Preparation and application of target LYPD3 chimeric antigen receptor T cell

Abstract

The invention discloses preparation and application of a targeting LYPD3 chimeric antigen receptor T cell. The chimeric antigen receptor comprises an LYPD3 antigen binding domain, a transmembrane domain, a costimulatory signal transduction domain and an intracellular signal transduction domain, the LYPD3 antigen binding domain comprises a heavy chain variable region with CDR-H1, CDR-H2 and CDR-H3 and a light chain variable region with CDR-L1, CDR-L2 and CDR-L3, and the CDR-H1, the CDR-H2 and the CDR-H3 are respectively composed of amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. And the CDR-L1, the CDR-L2 and the CDR-L3 are respectively composed of amino acid sequences as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to chimeric antigen receptor T cells targeting LYPD3. Background Art

[0002] Lung cancer is one of the most common malignant tumors globally and 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, becoming the number one cancer "killer" in China (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 extended the survival period of NSCLC patients, the prognosis is still very 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 tumor treatment and is also a research and development hotspot in current tumor treatment. CAR T therapy (chimeric antigen receptor T cell therapy) is one of the most promising tumor immunotherapies, achieving remarkable efficacy in the treatment of leukemia (Blood, 2021, 138(4): 318 - 330), and is also applied to the treatment of various solid tumors. However, due to the limitations of target selection, tumor heterogeneity, and patient individual differences, CAR T therapy shows low reactivity or non-reactivity in clinical patients; in addition, the lack of specific targets and the resulting off-target effects are also the main problems faced by current CAR T therapy. Therefore, targeting specific targets and developing highly efficient CAR T cells are of great significance for improving immune efficacy and reducing side effects.

[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] The preparation of high-performance CAR T effector cells targeting LYPD3 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 CAR T therapy, the present application provides a chimeric antigen receptor (CAR), which includes a LYPD3 antigen-binding domain, a transmembrane domain, a co-stimulatory signaling domain, and an intracellular signaling domain. The LYPD3 antigen-binding domain includes a heavy-chain variable region with CDR-H1, CDR-H2, and CDR-H3, and a light-chain variable region with CDR-L1, CDR-L2, and CDR-L3. 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 a CAR, the CAR comprising a LYPD3 antigen-binding domain, a transmembrane domain, a co-stimulatory signaling domain, and an intracellular signaling domain, wherein the LYPD3 antigen-binding domain 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 a CAR, the CAR comprising a LYPD3 antigen-binding domain, a transmembrane domain, a co-stimulatory signaling domain, and an intracellular signaling domain, wherein the LYPD3 antigen-binding domain 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] In another aspect, the present application also provides a cell comprising a vector or a CAR, the vector comprising a nucleic acid encoding the CAR, the CAR comprising a LYPD3 antigen-binding domain, a transmembrane domain, a co-stimulatory signaling domain, and an intracellular signaling domain, wherein the LYPD3 antigen-binding domain 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.

[0010] For the treatment regimen, the present application provides the use of a CAR or a cell in the preparation of a medicament for treating cancer, wherein the CAR comprises a LYPD3 antigen-binding domain, a transmembrane domain, a co-stimulatory signaling domain, and an intracellular signaling domain, wherein the LYPD3 antigen-binding domain 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; and the cell comprises a vector or the CAR, and the vector comprises a nucleic acid encoding the CAR. Brief Description of the Drawings

[0011] The present application will be described in more detail below with reference to the accompanying drawings, in which:

[0012] Figure 1 Electrophoresis diagram for the identification of anti-LYPD3 antibody;

[0013] Figure 2 Result diagram for detecting the antibody binding ability by ELISA method;

[0014] Figure 3 Result diagram for detecting the affinity of anti-LYPD3 #22 antibody (Biacore method);

[0015] Figure 4 Result diagram for detecting the expression of LYPD3 target cell antigen;

[0016] Figure 5 Result diagram for detecting the binding of anti-human LYPD3 antibody to target cells;

[0017] Figure 6 Schematic diagram of the chimeric antigen receptor gene targeting LYPD3;

[0018] Figure 7 Schematic diagram of the LYPD3-CAR lentiviral vector structure;

[0019] Figure 8 Result diagram for detecting the infection rate of LYPD3-CAR lentiviral T cells by flow cytometry;

[0020] Figure 9 Result diagram for detecting the in vitro killing effect of LYPD3-CAR T cells;

[0021] Figure 10This is a graph showing the in vivo anti-tumor detection results of LYPD3-CAR T cells. Detailed implementation mode

[0022] The present application relates to a chimeric antigen receptor (CAR), the CAR comprising a LYPD3 antigen-binding domain, a transmembrane domain, a co-stimulatory signaling domain, and an intracellular signaling domain, wherein the LYPD3 antigen-binding domain 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. In one embodiment, the LYPD3 antigen-binding domain comprises a Fab, F(ab’), F(ab’)2, Fv, or single-chain variable fragment (scFv). In a preferred embodiment, the LYPD3 antigen-binding domain comprises an scFv. 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 (QVS), 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 regions 1, 2, 3, and 4 of the heavy chain variable region consist 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 comprises framework regions (FR) 1, framework region 2, framework region 3, and framework region 4 separated by its 3 CDRs.In one embodiment, Framework Region 1, Framework Region 2, Framework Region 3, and Framework Region 4 of the light chain variable region comprise 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 consist 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 LYPD3 antigen-binding domain is humanized. In the present application, unless otherwise specified, the CDR sequences are defined according to the IMGT numbering scheme. In one embodiment, the transmembrane domain comprises a domain derived from the α, β, or γ chain of the T cell receptor. In a preferred embodiment, the transmembrane domain comprises the CD8α domain. In a preferred embodiment, the transmembrane domain comprises the human CD8α domain. In a preferred embodiment, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 40. In a preferred embodiment, the transmembrane domain consists of the amino acid sequence of SEQ ID NO: 40. In one embodiment, the intracellular signaling domain comprises the CD3ζ signaling domain. In a preferred embodiment, the intracellular signaling domain comprises the human CD3ζ intracellular signal peptide. In a preferred embodiment, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 44. In a preferred embodiment, the intracellular signaling domain consists of the amino acid sequence of SEQ ID NO: 44. In one embodiment, the co-stimulatory signaling domain comprises the intracellular signaling domain of one or more co-stimulatory molecules selected from the group consisting of: CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83. In a preferred embodiment, the co-stimulatory molecule comprises 4-1BB. In a preferred embodiment, the co-stimulatory molecule comprises human 4-1BB. In a preferred embodiment, the co-stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 42. In a preferred embodiment, the co-stimulatory signaling domain consists of the amino acid sequence of SEQ ID NO: 42. In one embodiment, the CAR comprises, from the N-terminus to the C-terminus in sequence: the LYPD3 antigen-binding domain, the transmembrane domain, the co-stimulatory signaling domain, and the intracellular signaling domain. In one embodiment, the CAR further comprises a signal peptide. In one embodiment, the signal peptide comprises the CD8α signal peptide. In one embodiment, the signal peptide comprises the human CD8α signal peptide.In a preferred embodiment, the signal peptide comprises the amino acid sequence of SEQ ID NO: 34. In a preferred embodiment, the signal peptide consists of the amino acid sequence of SEQ ID NO: 34. In one embodiment, the signal peptide is at the N-terminus of the LYPD3 antigen-binding domain. In one embodiment, the CAR further comprises a hinge region. In one embodiment, the hinge region comprises the CD8α hinge region. In one embodiment, the hinge region comprises the human CD8α hinge region. In a preferred embodiment, the hinge region comprises the amino acid sequence of SEQ ID NO: 38. In a preferred embodiment, the hinge region consists of the amino acid sequence of SEQ ID NO: 38. In one embodiment, the hinge region is between the LYPD3 antigen-binding domain and the transmembrane domain. In one embodiment, the LYPD3 antigen-binding domain comprises a heavy chain variable region and a light chain variable region, and the light chain variable region is at the N-terminus of the heavy chain variable region. In one embodiment, a hinge linker is included between the heavy chain variable region and the light chain variable region. In a preferred embodiment, the hinge linker comprises the amino acid sequence of SEQ ID NO: 36. In a preferred embodiment, the hinge linker consists of the amino acid sequence of SEQ ID NO: 36.

[0023] The present application also relates to a nucleic acid encoding a CAR, and the CAR can be the CAR of 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. In one embodiment, the nucleic acid comprises nucleotide sequences encoding a transmembrane domain, a co-stimulatory signaling domain and an intracellular signaling domain. In one embodiment, the nucleotide sequence encoding the transmembrane domain comprises SEQ ID NO: 39. In one embodiment, the nucleotide sequence encoding the intracellular signaling domain comprises SEQ ID NO: 43.In one embodiment, the nucleotide sequence encoding the co-stimulatory signaling domain comprises SEQ ID NO: 41. In one embodiment, the nucleic acid further comprises a nucleotide sequence encoding a signal peptide, a hinge region, and / or a hinge linker. In one embodiment, the nucleotide sequence encoding the signal peptide comprises SEQ ID NO: 33. In one embodiment, the nucleotide sequence encoding the hinge region comprises SEQ ID NO: 37. In one embodiment, the nucleotide sequence encoding the hinge linker comprises SEQ ID NO: 35.

[0024] This application also relates to a vector, which comprises a 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. In a preferred embodiment, the vector comprises a virus. In a more preferred embodiment, the vector comprises a lentivirus.

[0025] This application also relates to a cell, which contains a vector or a CAR, the vector can be the vector of any of the above embodiments, and the CAR can be the CAR of any of the above embodiments. In one embodiment, the cell comprises an immune effector cell. In a preferred embodiment, the cell comprises a T cell. In one embodiment, the immune effector cell is selected from one or more of the following group: αβ T cell, natural killer (NK) cell, natural killer T (NKT) cell, cytotoxic T lymphocyte (CTL), or any combination thereof. In one embodiment, the immune effector cell comprises a human immune effector cell.

[0026] This application also relates to the use of the CAR or the cell in the preparation of a drug for treating cancer, the CAR can be the CAR of any of the above embodiments, and the cell can be the cell of any of the above embodiments. In one embodiment, the cancer comprises a cancer expressing LYPD3. In one embodiment, the cancer comprises hematological malignancies and solid tumors. In one embodiment, the hematological malignancy comprises leukemia. In a preferred embodiment, the leukemia comprises AML. In a preferred embodiment, the solid tumor comprises lung cancer, breast cancer, renal cell carcinoma, liver cancer, and / or colorectal cancer. In a preferred embodiment, the solid tumor comprises lung cancer. In a more preferred embodiment, the lung cancer comprises non-small cell lung cancer.

[0027] Examples

[0028] This 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 this application. It should be pointed out that the spirit of this application and the protection scope of the claims are not limited by the following specific examples.

[0029] Example 1

[0030] Immunize mice with human LYPD3 protein, flow-sort LYPD3-specific memory B cells, and perform single-cell sequencing to obtain antibody sequences

[0031] (I) Experimental materials

[0032] 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 Sangon Biotech Co., Ltd., and FITC was purchased from Thermo Fisher Scientific (product number: 46410).

[0033] (II) Experimental methods

[0034] Mouse immunization: Select 6-8-week-old female BALB / c mice. Mix 50 μg of LYPD3 protein and complete Freund's adjuvant in equal volume for the first subcutaneous immunization; at 21 days and 42 days respectively, mix 50 μg of antigen and incomplete Freund's adjuvant in equal volume for the second and third immunizations; at 70 days, use 50 μg of antigen for the fourth immunization.

[0035] Sorting of LYPD3-specific memory B cells: 3 days later, decapitate the mice to death by cervical dislocation, take fresh spleens and lymph nodes, place them on a 70 μm sieve, grind the tissues, filter through a 70 μm sieve, resuspend and mix the cells and centrifuge. 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 to pass through the column to collect 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 times the volume of sorting buffer, mix well and centrifuge at 300 g for 10 min to discard the supernatant. Resuspend and mix with 500 μl of buffer and pass through the separation column for washing and separation to collect IgG1 +Cells, centrifuged at 300 g for 10 min, and the supernatant was discarded. Resuspend the cells with antibody incubation solution (PBS + 2% FBS) and adjust the cell density to 10 7 cells / ml. Take a small amount of cells as the control group. Add FITC-labeled LYPD3 antibody at 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 to 10 5 -10 6 cells / ml for flow sorting (BD AriaIII cell sorter).

[0036] (III) Experimental Results

[0037] We immunized mice with human LYPD3 protein as the immunogen. After the above four immunizations, 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 Genomics 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.

[0038] Example 2

[0039] Construction of VH and VL sequence vectors of anti-LYPD3 antibody and identification of antibody expression

[0040] (I) Experimental Materials

[0041] 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.

[0042] (II) Experimental Methods

[0043] Construction of VH and VL sequence vectors of LYPD3 antibody: The scFv genes of the light and heavy chains of the antibody were synthesized by Suzhou GeneCreate Biotechnology Co., Ltd. Subcloning was performed using the synthesized plasmid as a template to obtain PCR fragments, which were purified using a gel extraction kit. The PCR fragments were ligated into 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.

[0044] Expression of antibody in CHO-S cell system: Prepare reaction mixture A: 2 ml OptiPro-SFM + plasmid (50 μg each of light chain and heavy chain expression plasmids) and mixture B: 1.84 ml OptiPro-SFM + 160 μl ExpiFectamine TM CHO reagent, mix well by shaking, let stand at room temperature for 5 min, then add mixture B to mixture A, mix well by shaking, let stand at room temperature for 10 - 20 min, and slowly add 50 ml CHO-S cell system. Transfect for 18 - 22 h, then supplement 300 μl ExpiCHO TM Enhancer and 12 ml ExpiCHO TM Feed, culture at 37 °C, and after 7 days, collect the cell culture supernatant.

[0045] Antibody purification: Use the AKTA protein purification system for antibody purification. The specific method is as follows: Centrifuge or filter the cell culture supernatant to remove cell debris. Wash the protein G column with 1×PBS for 10 CV until the baseline is stable, load the cell supernatant containing the antibody onto AKTA, and re-equilibrate the column with PBS to wash away unbound impurities; elute the antibody on the column with 0.1 M glycine, pH 2.8, and collect it into a tube containing neutralizing solution (1 M Tris, pH 9.0) to neutralize the pH. The eluted antibody solution is centrifuged and replaced into PBS using an ultrafiltration tube.

[0046] (III) Experimental results

[0047] Synthesize the obtained heavy and light chain scFv sequence genes of the antibody, and construct them into VH and VL expression vectors by homologous recombination. Express the antibody using the CHO-S expression system and purify the antibody using the AKTA protein purification system. The purified antibody is detected for its purity and molecular weight by 10% polyacrylamide gel electrophoresis. Under completely reducing conditions, the anti-LYPD3 antibody shows two bands with molecular weights of approximately 55 kDa and 30 kDa, which are the heavy chain and light chain bands of the antibody respectively, and the purity is above 95% ( Figure 1 ). The above results indicate that we have successfully prepared and expressed a high-purity antibody targeting LYPD3.

[0048] Example 3

[0049] Determination of the affinity of anti-LYPD3 antibody

[0050] (I) Experimental materials

[0051] The antigen-coated 96-well plate 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, and the S series CM5 sensor chips were purchased from Cytiva.

[0052] (II) Experimental methods

[0053] ELISA method for detecting antibody affinity: Dilute the LYPD3 protein with PBS, coat a 96-well plate (100 ng / well), incubate overnight at 4 °C, wash the plate four times with PBST, block at room temperature with 3% BSA for 1 h, wash the plate four times with PBST, add 100 μl of the sample (test sample and control sample, 1 μg / ml) respectively, after incubating at room temperature for 1 h, wash the plate four times with PBST, add 100 μl of the secondary antibody, incubate at room temperature for 1 h, wash the plate four times with PBST, add 100 μl of TMB chromogenic solution, add 100 μl of the termination solution after color change, and detect the OD value at a single wavelength of 450 nm.

[0054] SPR method for detecting antibody affinity: Place the chip on a Biacore T200 (Cytiva) instrument, use the buffer HBSEP (10 mM HEPES, pH 7.5, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20), and the experimental temperature is 25 °C. Covalently link the antigen protein LYPD3 to the experimental channel by the method of amino coupling, serially dilute the antibody as the analyte and flow through the control channel and the experimental channel at a rate of 30 μl / min, the binding time is 120 s, the dissociation time is 400 s, and the regeneration buffer is Glycine 2.0. Use the Biacore T200 evaluation software 3.1 (Cytiva) to perform affinity (K D ) analysis, using the 1:1 binding mode.

[0055] (III) Experimental results

[0056] We first detected the binding of the antibody to the antigen by ELISA. The results showed that the anti-LYPD3 antibodies prepared and expressed by us could all bind to the 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 ).

[0057] Example 4

[0058] As Figure 4 、 5 shown, the binding of the anti-LYPD3 antibody to NCI-H2126 cells of the lung cancer cell line was detected by flow cytometry

[0059] (I) Experimental materials

[0060] The PE anti-human LYPD3 antibody, FITC anti-human IgG Fc antibody, and PE isotype antibody were purchased from Biolegend, and the IgG1 negative control antibody was purchased from Abcam. The antibody incubation solution was PBS + 2% FBS. The lung cancer cell line NCI-H2126 was purchased from Shanghai Fuheng Biotechnology Co., Ltd. (product number: FH0582).

[0061] (II) Experimental methods

[0062] Detection of LYPD3 membrane expression in NCI-H2126 cells: Take 1×10 5 NCI-H2126 cells, add 2 μl of the PE anti-human LYPD3 antibody and the isotype control antibody respectively, incubate in the dark at room temperature for 20 min, wash twice with PBS, add 200 μl of PBS to resuspend and mix the cells evenly, and detect the expression of the LYPD3 antigen by flow cytometry

[0063] Detection of the binding of the anti-LYPD3 antibody to target cells: Take 1×10 5 NCI-H2126 cells, add 20 μg / ml of the anti-LYPD3 antibody to resuspend and mix evenly. At the same time, set up a blank control group and an IgG1 negative control group, incubate at 37°C for 30 min, add PBS to resuspend and wash, centrifuge at 1200 rpm for 5 min to discard the supernatant; add 100 μl of the antibody incubation solution to resuspend and mix evenly, add 2 μl of the FITC anti-human IgG Fc antibody, incubate at room temperature for 20 min, add PBS to resuspend and wash twice, centrifuge at 1200 rpm for 5 min to discard the supernatant, add 200 μl of PBS to resuspend and mix evenly, and detect the antibody binding situation by flow cytometry

[0064] (III) Experimental results

[0065] We detected the expression of LYPD3 on the surface of NCI-H2126 cells of 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 the anti-LYPD3 antibody to NCI-H2126 cells. Flow cytometry found that among all the LYPD3-labeled antibodies of NCI-H2126 cells, the #22 antibody had the strongest specific binding, and the labeling rate was 88%( Figure 5) The above description indicates that the anti-LYPD3#22 antibody can specifically bind to NCI-H2126 cells with high expression of LYPD3. We selected the scFv of the anti-LYPD3#22 antibody for the subsequent design of CAR, and NCI-H2126 cells can be used as target cells for the subsequent CAR T research and development.

[0066] Example 5

[0067] As Figure 6 、 7 、shown in Figure 8, construction of the CAR lentiviral expression vector based on the anti-LYPD3#22 antibody scFv, lentivirus packaging, preparation, and preparation of LYPD3-CAR T cells

[0068] (I) Experimental materials

[0069] DMEM and Opti-MEM were purchased from Gibco. Polybrene was purchased from Yeasen Biotechnology (Shanghai) Co., Ltd. The WPRE primer was synthesized by Suzhou Anshengda Co., Ltd. The genomic DNA extraction kit was purchased from Nanjing Novozymes Biotech Co., Ltd. Lymphocyte separation medium was purchased from GE HealthCare. The magnetic stand, sorting magnetic beads, sorting column, sorting buffer, and human T cell activation beads were purchased from Miltenyi. The T cell medium X-VIVO 15 was purchased from Lonza. IL-2 was purchased from Peprotech. Retronectin was purchased from Takara. FBS was purchased from Gibco. FITC-labeled PL was purchased from ACRO.

[0070] (II) Experimental methods

[0071] Construction, packaging, and preparation of the CAR lentiviral expression vector for LYPD3: The CAR was successively connected with the following structures (see Figure 6 ): human CD8α signal peptide, antibody scFv targeting LYPD3, human CD8α hinge region (CD8α Hinge), human CD8α transmembrane region (CD8αTM), human co-stimulatory factor 4-1BB, and human CD3ζ intracellular signal peptide (CD3ζsignal). The single-chain antibody sequence of LYPD3 was derived from the VH and VL of the anti-LYPD3#22 antibody, and the remaining sequences were obtained by searching the NCBI website database. The CAR gene sequence was synthesized by Shanghai Heyuan Biotechnology Co., Ltd., and the lentiviral expression vector LV-CAR-LYPD3 (Heyuan Biotechnology (Shanghai) Co., Ltd., product number: HYKY-230630007-DLV) was constructed and subjected to lentivirus packaging and preparation (see Figure 7 ).

[0072] Preparation of LYPD3-CAR T cells: Peripheral blood mononuclear cells (PBMC) were isolated from healthy human volunteers. CD3 T cells were obtained using CD3 sorting magnetic beads. + The cells were cultured with IL-2 (10 ng / ml), and human T cell activation beads were used to activate the T cells. After 24 hours of activation, the cells were seeded into 24-well plates at a density of 5×10 5 cells / well. Lentiviral solutions of NC-CAR and LYPD3-CAR (MOI = 20) were added respectively, and then the infection-promoting reagent polybrene (8 μg / ml) was added. After resuspending and mixing well, the cells were centrifuged at 1000 g for 30 minutes and then placed in an incubator for amplification culture. Four days after viral infection, 1×10 6 cells of NT (control T cell group), NC-CAR T, and LYPD3-CAR T were taken, resuspended and washed twice with PBS, centrifuged to discard the supernatant, resuspended in 100 μl of PBS with 5 μl of FITC-labeled protein L, mixed well and incubated in the dark for 20 minutes. The positive expression rate of CAR recognized on the surface of CAR T cells was detected using a flow cytometer.

[0073] (III) Experimental results

[0074] We constructed a lentiviral expression vector of LYPD3-CAR targeting LYPD3 based on the sequence of anti-LYPD3#22 antibody scFv, and used the 293T / 17 cell system for lentivirus packaging and preparation. The obtained virus titer was 5E+08 TU / ml. The prepared LYPD3-CAR and control NC-CAR lentiviruses were used to infect activated CD3 + T cells (MOI = 20) respectively, and then amplified. Four days later, flow cytometry detection showed that the infection rate of T cells with LYPD3-CAR lentivirus was 52.1% (see Figure 8 ), indicating that we successfully prepared LYPD3-CAR T cells.

[0075] Example 6

[0076] As Figure 9 shown, detection of the in vitro killing of NCI-H2126 cells by LYPD3-CAR T cells

[0077] (I) Experimental materials

[0078] 1640 was purchased from Gibco, and the LDH detection kit was purchased from Promega. Killing medium: 1640 + 4% FBS.

[0079] (II) Experimental methods

[0080] The cell density of LYPD3-CAR T cells was adjusted to 2×10 6cells / ml (10:1 group), and were serially diluted in killing medium at ratios of 10:1, 5:1, and 2.5:1 to serve as effector cells, with NC-CAR T cells as the control; NCI-H2126 cells were adjusted to a cell density of 2×10 5 cells / ml as target cells. 50 μl of effector cells and target cells were respectively placed in a 96-well plate for co-culture. Each group had three replicates (E:T ratios were 10:1, 5:1, and 2.5:1). Control groups were set up: spontaneous release of target cells (50 μl of target cells + 50 μl of killing medium), maximum release of target cells (50 μl of target cells + 50 μl of killing medium), spontaneous release of effector cells (50 μl of effector cells + 50 μl of killing medium), background (100 μl of killing medium), and volume correction control group (100 μl of killing medium); the 96-well plate was sealed with a sealing film, centrifuged at 300 g for 5 min in a horizontal centrifuge, and then cultured in a 37 °C, 5% CO2 incubator for 16 h; 10 μl of 10X lysis solution was added to each well of the maximum release group of target cells and the volume correction control group, and the 96-well plate was placed back in the incubator and cultured for another 45 min, then sealed with a sealing film and centrifuged horizontally at 250 g for 5 min; a new 96-well plate was prepared, and 50 μl of the cell killing supernatant after the above centrifugation was added to each well, and at the same time, 50 μl of LDH detection substrate solution was added, and incubated in the dark at room temperature for 20 min; 50 μl of stop solution was added to each well, and the absorbance at 492 nm was measured with an ELISA reader.

[0081] Result statistics: The absorbance values of all experimental groups, the spontaneous release group of effector cells, and the spontaneous release group of target cells should be subtracted by the average absorbance value of the background; the absorbance value of the maximum release group of target cells should be subtracted by the average absorbance value of the volume correction control group; the corrected values were used for the statistics of the killing rate: Cell killing rate (%) = [(release of experimental group - spontaneous release of effector cells - spontaneous release of target cells) / (maximum release of target cells - spontaneous release of target cells)]×100%.

[0082] (III) Experimental results

[0083] To detect the killing effect of LYPD3-CAR T cells on target cells, we co-cultured LYPD3-CAR T cells with NCI-H2126 cells at different effector-to-target ratios for 16 hours. NC-CAR T cells were used as negative control CAR T cells, and the killing rate of CAR T cells on target cells was detected by the LDH method. The results showed that the control CAR T cells had no killing effect on target cells, while LYPD3-CAR T cells had a significant killing effect on target cells NCI-H2126: when the effector-to-target ratio was 2.5:1, the killing rate was 25.58%; when the effector-to-target ratio was 5:1, the killing rate was 26.38%; when the effector-to-target ratio was 10:1, the killing rate was 37.15% (see Figure 9) The above results indicate that the LYPD3-CAR T cells we prepared can significantly kill NCI-H2126 cells in vitro.

[0084] Example 7

[0085] As Figure 10 shown, a mouse lung cancer tumor model was constructed to detect the in vivo anti-tumor effect of LYPD3-CAR T cells

[0086] (I) Experimental materials

[0087] Severely immunodeficient B-NDG mice were purchased from Beijing Biocytogen Co., Ltd.

[0088] (II) Experimental methods

[0089] Establishment of mouse lung cancer model: Female B-NDG mice at 6-8 weeks old were subcutaneously injected with NCI-H2126 cells (1×10 7 cells / mouse) to construct a subcutaneous tumor model of mouse lung cancer. Detection of in vivo anti-tumor effect of LYPD3-CAR T cells: When the tumor volume reached about 100 mm 3 3, the prepared LYPD3-CAR T cells and NC-CAR T cells were respectively injected into the tail veins of mice (4×10 6 cells / mouse, and the second infusion was performed 4 days later, n = 4); after infusion, the length and width of the tumor mass were measured with a vernier caliper every 2-3 days. The tumor volume was evaluated according to V = 1 / 2AB 2 (V = tumor volume; A = tumor length; B = tumor width), and the results were plotted into a graph. When the maximum diameter of the tumor reached 1-2 cm, the mice were sacrificed to obtain the tumor, and the in vivo anti-tumor effect of CAR-T cells was evaluated.

[0090] (III) Experimental results

[0091] To further detect the in vivo anti-tumor effect of LYPD3-CAR T cells, we constructed a B-NDG mouse model with subcutaneous tumors of NCI-H2126 cells. When the tumor volume reached about 100 mm 3 3, LYPD3-CAR T cells and control CAR T cells were adoptively transferred respectively, and the tumor growth was measured. We found that the control CAR T cells had no tumor inhibitory effect, and LYPD3-CAR T cells could significantly inhibit the in vivo tumor growth of lung cancer cells (see Figure 10 ). This indicates that adoptive transfer of LYPD3-CAR T cells can effectively inhibit the growth of lung cancer cells in vivo.

[0092] In summary, compared with the prior art, the present invention has the following advantages:

[0093] The present invention provides a LYPD3 scFv nucleotide sequence, a vector, a host cell for treating pulmonary malignant tumors and their application in anti-tumor. Through the design of the CAR structure, a novel CAR T cell is prepared, which has a significant killing effect on tumor cells and inhibits the growth of tumors in vivo, providing an effective strategy for lung cancer immunotherapy.

Claims

1. A chimeric antigen receptor (CAR), comprising a LYPD3 antigen binding domain, a transmembrane domain, a co-stimulatory signaling domain, and an intracellular signaling domain, wherein the LYPD3 antigen binding domain 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 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 CAR of claim 1, wherein: The antibody or antigen-binding fragment thereof or the LYPD3 antigen-binding domain comprises Fab, F(ab'), F(ab')2, Fv or single-chain variable fragment (scFv), preferably scFv; and / or The isotype of the antibody or antigen-binding fragment thereof includes IgA, IgD, IgE, IgG or IgM.

3. The CAR 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.

4. The CAR of any one of claims 1 to 3, wherein: The transmembrane domain includes a domain derived from the α, β or γ chain of a T cell receptor, preferably a CD8 α domain; The intracellular signaling domain comprises a CD3ζ signaling domain, preferably a human CD3ζ intracellular signal peptide; and / or The co-stimulatory signaling domain comprises an intracellular signaling domain of one or more co-stimulatory molecules selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83, preferably 4-1BB.

5. A nucleic acid encoding the CAR according to any one of claims 1 to 4. A vector comprising the nucleic acid according to claim 5 .

7. A cell comprising the vector of claim 6 or the CAR of any one of claims 1-4.

8. The vector of claim 6 or the cell of claim 7, wherein: The vector comprises one or more selected from the group consisting of: a plasmid, a phagemid, a phage or a derivative thereof, a virus and a cosmid, preferably a virus, more preferably a lentivirus; and / or The cells include immune effector cells, such as T cells, and optionally, the immune effector cells include one or more selected from the group consisting of αβT cells, natural killer (NK) cells, natural killer T (NKT) cells, cytotoxic T lymphocytes (CTLs), or any combination thereof.

9. Use of the CAR as described in any one of claims 1-4 or the cell as described in claim 7 or 8 in the preparation of a medicament for treating cancer.

10. The use of claim 9, wherein the cancer comprises a cancer expressing LYPD3, preferably comprises a hematological malignancy and a solid tumor, for example, the hematological malignancy comprises a leukemia, more preferably, the leukemia comprises acute myeloid leukemia (AML), and preferably, the solid tumor comprises lung cancer, breast cancer, renal cell carcinoma, liver cancer and / or colorectal cancer, more preferably, the solid tumor comprises lung cancer, for example, non-small cell lung cancer.

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