Preparation of lypd3-targeting antibody and application thereof in tumor immunotherapy

Abstract

This invention discloses the preparation of an antibody targeting LYPD3 and its application in tumor immunotherapy. The antibody or its antigen-binding fragment includes a heavy chain variable region having CDR-H1, CDR-H2, and CDR-H3, and a light chain variable region having CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprise the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to pharmaceutical products containing immunoglobulins, and more specifically to anti-LYPD3 antibodies. Background Technology

[0002] Lung cancer is one of the most common malignant tumors worldwide and the leading cause of cancer-related deaths, with its incidence and mortality rates rapidly increasing. In my country, non-small cell lung cancer (NSCLC) ranks first in both incidence and mortality among malignant tumors (Biomed Pharmacother, 2023, 31, 169: 115891). Although surgery, chemotherapy, radiotherapy, and targeted drug therapy have significantly improved the quality of life and prolonged survival of NSCLC patients, the prognosis remains poor, with a 5-year survival rate of only about 20% (Lung Cancer, 2021, 159: 34-41). Therefore, there is an urgent need to seek new treatment methods.

[0003] In recent years, immunotherapy has achieved remarkable efficacy in cancer treatment and is currently a hot research topic in cancer treatment. Antibody immunotherapy, especially the development of monoclonal antibody (mAb) drugs, has become the focus of the current cancer treatment field (Thorax, 2022, 77(12): 1163-1174). However, due to the limitations of target selection, the heterogeneity of tumors, and the low or no responsiveness of antibody immunotherapy in clinical patients, the widespread application of this therapy is greatly limited. In addition, due to the lack of specific targets, antibodies are prone to off-target effects during treatment, which not only reduces the therapeutic effect but may also cause a series of adverse reactions and toxic side effects to patients. Therefore, precise targeting of specific targets and the development of highly efficient antibodies have become the key to breaking through the bottleneck of antibody immunotherapy. This will not only help to significantly improve the efficacy of immunotherapy but also effectively reduce toxic side effects, providing a safer and more effective treatment option for cancer patients.

[0004] LY6 / PLAUR Domain Containing 3 (LYPD3) is a highly glycosylated membrane protein that is highly expressed in various tumors such as lung cancer, breast cancer, renal cell carcinoma, liver cancer, colorectal cancer, and acute myeloid leukemia (Lung Cancer, 2007, 58(2): 260-266; Oncol Rep, 2017, 38(5): 2697-2704; Br J Cancer, 2007, 97(8): 1146-1156; Front Genet, 2022, 13: 795820). It participates in the occurrence, development, and progression of tumors and can serve as a potential biomarker for tumors. In particular, studies have found that approximately 50% of lung cancer patients and 75% of lung cancer metastases highly express LYPD3, but it is not expressed in normal lung tissue (Oncogene, 2002, 21: 7749-7763), suggesting that LYPD3 could serve as a biomarker for lung cancer prognosis and immunotherapy (Transl Cancer Res, 2024, 13(3): 1394-1405). Therefore, targeting LYPD3 could be a potential strategy for lung cancer immunotherapy.

[0005] Screening for novel high-affinity antibodies targeting LYPD3 has significant application prospects and implications in the immunotherapy of tumors such as lung cancer and breast cancer. Summary of the Invention

[0006] To address the aforementioned technical problems, this application provides an anti-LYPD3 antibody or its antigen-binding fragment thereof, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region having CDR-H1, CDR-H2, and CDR-H3 and a light chain variable region having CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1, CDR-H2, and CDR-H3 respectively comprise the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and CDR-L1, CDR-L2, and CDR-L3 respectively comprise the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7 (QVS), and SEQ ID NO: 8.

[0007] In another aspect, this application also provides a nucleic acid encoding an anti-LYPD3 antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising a heavy chain variable region having CDR-H1, CDR-H2 and CDR-H3 and a light chain variable region having CDR-L1, CDR-L2 and CDR-L3, wherein CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7 (QVS) and SEQ ID NO: 8, respectively.

[0008] In another aspect, this application also provides a vector comprising a nucleic acid encoding an anti-LYPD3 antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising a heavy chain variable region having CDR-H1, CDR-H2 and CDR-H3 and a light chain variable region having CDR-L1, CDR-L2 and CDR-L3, wherein CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7 (QVS) and SEQ ID NO: 8, respectively.

[0009] Regarding treatment options, this application provides the use of an anti-LYPD3 antibody or its antigen-binding fragment in the preparation of a medicament for treating cancer, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region having CDR-H1, CDR-H2, and CDR-H3 and a light chain variable region having CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprise the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7 (QVS), and SEQ ID NO: 8, respectively. Attached Figure Description

[0010] The present application will now be described in more detail with reference to the accompanying drawings, in which:

[0011] Figure 1 Electrophoresis image for identification of anti-LYPD3 antibody;

[0012] Figure 2This is a graph showing the results of antibody binding assay using ELISA.

[0013] Figure 3 The image shows the results of the affinity assay for anti-LYPD3#22 antibody (Biacore method).

[0014] Figure 4 This is a graph showing the results of LYPD3 target cell antigen expression detection.

[0015] Figure 5 This is a graph showing the results of the detection of the binding of anti-human LYPD3 antibody to target cells;

[0016] Figure 6 This is a graph illustrating the in vitro ADCC effect induced by anti-LYPD3 antibody.

[0017] Figure 7 This is a diagram illustrating the in vivo ADCC effect induced by anti-LYPD3 antibody. Detailed Implementation

[0018] This application relates to an anti-LYPD3 antibody or its antigen-binding fragment, said antibody or antigen-binding fragment comprising heavy chain variable regions having CDR-H1, CDR-H2, and CDR-H3 and light chain variable regions having CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprise the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7 (QVS), and SEQ ID NO: 8, respectively. In one embodiment, the antibody or its antigen-binding fragment comprises Fab, F(ab'), F(ab')2, Fv, or a single-chain variable fragment (scFv). In a preferred embodiment, the antibody or its antigen-binding fragment comprises scFv. In one embodiment, the isotype of the antibody or its antigen-binding fragment comprises IgA, IgD, IgE, IgG, or IgM. In a preferred embodiment, the isotype of the antibody or its antigen-binding fragment includes IgG. In a preferred embodiment, the antibody or its antigen-binding fragment includes a subtype of IgG isotype IgG1, IgG2, IgG3, or IgG4. In a preferred embodiment, the antibody or its antigen-binding fragment includes subtype IgG1. In a preferred embodiment, the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 1. In a preferred embodiment, the heavy chain variable region is composed of the amino acid sequence of SEQ ID NO: 1. In a preferred embodiment, the light chain variable region includes the amino acid sequence of SEQ ID NO: 2. In a preferred embodiment, the light chain variable region is composed of the amino acid sequence of SEQ ID NO: 2. In a preferred embodiment, CDR-H1, CDR-H2, and CDR-H3 are composed of the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively, and CDR-L1, CDR-L2, and CDR-L3 are composed of the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. In one embodiment, the heavy chain variable region includes frame regions (FR) 1, FR 2, FR 3, and FR 4 separated by its three CDRs. In one embodiment, frame regions 1, FR 2, FR 3, and FR 4 of the heavy chain variable region respectively include the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.In one preferred embodiment, frame regions 1, 2, 3, and 4 of the heavy chain variable region are composed of the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively. In one embodiment, the light chain variable region includes frame regions (FR) 1, 2, 3, and 4 separated by its three CDRs. In one embodiment, frame regions 1, 2, 3, and 4 of the light chain variable region include the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively. In one preferred embodiment, frame regions 1, 2, 3, and 4 of the light chain variable region are composed of the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively. In one embodiment, the antibody or its antigen-binding fragment is humanized. In this application, unless otherwise stated, the CDR sequences are defined according to the IMGT numbering scheme.

[0019] This application also relates to a nucleic acid encoding an anti-LYPD3 antibody or an antigen-binding fragment thereof, wherein the anti-LYPD3 antibody or antigen-binding fragment thereof may be the anti-LYPD3 antibody or antigen-binding fragment thereof described in any of the above embodiments. In one embodiment, the nucleic acid includes nucleotide sequences encoding a heavy chain variable region and a light chain variable region. In one embodiment, the nucleotide sequence encoding the heavy chain variable region includes SEQ ID NO: 17. In one embodiment, the nucleotide sequence encoding the light chain variable region includes SEQ ID NO: 18. In one embodiment, the nucleotide includes nucleotide sequences encoding CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. In one embodiment, the nucleotide sequence encoding CDR-H1 includes SEQ ID NO: 19. In one embodiment, the nucleotide sequence encoding CDR-H2 includes SEQ ID NO: 20. In one embodiment, the nucleotide sequence encoding CDR-H3 includes SEQ ID NO: 21. In one embodiment, the nucleotide sequence encoding CDR-L1 includes SEQ ID NO: 22. In one embodiment, the nucleotide sequence encoding CDR-L2 includes SEQ ID NO: 23 (CAGGTGTCT). In one embodiment, the nucleotide sequence encoding CDR-L3 includes SEQ ID NO: 24. In one embodiment, the nucleotide sequence includes nucleotide sequences encoding FR1, FR2, FR3, and FR4 of the heavy chain variable region and FR1, FR2, FR3, and FR4 of the light chain variable region. In one embodiment, the nucleotide sequence encoding FR1 of the heavy chain variable region includes SEQ ID NO: 25. In one embodiment, the nucleotide sequence encoding FR2 of the heavy chain variable region includes SEQ ID NO: 26. In one embodiment, the nucleotide sequence encoding FR3 of the heavy chain variable region includes SEQ ID NO: 27. In one embodiment, the nucleotide sequence encoding FR4 of the heavy chain variable region includes SEQ ID NO: 28. In one embodiment, the nucleotide sequence encoding FR1 of the light chain variable region includes SEQ ID NO: 29. In one embodiment, the nucleotide sequence encoding FR2 of the light chain variable region includes SEQ ID NO: 30. In one embodiment, the nucleotide sequence encoding FR3 of the light chain variable region includes SEQ ID NO: 31. In one embodiment, the nucleotide sequence encoding FR4, the variable region of the light chain, includes SEQ ID NO: 32.

[0020] This application also relates to a vector comprising a nucleic acid, which may be a nucleic acid of any of the embodiments described above. In one embodiment, the vector comprises one or more selected from the group consisting of plasmids, phage particles, bacteriophages or derivatives thereof, viruses, and granules.

[0021] This application also relates to the use of anti-LYPD3 antibodies or antigen-binding fragments thereof in the preparation of medicaments for treating cancer, wherein the antibody or antigen-binding fragment may be any of the antibodies or antigen-binding fragments described in the foregoing embodiments. In one embodiment, the cancer includes cancers expressing LYPD3. In one embodiment, the cancer includes hematologic malignancies and solid tumors. In one embodiment, the hematologic malignancies include leukemia. In a preferred embodiment, the leukemia includes AML. In a preferred embodiment, the solid tumor includes lung cancer, breast cancer, renal cell carcinoma, liver cancer, and / or colorectal cancer. In a preferred embodiment, the solid tumor includes lung cancer. In a more preferred embodiment, the lung cancer includes non-small cell lung cancer.

[0022] Example

[0023] This application will be described in detail through the following exemplary embodiments. These embodiments are only intended to help those skilled in the art better understand the invention of this application. It should be noted that the spirit and scope of protection of this application are not limited to the following specific embodiments.

[0024] Example 1

[0025] Mice were immunized with human LYPD3 protein, and LYPD3-specific memory B cells were sorted by flow cytometry and antibody sequences were obtained by single-cell sequencing.

[0026] (I) Experimental Materials

[0027] BALB / c mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., Freund's adjuvant was purchased from Sigma-Aldrich, the immunogen LYPD3 protein was purchased from Nanjing Youai Biotechnology R&D Co., Ltd. (catalog number: UA010207), mouse memory B cell isolation kit and QuadroMACS Starting kit were purchased from Miltenyi, PBS was purchased from Hyclone, 0.4% trypan blue was purchased from Sangon Biotech Co., Ltd., and FITC was purchased from Thermo Fisher Scientific (catalog number: 46410).

[0028] (II) Experimental Methods

[0029] Mouse immunization: Female BALB / c mice aged 6-8 weeks were selected, and 50 μg of LYPD3 protein and an equal volume of complete Freund's adjuvant were mixed and administered as the first subcutaneous injection. The second and third immunizations were administered on days 21 and 42, respectively, using an equal volume of 50 μg of antigen and an equal volume of incomplete Freund's adjuvant. The fourth immunization was administered on day 70 using 50 μg of antigen.

[0030] LYPD3-specific memory B cell sorting: Three days later, mice were euthanized by cervical dislocation. Fresh spleens and lymph nodes were harvested, placed on a 70μm sieve, and the tissues were ground, filtered through the 70μm sieve, resuspended, mixed, and centrifuged. The cells were resuspended in PBS and sorting buffer was added. Every 10... 8 Add 100 μl of memory B cell biotin-antibody mixture, 10 μl of anti-IgG1-APC, and 50 μl of sorting buffer to each cell line, and incubate at 4°C for 5 min. Add 300 μl of buffer and 200 μl of anti-biotin MicroBeads, incubate at 4°C for 10 min, centrifuge at 300g for 10 min, and discard the supernatant. Add 500 μl of buffer and pass through a column to collect negative cells. Centrifuge the collected cells at 300g for 10 min, discard the supernatant, add 400 μl of buffer and 100 μl of anti-APC MicroBeads, incubate at 4°C for 15 min, add 10 times the volume of sorting buffer, mix well, centrifuge at 300g for 10 min, and discard the supernatant. Resuspend in 500 μl of buffer, mix well, and pass through a separation column for washing and separation to collect IgG1 cells. + Centrifuge cells at 300g for 10 minutes and discard the supernatant. Resuspend cells in antibody incubation buffer (PBS + 2% FBS) and adjust cell density to 102. 7 Cells were collected at a density of 103 / ml as a control group. FITC-labeled LYPD3 antibody was added at a concentration of 2 μg / ml, and the cells were incubated at 4°C for 20 min. The cells were washed twice with PBS, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The cell density was adjusted to 103 cells / 5 -10 6 Cells / ml were sorted by flow cytometry (BD AriaIII sorting flow cytometer).

[0031] (III) Experimental Results

[0032] We immunized mice with human LYPD3 protein as an immunogen. After four immunizations, we isolated the spleen and lymph nodes of the mice; and obtained LYPD3-specific IgG1 by flow cytometry. + LYPD3 + Memory B cells. We commissioned Shanghai Jingneng Biotechnology Co., Ltd. to perform 10x Genomics BCR gene sequencing on single B cells. Based on the sequencing results, antibody sequences with a frequency ≥2 were selected for cloning and expression.

[0033] Example 2

[0034] Construction of VH and VL sequence vectors for anti-LYPD3 antibody and identification of antibody expression

[0035] (I) Experimental Materials

[0036] The heavy and light chain expression plasmids of the antibody were purchased from InvivoGen, the heavy and light chain genes of the antibody were synthesized by Suzhou Genewiz Biotechnology Co., Ltd., the gel extraction kit was purchased from Takara, the homologous recombinase was purchased from Nanjing Novizan Biotechnology Co., Ltd., the expiCHO-S cell line and transfection reagent were purchased from Thermo Fisher Scientific, and the protein G column was purchased from GE.

[0037] (II) Experimental Methods

[0038] Construction of VH and VL sequence vectors for LYPD3 antibody: The light and heavy chain scFv genes of the antibody were synthesized by Suzhou Genewiz Biotechnology Co., Ltd. Subcloning was performed using the gene synthesis plasmid as a template to obtain PCR fragments. The fragments were purified using a gel extraction kit and ligated into VH (pFUSEss-CHIg-hG1) and VL (pFUSE2ss-CLIg-hk) expression vectors via homologous recombination. The vectors were then transformed into DH5α competent cells, and positive clones were obtained by sequencing, yielding correctly paired light and heavy chain expression plasmids for the antibody.

[0039] CHO-S cell system expression antibody: Prepare reaction mixture A: 2 ml OptiPro-SFM + plasmid (50 μg each of light chain and heavy chain expression plasmid) and mixture B: 1.84 ml OptiPro-SFM + 160 μl ExpiFectamine. TM After mixing CHO reagent by vortexing and incubating at room temperature for 5 minutes, add mixture B to mixture A by vortexing and incubating at room temperature for 10-20 minutes. Slowly add 50 ml of CHO-S cell system. 18-22 hours after transfection, add 300 μl of ExpiCHO to the system. TTM Enhancer, 12ml ExpiCHO TM Feed the cells and incubate them at 37°C. After 7 days, collect the cell culture supernatant.

[0040] Antibody purification: Antibodies were purified using the AKTA protein purification system. The specific method was as follows: Cell culture supernatant was centrifuged or filtered to remove cell debris. The Protein G column was washed with 1×PBS for 10 CV until the baseline stabilized. The cell supernatant containing antibodies was loaded onto the AKTA system, and the column was equilibrated again with PBS to wash away unbound impurities. The antibodies on the column were eluted with 0.1M glycine (pH 2.8) and collected in a tube containing neutralization buffer (1M Tris, pH 9.0) to neutralize the pH. The eluted antibody solution was centrifuged using an ultrafiltration tube and then transferred to PBS.

[0041] (III) Experimental Results

[0042] The obtained antibody heavy and light chain scFv sequences were synthesized and constructed into VH and VL expression vectors via homologous recombination. The antibody was expressed using the CHO-S expression system and purified using the AKTA protein purification system. The purified antibody was analyzed for purity and molecular weight by 10% polyacrylamide gel electrophoresis. Under complete reduction conditions, the anti-LYPD3 antibody showed two bands with molecular weights of approximately 55 kDa and 30 kDa, representing the heavy and light chain bands respectively, with a purity exceeding 95%. Figure 1 The above results demonstrate that we have successfully prepared and expressed a high-purity antibody targeting LYPD3.

[0043] Example 3

[0044] Anti-LYPD3 antibody affinity assay

[0045] (I) Experimental Materials

[0046] Antigen-coated 96-well plates were purchased from Thermo Fisher Scientific, LYPD3 protein was purchased from Nanjing Youai Biotechnology R&D Co., Ltd. (catalog number: UA010207), secondary antibody (human IgG H&L-HRP, catalog number: ab6759), TMB chromogenic solution and stop solution were purchased from Abcam, and S-series CM5 sensor chips were purchased from Cytiva.

[0047] (II) Experimental Methods

[0048] ELISA assay for antibody affinity: LYPD3 protein was diluted with PBS and coated into 96-well plates (100 ng / well). The plates were incubated overnight at 4°C. The plates were washed four times with PBST, blocked with 3% BSA at room temperature for 1 h, washed four times with PBST, and 100 μl of sample (test sample and control sample, 1 μg / ml) was added. The plates were incubated at room temperature for 1 h, washed four times with PBST, and 100 μl of secondary antibody was added. The plates were incubated at room temperature for 1 h, washed four times with PBST, and 100 μl of TMB chromogenic solution was added. After color change, 100 μl of stop solution was added, and the OD value was detected at 450 nm using a single wave.

[0049] SPR method for antibody affinity detection: A chip was placed on a Biacore T200 (Cytiva) instrument, using HBSEP buffer (10 mM HEPES, pH 7.5, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20) at 25°C. The antigen protein LYPD3 was covalently linked to the experimental channel using amino-coupled coupling. Serially diluted antibodies were used as analytes and flowed through both the control and experimental channels at a rate of 30 μl / min. Binding time was 120 seconds, dissociation time was 400 seconds, and regeneration buffer was Glycine 2.0. Affinity (K0.05) was measured using Biacore T200 evaluation software 3.1 (Cytiva). D Analysis shows that a 1:1 combination of action mode is used.

[0050] (III) Experimental Results

[0051] We first detected the binding of antibodies to antigens using ELISA. The results showed that all the anti-LYPD3 antibodies we prepared and expressed could bind to human LYPD3 protein, comparable to the positive control. Figure 2 We further tested the affinity of the anti-LYPD3#22 antibody using Biacore, and the results showed that the anti-LYPD3#22 antibody had high affinity, K D =2.8×10 -10 M( Figure 3 ).

[0052] Example 4

[0053] like Figure 4 , 5 As shown, flow cytometry was used to detect the binding of anti-LYPD3 antibody to the lung cancer cell line NCI-H2126.

[0054] (I) Experimental Materials

[0055] PE anti-human LYPD3 antibody, FITC anti-human IgG Fc antibody, and PE isotype antibody were purchased from Biolegend, IgG1 negative control antibody was purchased from Abcam, antibody incubation solution: PBS + 2% FBS, and lung cancer cell line NCI-H2126 was purchased from Shanghai Fuheng Biotechnology Co., Ltd. (Catalog No.: FH0582).

[0056] (II) Experimental Methods

[0057] NCI-H2126 cell LYPD3 membrane expression detection: 1×10⁻⁶ cells were used. 5NCI-H2126 cells were incubated with 2 μl of PE anti-human LYPD3 antibody and isotype control antibody at room temperature in the dark for 20 min. After washing twice with PBS, the cells were resuspended and mixed with 200 μl of PBS. The expression of LYPD3 antigen was detected by flow cytometry.

[0058] Detection of anti-LYPD3 antibody binding to target cells: 1×10⁶ NCI-H2126 cells were used. 5 Add 20 μg / ml anti-LYPD3 antibody, resuspend and mix well. A blank control group and an IgG1 negative control group were also set up. Incubate at 37℃ for 30 min, resuspend and wash with PBS, centrifuge at 1200 rpm for 5 min and discard the supernatant. Add 100 μl of antibody incubation buffer, resuspend and mix well, add 2 μl of FITC anti-human IgG Fc antibody, incubate at room temperature for 20 min, resuspend and wash twice with PBS, centrifuge at 1200 rpm for 5 min and discard the supernatant, add 200 μl of PBS, resuspend and mix well, and detect antibody binding by flow cytometry.

[0059] (III) Experimental Results

[0060] We used flow cytometry to detect the expression of LYPD3 on the surface of the lung cancer cell line NCI-H2126. The results showed that the LYPD3 expression rate in NCI-H2126 cells was 97.9%, indicating that NCI-H2126 cells highly express LYPD3. Figure 4 We further examined the specific binding of anti-LYPD3 antibodies to NCI-H2126 cells. Flow cytometry revealed that among all LYPD3-labeled antibodies used on NCI-H2126 cells, antibody #22 exhibited the strongest specific binding, with a labeling rate of 88%. Figure 5 The above demonstrates that the anti-LYPD3#22 antibody can specifically bind to NCI-H2126 cells that highly express LYPD3.

[0061] Example 5

[0062] In vitro ADCC effect detection of anti-LYPD3 antibody

[0063] (I) Experimental Materials

[0064] Jurkat / NFAT-luc cells were purchased from ICON Biotech (Suzhou) Co., Ltd.; Bio-Glo TTM The luciferase assay reagent was purchased from Promega; the RPMI 1640, FBS, and Thermo Varioskan Flash microplate reader were purchased from Thermo Fisher Scientific; the IgG1 antibody was purchased from Southern Biotech; and PBS was purchased from Sangon Biotech Co., Ltd.

[0065] (II) Experimental Methods

[0066] Anti-LYPD3#22 antibody was serially diluted in RPMI 1640 medium at concentrations of 500 μg / ml, 300 μg / ml, and 3-fold dilutions starting from 300 μg / ml, for a total of 10 concentration gradients. IgG1 antibody was also serially diluted in RPMI 1640 medium, starting at 100 μg / ml and then 3-fold dilutions for a total of 8 concentration gradients. 25 μl of each serially diluted antibody was added to each well in a white, clear, flat-bottomed 96-well plate, with two replicates. A blank control group (without antibody) was also included. 25 μl of ADCC buffer (RPMI 1640 medium + 4% FBS) was added to each well. Jurkat / NFAT-luc effector cells and NCI-H2126 target cells were centrifuged at 1000 rpm for 5 min, resuspended in ADCC buffer, washed once, counted, and the cell density was adjusted. The NCI-H2126 cell density was adjusted to 5 x 10⁻⁶ cells / well. 5 Cells / ml, Jurkat / NFAT-luc density adjusted to 3 x 10-1 6 Cells / ml were gently pipetted between the effector cells (Jurkat / NFAT-luc) and target cells (NCI-H2126). 25 μl of cell suspension was added to each well of a 96-well plate containing the antibody (total volume 75 μl). The plates were incubated at 37°C with 5% CO2 for 6 hours. The substrate solution was equilibrated to room temperature beforehand. The plates were removed from the 37°C incubator and allowed to equilibrate at room temperature for 15 minutes. 25 μl of the detection substrate was added to each well, and the plates were incubated at room temperature for 5 minutes. Fluorescence was detected using the chemiluminescence module of a microplate reader. After background subtraction, the x-axis represents the logarithm of the antibody concentration, and the y-axis represents the fluorescence value. The EC50 was calculated using a curve fitted with GraphPadPrism 8 data analysis software. 50 .

[0067] (III) Experimental Results

[0068] Using NCI-H2126 cells as target cells and Jurkat / NFAT-luc cells as effector cells, we examined the antibody-mediated ADCC effect. We found that the anti-LYPD3#22 antibody had a significant ADCC effect on NCI-H2126 cells compared to the isotype control antibody IgG1, with EC50% higher activity. 50 =8.12nM( Figure 6 ).

[0069] Example 6

[0070] In vivo ADCC effect detection of anti-LYPD3 antibody

[0071] (I) Experimental Materials

[0072] hIgG1 isotype antibody (#BE0297) and mouse IgG1 isotype antibody (#BE0083) were purchased from Bioxcell, rIL2 (#GMP-CD66) was purchased from Suzhou Nearshore Protein Technology Co., Ltd., and NCG mice were purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.

[0073] (II) Experimental Methods

[0074] Female NCG mice with severe immunodeficiency, aged 6-8 weeks, were selected and subcutaneously injected with 1×10⁻⁶ NCG mice. 7 A mouse tumor model was constructed using NCI-H292 tumor cells, and the cells were grouped as follows: IgG1 group and anti-LYPD3 antibody group (n=7 in each group). The tumors were allowed to grow to 50-80 mm. 3 100 μg of control hIgG1 isotype or anti-LYPD3#22 antibody was injected via tail vein, and the antibody was resuspended in 1×10⁻⁶ solution. 7 300 μl HBSS cells containing 5 μg rIL2 and 5 μg mouse IgG1 isotype antibody were used in NK92 cells. Antibody treatment and tumor size measurement were performed every three days for a total of 7 treatments.

[0075] (III) Experimental Results

[0076] We first constructed an NCI-H292 NCG tumor-bearing mouse model, and waited for the tumor to grow to 50-80 mm. 3 In mice, anti-LYPD3 antibody and NK92 cells were adopted and reinfused, respectively, with IgG1 as a negative control. The ADCC effect was observed in vivo. Results showed that, compared with the isotype IgG1 control, anti-LYPD3 antibody significantly inhibited the growth of lung cancer cells in mice and exhibited a significant ADCC effect in vivo. Figure 7 ).

[0077] In summary, compared with the prior art, the present invention has the following advantages:

[0078] This invention develops a novel single-chain antibody sequence targeting LYPD3, which has a significant inhibitory effect on LYPD3-positive lung cancer cells, providing a new immunotherapy strategy for the clinical treatment of patients with lung cancer, breast cancer, renal cell carcinoma, liver cancer, colorectal cancer, etc.

Claims

1. An anti-LYPD3 antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain variable region having CDR-H1, CDR-H2 and CDR-H3 and a light chain variable region having CDR-L1, CDR-L2 and CDR-L3, wherein CDR-H1, CDR-H2 and CDR-H3 are composed of the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, and CDR-L1, CDR-L2 and CDR-L3 are composed of the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

2. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the antibody or antigen-binding fragment thereof comprises Fab, F(ab'), F(ab')2 or Fv.

3. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the antibody or antigen-binding fragment thereof comprises scFv.

4. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the isotype of the antibody or antigen-binding fragment thereof includes IgA, IgD, IgE, IgG or IgM.

5. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

2.

6. A nucleic acid that encodes an antibody or an antigen-binding fragment thereof as described in any one of claims 1-5.

7. A vector comprising the nucleic acid as described in claim 6.

8. Use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-5 in the preparation of a medicament for treating solid tumors, wherein the solid tumors include lung cancer, breast cancer, renal cell carcinoma, liver cancer, or colorectal cancer.

9. The use as claimed in claim 8, wherein the solid tumor includes non-small cell lung cancer.

10. Use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-5 in the preparation of a medicament for treating leukemia.

11. The use as claimed in claim 10, wherein the leukemia includes acute myeloid leukemia (AML).

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