MUC18-targeting chimeric antigen receptor and use thereof

Abstract

The present application relates to an MUC18-targeting chimeric antigen receptor, which comprises an antibody fragment that specifically recognizes MUC18, an IgG4 hinge region, a CD8 transmembrane region or a CD28 transmembrane region, a CD28 costimulatory signaling domain or a 4-1BB costimulatory signaling domain, and a CD3ζ signaling domain. The antibody fragment contains a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH contains HC CDR1 having an amino acid sequence as shown in SEQ ID NO: 7, HC CDR2 having an amino acid sequence as shown in SEQ ID NO: 8, and HC CDR3 having an amino acid sequence as shown in SEQ ID NO: 9; and the VL contains LC CDR1 having an amino acid sequence as shown in SEQ ID NO: 10, LC CDR2 having an RAS amino acid sequence, and LC CDR3 having an amino acid sequence as shown in SEQ ID NO: 11.

Description

Chimeric antigen receptor targeting MUC18 and its use

[0001] This application claims priority to a Chinese application filed on February 7, 2024, with application number 202410172661.6, entitled “Chimeric Antigen Receptor Targeting MUC18 and Uses Thereof.” The entire contents of that priority application are incorporated herein by reference. The contents of all references cited in this application are incorporated herein by reference. Technical Field

[0002] The present application relates to a chimeric antigen receptor targeting MUC18, isolated cells containing the chimeric antigen receptor, and their use in the preparation of medicaments. Background Art

[0003] Melanoma is the fastest-growing malignant tumor worldwide, with the number of cases increasing by 3%-5% annually. According to global cancer data for 2020, there were 325,000 new cases of melanoma and 57,000 deaths [1]. In China, there are approximately 20,000 new cases of melanoma each year [2].

[0004] Surgery is the primary means of achieving a radical cure for early-stage melanoma. However, due to low awareness of the disease among the Chinese population, most melanoma patients are initially diagnosed in the middle or late stages. A survey found that 56% of patients were diagnosed in the middle or late stages (stages III-IV), while only 13% were in the early stages (stages I-II) [Reference 2]. Late-stage patients often lack effective treatments and have poor survival. Once metastasis develops, the 5-year survival rate is only 4.6%. Even with more precise targeted therapies, 90% of patients develop drug resistance after 5 to 7 months of treatment. Previous surveys have shown significant differences between Chinese and Western melanoma classifications. Melanoma types in China exhibit "Chinese characteristics," with a higher prevalence of acral (41.8%) and mucosal (22.6%) melanomas. These types of melanomas are less responsive to the currently effective immune checkpoint inhibitor PD-1, with an efficacy rate of only 14%-15% [References 2, 3, 4]. This may be related to the lower mutation burden of melanoma genes in the Chinese population [Reference 4]. The key to solving this dilemma lies in finding efficient targets for the treatment of malignant melanoma, and then combining immune and targeted therapies to improve treatment response rates and reduce recurrence rates.

[0005] MUC18, also known as CD146, is a type I single-chain transmembrane glycoprotein belonging to the immunoglobulin superfamily. It is divided into three subtypes: long, short, and soluble. Its protein structure consists of five immunoglobulin-like domains in the extracellular region, a single hydrophobic transmembrane domain, and a short cytoplasmic region. It is located on the cell surface as a monomer and dimer, and dimerization transmits downstream signals [Reference 5]. In 1987, Johnson's team first discovered the MUC18 protein in malignant melanoma [Reference 6]. The main physiological function of MUC18 is its adhesion molecule properties and its involvement in angiogenesis [Reference 7]. MUC18 expression levels are closely correlated with malignant tumor progression, tumor angiogenesis, metastasis, and prognosis. Studies have found that MUC18 is highly expressed in metastatic melanoma and advanced primary cancer tissues, accounting for approximately 70% of expression. In primary tumors, MUC18 expression increases with increasing vertical thickness. In most metastatic tumors, MUC18 expression gradually increases with increasing malignancy, but expression is lower in shallow, low-metastatic tumors (thickness <0.75 mm) [References 5, 6]. In addition to melanoma, MUC18 is highly expressed in a variety of tumor tissues, including sarcomas, triple-negative breast cancer, ovarian cancer, placental trophoblast tumor, choriocarcinoma, small cell lung cancer, renal clear cell carcinoma, thyroid cancer, liver cancer, head and neck squamous cell carcinoma, and bile duct carcinoma [Reference 5]. In normal tissues, MUC18 is primarily expressed in embryonic tissue, intermediate trophoblast cells, activated vascular endothelial cells, and smooth muscle cells [Reference 5]. Therefore, MUC18 can be used as a target for melanoma and in the development of targeted therapeutics.

[0006] Chimeric antigen receptor T (CAR-T) cell therapy, an innovative immunotherapy, plays a significant role in current cancer treatment. The concept of CAR-T was first proposed in the late 1980s. It combines the advantages of antibody recognition specificity with the cytotoxicity of T cells to form an adoptive immune response [Reference 8]. The CAR molecular structure primarily consists of a single-chain antibody fragment (scFv) that specifically recognizes an antigen, a hinge region, a transmembrane region, a costimulatory signaling domain, and a CD3ζ signaling domain. In recent years, the use of anti-CD19 and anti-CD20 chimeric antigen receptor T cells has achieved promising results in the treatment of lymphoid malignancies (B-cell acute lymphoblastic leukemia and non-Hodgkin's lymphoma) [Reference 9]. Currently, 10 CAR-T drugs for hematological malignancies have been approved for marketing worldwide, but there is still a lack of CAR-T drugs for solid tumors on the market. This may be related to factors such as the heterogeneity of antigen expression in solid tumor cells, residual tumor stem cells, a highly immunosuppressive tumor microenvironment, and an extracellular matrix that is difficult for CAR-T cells to penetrate. However, research data has reported that some early clinical trials in the past two years have demonstrated the potential of CAR-T therapy for the treatment of solid tumors. These include studies targeting mesothelin CAR-T at Memorial Sloan Kettering Cancer Center [Reference 10], GD2 CAR-T at Stanford University [Reference 11], prostate-specific membrane antigen (PSMA) CAR-T at the University of Pennsylvania [Reference 12], and claudin 18.2 CAR-T at Beijing Cancer Hospital and Cogen Biosciences [Reference 13]. Therefore, CAR-T therapy is also promising for malignant solid tumors like melanoma. Most CAR-T clinical trials for advanced melanoma are in Phase I or II, targeting targets such as VEGFR2, GD2, cMet, and hCD70. Most of these trials are still recruiting or in progress. Only the VEGFR2-targeting CAR-T trial has published clinical results: 23 of 24 enrolled patients with advanced melanoma experienced disease progression [Reference 14]. Identifying effective CAR-T targets in patients with advanced melanoma is an urgent issue that needs to be addressed.

[0007] Document 1: Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F.Global Cancer Statistics 2020: GLOBOCAN Estimates of Incience and Mortality Worldwide for 36 Cancers in 185 Countries.CA Cancer J Clin.2021 May;71(3):209-249.doi:10.3322 / caac.21660.

[0008] Reference 2: White Paper on the Current Status of Melanoma Patient Behavior in China. 2021. CSCO.

[0009] Document 3: Alexander N. Shoushtari; Riyue Bao; Jason J. Luke. PD-1 Blockade in Chinese versus Western Patients with Melanoma. Clin Cancer kes (2020) 26(16): 4171-4173.

[0010] Literature 4: Cui C, Lian B, Zhang X, Wu D, Li K, Si L, Yang Y, Tian H, Zhou L, Chi Z, Sheng X, Kong Y, Mao L, Wang X, Bai X, Yan X, Li S, Dai J, Tang B, Wei Proposal.Ann Surg Oncol.2022 Aug;29(8):5221-5234.doi:10.1245 / s10434-022-11670-6.

[0011] Document 5: Duan HX, Xiong CL, Jing L, et al. Review and prospect of CD146 research(in Chinese). Sci Sin Vitae. 2020, 50: 1339-1387.

[0012] Reference 6: Lehmann JM, Holzmann B, Breitbart EW, Schmiegelow P, Riethmüller G, Johnson JP. Discrimination between benign and malignant cells of melanocytic lineage by two novel antigens, a glycoprotein with a molecular weight of 113,000 and a protein with a molecular weight of 76,000. Cancer Res. 1987 Feb 1;47(3):841-5.

[0013] Reference 7: Wang Z, Xu Q, Zhang N, Du X, Xu G, Yan X. CD146, from a melanoma cell adhesion molecule to a signaling receptor. Signal Transduct Target Ther. 2020 Aug 11;5(1):148.

[0014] Reference 8: Gross G, Waks T, Eshhar Z. Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci U S A. 1989 Dec;86(24):10024-8.

[0015] Reference 9: Khalil DN, Smith EL, Brentjens RJ. Wolchok JD. The future of cancer treatment: immunomodulation. CARs and combination immunotherapy. Nat Rev Clin Oncol 2016;13(6):394.

[0016] Reference 10: Adusumilli, P.S., et al., A Phase I Trial of Regional Mesothelin-Targeted CAR T-cell Therapy in Patients with Malignant Pleural Disease, in Combination with the Anti-PD-1 Agent Pembrolizumab. Cancer Discov, 2021. 11(11): p. 2748-2763.

[0017] Reference 11: Majzner, R.G., et al., GD2-CAR T cell therapy for H3K27M-mutated diffuse midline gliomas. Nature, 2022. 603(7903): p. 934-941.

[0018] Reference 12: Narayan, V., et al., PSMA-targeting TGFbeta-insensitive armored CAR T cells in metastatic castration-resistant prostate cancer: a phase l trial. Nat Med, 2022. 28(4): p. 724-734.

[0019] Reference 13: Qi, C., et al., Claudinl8.2-specific CAR T cells in gastrointestinal cancers: phase ltrial interim results. Nat Med, 2022.

[0020] Reference 14: Yu J, Wu X, Yan J, Yu H, Xu L, Chi Z, Sheng X, Si L, Cui C, Dai J, Ma M, Xu T, Kong Y, Guo J. Anti-GD2 / 4-1BB chimeric antigen receptor T cell therapy for the treatment of Chinese melanoma patients. J Hematol Oncol. 2018 Jan 3;11(1):1. Summary of the Invention

[0021] The cure rate for patients with advanced melanoma is low, and there are currently no reports of clinically effective CAR-T drugs for the treatment of melanoma.

[0022] In view of the above problems, the present invention provides a chimeric antigen receptor (CAR) targeting MUC18, a nucleic acid encoding the CAR, and a vector or isolated cell containing the CAR, which can effectively kill MUC18-positive tumor cells.

[0023] Specifically, the present invention provides:

[0024] (1) A chimeric antigen receptor targeting MUC18, comprising: a scFv that specifically recognizes MUC18, an IgG4 hinge region, a CD8 transmembrane region or a CD28 transmembrane region, a CD28 co-stimulatory signal domain or a 4-1BB co-stimulatory signal domain, and a CD3 ζ signal domain; the scFv comprises a heavy chain variable region VH and a light chain variable region VL, the VH comprises an HC CDR1 with an amino acid sequence as shown in SEQ ID NO: 7, an HC CDR2 with an amino acid sequence as shown in SEQ ID NO: 8, and an HC CDR3 with an amino acid sequence as shown in SEQ ID NO: 9, and the VL comprises an LC CDR1 with an amino acid sequence as shown in SEQ ID NO: 10, an LC CDR2 with an amino acid sequence as shown in SEQ ID NO: 11, and an LC CDR3 with an amino acid sequence as shown in SEQ ID NO: 11.

[0025] (2) The chimeric antigen receptor according to (1) above, wherein the VH is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence of SEQ ID NO: 5, and the VL is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence of SEQ ID NO: 6.

[0026] (3) The chimeric antigen receptor according to (1) or (2) above, wherein the VH comprises the amino acid sequence of SEQ ID NO: 5, and the VL comprises the amino acid sequence of SEQ ID NO: 6.

[0027] (4) The chimeric antigen receptor according to any one of (1) to (3) above, further comprising a signal peptide having an amino acid sequence as shown in SEQ ID NO: 1, and optionally further comprising a tag having an amino acid sequence as shown in SEQ ID NO: 2.

[0028] (5) The chimeric antigen receptor according to any one of (1) to (4) above, wherein the hinge region is an IgG4 hinge region having the amino acid sequence shown in SEQ ID NO: 15, the transmembrane region is a CD8 transmembrane region having the amino acid sequence shown in SEQ ID NO: 16 or a CD28 transmembrane region having the amino acid sequence shown in SEQ ID NO: 17, the costimulatory signal domain is a CD28 costimulatory signal having the amino acid sequence shown in SEQ ID NO: 18 or a 4-1BB costimulatory signal domain having the amino acid sequence shown in SEQ ID NO: 19, and the CD3ζ signal domain is a CD3ζ signal domain having the amino acid sequence shown in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23.

[0029] (6) The chimeric antigen receptor according to (5) above, wherein the hinge region is an IgG4 hinge region having the amino acid sequence shown in SEQ ID NO: 15, the transmembrane region is a CD8 transmembrane region having the amino acid sequence shown in SEQ ID NO: 16, the costimulatory signal domain is a CD28 costimulatory signal having the amino acid sequence shown in SEQ ID NO: 18, and the CD3ζ signal domain has the amino acid sequence shown in SEQ ID NO: 19.

[0030] (7) The chimeric antigen receptor according to any one of (1) to (6) above, which has the amino acid sequence shown in SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34, or an amino acid sequence that is at least 85%, preferably at least 90%, and more preferably at least 95% identical to these sequences.

[0031] (8) An isolated nucleic acid encoding the chimeric antigen receptor according to any one of (1) to (7) above.

[0032] (9) A vector comprising the nucleic acid according to (8) above.

[0033] (10) An isolated cell comprising the chimeric antigen receptor according to any one of (1) to (7) above, the nucleic acid according to (8) above, or the vector according to (9) above.

[0034] (11) Use of the chimeric antigen receptor according to any one of (1) to (7), the nucleic acid according to (8), the vector according to (9), or the cell according to (10) in the preparation of a drug for treating MUC18-positive tumors.

[0035] (12) The use according to (11) above, wherein the drug is used to treat melanoma, sarcoma, triple-negative breast cancer, ovarian cancer, placental trophoblastoma, choriocarcinoma, small cell lung cancer, renal clear cell carcinoma, thyroid cancer, liver cancer, head and neck squamous cell carcinoma or bile duct cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 shows the transduction efficiency of MUC18 CAR molecules containing strep tag II, humanized scFv, different hinge regions, transmembrane regions and signaling domains in T cells.

[0037] Figure 2 shows the total cell proliferation of MUC18 CAR-T cells containing strep tag II, humanized scFv, different hinge regions, transmembrane regions, and signaling domains.

[0038] Figure 3 shows the proliferation rate of MUC18 CAR-T cells containing strep tag II, humanized scFv, different hinge regions, transmembrane regions, and signaling domains.

[0039] Figure 4 shows the expression of MUC18 CAR-T cell differentiation-depleting molecules containing strep tag II, humanized scFv, different hinge regions, transmembrane regions, and signaling domains.

[0040] Figure 5 shows the killing efficiency of target cells by MUC18 CAR-T cells containing strep tag II, humanized scFv, different hinge regions, transmembrane regions and signaling domains.

[0041] Figure 6 shows the real-time killing efficiency of target cells by MUC18 CAR-T cells containing strep tag II, humanized scFv, different hinge regions, transmembrane regions, and signaling domains.

[0042] Figure 7 shows the tumor killing and CAR-T expansion results of MUC18 CAR-T cells containing strep tag II, humanized scFv, different hinge regions, transmembrane regions and signaling domains under repeated stimulation of target cells.

[0043] Figure 8 shows the proliferation and tumor inhibition effects of MUC18 CAR-T cells containing strep tag II, humanized scFv, different hinge regions, transmembrane regions, and signaling domains in tumor-bearing mice.

[0044] Figure 9 shows the transduction efficiency of MUC18 CAR molecules containing humanized scFv, IgG4 hinge region, different transmembrane regions and signaling domains in T cells.

[0045] Figure 10 shows the total cell proliferation of MUC18 CAR-T containing humanized scFv, IgG4 hinge region, different transmembrane regions and signaling domains.

[0046] Figure 11 shows the proliferation rate of MUC18 CAR-T cells containing humanized scFv, IgG4 hinge region, different transmembrane regions and signaling domains.

[0047] FIG12 shows the real-time killing efficiency of target cells by MUC18 CAR-T cells containing humanized scFv, IgG4 hinge region, different transmembrane regions and signaling domains.

[0048] FIG13 shows the results of five rounds of repeated stimulation of MUC18 CAR-T cells containing humanized scFv, IgG4 hinge region, different transmembrane regions and signaling domains by target cells starting from day 8 of T cell culture.

[0049] FIG14 shows the results of four rounds of repeated stimulation of MUC18 CAR-T cells containing humanized scFv, IgG4 hinge region, different transmembrane regions and signaling domains by target cells starting from day 11 of T cell culture.

[0050] FIG15 shows the proliferation and tumor inhibition effects of MUC18 CAR-T cells containing humanized scFv, IgG4 hinge region, different transmembrane regions and signaling domains in tumor-bearing mice. DETAILED DESCRIPTION

[0051] One aspect of the present application relates to chimeric antigen receptors targeting MUC18.

[0052] The chimeric antigen receptor of the present invention uses an scFv that specifically recognizes MUC18, wherein the scFv comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises an HC CDR1 with the amino acid sequence shown in SEQ ID NO: 7, an HC CDR2 with the amino acid sequence shown in SEQ ID NO: 8, and an HC CDR3 with the amino acid sequence shown in SEQ ID NO: 9, and the VL comprises an LC CDR1 with the amino acid sequence shown in SEQ ID NO: 10, an LC CDR2 with the amino acid sequence of RAS, and an LC CDR3 with the amino acid sequence shown in SEQ ID NO: 11.

[0053] In some embodiments, the VH of the scFv that specifically recognizes MUC18 is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence of SEQ ID NO: 5, and the VL is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence of SEQ ID NO: 6. Preferably, the VH comprises the amino acid sequence of SEQ ID NO: 5, and the VL comprises the amino acid sequence of SEQ ID NO: 6. More preferably, the scFv comprises the VH set forth in SEQ ID NO: 5 and the VL set forth in SEQ ID NO: 6.

[0054] The hinge regions used in the chimeric antigen receptors of the present invention include the following three types: a 45-amino acid CD8 hinge region, whose amino acid sequence is SEQ ID NO: 13 and whose nucleotide sequence is SEQ ID NO: 47; a 39-amino acid CD28 hinge region, whose amino acid sequence is SEQ ID NO: 14 and whose nucleotide sequence is SEQ ID NO: 48; and a 12-amino acid IgG4 hinge region, whose amino acid sequence is SEQ ID NO: 15 and whose nucleotide sequence is SEQ ID NO: 49.

[0055] In some more specific embodiments, the chimeric antigen receptor of the present invention preferably uses the IgG4 hinge region.

[0056] The transmembrane regions used in the chimeric antigen receptor of the present invention include the following two types: a 24-amino acid CD8 transmembrane region, whose amino acid sequence is SEQ ID NO: 16 and nucleotide sequence is SEQ ID NO: 50; and a 27-amino acid CD28 transmembrane region, whose amino acid sequence is SEQ ID NO: 17 and nucleotide sequence is SEQ ID NO: 51.

[0057] In some more specific embodiments, the chimeric antigen receptor of the present invention preferably utilizes the CD8 transmembrane region.

[0058] The costimulatory signaling domains used in the chimeric antigen receptor of the present invention include the following two types: a 41-amino acid CD28 costimulatory signaling domain, whose amino acid sequence is SEQ ID NO: 18 and the nucleotide sequence is SEQ ID NO: 52; and a 42-amino acid 4-1BB costimulatory signaling domain, whose amino acid sequence is SEQ ID NO: 19 and the nucleotide sequence is SEQ ID NO: 53.

[0059] In some more specific embodiments, the chimeric antigen receptors of the present invention preferably utilize the CD28 co-stimulatory signaling domain.

[0060] The CD3ζ signaling domain used in the chimeric antigen receptor of the present invention includes the following four types: one wild-type and three mutant types. The amino acid sequence of the wild-type CD3ζ intracellular signaling domain is SEQ ID NO: 20, and the nucleotide sequence is SEQ ID NO: 54. Based on the wild-type CD3ζ, the three mutant designs are: CD3ζ mutant type 1, which has one amino acid mutation, Q14 to K, with an amino acid sequence of SEQ ID NO: 21 and a nucleotide sequence of SEQ ID NO: 55; CD3ζ mutant type 2, which has four amino acid mutations, V2 to L, D9 to E, Q15 to K, and Y90 to F, with an amino acid sequence of SEQ ID NO: 22 and a nucleotide sequence of SEQ ID NO: 56; and CD3ζ mutant type 3, which has five amino acid mutations, V2 to L, D9 to E, Q14 to K, Q15 to K, and Y90 to F, with an amino acid sequence of SEQ ID NO: 23 and a nucleotide sequence of SEQ ID NO: 57.

[0061] In some embodiments, the chimeric antigen receptor comprises different sequence design combinations of the above four regions.

[0062] In some more specific embodiments, the chimeric antigen receptors of the invention utilize the wild-type CD3zeta signaling domain.

[0063] In some more specific embodiments, the chimeric antigen receptor of the present invention has an amino acid sequence as shown in SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or an amino acid sequence that is at least 85%, preferably at least 90%, and more preferably at least 95% identical to these sequences.

[0064] In some more specific embodiments, the nucleotide sequences of the chimeric antigen receptors of the present invention are SEQ ID NOs: 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, and 68, respectively.

[0065] In some more specific embodiments, the chimeric antigen receptor of the present invention comprises the signal peptide sequence shown in SEQ ID NO:1.

[0066] The chimeric antigen receptor of the present invention may contain other genes such as tags, cytokines, or regulatory genes as needed.

[0067] Specific examples of tags include the detection tag Strep tag II, which has the amino acid sequence shown in SEQ ID NO: 2 and the nucleotide sequence shown in SEQ ID NO: 36. The tag's attachment position and connecting peptide are not particularly limited. For example, the tag can be linked to the scFv via a connecting peptide having the amino acid sequence shown in SEQ ID NO: 3.

[0068] The present application also relates to isolated nucleic acids encoding the above chimeric antigen receptors, and vectors comprising the nucleic acids.

[0069] The present application further relates to an isolated cell comprising the above-mentioned chimeric antigen receptor, nucleic acid or vector, wherein the cell is preferably not a germ cell or a fertilized egg. The cell is preferably a lymphocyte, including, for example, a T cell, a B cell, a K cell, a NK cell, etc.

[0070] The chimeric antigen receptors, vectors, or cells of the present application can be used to treat MUC18-positive tumors or to prepare medicaments for treating MUC18-positive tumors. Tumors include, but are not limited to, melanoma, sarcoma, triple-negative breast cancer, ovarian cancer, placental trophoblastoma, choriocarcinoma, small cell lung cancer, clear cell renal carcinoma, thyroid cancer, liver cancer, head and neck squamous cell carcinoma, or bile duct cancer, all of which are known to overexpress MUC18.

[0071] The amino acid sequences and nucleotide sequences described in this application are shown in Tables 1 and 2 below, respectively.

[0072] Table 1 Amino acid sequence

[0073] Table 2 Nucleotide sequences

[0074] The specific CAR molecular compositions of this application are shown in Table 3 below.

[0075] Table 3

[0076] Example

[0077] Example 1: Construction of CAR molecules containing humanized MUC18 scFv and preparation of CAR-T

[0078] The nucleotide sequence containing the signal peptide sequence, tag sequence, linker peptide sequence between the tag and scFv, scFv sequence, hinge region, transmembrane region, co-stimulatory signal domain, and CD3ζ intracellular region in Table 2 was synthesized by gene synthesis. Then, using homologous recombination and enzyme ligation, each sequence fragment was constructed into the lentiviral vector pLenti6.3 / V5 (Thermo Fisher, Waltham, MA, USA) to form a complete CAR molecular vector. Table 3 shows the various components of the CAR molecular structure of CAR-T-1 to 6.

[0079] T cells were isolated from peripheral blood mononuclear cells of healthy volunteers using CD3 / CD28 antibody-coupled magnetic beads (Thermo Fisher, Cat#40203D). The isolated and purified T cells were cultured at a concentration of 1.5×10 6 The cells were inoculated into an X-VIVO 15 (Lonza, Switzerland) culture system containing 500 IU / mL IL-2 (Shandong Jintai Bioengineering Co., Ltd., China) at a density of 10 cells / mL. After 24 hours of culture, the lentiviral vector carrying the MUC18 CAR molecule sequence was transfected into T cells at an MOI of 1. 24 hours after lentiviral infection, the virus in the suspension was removed by changing the cell medium, and the cells were inoculated into a fresh X-VIVO 15 culture system containing 500 IU / mL IL-2 and continued to be cultured. Four days after lentiviral infection, the cells in the culture system were repeatedly pipetted and collected into a centrifuge tube, and then placed on a magnetic stand for 5 minutes to remove the CD3 / CD28 magnetic beads. The T cells were centrifuged and counted, and approximately 5×10 5 The cells were stained, and the CAR molecule transduction efficiency of each group of cells was detected using a full-spectrum flow cytometer (N / -00008-0A, Cytek Biosciences, Inc. Fremont).

[0080] Figure 1 shows the CAR transduction efficiency of T cells in each group after infection with lentivirus containing the CAR molecule sequence for 4, 6, 8, 11, and 13 days. The expression rate of the CAR molecule in each group increased with prolonged culture time. CAR-T-6 and CAR-T-5 had slightly lower expression rates, while CAR-T-3 had the highest expression rate.

[0081] Example 2: In vitro proliferation capacity of MUC18 CAR-T cells containing strep tag II, humanized scFv, different hinge regions, transmembrane regions and signaling domains

[0082] During the MUC18 CAR-T cell culture process, lentiviral infection of T cells was considered the second day of culture. Thereafter, every 2 to 3 days, CAR-T cells were harvested and centrifuged, and resuspended in 1-2 ml of X.VIVO 15 medium containing 500 IU / mL IL-2. Ten microliters of cells were diluted to a certain multiple and stained with trypan blue (Solarbio, Cat#C0040) at a 1:1 volume ratio. Cell viability and viable cell concentration were recorded using a cell counter (Countess 3FL, Invitrogen by Thermo Fisher Scientific), and the total cell number in each group was calculated for statistical analysis of cell proliferation in each group.

[0083] Figure 2 shows that the total cell proliferation of CAR-T-1 and CAR-T-2 was similar to that of the control T group (T cells not infected with CAR molecules), and the total cell proliferation of CAR-T-3, CAR-T-4, CAR-T-5, and CAR-T-6 was slightly lower, but the overall difference between the groups was not significant;

[0084] Figure 3 shows that the CAR-T cell proliferation rates of CAR-T-2 and CAR-T-5 were relatively low, indicating that CD3ζ mutants 2 and 3 reduced the proliferation rate of CAR-T cells under these scFvs. The proliferation rate of CAR-T-3 was higher than that of CAR-T-1, and the proliferation rate of CAR-T-6 was higher than that of CAR-T-4, indicating that CAR molecules using the IgG4 hinge region achieved better CAR-T cell proliferation than CAR molecules using the CD8 and CD28 hinge regions.

[0085] Example 3: Expression of differentiation-related molecules and exhaustion-related molecules in MUC18 CAR-T cells containing s.rep tag II, humanized scFv, different hinge regions, transmembrane regions, and signaling domains

[0086] On the 8th day of CAR-T cell culture, 1×10 6 The cells were centrifuged at 400 g for 5 minutes, the supernatant was discarded and resuspended in 100 μL of DPBS (HyClone, CAT#SH30028.02); fluorescent antibodies related to T cell differentiation and exhaustion were then added to each group of CAR-T cells for labeling, and the cells were detected using a full-spectrum flow cytometer (N7-00008-0A, Cytek Biosciences, Inc. Fremont).

[0087] In the results of Figure 4-A, the marker of the differentiation phenotype of naive T cells and stem memory T cells (S&N) is CD45RA + CD62L +The cell population, the marker of the differentiation phenotype of central memory T cells (CM) is CD45RA - CD62L + The cell population, the marker of the differentiated phenotype of effector memory T cells (EM) is CD45RA - CD62L - The cell population, the marker of the differentiation phenotype of effector T cells (EFF) is CD45RA + CD62L - Compared with the T group, the proportion of EFF cell populations in each CAR-T group was slightly higher. Among the CAR-T groups, the proportion of S&N cell populations was slightly higher in CAR-T-3 and CAR-T-6. This indicates that the degree of cell differentiation in the CAR-T-3 and CAR-T-6 groups is lower than that in other CAR-T groups. The high proportion of naive T cells and stem memory T cells indicates that T cells have the potential for continued differentiation and proliferation, further indicating that these two CAR-T cells can continue to differentiate and proliferate and have good persistence in vivo.

[0088] Figure 4-B uses two exhaustion molecular markers, PD-1 and LAG-3, to represent the T cell exhaustion phenotype of each group. The results show that the exhaustion levels of CAR-T-1, CAR-T-2, and CAR-T-3 are lower than those of the T cell group; the exhaustion levels of CAR-T-4, CAR-T-5, and CAR-T-6 are higher than those of the T cell group, among which the exhaustion levels of CAR-T-5 and CAR-T-6 are lower than those of the CAR-T-4 group. Example 4: Short-term killing efficiency of MUC18 CAR-T cells containing strep tag II, humanized scFv, different hinge regions, transmembrane regions, and signaling domains against MUC18-positive tumor cells in vitro

[0089] The MUC18-expressing tumor cell line A375 (Cat#CRL-1619, ATCC) was passaged every 2 days and cultured in DMEM medium (Cat#sh30022.01, Hyclone) containing 10% serum (Cat#04-001-1C04001-500, Bioind). When A375 cells reached sufficient numbers, they were digested with 0.25% trypsin (Cat#03-050-1BCS, Bioind). After the cells were detached, digestion was terminated with 2 volumes of serum-containing medium. The cells were collected and centrifuged in a centrifuge tube. The tumor cells were resuspended in DMEM medium containing 10% serum to a concentration of 2×10 cells per ml. 5 The remaining cells were used for subculture.

[0090] On the 8th day of CAR-T culture, 1×10 7A375 cells were resuspended in 1 ml of normal saline (Hebei Tiancheng Pharmaceutical Co., Ltd., China), and 10 μL of Calcein-AM (1 μg / μL, Cat#C3100MP, ThermoFisher, USA) was added. The cells were gently mixed and incubated in a 37°C incubator for 30 minutes to label the target cells. After incubation, the cells were washed twice with normal saline, resuspended in X-VIVO 15 medium, and counted. 1×10 cells were added to each well of a 48-well cell culture plate (Cat#150687, Thermo). 5 CAR-T cells were added to each well of the positive control group at effector-target ratios (CAR-T cell: tumor cell, E:T) of 1:1 and 3:1, respectively, to achieve the desired total T cell count. The volume was then adjusted to 500 μl using X-VIVO 15 medium, and the 48-well plate was incubated in a 37°C, 5% CO2 incubator for 5.5 hours. Following incubation, the positive control group was treated with 2% Triton-X-100 (Cat# T8787-100ML, Sigma, Germany) to completely lyse the target cells. The supernatant was collected and centrifuged at 500 g for 5 minutes. 100 μl of the supernatant from the killing system was transferred to each well of the microplate and fluorescence was measured using a multi-function microplate reader (Varioscan Lux, ThermoFisher) (excitation wavelength: 495 nm, emission wavelength: 515 nm).

[0091] Figure 5 shows that compared to the control T cell group, all CAR-T groups had a killing effect on MUC18-positive tumor cell lines. The killing efficiency of CAR-T-4, CAR-T-5, and CAR-T-6 with the CD28 costimulatory domain was higher than that of CAR-T-1, CAR-T-2, and CAR-T-3 with the 4-1BB costimulatory domain. The difference in killing efficiency was even greater at an E:T ratio of 3:1, indicating that MUC18 CAR-T with the CD28 costimulatory signaling domain is more efficient in killing target cells in a shorter period of time than MUC18 CAR-T with the 4-1BB costimulatory signaling domain.

[0092] Example 5: Real-time killing efficiency of MUC18 CAR-T cells containing strep tag II, humanized scFv, different hinge regions, transmembrane regions and signaling domains against MUC18-positive tumor cells in vitro

[0093] The tumor cells treated in Example 4 were plated as follows: 50 μl of tumor cell culture medium was added to the wells of the 96-well plate E-Plate (E-plate96) equipped with a real-time killing instrument (Agilent xCELLigence RTCA SP), and the plate was placed on the real-time killing instrument for instrument self-check; E-plate96 was removed, and 100 μl (2×10 4 The tumor cell suspension was mixed evenly with 100 μL of cells / well and placed at room temperature for 30 minutes. The E-plate 96 was placed on a real-time killing instrument in a 37°C, 5% CO2 incubator to detect the cell proliferation curve overnight. When the cell proliferation index (Index value) reached 0.6 or above, the instrument was stopped, the E-plate 96 was removed, and placed in a biosafety cabinet. 50 μL of supernatant was aspirated from each well with a pipette and discarded. 100 μL (2×10 3 The total number of T cells prepared with X-VIVO 15 medium was consistent and the concentration was 2×10 per ml. 4 The E-Plate96 assay plate was placed on a real-time killing instrument for continuous monitoring for 32 hours to observe the real-time killing effect of each group of CAR-T cells on tumor cells.

[0094] The experimental results are shown in Figure 6. The tumor cell proliferation in the T cell group is consistent with that in the tumor cell group alone, indicating that the T cell group has no tumor killing ability. Among the CAR-T groups, the killing ability of CAR-T-4, CAR-T-5, and CAR-T-6 with CD28 costimulatory domains is higher than that of CAR-T-1, CAR-T-2, and CAR-T-3 with 4-1BB costimulatory domains, and is consistent with the killing efficiency trend of short-term killing in Example 4. Among them, the killing ability of CAR-T-3 is higher than that of CAR-T-1 and CAR-T-2, and the killing ability of CAR-T-6 is higher than that of CAR-T-4 and CAR-T-5, indicating that in terms of the in vitro killing ability of CAR-T cells, CAR molecules with IgG4 hinge regions are superior to CAR molecules with CD8 or CD28 hinge regions. Moreover, the tumor killing rate of CAR-T-6 is faster, and the killing efficiency is also the best among the six groups of CAR-T cells. Therefore, MUC18 CAR-T with IgG4 hinge region and CD28 costimulatory signaling domain has higher killing efficiency of target cells.

[0095] Example 6: Long-term killing efficiency of MUC18 CAR-T cells containing strep tag II, humanized scFv, different hinge regions, transmembrane regions and signaling domains under repeated stimulation of MUC18-positive tumor cells

[0096] By gene synthesis and homologous recombination, the luciferase sequence and enhanced green fluorescent protein (EGFP) tag sequence were constructed into the lentiviral vector pLenti6.3 / V5. This lentiviral vector was used to infect the MUC18-positive A375 tumor cell line, and an A375 tumor cell line overexpressing luciferase and EGFP fluorescent tags was screened and named A375-LAE. After reviving A375-LAE cells, they were passaged every 2 days and cultured in DMEM medium containing 10% serum. When A375-LAE cells reached a sufficient number, they were digested with 0.25% trypsin. After the cells were detached, the digestion was terminated with 2 volumes of serum-containing medium. The cells were collected and centrifuged in a centrifuge tube and resuspended in DMEM medium containing 10% serum to adjust the cell density to 3×10 cells per ml. 5 3×10 cells were added to each well of a 24-well cell culture plate (Cat#142475, Thermo). 5 Target cells A375-LAE were added, and three replicate wells were set up for each group. After 24 hours, the tumor cells adhered to the wall and the supernatant was removed. 3×10 resuspended in X-VIVO 15 medium was added to each well at an E:T ratio of 1:1. 5 CAR-T cells were added to make up the total number of T cells and the volume was adjusted to 2 ml with X-VIVO 15 medium. The 24-well plate was placed in a 37°C, 5% CO2 incubator for co-incubation. After co-incubation for 48 hours, the tumor cells were digested and plated. 3×10 5 A375-LAE target cells were plated 24 hours after tumor cell attachment, i.e., 72 hours after CAR-T cells were co-incubated with A375-LAE. The tumor cells plated the previous day had completed attachment and the supernatant was removed. Simultaneously, 1 ml of supernatant from each well of the 24-well plate from the first round of co-incubation was gently aspirated and discarded without agitation. The remaining 1 ml of supernatant in the plate was used to resuspend the CAR-T cells. The cells were then rinsed once with 1 ml of fresh X-VIVO 15 medium and transferred to the corresponding second batch of tumor-containing plates for the second round of co-incubation. The above plating and transfer process was repeated. If the CAR-T cells failed to completely kill the tumor cells within 48 hours of co-incubation with tumor cells, the CAR-T cells and digested tumor cells from each well were collected into a centrifuge tube and centrifuged at 400g for 5 minutes. The supernatant was discarded and resuspended in 100 μl of DPBS. Each group of cells was then fluorescently labeled with antibodies detecting CD3 and CAR expression and analyzed using a full-spectrum flow cytometer.

[0097] Figure 7 shows the residual tumor cell and CAR-T cell counts in each group after five rounds of repeated stimulation of MUC18 CAR-T cells with MUC18-positive tumor cells. Figure 7-A shows that CAR-T-6, CAR-T-3, CAR-T-5, and CAR-T-4 have relatively few residual tumor cells, while Figure 7-B shows that CAR-T-4, CAR-T-5, and CAR-T-6 have relatively large residual CAR-T cells. Overall, within the 4-1BB costimulatory domain group, CAR-T-3 exhibited significantly better tumor suppression than CAR-T-1 and CAR-T-2, but the number of residual CAR-T cells was slightly lower than that of CAR-T-1. Within the CD28 costimulatory domain group, CAR-T-6 exhibited slightly better tumor suppression than CAR-T-4 and CAR-T-5, but the difference was not significant. There was no significant difference in CAR-T cell proliferation among CAR-T-4, CAR-T-5, and CAR-T-6.

[0098] Example 7: Tumor Suppression Ability of MUC18 CAR-T Cells Containing Strep Tag II, Humanized scFv, Different Hinge Regions, Transmembrane Regions, and Signaling Domains in Tumor-Bearing Mice

[0099] T cells were isolated from peripheral blood mononuclear cells of healthy volunteers using CD3 / CD28 antibody-coupled magnetic beads. The isolated and purified T cells were cultured at a concentration of 1.5×10 6 cells / ml and inoculated into X-VIVO 15 medium containing 500IU / mL IL-2. After 24 hours of culture, the lentiviral vector carrying the MUC18 CAR molecule sequence was transfected into T cells at an MOI of 1. 24 hours after lentiviral infection, the cell medium was changed to remove the virus in the suspension, and the cells were inoculated into a fresh X-VIVO 15 culture system containing 500IU / mL IL-2 and continued to be cultured. Five days after lentiviral infection, the cells in the culture system were repeatedly blown up and down with a pipette and collected into a centrifuge tube, and then placed on a magnetic stand for 5 minutes to remove the CD3 / CD28 magnetic beads. After that, the medium was changed every 48 hours at 5.0×10 5 The cells were inoculated into fresh X-VIVO 15 culture system containing 500 IU / mL IL-2 and continued to be cultured. On the 9th day of culture, T cells were harvested, centrifuged and counted. Approximately 5.0×10 cells / mL were taken from each group. 5 The cells were stained and the CAR molecule transduction efficiency of each group of cells was detected using full-spectrum flow cytometry.

[0100] A total of 36 5-6 week-old NCG mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd., China) were divided into 6 groups of 6 mice each. Each mouse was subcutaneously inoculated with 1.0×10 6A375 cells were inoculated, and the tumor volume of the mice was measured and analyzed within 13 days after inoculation to verify whether the mouse melanoma model was successfully established. After the mouse melanoma model was successfully established, each group of mice was injected with MUC18 CAR-T cells (3.0×10 6 CAR-T cells / mouse). Control mice were injected with the corresponding number of T cells and the corresponding volume of PBS as a negative control. The day of CAR-T cell injection was marked as day 1. CAR-T detection was performed on peripheral blood of mice twice a week for the first two weeks and once a week thereafter. The tumor volume of the mice was measured on the day before or after blood collection.

[0101] Figure 8-A shows that compared to the control T cell group and the PBS group, the CAR-T-3, CAR-T-4, CAR-T-5, and CAR-T-6 groups significantly inhibited tumor growth. On day 18 after CAR-T infusion, the tumor volume of the CAR-T mice in the CAR-T groups decreased to zero. The CAR-T-6 group showed a faster tumor suppression rate than the other groups, indicating that MUC18 CAR-T cells with humanized scFv can quickly and effectively eliminate inoculated A375 melanoma tumors in mice. Figure 8-B shows the trend of CAR-T cell proliferation in the peripheral blood of mice in each group. CAR-T-5 cells showed the fastest proliferation, indicating that MUC18 CAR-T cells can effectively inhibit the growth of MUC18-positive tumors in tumor-bearing mice and have good CAR-T cell proliferation ability in vivo.

[0102] Example 8: Construction of MUC18 CAR molecules containing humanized scFv, IgG4 hinge region, different transmembrane regions and different signaling domains and preparation of CAR-T

[0103] Based on the tag-sequenced CAR vector described in Example 1, a gene cloning method was used to replace the CD28 transmembrane region of CAR-T-6 with that of CD8, thereby generating CAR-T-8. Furthermore, the wild-type CD3ζ was replaced with CD3ζ mutant 2, thereby generating CAR-T-7. Using gene cloning, signal peptide and tag-removed CAR sequence fragments were obtained. These sequence fragments were homologously recombined with the CAR gene fragments of CAR-T-6, CAR-T-7, and CAR-T-8, respectively. The resulting homologous recombinant gene fragments were then constructed into the lentiviral vector pLenti6.3 / V5 using enzyme digestion and ligation, resulting in the complete tag-free MUC18 CAR vectors CAR-T-9, CAR-T-10, and CAR-T-11. The CAR molecular structures of CAR-T-7 to CAR-T-11 are shown in Table 3.

[0104] T cells were isolated from peripheral blood mononuclear cells of healthy volunteers using CD3 / CD28 antibody-coupled magnetic beads. The isolated and purified T cells were cultured at a concentration of 1.5×10 6 cells / mL and inoculated into the X-VIVO 15 culture system containing 500IU / mL IL-2. After 24 hours of culture, the lentiviral vector carrying the MUC18 CAR molecule sequence was transfected into the T cells at an MOI of 1.5. 24 hours after lentiviral infection, the cell medium was changed, the virus in the suspension was removed, and the cells were inoculated into a fresh X-VIVO 15 culture system containing 500IU / mL IL-2 and continued to be cultured. Four days after lentiviral infection, the cells in the culture system were repeatedly blown up and down with a pipette and collected into a centrifuge tube, then placed on a magnetic stand for 5 minutes to remove the CD3 / CD28 magnetic beads, and the T cells were centrifuged and counted. On the 6th day of lentiviral infection and every 2 days thereafter, about 5×10 5 The cells were stained and the CAR molecule transduction efficiency of each group of cells was detected using full-spectrum flow cytometry.

[0105] Figure 9 shows the CAR transduction efficiency of each group of cells 6, 8, and 11 days after lentivirus infection. The CAR molecule expression positivity of CAR-T-7 and CAR-T-8 was significantly higher than that of CAR-T-6, and the CAR molecule expression positivity of CAR-T-10 and CAR-T-11 was also significantly higher than that of CAR-T-9. This suggests that the CD8 transmembrane region significantly improves the transduction efficiency of CAR molecules compared to the CD28 transmembrane region.

[0106] Example 9: In vitro proliferation capacity of CAR-T containing humanized scFv, IgG4 hinge region, different transmembrane regions and different signaling domains

[0107] During the CAR-T-7~11 cell culture process, lentiviral infection of T cells was considered the second day of culture. Thereafter, every 2 to 3 days, the CAR-T cells were collected and centrifuged, resuspended in 1-2 mL of culture medium, and 10 μL of cells were diluted a certain multiple and stained with trypan blue in a one-to-one ratio. The cell viability and viable cell concentration were recorded using a cell counter, and the total cell number of each group was calculated to statistically analyze the proliferation of cells in each group.

[0108] Figure 10 shows that the total cell proliferation of CAR-T-7 and CAR-T-10 is similar to that of the control T group, while the total cell proliferation of CAR-T-6 and CAR-T-11 is slightly lower; Figure 11 shows the proliferation rate of CAR-T cells from the sixth to the eighth day of culture. The results show that the CAR-T cell proliferation rate of CAR-T-10 is the fastest, indicating that the sequence combination of this CAR molecule increases the proliferation rate of CAR-T cells, but overall there is little difference in the proliferation rate of CAR-T cells in each group.

[0109] Example 10: Real-time killing efficiency of MUC18 CAR-T cells containing humanized scFv, IgG4 hinge region, different transmembrane regions and different signaling domains against MUC18-positive tumor cell lines in vitro

[0110] The MUC18-expressing tumor cell line A375 was passaged every 2 days and cultured in DMEM medium containing 10% serum. When A375 cells reached sufficient numbers, they were digested with 0.25% trypsin. After the cells detached, digestion was terminated with 2 volumes of serum-containing medium. The cells were collected and centrifuged in a centrifuge tube. The tumor cells were resuspended in DMEM medium containing 10% serum to a concentration of 2 × 10 cells per ml. 5 The concentration of cells was set aside and a portion was subcultured.

[0111] The treated tumor cells were plated as follows: 50 μl of tumor cell culture medium was added to each well of the 96-well plate (E-Plate96) equipped with the real-time killing instrument, and the plate was placed on the real-time killing instrument for instrument self-check; the E-plate96 was removed and 100 μl (2×10 4 The tumor cell suspension was mixed evenly with 100 μL of cells / well and placed at room temperature for 30 minutes. The E-plate 96 was placed on a real-time killing instrument in a 37°C, 5% CO2 incubator to detect the cell proliferation curve overnight. When the cell index value reached 0.6 or above, the instrument was stopped, the E-plate 96 was removed and placed in a biosafety cabinet. 50 μL of supernatant was aspirated from each well with a pipette and discarded. 100 μL of 2×10 3 The total number of T cells prepared with X-VIVO 15 medium was consistent and the concentration was 2×10 per ml. 4 The E-Plate96 detection plate was placed on the real-time killing instrument to continue monitoring and observe the killing effect of each group of CAR-T cells on tumor cells.

[0112] The real-time killing results are shown in Figure 12. Tumor cell proliferation in the T cell group was consistent with that in the tumor cell-alone group, indicating that the T cells lacked tumor-killing ability. The real-time killing results of T cell culture performed on day 8 are shown in Figure 12-A. All CAR-T cells in each group demonstrated significant tumor-killing ability, with CAR-T-11 and CAR-T-6 showing the fastest killing rates. The real-time killing results of T cell culture performed on day 11 are shown in Figure 12-B. CAR-T-8 demonstrated a higher tumor-killing rate than CAR-T-6 and CAR-T-7; CAR-T-11 demonstrated a higher tumor-killing rate than CAR-T-9 and CAR-T-10. CAR-T-8 demonstrated the greatest tumor-killing effect, while CAR-T-11, CAR-T-6, and CAR-T-7 demonstrated similar tumor-killing rates. These results indicate that MUC18 CAR-T containing humanized scFv and IgG4 hinge region has a strong killing effect on MUC18-positive tumor cells, and when the CAR molecule contains the CD8 transmembrane region, it can enhance the real-time killing ability of CAR-T cells in vitro.

[0113] Example 11: Long-term killing efficiency of MUC18 CAR-T cells containing humanized scFv, IgG4 hinge region, different transmembrane regions and different signaling domains against MUC18-positive tumor cell lines in vitro

[0114] After A375-LAE cells recovered, they were passaged every 2 days and cultured in DMEM medium containing 10% serum. When A375-LAE cells reached a sufficient number, they were digested with 0.25% trypsin. After the cells fell off, the digestion was terminated with 2 volumes of serum-containing medium. The cells were collected and centrifuged in a centrifuge tube and resuspended in DMEM medium containing 10% serum to adjust the cell density to 5 × 10 cells per ml. 4 5×10 cells were added to each well of a 48-well cell culture plate. 4 Target cells A375-LAE were added, and three replicates were set up for each group. After 24 hours, the tumor cells adhered to the wall and the supernatant was removed. 5×10 resuspended in X-VIVO 15 medium was added to each well at an E:T ratio of 1:1. 4 CAR-T cells were added to each group to make the total number of T cells in each group the same. The volume was adjusted to 1 mL with X-VIVO 15 medium and the 48-well plate was placed in a 37°C, 5% CO2 incubator for co-incubation. After co-incubation for 48 hours, the tumor cells were digested and plated. 5×10 4Resuspend A375-LAE target cells in DMEM medium containing 10% serum. After 24 hours of tumor cell attachment, that is, when CAR-T cells are co-incubated with A375-LAE for 72 hours, the tumor cells plated the previous day have completed attachment and the supernatant is removed. At the same time, gently aspirate 500 μl of supernatant from each well of the culture plate from the first round of co-incubation and discard it. Do not shake or pipette. Use the remaining 500 μl in the well plate to resuspend the CAR-T cells, then rinse once with 500 μl of fresh X-VIVO 15 medium and transfer to the corresponding second batch of tumor-containing well plates for the second round of co-incubation. The above plating and transfer experimental process is repeated. When the CAR-T cells cannot completely kill the tumor within 48 hours of co-incubation with tumor cells, the CAR-T cells in each well and the digested tumor cells are mixed and recovered into the corresponding centrifuge tube, centrifuged at 400g for 5 minutes, the supernatant is discarded and resuspended in 100 microliters of DPBS; then, CD3 and CAR expression detection antibodies are added to each group of cells for fluorescent labeling, and classification and counting are detected using a full-spectrum flow cytometer.

[0115] Figures 13 and 14 show the residual numbers of tumor cells and CAR-T cells collected from each group after five rounds of repeated stimulation with MUC18-positive tumor cells, starting on day 8 of T cell culture, and after four rounds of repeated stimulation with MUC18-positive tumor cells, starting on day 11 of T cell culture. As shown in Figures 13-A and 14-A, the residual number of tumor cells in each CAR-T group was significantly reduced compared to the T cell group, with no significant differences between the CAR-T groups. As shown in Figures 13-B and 14-B, the residual number of CAR-T cells in the unlabeled CAR-T-11 and CAR-T-10 groups was significantly greater than that in the CAR-T-9 group, with no significant difference in the residual number of CAR-T cells between the CAR-T-11 and CAR-T-10 groups. The remaining CAR-T cells in the labeled CAR-T-6, CAR-T-7, and CAR-T-8 groups were not significantly different from those in the unlabeled CAR-T-11 and CAR-T-10 groups, indicating that MUC18 CAR-T cells can still effectively kill tumor cells and maintain a certain number of CAR-T cells after multiple rounds of stimulation with MUC18-positive tumor cells.

[0116] Example 12: Tumor Suppression Ability of MUC18 CAR-T Cells Containing Humanized scFv, IgG4 Hinge Region, Different Transmembrane Regions and Signaling Domains in Tumor-Bearing Mice

[0117] T cells were isolated from peripheral blood mononuclear cells of healthy volunteers using CD3 / CD28 antibody-coupled magnetic beads. The isolated and purified T cells were cultured at a concentration of 1.5×10 6cells / mL and inoculated into the X-VIVO 15 culture system containing 500IU / mL IL-2. After 24 hours of culture, the lentiviral vector carrying the MUC18 CAR molecule sequence was used to infect T cells at an MOI of 1.5. 24 hours after lentiviral infection, the cell medium was changed, the virus in the suspension was removed, and the cells were inoculated into a fresh X-VIVO 15 culture system containing 500IU / mL IL-2 and continued to be cultured. Four days after lentiviral infection, the cells in the culture system were repeatedly blown up and down with a pipette and collected into a centrifuge tube, and then placed on a magnetic stand for 5 minutes to remove the CD3 / CD28 magnetic beads. Thereafter, the medium was changed every 48 hours at 5.0×10 5 The cells were inoculated into fresh X-VIVO 15 culture system containing 500 IU / mL IL-2 and continued to be cultured. On the 8th day of culture, T cells were harvested, centrifuged and counted. Approximately 5.0×10 cells / ml were taken from each group. 5 The cells were stained and the CAR molecule transduction efficiency of each group of cells was detected using full-spectrum flow cytometry.

[0118] A total of 48 5-6 week old NCG mice were divided into 8 groups of 6 mice each. Each mouse was subcutaneously inoculated with 1×10 6 A375 cells were inoculated. Within 13 days after inoculation, the tumor volume of the mice was measured and analyzed to verify whether the mouse melanoma model was successfully established. After the mouse melanoma model was successfully established, each group of mice was injected with MUC18 CAR-T cells (2×10 6 CAR-T cells / mouse). Control mice were injected with the corresponding number of T cells and the corresponding volume of PBS as a negative control. The day of CAR-T cell injection was marked as day 1. CAR-T detection was performed on peripheral blood of mice twice a week for the first two weeks and once a week thereafter. The tumor volume of the mice was measured on the day before or after blood collection.

[0119] Figure 15-A shows that compared to the control T cell group and the PBS group, each CAR-T group significantly inhibited tumor growth. On day 18 after CAR-T infusion, the tumor volume of mice in the CAR-T-7 group decreased to zero. On day 22 after CAR-T infusion, the tumor volume of mice in the CAR-T-9 and CAR-T-11 groups decreased to zero. On day 22 after CAR-T infusion, the tumor volume of 5 mice in the CAR-T-10 group decreased to zero. Figure 15-B shows that all CAR-T cells in each group expanded to some extent in the mice after infusion. This indicates that both tagged and untagged MUC18 CAR-T cells can significantly inhibit the growth of MUC18-positive tumors in tumor-bearing mice and can induce a certain amount of CAR-T cell expansion.

Claims

1. A chimeric antigen receptor targeting MUC18, comprising: Antibody fragments that specifically recognize MUC18, IgG4 hinge region, CD8 transmembrane region or CD28 transmembrane region, CD28 costimulatory signaling domain or 4-1BB costimulatory signaling domain, CD3ζ signaling domain; The antibody fragment comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises an HC CDR1 with the amino acid sequence shown in SEQ ID NO: 7, an HC CDR2 with the amino acid sequence shown in SEQ ID NO: 8, and an HC CDR3 with the amino acid sequence shown in SEQ ID NO: 9, and the VL comprises an LC CDR1 with the amino acid sequence shown in SEQ ID NO: 10, an LC CDR2 with the amino acid sequence of RAS, and an LC CDR3 with the amino acid sequence shown in SEQ ID NO:

11.

2. The chimeric antigen receptor of claim 1, wherein the VH is at least 85% identical to the amino acid sequence of SEQ ID NO: 5, and the VL is at least 85% identical to the amino acid sequence of SEQ ID NO: 6; Preferably, the VH comprises the amino acid sequence of SEQ ID NO: 5, and the VL comprises the amino acid sequence of SEQ ID NO:

6.

3. The chimeric antigen receptor of claim 1 or 2, wherein the hinge region is an IgG4 hinge region having the amino acid sequence of SEQ ID NO: 15, the transmembrane region is a CD8 transmembrane region having the amino acid sequence of SEQ ID NO: 16 or a CD28 transmembrane region having the amino acid sequence of SEQ ID NO: 17, the costimulatory signaling domain is a CD28 costimulatory signal having the amino acid sequence of SEQ ID NO: 18 or a 4-1BB costimulatory signaling domain having the amino acid sequence of SEQ ID NO: 19, and the CD3 zeta signaling domain is a CD3 zeta signaling domain having the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23; Preferably, the hinge region is an IgG4 hinge region having the amino acid sequence shown in SEQ ID NO: 15, the transmembrane region is a CD8 transmembrane region having the amino acid sequence shown in SEQ ID NO: 16, the costimulatory signal domain is a CD28 costimulatory signal having the amino acid sequence shown in SEQ ID NO: 18, and the CD3ζ signal domain has the amino acid sequence shown in SEQ ID NO:

20.

4. The chimeric antigen receptor according to any one of claims 1 to 3, further comprising a signal peptide having an amino acid sequence as shown in SEQ ID NO: 1, and optionally further comprising a tag having an amino acid sequence as shown in SEQ ID NO:

2.

5. The chimeric antigen receptor according to any one of claims 1 to 4, which has an amino acid sequence as shown in SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34, or an amino acid sequence that is at least 85% identical to these sequences.

6. An isolated nucleic acid encoding the chimeric antigen receptor according to any one of claims 1 to 5.

7. A vector comprising the nucleic acid according to claim 6.

8. An isolated cell comprising the chimeric antigen receptor according to any one of claims 1 to 5, or the vector according to claim 7.

9. Use of the chimeric antigen receptor according to any one of claims 1 to 5, the nucleic acid according to claim 6, the vector according to claim 7, or the cell according to claim 8 in the preparation of a medicament for treating MUC18-positive tumors.

10. The use of claim 9, wherein the medicament is used to treat melanoma, sarcoma, triple-negative breast cancer, ovarian cancer, placental trophoblastic tumor, choriocarcinoma, small cell lung cancer, clear cell renal carcinoma, thyroid cancer, liver cancer, head and neck squamous cell carcinoma, or bile duct cancer.

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