Preparation of targeting LYPD3 antibody and application of targeting LYPD3 antibody in tumor immunotherapy

Abstract

The invention discloses preparation of a targeting LYPD3 antibody and application of the targeting LYPD3 antibody in tumor immunotherapy. The antibody or antigen binding fragment thereof 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 and SEQ ID NO: 8, respectively.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, specifically relates to pharmaceutical products of immunoglobulins, and more specifically relates to anti-LYPD3 antibodies. Background Art

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

[0003] In recent years, immunotherapy has achieved remarkable efficacy in tumor treatment and is also a current research and development hotspot in tumor treatment. Antibody immunotherapy, especially the research and development of monoclonal antibody (mAb) drugs, has become the focus in the current field of tumor treatment (Thorax, 2022, 77(12): 1163-1174). However, due to the limitations in target selection, tumor heterogeneity, and the low or non-responsiveness of antibody immunotherapy in clinical patients, the wide application of this therapy has been greatly restricted; in addition, due to the lack of specific targets, antibodies are prone to off-target effects during the treatment process, which not only reduces the treatment effect but may also trigger a series of adverse reactions, bringing toxic side effects to patients. Therefore, precisely targeting specific targets and developing highly effective antibodies have become the key to breaking through the bottleneck of antibody immunotherapy, which not only helps to significantly improve the efficacy of immunotherapy but also effectively reduces toxic side effects, providing a safer and more effective treatment plan for tumor 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 is involved in the occurrence, development, and progression of tumors and can be used as a potential tumor biomarker. In particular, it has been 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 can be used as a biomarker for lung cancer prognosis and immunotherapy (Transl Cancer Res, 2024, 13(3): 1394-1405). Therefore, targeting LYPD3 can be a potential strategy for lung cancer immunotherapy.

[0005] Targeting LYPD3 to screen for novel high-affinity antibodies has important application prospects and significance in the immunotherapy of tumors such as lung cancer and breast cancer. Summary of the Invention

[0006] To solve the above technical problems, in one aspect, for anti-LYPD3 antibodies, the present application provides an anti-LYPD3 antibody or its antigen-binding fragment. 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 respectively include 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 include the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7 (QVS), and SEQ ID NO: 8.

[0007] In another aspect, the present application also provides a nucleic acid encoding an anti-LYPD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof 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.

[0008] In another aspect, the present application also provides a vector comprising a nucleic acid encoding an anti-LYPD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof 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.

[0009] Regarding the treatment regimen, the present application provides the use of an anti-LYPD3 antibody or an antigen-binding fragment thereof in the preparation of a medicament for treating cancer, wherein the antibody or the antigen-binding fragment thereof 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. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0011] Figure 1 Electrophoresis diagram identified for anti-LYPD3 antibody;

[0012] Figure 2Result diagram of detecting antibody binding capacity by ELISA method;

[0013] Figure 3 Result diagram of detecting the affinity of anti-LYPD3#22 antibody (Biacore method);

[0014] Figure 4 Result diagram of detecting the antigen expression of LYPD3 target cells;

[0015] Figure 5 Result diagram of detecting the binding of anti-human LYPD3 antibody to target cells;

[0016] Figure 6 Analysis diagram of in vitro ADCC effect induced by anti-LYPD3 antibody;

[0017] Figure 7 Analysis diagram of in vivo ADCC effect induced by anti-LYPD3 antibody. Detailed implementation method

[0018] This application relates to 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 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 antigen-binding fragment thereof comprises Fab, F(ab’), F(ab’)2, Fv or single-chain variable fragment (scFv). In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises scFv. In one embodiment, the isotype of the antibody or antigen-binding fragment thereof comprises IgA, IgD, IgE, IgG or IgM. In a preferred embodiment, the isotype of the antibody or antigen-binding fragment thereof comprises IgG. In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a subclass of IgG isotype, IgG1, IgG2, IgG3 or IgG4. In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises subclass IgG1. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1. In a preferred embodiment, the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 2. In a preferred embodiment, the light chain variable region consists of the amino acid sequence of SEQ ID NO: 2. In a preferred embodiment, CDR-H1, CDR-H2 and CDR-H3 consist 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 consist 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 comprises framework regions (FR) 1, framework region 2, framework region 3 and framework region 4 separated by its 3 CDRs. In one embodiment, framework regions 1, 2, 3 and 4 of the heavy chain variable region comprise the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 respectively.In a preferred embodiment, Framework Region 1, Framework Region 2, Framework Region 3, and Framework Region 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 Framework Region (FR) 1, Framework Region 2, Framework Region 3, and Framework Region 4 separated by its three CDRs. In one embodiment, Framework Region 1, Framework Region 2, Framework Region 3, and Framework Region 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 a preferred embodiment, Framework Region 1, Framework Region 2, Framework Region 3, and Framework Region 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 specified, the CDR sequences are defined according to the IMGT numbering scheme.

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

[0020] The present application also relates to a vector, which comprises nucleic acid, and the nucleic acid can be the nucleic acid of any of the above embodiments. In one embodiment, the vector comprises one or more selected from the group consisting of: plasmid, phagemid, phage or its derivative, virus and cosmid.

[0021] The present application also relates to the use of an anti-LYPD3 antibody or an antigen-binding fragment thereof in the preparation of a medicament for treating cancer, and the antibody or the antigen-binding fragment thereof can be the antibody or the antigen-binding fragment thereof in any of the above embodiments. In one embodiment, the cancer includes cancers expressing LYPD3. In one embodiment, the cancer includes hematological malignancies and solid tumors. In one embodiment, the hematological malignancy includes 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] Examples

[0023] The present application will be described in detail by the following exemplary specific examples. The following examples are only used to help those skilled in the art better understand various inventions of the present application. It should be pointed out that the spirit of the present application and the protection scope of the claims are not limited by the following specific examples.

[0024] Example 1

[0025] Immunize mice with human LYPD3 protein, flow-sort LYPD3-specific memory B cells and perform single-cell sequencing to obtain antibody sequences

[0026] (I) Experimental materials

[0027] BALB / c mice were purchased from Jiangsu Jicui Yakang 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. (product number: UA010207), the 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 Shengong Biotech Co., Ltd., and FITC was purchased from Thermo Fisher Scientific (product number: 46410).

[0028] (II) Experimental methods

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

[0030] Sorting of LYPD3 - specific memory B cells: Three days later, the mice were sacrificed by cervical dislocation. Fresh spleens and lymph nodes were taken, placed on a 70 - μm sieve, ground the tissues, filtered through a 70 - μm sieve, resuspended and mixed the cells and centrifuged. Resuspend with PBS and add sorting buffer. Add 100 μl of memory B cell biotin - antibody mixture, 10 μl of anti - IgG1 - APC, and 50 μl of sorting buffer to every 10 8 cells, 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 300 g for 10 min and discard the supernatant. Add 500 μl of buffer and pass through the column to collect the negative cells. Centrifuge the collected cells at 300 g for 10 min, discard the supernatant, then add 400 μl of buffer and 100 μl of anti - APC MicroBeads, incubate at 4°C for 15 min, add 10 - fold volume of sorting buffer, mix well and centrifuge at 300 g for 10 min to discard the supernatant. Resuspend with 500 μl of buffer, mix well and pass through the separation column for washing and separation, collect the IgG1 + cells, centrifuge at 300 g for 10 min and discard the supernatant. Resuspend with the antibody incubation solution (PBS + 2% FBS) to adjust the cell density to 10 7 cells / ml, and take a small amount of cells as the control group. Add FITC - labeled LYPD3 antibody according to a concentration of 2 μg / ml, incubate at 4°C for 20 min, wash twice with PBS, centrifuge at 1500 rpm for 5 min and discard the supernatant. Adjust the cell density with PBS to 10 5 - 10 6 cells / ml for flow sorting (BD AriaIII cell sorter).

[0031] (III) Experimental results

[0032] We immunized mice with human LYPD3 protein as the immunogen. After the above four - time immunization, the spleens and lymph nodes of the mice were isolated; through flow sorting, LYPD3 - specific IgG1 + LYPD3 + memory B cells were obtained. We commissioned Shanghai Jingneng Biotechnology Co., Ltd. to perform single - B - cell 10x Genomics BCR gene sequencing. According to 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 of 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 GeneCreate Biotechnology Co., Ltd. The gel extraction kit was purchased from Takara. The homologous recombination enzyme was purchased from Nanjing Novoprotein Scientific Co., Ltd. The expiCHO-S cell line and transfection reagent were purchased from Thermo Fisher Scientific. The protein G column was purchased from GE.

[0037] (II) Experimental methods

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

[0039] Expression of antibody in CHO-S cell system: Prepare reaction mixture A: 2 ml OptiPro-SFM + plasmid (50 μg each of light and heavy chain expression plasmids) and mixture B: 1.84 ml OptiPro-SFM + 160 μl ExpiFectamine TM CHO reagent, shake well and let stand at room temperature for 5 min. Then add mixture B to mixture A, shake well and let stand at room temperature for 10 - 20 min, and slowly add to 50 ml CHO-S cell system. Transfect for 18 - 22 h, add 300 μl ExpiCHO TTM Enhancer, 12 ml ExpiCHO TM Feed, culture at 37°C, and after 7 days, collect the cell culture supernatant.

[0040] Antibody purification: The antibody was purified using an AKTA protein purification system. The specific method was as follows: The cell culture supernatant was centrifuged or filtered to remove cell debris. The protein G column was rinsed with 1×PBS for 10 column volumes (CV) until the baseline was stable. The cell supernatant containing the antibody was loaded onto the AKTA, and the column was re-equilibrated with PBS to wash away unbound impurities. The antibody on the column was eluted with 0.1 M glycine, pH 2.8, and collected into a tube containing a neutralizing solution (1 M Tris, pH 9.0) to neutralize the pH. The eluted antibody solution was centrifuged using an ultrafiltration tube to exchange it into PBS.

[0041] (III) Experimental results

[0042] The heavy and light chain scFv sequence genes of the obtained antibody were synthesized and constructed into VH and VL expression vectors by homologous recombination. The antibody was expressed using the CHO-S expression system and purified using the AKTA protein purification system. The purified antibody was detected for its purity and molecular weight by 10% polyacrylamide gel electrophoresis. Under fully reduced conditions, the anti-LYPD3 antibody showed two bands with molecular weights of approximately 55 kDa and 30 kDa, which were the heavy and light chain bands of the antibody, respectively, and the purity was above 95% ( Figure 1 ). The above results indicate that we successfully prepared and expressed a high-purity antibody targeting LYPD3.

[0043] Example 3

[0044] Determination of the affinity of the anti-LYPD3 antibody

[0045] (I) Experimental materials

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

[0047] (II) Experimental methods

[0048] ELISA method for detecting antibody affinity: The LYPD3 protein was diluted with PBS and used to coat a 96-well plate (100 ng / well), incubated overnight at 4°C. The plate was washed four times with PBST, blocked with 3% BSA at room temperature for 1 h, and then washed four times with PBST. 100 μl of samples (test samples and control samples, 1 μg / ml) were added respectively, incubated at room temperature for 1 h, followed by washing the plate four times with PBST. 100 μl of the secondary antibody was added and incubated at room temperature for 1 h. After washing the plate four times with PBST, 100 μl of TMB chromogenic solution was added. After color change, 100 μl of termination solution was added, and the OD value was detected at a single wavelength of 450 nm.

[0049] SPR method for detecting antibody affinity: Place the chip on a Biacore T200 (Cytiva) instrument, and use buffer HBSEP (10 mM HEPES, pH 7.5, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20). The experimental temperature is 25 °C. Covalently link the antigen protein LYPD3 to the experimental channel by the method of amino coupling. Dilute the antibody in a serial dilution as the analyte and flow it through the control channel and the experimental channel at a rate of 30 μl / min. The binding time is 120 seconds and the dissociation time is 400 seconds. The regeneration buffer is Glycine 2.0. Use Biacore T200 evaluation software 3.1 (Cytiva) for affinity (K D ) analysis, using the 1:1 binding mode.

[0050] (III) Experimental results

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

[0052] Example 4

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

[0054] (I) Experimental materials

[0055] PE anti-human LYPD3 antibody, FITC anti-human IgG Fc antibody, and PE isotype antibody were purchased from Biolegend. The IgG1 negative control antibody was purchased from Abcam. Antibody incubation solution: PBS + 2% FBS. The lung cancer cell line NCI-H2126 was purchased from Shanghai Fuheng Biotechnology Co., Ltd. (product number: FH0582).

[0056] (II) Experimental methods

[0057] Detection of LYPD3 membrane expression in NCI-H2126 cells: Take 1×10 5For NCI-H2126 cells, 2 μl of PE anti-human LYPD3 antibody and isotype control antibody were added respectively. After incubation in the dark at room temperature for 20 min, the cells were washed twice with PBS, resuspended in 200 μl of PBS and mixed well, and the expression of LYPD3 antigen was detected by flow cytometry.

[0058] Detection of the binding of anti-LYPD3 antibody to target cells: Take 1×10 5 of NCI-H2126 cells, resuspend and mix well with 20 μg / ml anti-LYPD3 antibody. At the same time, a blank control group and an IgG1 negative control group were set up. Incubate at 37 °C for 30 min, add PBS to resuspend and wash, centrifuge at 1200 rpm for 5 min and discard the supernatant; add 100 μl of antibody incubation solution to resuspend and mix well, add 2 μl of FITC anti-human IgG Fc antibody and incubate at room temperature for 20 min, add PBS to resuspend and wash twice, centrifuge at 1200 rpm for 5 min and discard the supernatant, add 200 μl of PBS to resuspend and mix well, and detect the antibody binding situation by flow cytometry.

[0059] (III) Experimental results

[0060] We detected the expression of LYPD3 on the surface of NCI-H2126 cells in the lung cancer cell line by flow cytometry. The results showed that the expression rate of LYPD3 in NCI-H2126 cells was 97.9%, indicating that NCI-H2126 cells highly expressed LYPD3 ( Figure 4 ). We further detected the specific binding of anti-LYPD3 antibody to NCI-H2126 cells. Flow cytometry found that among all LYPD3-labeled antibodies in NCI-H2126 cells, the #22 antibody had the strongest specific binding, and the labeling rate was 88% ( Figure 5 ). The above indicates that anti-LYPD3 #22 antibody can specifically bind to NCI-H2126 cells with high expression of LYPD3.

[0061] Example 5

[0062] Detection of the in vitro ADCC effect of anti-LYPD3 antibody

[0063] (I) Experimental materials

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

[0065] (II) Experimental methods

[0066] The anti-LYPD3#22 antibody was serially diluted with RPMI 1640 medium at concentrations of 500 μg / ml, 300 μg / ml, and then serially diluted 3-fold starting from 300 μg / ml, for a total of 10 concentration gradients. The IgG1 antibody was serially diluted with RPMI 1640 medium starting from an initial concentration of 100 μg / ml and serially diluted 3-fold, for a total of 8 concentration gradients. 25 μl of the serially diluted antibody was added successively to a white transparent flat-bottom 96-well plate, with 2 replicates. A blank group without antibody was set up, and 25 μl of ADCC buffer (RPMI 1640 medium + 4% FBS) was added; after centrifuging the Jurkat / NFAT-luc effector cells and NCI-H2126 target cells at 1000 rpm for 5 min, they were resuspended and washed once with ADCC buffer, then counted and the cell density was adjusted. The density of NCI-H2126 was adjusted to 5×10 5 cells / ml, and the density of Jurkat / NFAT-luc was adjusted to 3×10 6 cells / ml. The effector cell Jurkat / NFAT-luc and target cell NCI-H2126 cell suspensions were gently pipetted, and 25 μl of the cell suspension (total volume 75 μl) was added to each of the above 96-well plates containing the antibody; the plates were incubated in an incubator at 37 °C and 5% CO2 for 6 h; the substrate solution was pre-equilibrated to room temperature in advance, the detection plate was taken out of the 37 °C incubator and placed at room temperature for 15 min to equilibrate, and 25 μl of the detection substrate was added to each well of the detection plate and incubated at room temperature for 5 min. The fluorescence was detected using the chemiluminescence module of an ELISA reader. After subtracting the background value, the logarithm of the antibody concentration was plotted on the x-axis and the fluorescence value on the y-axis, and the curve was fitted using GraphPad Prism 8 data analysis software to calculate the EC 50 .

[0067] (III) Experimental results

[0068] Using NCI-H2126 cells as target cells and Jurkat / NFAT-luc as effector cells, we detected the antibody-mediated ADCC effect. It was found that compared with the isotype control antibody IgG1, the anti-LYPD3#22 antibody had a significant ADCC effect on NCI-H2126 cells, with an EC 50 = 8.12 nM ( Figure 6 ).

[0069] Example 6

[0070] Detection of the in vivo ADCC effect of anti-LYPD3 antibody

[0071] (I) Experimental materials

[0072] hIgG1 isotype antibody (#BE0297) and murine IgG1 isotype antibody (#BE0083) were purchased from Bioxcell, and rIL2 (#GMP-CD66) was purchased from Novoprotein Scientific Inc., Suzhou. NCG mice were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.

[0073] (II) Experimental methods

[0074] Six- to eight-week-old severely immunodeficient female NCG mice were selected and subcutaneously injected with 1×10 7 NCI-H292 tumor cells to establish a mouse tumor model, and were grouped as follows: IgG1 group and anti-LYPD3 antibody group (n = 7 for each group). When the tumors grew to 50-80 mm 3 , 100 μg of control hIgG1 isotype or anti-LYPD3 #22 antibody were injected via the tail vein respectively. The antibody was resuspended in 1×10 7 NK92 cells (300 μl HBSS containing 5 μg rIL2 and 5 μg murine IgG1 isotype antibody). Antibody treatment and tumor size measurement were performed every three days for a total of 7 times.

[0075] (III) Experimental results

[0076] We first established an NCI-H292 NCG tumor-bearing mouse model. When the tumors grew to about 50-80 mm 3 , anti-LYPD3 antibody and NK92 cells were adoptively transferred respectively, with IgG1 as a negative control, and the in vivo ADCC effect was observed. The results showed that compared with the isotype IgG1 control, the anti-LYPD3 antibody could significantly inhibit the growth of lung cancer cells in mice and had a significant in vivo ADCC effect ( Figure 7 ).

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

[0078] The present invention has developed a new single-chain antibody sequence targeting LYPD3, which has a significant inhibitory effect on LYPD3-positive lung cancer cells, and provides 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, 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 consist 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 consist 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 according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises Fab, F(ab'), F(ab')2, Fv or single-chain variable fragment (scFv), preferably scFv.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the isotype of the antibody or antigen-binding fragment thereof comprises IgA, IgD, IgE, IgG or IgM. 4 . The antibody or antigen-binding fragment thereof of 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.

5. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4. A vector comprising the nucleic acid according to claim 5 .

7. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in the preparation of a medicament for treating cancer.

8. The use of claim 7, wherein the cancer comprises a cancer expressing LYPD3.

9. The use according to claim 7, wherein the cancer comprises hematological malignancies and solid tumors.

10. The use of claim 9, wherein the hematological malignancy comprises leukemia, preferably, the leukemia comprises acute myeloid leukemia (AML); and / or the solid tumor comprises lung cancer, breast cancer, renal cell carcinoma, liver cancer and / or colorectal cancer, preferably, the solid tumor comprises lung cancer, e.g., non-small cell lung cancer.

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