gel (GenScript) for gel electrophoresis and the protein bands were visualized by Coomassie Brilliant Blue staining. The protein gel with the visualized protein bands was scanned using an EPSON V550 color scanner (decolorization with a destaining solution until the gel background was transparent), and the purity of the reducing and non-reducing bands was calculated using ImageJ according to the peak area normalization method. The results are shown in Figure 2. Figure 2 As described above, the bands of each antibody in non-reducing gel are around 150KD, and the bands of reducing gel are around 55KD and 25KD, which are in line with the expected size. The purity of all candidate antibodies detected by reducing gel is greater than 90%.

[0079] 4.2 SEC-HPLC determination of antibody monomer purity

[0080] The basic steps are as follows: dilute each antibody and IPI quality control product to a concentration of 0.5 mg / mL with a mobile phase solution (150 mmol / L phosphate buffer, pH 7.4). Use an Agilent HPLC 1100 column (XBridge BEH SEC 3.5 μm, 7.8 mm ID×30 cm) and set the relevant parameters: Waters flow rate is set to 0.8 mL / min, injection volume is 20 μL, and VWD detector wavelengths are 280 nm and 214 nm. Inject blank solution, IPI quality control solution, and sample solution in sequence. The area normalization method is used to calculate the percentage of high molecular weight polymers, antibody monomers, and low molecular weight substances in the sample. The results of the physical and chemical properties of the candidate antibodies are shown in Table 1.

[0081] Table 1 Physicochemical properties of candidate antibodies obtained in this application

[0082]

[0083]

[0084] Note: N / A means no testing was performed

[0085] Example 5 Antibody Antigen Binding Activity Detection

[0086] In this example, the affinity effect of candidate antibodies (A020, A049, A089, A092, A093, A098, A107, A108, A114, A115, A119, A124, A128, A159, A164, A167, A179, A181) and human GSV0-HERV-K102-SU-hFc antigen protein was detected based on the ELISA double antibody sandwich method. The specific experimental steps are as follows:

[0087] K102-SU-hFc (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 and the positive control antibody GN_mAb_Env_K01 were added with gradient dilutions and incubated for 1 hour. After washing three times with PBST, the secondary antibody Anti-Human-κ+λ-HRP (Millipore, AP502P+AP506P, 1:4000) was added and incubated for 50 minutes. 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 measured at OD using an enzyme reader (Molecular Devices, SpecterMax190). 450 Read the data below.

[0088] The results are as follows Figure 3A to Figure 3B As shown, 11 antibody molecules, including A020, A049, A093, A098, A108, A119, A159, A167, A179, A181, and A128, have good affinity activity with the antigen protein K102-SU-hFc, which is better than the control antibody; the antigen binding activity of one antibody molecule, A092, is equivalent to that of the control antibody; 5 antibody molecules, including A089, A107, A114, A115, and A164, have poor binding activity with the antigen protein K102-SU-hFc, and are worse than the control antibody.

[0089] Example 6 ELISA double antibody sandwich pairing

[0090] In this example, affinity sorting and biotin labeling were performed on the 18 antibody molecules and 1 positive antibody molecule obtained above, and antibody epitope grouping was performed by competition method. Antibody pairing and maximum sensitivity detection were performed at the same time, aiming to establish a double antibody sandwich ELISA detection kit specific for HERV-K102-SU-hFc antigen protein.

[0091] 6.1 Biotin labeling of antibodies

[0092] This experiment uses the coupling method and enzyme-linked immunosorbent assay (ELISA) to biotinylate 19 antibody molecules and detect the activity of antibody molecules before and after labeling for quality control testing. The basic steps are as follows:

[0093] The required volume of Biotin was calculated based on the concentration, volume and relative molecular mass of the labeled protein. Biotin was added to each protein to be labeled, mixed and placed in an ice bath for 2 hours. After labeling, ultrapure water was added to the ultrafiltration tube for desalination. A280 was used to determine the protein concentration before and after labeling, and the labeling efficiency was determined at the same time. ELISA was used to detect the residual Biotin and the labeling effect, and the activity of the protein before and after biotin labeling and the system adaptability were also detected.

[0094] The results are as follows Figure 4A -L shows that 19 proteins were successfully labeled with biotin, the residual biotin in the solution was qualified, and the biotin labeling effect on the protein was good. The binding of 15 antibody proteins to K102-SU-hFc after labeling was close to that before labeling. The binding activity of GN-mAb-Env-K01 to K102-SU-hFc after labeling was 45.6% before labeling; the binding activity of A093 to K102-SU-hFc after labeling was 209.2% before labeling; the binding activity of A115 to K102-SU-hFc after labeling was 49.7% before labeling; the binding activity of A128 to K102-SU-hFc after labeling was 63.9% before labeling. Conclusion: All 19 antibody proteins were successfully labeled with biotin.

[0095] 6.2 Antibody epitope grouping

[0096] This experiment used ELISA to group the epitopes of the 18 full-length antibodies obtained above. The basic steps are as follows:

[0097] Dilute the antigen sample K102-SU-hFc to 1μg / mL with PBS, add it to a 96-well ELISA plate at 30μL / well, and coat overnight at 4°C. Wash the plate 3 times with PBST, add blocking solution (5% PBSM) and block at room temperature for 2h. Wash the plate 3 times with PBST, dilute the antibody to be tested to 10μg / mL with 1% PBSM, add it to a 96-well ELISA plate, 30uL / well, and incubate at room temperature for 1h. Competitive antibody incubation: Do not wash the plate, dilute the biotinylated antibody to 0.1μg / mL with 1% PBSM, add it to a 96-well ELISA plate, 30uL / well, and incubate with competitive antibodies. Wash the plate 3 times with PBST, wash the NeutrAvidin-HRP with PBS, add TMB for color development, and terminate the reaction with 2M sulfuric acid. Use an enzyme reader to read the OD 450 Absorbance value. Result calculation: Inhibition rate = (antibody competition OD 450 Value / antibody affinity OD 450 value) x 100%

[0098] The results showed that the above 18 antibodies were divided into 3 epitopes, namely epitope 1: A049, A020, A092, A093, A098, A108, A119, A159, A164, A167, A179, A181, A124; epitope 2: A089, A114; epitope 3: A107, A115, A128.

[0099] 6.3 Antibody epitope pairing detection

[0100] This experiment uses enzyme-linked immunosorbent assay to pair the 18 antibodies screened above. The experimental principle is: bind the candidate antibody (unlabeled) to the surface of the solid phase carrier, then add the known antigen for incubation, then add the biotinylated antibody for incubation, then use the enzyme-labeled antibody to incubate with it, and finally use the color developer to develop the color, and the color depth is proportional to the content of the substance to be tested. The basic steps are as follows:

[0101] The unlabeled candidate antibody sample was diluted to 4 μg / mL with PBS, added to a 96-well ELISA plate at 30 μL / well, and coated overnight at 4°C. The plate was washed 3 times with PBST, and a blocking solution (5% PBSM) was added to block at room temperature for 2 hours. The plate was washed 3 times with PBST, and the antigen sample K102-SU-hFc was diluted to 10 μg / mL with 1% PBSM as the first concentration point, and 7 concentration points were diluted 3 times and added to a 96-well ELISA plate at 30 μL / well, and incubated at room temperature for 1 hour. The biotinylated antibody was diluted to 4 μg / mL with 1% PBSM, and added to a 96-well ELISA plate at 30 μL / well, and incubated at room temperature for 1 hour. The plate was washed 3 times with PBST, and NeutrAvidin-HRP was diluted 1:2000 with PBS, and 30 μL per well was added to the plate, and incubated at room temperature for 50 minutes. The plate was washed, TMB was added for color development, and the reaction was terminated with 2M sulfuric acid. Use a microplate reader to read the OD 450 The absorbance value was used for data collection.

[0102] The results are as follows Figure 5A -L, the above 8 antibodies were taken for orthogonal detection, and 8 pairs of good pairing relationships were selected, namely: A115 and A108-Biotin, A089 and A108-Biotin, A114 and A108-Biotin, A115 and A181-Biotin, A115 and A164-biotin, A089 and A115-Biotin, A114 and A115-Biotin, A089 and A093-Biotin. These 8 pairs of paired antibodies have excellent effects in detecting K102-SU-hFc antigen, and their EC 50 The value range is: 0.0330~0.05173μg / mL.

[0103] 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: 91-105 (among which A049, A092 and A093 have no LCDR3 sequence).

[0104] Table 2 Amino acid sequences of the CDR regions of delivered antibodies

[0105]

[0106]

[0107] In Table 3, according to the clone number, the VH sequences are SEQ ID NOs: 106 to 123, and the VL sequences are SEQ ID NOs: 124 to 141.

[0108] Table 3 Light and heavy chain sequences of delivered antibodies

[0109]

[0110]

[0111]

[0112]

[0113] Table 4 Antibody pairing table

[0114]

[0115]

[0116]

[0117] The HERV-K102-SU sequence is shown in SEQ ID NO: 146:

[0118] MVTPVTWMDNPIEIYVNDSVWVPGPIDDRCPAKPEEEGMMINISIGYRYPPICLGRAPGCL

[0119] MPAVQNWLVEVVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQRSLKFRPKGKPCPKEIP

[0120] KESKNTEVLVWEECVANSAVILQNNEFGTIIDWAPRGQFYHNCSGQTQSCPSAQVSPAVD

[0121] SDLTESLDKHKHKKLQSFYPWEWGEKRISTPRPKIVSPVSGPEHPELWRLTVASHHIRIWS

[0122] GNQTLETRDCKPFYTIDLNSSLTVPLQSCVKPPYMLVVGNIVIKPDSQTITCENCRLLSCIDS

[0123] TFNWQHRILLVRAREGVWIPVSMDRPWEASPSVHILTEVLKGVLNRSKR

[0124] 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 105.

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: 106 to 123, and the amino acid sequence of VL is shown in sequence as SEQ ID NOs: 124 to 141.

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: 106 to 123, respectively, and the VL amino acid sequences of the antibodies or antigen-binding fragments thereof 1 to 18 are shown in SEQ ID NOs: 124 to 141, 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-SU. The amino acid sequence of HERV-K102-SU is shown in SEQ ID NO:

146.

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 mouse 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 murine 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 mouse 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.

13. A kit for detecting HERV-K102-Env expression according to claim 9, characterized in that: The antibodies or antigen-binding fragments thereof include 18, whose monoclonal numbers are A020, A049, A089, A092, A093, A098, A107, A108, A114, A115, A119, A124, A128, A159, A164, A167, A179, and A181, and the ELISA double antibody sandwich pairing includes any one of the 8 pairs, specifically: A115 and A108-Biotin, A089 and A108-Biotin, A114 and A108-Biotin, A115 and A181-Biotin, A115 and A164-biotin, A089 and A115-Biotin, A114 and A115-Biotin, A089 and A093-Biotin.

Images