Preparation and application of lypd3 chimeric antigen receptor t cells

Abstract

The application discloses a preparation method and application of a LYPD3 chimeric antigen receptor T cell. The chimeric antigen receptor comprises an LYPD3 antigen binding domain, a transmembrane domain, a costimulatory signaling domain and an intracellular signaling domain, wherein 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, wherein CDR-H1, CDR-H2 and CDR-H3 are respectively composed of 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 are respectively composed of the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically involving T cells that target LYPD3 chimeric antigen receptor. 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), in particular, ranks first in both incidence and mortality among malignant tumors, becoming the country's leading cancer killer (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. CAR T therapy (chimeric antigen receptor T cell therapy) is one of the most promising cancer immunotherapies, achieving remarkable efficacy in the treatment of leukemia (Blood, 2021, 138(4): 318-330), and has also been applied to the treatment of various solid tumors. However, due to limitations in target selection, tumor heterogeneity, and individual patient differences, CAR T therapy has low or no response in clinical patients; in addition, the lack of specific targets and the resulting off-target effects are also major problems currently facing CAR T therapy. Therefore, targeting specific targets and developing high-efficiency CAR T cells is of great significance for improving immunotherapy efficacy and reducing toxic 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 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] The preparation of high-efficiency 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 address the aforementioned technical problems, and in one aspect of CAR T therapy, this application provides a chimeric antigen receptor (CAR), the CAR comprising a LYPD3 antigen-binding domain, a transmembrane domain, a co-stimulatory signal transduction domain, and an intracellular signal transduction 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.

[0007] In another aspect, this application also provides a nucleic acid encoding a CAR, the CAR comprising a LYPD3 antigen-binding domain, a transmembrane domain, a co-stimulatory signal transduction domain, and an intracellular signal transduction 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.

[0008] In another aspect, this 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 signal transduction domain, and an intracellular signal transduction 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.

[0009] In another aspect, this application also provides a cell comprising a vector or CAR, the vector comprising nucleic acid encoding the CAR, the CAR comprising a LYPD3 antigen-binding domain, a transmembrane domain, a co-stimulatory signal transduction domain, and an intracellular signal transduction 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.

[0010] Regarding treatment options, this application provides the use of a CAR or cell in the preparation of a drug for treating cancer, wherein the CAR comprises a LYPD3 antigen-binding domain, a transmembrane domain, a co-stimulatory signal transduction domain, and an intracellular signal transduction 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, wherein the vector comprises a nucleic acid encoding the CAR. Attached Figure Description

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

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

[0013] Figure 2 This is a graph showing the results of antibody binding assay using ELISA.

[0014] Figure 3 The image shows the results of the affinity assay for anti-LYPD3#22 antibody (Biacore method).

[0015] Figure 4 This is a graph showing the results of LYPD3 target cell antigen expression detection.

[0016] Figure 5 This is a graph showing the results of the detection of the binding of anti-human LYPD3 antibody to target cells;

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

[0018] Figure 7 This is a schematic diagram of the LYPD3-CAR lentiviral vector structure.

[0019] Figure 8 Figure showing the results of flow cytometry detection of LYPD3-CAR lentiviral T-cell infection rate;

[0020] Figure 9 The image shows the results of in vitro killing effect assay 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

[0022] This application relates to a chimeric antigen receptor (CAR), the CAR comprising a LYPD3 antigen-binding domain, a transmembrane domain, a co-stimulatory signal transduction domain, and an intracellular signal transduction 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 Fab, F(ab'), F(ab')2, Fv, or a single-chain variable fragment (scFv). In a preferred embodiment, the LYPD3 antigen-binding domain comprises 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 is composed 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 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 (QVS), and SEQ ID NO: 8, respectively. In one embodiment, the heavy chain variable region comprises frame regions (FR) 1, FR 2, FR 3, and FR 4 separated by its three CDRs. In one embodiment, frame 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, 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 comprises 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 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, 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 LYPD3 antigen-binding domain is humanized. In this application, unless otherwise stated, the CDR sequence is 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 a CD8α domain. In a preferred embodiment, the transmembrane domain comprises a 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 is composed of the amino acid sequence of SEQ ID NO: 40. In one embodiment, the intracellular signal transduction domain includes a CD3ζ signal transduction domain. In a preferred embodiment, the intracellular signal transduction domain includes a human CD3ζ intracellular signal peptide. In a preferred embodiment, the intracellular signal transduction domain includes the amino acid sequence of SEQ ID NO: 44. In a preferred embodiment, the intracellular signal transduction domain is composed of the amino acid sequence of SEQ ID NO: 44. In one embodiment, the co-stimulatory signal transduction domain includes an intracellular signal transduction 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 includes 4-1BB. In a preferred embodiment, the co-stimulatory molecule includes human 4-1BB. In a preferred embodiment, the co-stimulatory signal transduction domain includes the amino acid sequence of SEQ ID NO: 42. In a preferred embodiment, the co-stimulatory signal transduction domain is composed of the amino acid sequence of SEQ ID NO: 42. In one embodiment, the CAR comprises, from the N-terminus to the C-terminus, a LYPD3 antigen-binding domain, a transmembrane domain, a co-stimulatory signal transduction domain, and an intracellular signal transduction 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 a preferred embodiment, the signal peptide is located at the N-terminus of the LYPD3 antigen-binding domain. In a preferred embodiment, the CAR further comprises a hinge region. In a preferred embodiment, the hinge region comprises a CD8α hinge region. In a preferred embodiment, the hinge region comprises a 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 a preferred embodiment, the hinge region is located between the LYPD3 antigen-binding domain and the transmembrane domain. In a preferred 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 located at the N-terminus of the heavy chain variable region. In a preferred embodiment, a hinge header is included between the heavy chain variable region and the light chain variable region. In a preferred embodiment, the hinge header comprises the amino acid sequence of SEQ ID NO: 36. In a preferred embodiment, the hinge joint consists of the amino acid sequence of SEQ ID NO: 36.

[0023] This application also relates to a nucleic acid encoding a CAR, which can be the CAR of any of the above embodiments. In one embodiment, the nucleic acid includes a nucleotide sequence 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 a nucleotide sequence 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 of the light chain variable region includes SEQ ID NO: 32. In one embodiment, the nucleic acid includes a nucleotide sequence encoding a transmembrane domain, a co-stimulatory signal transduction domain, and an intracellular signal transduction domain. In one embodiment, the nucleotide sequence encoding the transmembrane domain includes SEQ ID NO: 39. In one embodiment, the nucleotide sequence encoding the intracellular signal transduction domain includes SEQ ID NO: 43.In one embodiment, the nucleotide sequence encoding the co-stimulatory signal transduction domain includes SEQ ID NO: 41. In one embodiment, the nucleic acid further includes a nucleotide sequence encoding a signal peptide, a hinge region, and / or a hinge head. In one embodiment, the nucleotide sequence encoding the signal peptide includes SEQ ID NO: 33. In one embodiment, the nucleotide sequence encoding the hinge region includes SEQ ID NO: 37. In one embodiment, the nucleotide sequence encoding the hinge head includes SEQ ID NO: 35.

[0024] This application also relates to a vector comprising nucleic acid, which may 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 plasmids, phage particles, bacteriophages or derivatives thereof, viruses, and granules. 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 comprising a vector or CAR, the vector being a vector of any of the above embodiments, and the CAR being a CAR of any of the above embodiments. In one embodiment, the cell comprises an immune effector cell. In a preferred embodiment, the cell comprises T cells. In one embodiment, the immune effector cell is selected from one or more of the group consisting of αβT cells, natural killer (NK) cells, natural killer T (NKT) cells, cytotoxic T lymphocytes (CTLs), 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 CARs or cells in the preparation of medicaments for treating cancer, wherein the CAR can be a CAR of any of the above embodiments, and the cell can be a cell of any of the above embodiments. In one embodiment, the cancer includes cancer 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.

[0027] Example

[0028] 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.

[0029] Example 1

[0030] 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.

[0031] (I) Experimental Materials

[0032] 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).

[0033] (II) Experimental Methods

[0034] 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.

[0035] 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).

[0036] (III) Experimental Results

[0037] 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.

[0038] Example 2

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

[0040] (I) Experimental Materials

[0041] 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.

[0042] (II) Experimental Methods

[0043] 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.

[0044] 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. TM Enhancer, 12ml ExpiCHO TM Feed the cells and incubate them at 37°C. After 7 days, collect the cell culture supernatant.

[0045] 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.

[0046] (III) Experimental Results

[0047] 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.

[0048] Example 3

[0049] Anti-LYPD3 antibody affinity assay

[0050] (I) Experimental Materials

[0051] 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.

[0052] (II) Experimental Methods

[0053] 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.

[0054] 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.

[0055] (III) Experimental Results

[0056] 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 ).

[0057] Example 4

[0058] 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.

[0059] (I) Experimental Materials

[0060] 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).

[0061] (II) Experimental Methods

[0062] NCI-H2126 cell LYPD3 membrane expression detection: 1×10⁻⁶ cells were used. 5 NCI-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.

[0063] 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.

[0064] (III) Experimental Results

[0065] 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 5The above demonstrates that the anti-LYPD3#22 antibody can specifically bind to NCI-H2126 cells that highly express LYPD3. We selected the scFv of the anti-LYPD3#22 antibody for subsequent CAR design, and NCI-H2126 cells can serve as target cells for subsequent CAR T cell development.

[0066] Example 5

[0067] like Figure 6 , 7 As shown in Figures 8 and 9, the construction, lentivirus packaging, preparation, and LYPD3-CAR T cell preparation of CAR lentiviral expression vector based on anti-LYPD3#22 antibody scFv are described.

[0068] (I) Experimental Materials

[0069] DMEM and Opti-MEM were purchased from Gibco, polygluconate from Yisheng Biotechnology (Shanghai) Co., Ltd., WPRE primers were synthesized by Suzhou Anshengda Co., Ltd., the genome extraction kit was purchased from Nanjing Novizan Biotechnology Co., Ltd., lymphocyte separation medium was purchased from GE HealthCare, magnetic rack, sorting beads, sorting column, sorting buffer and human T cell activation beads were purchased from Miltenyi, T cell culture medium X-VIVO 15 was purchased from Lonza, IL-2 was purchased from Peprotech, retrotronectin was purchased from Takara, FBS was purchased from Gibco, and FITC-labeled PL was purchased from ACRO.

[0070] (II) Experimental Methods

[0071] Construction, packaging, and preparation of LYPD3 CAR lentiviral expression vectors: CAR sequential linking structures (see...) Figure 6 The sequence consists of: human CD8α signal peptide, LYPD3-targeting antibody scFv, 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 LYPD3 single-chain antibody sequence was derived from the VH and VL sequences of the anti-LYPD3#22 antibody, and the remaining sequences were obtained from the NCBI 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., catalog number: HYKY-230630007-DLV) was constructed for lentiviral packaging and preparation (see [link to documentation]). Figure 7 ).

[0072] Preparation of LYPD3-CAR T cells: Peripheral blood cells (PBMCs) were isolated from healthy volunteers and sorted with CD3 magnetic beads to obtain CD3. + T cells. Cells were cultured with IL-2 (10 ng / ml) and activated with human T cell activation beads; after 24 hours of activation, T cells were incubated at 5 × 10⁻⁶ cells / ml. 5 Seeds were seeded per well in 24-well plates. Lentiviral solutions of NC-CAR and LYPD3-CAR (MOI = 20) were added, followed by the addition of polyglobulin (8 μg / ml). The cells were resuspended and mixed, then centrifuged at 1000g for 30 min and placed in an incubator for amplification culture. Four days after viral infection, 1 × 10⁶ NT (control T cell group), NC-CAR T, and LYPD3-CAR T cells were collected. 6 Each cell was resuspended in PBS and washed twice. After centrifugation and discarding of the supernatant, 5 μl of FITC-labeled protein L was added and resuspended in 100 μl of PBS. The mixture was mixed and incubated in the dark for 20 min. The positive expression rate of CAR recognition on the surface of CAR T cells was detected by flow cytometry.

[0073] (III) Experimental Results

[0074] We constructed a LYPD3-CAR lentiviral expression vector targeting LYPD3 based on the sequence of the anti-LYPD3#22 antibody scFv, and packaged and prepared the lentivirus using a 293T / 17 cell system. The obtained viral titer was 5E+08TU / ml. The prepared LYPD3-CAR and control NC-CAR lentiviruses were then used to infect activated CD3 cells. + T cells (MOI=20) were collected and expanded. Four days later, flow cytometry analysis revealed an infection rate of 52.1% for T cells infected with LYPD3-CAR lentivirus (see [link to article]). Figure 8 This indicates that we have successfully prepared LYPD3-CAR T cells.

[0075] Example 6

[0076] like Figure 9 As shown, the in vitro killing effect of LYPD3-CAR T cells on NCI-H2126 cells was detected.

[0077] (I) Experimental Materials

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

[0079] (II) Experimental Methods

[0080] The LYPD3-CAR T cells were adjusted to a cell density of 2×10⁻⁶. 6Cells / ml (10:1 group) were serially diluted with killing medium to 10:1, 5:1, and 2.5:1 as effector cells, and NC-CAR T cells as controls; NCI-H2126 cells were adjusted to a cell density of 2×10⁶. 5 The target cells were defined as cells per ml. 50 μl of effector cells and target cells were placed in 96-well plates for co-culture, with three replicates per group (E:T ratios of 10:1, 5:1, and 2.5:1). Control groups were set up: spontaneous release of target cells (50 μl target cells + 50 μl killing medium), maximum release of target cells (50 μl target cells + 50 μl killing medium), spontaneous release of effector cells (50 μl effector cells + 50 μl killing medium), background (100 μl killing medium), and volume correction control group (100 μl killing medium). The 96-well plates were sealed with sealing film, centrifuged at 300 g for 5 min, and then incubated at 37°C in a 5% CO2 incubator for 16 h. 10 μl of the target cell maximum release group and volume correction control group were added to each well. 10X lysis solution was used to incubate the 96-well plate in an incubator for 45 min. The plate was then sealed with sealing film and centrifuged at 250g for 5 min. A new 96-well plate was prepared, and 50 μl of the centrifuged cell-killing supernatant and 50 μl of LDH detection substrate solution were added to each well. The plate was incubated at room temperature in the dark for 20 min. 50 μl of stop solution was added to each well, and the absorbance was measured at 492 nm using a microplate reader.

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

[0082] (III) Experimental Results

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

[0084] Example 7

[0085] like Figure 10 As 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] Severe immunodeficient B-NDG mice were purchased from Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd.

[0088] (II) Experimental Methods

[0089] Establishment of a mouse lung cancer model: 6-8 week old female B-NDG mice were selected and subcutaneously injected with NCI-H2126 cells (1×10⁻⁶). 7 A mouse subcutaneous lung cancer tumor model was constructed using cells / mouse. The in vivo antitumor effect of LYPD3-CAR T cells was assessed: when the tumor volume reached 100 mm². 3 Around 1000 cells, LYPD3-CAR T cells and NC-CAR T cells were injected into mice via the tail vein (4 × 10⁻⁶ cells). 6 Cells / animal, second infusion after 4 days, n=4); after infusion, the length and width of the mass are measured with calipers every 2-3 days. V=1 / 2AB 2 (V = tumor volume; A = tumor length; B = tumor width) Assess the tumor volume and plot it on a graph. When the maximum diameter of the tumor reaches 1-2 cm, sacrifice the mouse and remove the tumor to evaluate the in vivo anti-tumor effect of CAR-T cells.

[0090] (III) Experimental Results

[0091] To further investigate the in vivo antitumor effect of LYPD3-CAR T cells, we constructed a B-NDG mouse model with NCI-H2126 cells bearing tumors subcutaneously. The tumor volume was increased to 100 mm². 3 On the left and right sides, LYPD3-CAR T cells and control CAR T cells were adopted and infused respectively, and tumor growth was measured. We found that control CAR T cells had no tumor-suppressive effect, while LYPD3-CAR T cells significantly inhibited the in vivo tumor growth of lung cancer cells (see...). Figure 10 This indicates that adoptive infusion 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] This invention provides a LYPD3 scFv nucleotide sequence, vector, host cell, and its application in anti-tumor therapy for treating malignant lung tumors. Through the design of the CAR structure, novel CAR T cells were prepared, exhibiting a significant killing effect on tumor cells and inhibiting tumor growth in vivo, thus providing an effective strategy for lung cancer immunotherapy.

Claims

1. A chimeric antigen receptor (CAR), said CAR comprising a LYPD3 antigen-binding domain, a hinge region, a transmembrane domain, a co-stimulatory signal transduction domain, and an intracellular signal transduction 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 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, QVS, and SEQ ID NO: 8, respectively. The heavy chain variable region is composed of the amino acid sequence of SEQ ID NO: 1, and the light chain variable region is composed of the amino acid sequence of SEQ ID NO:

2. The hinge region is composed of the amino acid sequence of SEQ ID NO:

38. The transmembrane domain consists of the amino acid sequence of SEQ ID NO:

40. The co-stimulatory signal transduction domain is composed of the amino acid sequence of SEQ ID NO: 42, and The intracellular signal transduction domain consists of the amino acid sequence of SEQ ID NO:

44.

2. The CAR as claimed in claim 1, wherein: The LYPD3 antigen-binding domain is Fab, F(ab'), F(ab')2, Fv, or a single-stranded variable fragment (scFv); and / or The isotypes of the LYPD3 antigen-binding domain include IgA, IgD, IgE, IgG, or IgM.

3. The CAR of claim 2, wherein the LYPD3 antigen-binding domain is scFv.

4. A nucleic acid that encodes a CAR as described in any one of claims 1-3.

5. A vector comprising the nucleic acid as described in claim 4, wherein the vector is a lentivirus.

6. A cell comprising the vector as described in claim 5 or the CAR as described in any one of claims 1-3, wherein the cell is a T cell.

7. Use of the CAR as described in any one of claims 1-3 or the cell as described in claim 6 in the preparation of a medicament for treating lung cancer.

8. The use as described in claim 7, wherein the lung cancer is non-small cell lung cancer.

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