Chimeric antigen receptor against glypican-3 and use thereof

Abstract

The present application relates to a chimeric antigen receptor, comprising genes encoding a GPC3-targeting scFv, hinge region, transmembrane region, CD3ζ signaling domain, costimulatory signaling domain, and CD40L protein, wherein the scFv has an amino acid sequence as shown in SEQ ID NO: 1; the costimulatory signaling domain is derived from CD28; the CD40L has an amino acid sequence as shown in SEQ ID NO: 5; and the CD3ζ signaling domain is one of the following variants of a natural CD3ζ signaling domain sequence (corresponding to positions 52-163 of an amino acid sequence listed in NP_000725.1): 1) substitutions of the membrane-proximal amino acid V2 with L, D9 with E, and Q15 with K in the natural CD3ζ signaling domain sequence; 2) a substitution of a tyrosine phosphorylation site Y90 with F in the natural CD3ζ signaling domain sequence; and 3) substitutions of the membrane-proximal amino acid V2 with L, D9 with E, Q15 with K, and the tyrosine phosphorylation site Y90 with F in the natural CD3ζ signaling domain sequence.

Description

Chimeric antigen receptor against glypican 3 and its use

[0001] This application claims priority to the Chinese patent application with application date of November 20, 2023 and application number 202311541466.8. Technical Field

[0002] The present invention relates to a novel isolated anti-Glypican-3 (GPC3) antibody, a chimeric antigen receptor (CAR) containing the antibody, a cell containing the chimeric antigen receptor, and uses thereof. Background Art

[0003] Cancer immune cell therapy, particularly chimeric antigen receptor T cells (CAR-T), has achieved remarkable efficacy in the treatment of hematologic malignancies. Major breakthroughs have been achieved in multiple clinical studies targeting CD19- and BCMA-positive hematologic malignancies. Preclinical and exploratory clinical studies of solid tumors, such as liver cancer, have also shown promising results, bringing new hope to cancer patients. Seven CAR-T drugs targeting hematologic malignancies are currently marketed worldwide.

[0004] CAR-T technology combines the specificity of antibodies with the cytotoxicity of T cells, creating an effective adoptive immunity pathway. CARs typically consist of an extracellular antigen-binding domain, a hinge region, a transmembrane region, a costimulatory signaling domain, and an intracellular CD3ζ signaling domain. However, CAR-T therapy often faces multiple obstacles in the treatment of solid tumors, including limited efficacy and off-target effects. Currently, global CAR-T drug development for malignant solid tumors like liver cancer is still in the clinical trial stage, and no related products have been marketed domestically or internationally.

[0005] GPC3 is highly expressed in hepatocellular carcinoma (HCC) (expression rate approximately 74.8%) and in various malignant solid tumors, including lung squamous cell carcinoma, yolk sac tumor, melanoma, and osteosarcoma. It is not expressed in normal tissues, such as the liver, kidney, and stomach, of healthy individuals (Bi et al., Oncotarget 8:52866, 2017; Gao et al., Clin Cancer Res 20:6418, 2014). Monoclonal antibodies targeting GPC3 have shown good safety in clinical trials (Abou-Alfa et al., J Hepatol 65:289, 2016; Zhu et al., Clin Cancer Res 19:920, 2013). The results of the world's first early clinical study of GPC3-targeted CAR-T cells for the treatment of advanced liver cancer patients (Shi et al., Clin Cancer Res 19:3259, 2020) showed that CAR-T therapy has good safety in hepatocellular carcinoma, with two partial responses observed among 13 patients, demonstrating certain clinical application potential. However, there is still much room for improvement in efficacy. Therefore, how to improve the efficacy of CAR-T in the treatment of solid tumors such as liver cancer through rational drug design is an important technical issue that needs to be addressed.

[0006] Compared with the treatment of hematological tumors, the probability of CAR-T cells contacting target antigens in patients with solid tumors is low, and the solid tumor microenvironment is highly immunosuppressive. The peak expansion of CAR-T cells in patients with solid tumors is much lower than that in patients with hematological tumors. This is an important reason for the poor effect of CAR-T cells in treating solid tumors. Therefore, the treatment of solid tumors requires CAR-T cells to have stronger killing activity, in vivo expansion ability and sustainability.

[0007] Summary of the Invention

[0008] Currently known CAR molecules targeting GPC3 typically use a simple second-generation CAR design, with both the costimulatory signaling domain and the intracellular CD3ζ signaling domain using wild-type molecules. The inventors of this application have discovered that these molecular designs may result in the CAR-T cell being unable to achieve the optimal binding state for immune synapse formation when binding to the target molecule, or that the signal transmission to the cell after immune synapse formation is too strong or too weak, thereby affecting the survival and proliferation of CAR-T cells in the body, the persistence of their tumor cell killing ability, and the regulation of the entire immune system function, ultimately manifesting as differences in clinical efficacy and safety.

[0009] The intracellular signaling domain is a fundamental element for CAR-T activation and its killing effect. Its structural design is crucial for the full activation and sustained survival of CAR-T cells. Each CD3ζ signaling domain has three immunoreceptor tyrosine-based activation motifs (ITAMs), which can amplify the activation signal. The three ITAMs in the CD3ζ signaling domain have a total of six tyrosine phosphorylation sites. The order and number of phosphorylation of these six tyrosine sites play a key role in whether T cells can be activated and play an important role in the development and differentiation of T cells after activation (Kersh et al., Science 281:572, 2018). Redundant ITAM activation in the CD3ζ signaling domain can promote T cell differentiation and exhaustion. In addition, during the activation process of the CD3ζ signaling domain, the spatial conformation of its juxta-membrane region also plays an important role in the formation of the immune synapse (Guy et al., Immunol Rev 232:7, 2009).

[0010] Therefore, the applicant of the present application mutated the amino acids of the membrane-proximal end and immune receptor tyrosine activation motif of the CD3ζ signaling domain to improve the signal activation intensity and persistence of the CD3ζ signaling domain (refer to patent PCT / CN2021 / 076247).

[0011] Furthermore, the present invention co-expresses the co-stimulatory ligand CD40L in CAR-T cells to identify and kill CD40-positive tumor cells and activate CD40-positive antigen-presenting cells, ultimately obtaining CAR-T cells with better therapeutic effects.

[0012] Therefore, the present invention provides a novel CAR targeting GPC3, which comprises genes encoding a humanized scFv targeting GPC3, a CD3ζ signaling domain, and a CD40L protein.

[0013] Specifically, the present invention provides:

[0014] (1) A chimeric antigen receptor comprising genes encoding a scFv targeting GPC3, a hinge region, a transmembrane region, a CD3ζ signaling domain, a co-stimulatory signaling domain, and a CD40L protein; wherein,

[0015] The scFv has the amino acid sequence shown in SEQ ID NO: 1;

[0016] The co-stimulatory signal domain is derived from CD28;

[0017] The CD40L has the amino acid sequence shown in SEQ ID NO: 5;

[0018] The CD3ζ signaling domain is one of the following variants of the native CD3ζ signaling domain sequence:

[0019] 1) The membrane-proximal amino acid V2 in the native CD3ζ signaling domain sequence was replaced with L, D9 with E, and Q15 with K;

[0020] 2) replacing the tyrosine phosphorylation site Y90 in the native CD3ζ signaling domain sequence with F;

[0021] 3) replacing the membrane-proximal amino acid V2 with L, D9 with E, Q15 with K, and the tyrosine phosphorylation site Y90 with F in the native CD3ζ signaling domain sequence;

[0022] The natural CD3ζ signal domain sequence corresponds to positions 52-163 of the amino acid sequence listed in NP_000725.1.

[0023] (2) The chimeric antigen receptor according to (1) above, wherein the hinge region or transmembrane region is derived from CD8α, CD28, IgG1, or IgG4.

[0024] (3) The chimeric antigen receptor according to (1) or (2) above, further comprising a gene encoding a signal peptide.

[0025] (4) The chimeric antigen receptor according to any one of (1) to (3) above, wherein the gene encoding the CD40L protein is linked to the C-terminus of the CD3ζ signaling domain via a linker peptide.

[0026] (5) The chimeric antigen receptor according to any one of (1) to (4) above, wherein the CD3ζ signaling domain has the amino acid sequence shown in SEQ ID NO: 3.

[0027] (6) The chimeric antigen receptor according to any one of (1) to (5) above, which comprises or has the amino acid sequence shown in SEQ ID NO: 9.

[0028] (7) An isolated nucleic acid encoding the chimeric antigen antibody according to any one of (1) to (6) above.

[0029] (8) A vector comprising the nucleic acid described in (7) above.

[0030] (9) An isolated cell comprising the chimeric antigen antibody according to any one of (1) to (6) above or the vector according to (8) above, wherein the cell is not a germ cell or a fertilized egg.

[0031] (10) Use of the chimeric antigen receptor according to any one of (1) to (6), the nucleic acid according to (7), the vector according to (8), or the cell according to (9) in the preparation of a drug for treating GPC3-positive malignant tumors.

[0032] (11) The use according to (10) above, wherein the drug is used to prepare a drug for treating liver cancer, lung cancer, osteosarcoma or melanoma.

[0033] Compared with existing CAR-T, CAR-T cells containing the novel CAR of the present invention can significantly improve the killing ability, expansion efficiency and duration of CAR-T cells targeting GPC3 in vivo, improve the efficacy of killing tumors, and ultimately achieve the purpose of improving clinical efficacy and reducing disease recurrence. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 shows the transduction efficiency of GPC3 CAR lentivirus.

[0035] Figure 2 shows the proliferation curves of total T cells and CAR-T cells during the CAR-T preparation process.

[0036] FIG3 shows the expression of GPC3 and CD40 on the surface of HepG2 and genetically engineered HepG2 cells.

[0037] Figure 4 is the killing curve of CAR-T-0 and CAR-T-1 on HepG2 cells.

[0038] Figure 5 shows the killing effects of CAR-T-1 and CAR-T-2 on target cells with different GPC3 and CD40 expression levels.

[0039] Figure 6 shows the expansion of GPC3 CAR-T cells under repeated stimulation of HepG2 cells.

[0040] Figure 7 shows cytokine secretion by GPC3 CAR-T cells upon stimulation with Huh7 and HepG2 cells.

[0041] Figure 8 shows the effects of CAR-T-1 and CAR-T-2 on the expression of antigen presentation-related proteins on the surface of Daudi cells.

[0042] Figure 9 shows the total T cells, CAR expression rate, and CAR-T cell proliferation curve during the CAR-T preparation process.

[0043] FIG10 shows the results of detecting the differentiation and exhaustion levels of CAR-T cells in a resting state.

[0044] Figure 11 shows the number of tumor cells and CAR-T cells after repeated stimulation of target cells.

[0045] FIG12 shows the tumor inhibition effect (A) and in vivo expansion (B) of CAR-T-2 in tumor-bearing mice. DETAILED DESCRIPTION

[0046] 1. scFv of GPC3 CAR molecule

[0047] The scFv of the GPC3 CAR molecule of the present invention is composed of a light chain variable region (VL), a linker, and a heavy chain variable region (VH), wherein VL and VH can be derived from a monoclonal antibody that specifically recognizes the GPC3 target (reference patent CN200580000807.4), the amino acid sequence of scFv is such as SEQ ID NO: 1, and the nucleotide sequence of scFv is such as SEQ ID NO: 2.

[0048] 2. Design of the GPC3 CAR Molecular Signaling Domain

[0049] Based on the natural CD3ζ signal domain sequence (corresponding to amino acid sequence positions 52-163 listed in NP_000725.1), the CD3ζ signal domain of the CAR molecule was designed, including:

[0050] 1) The membrane-proximal amino acid V2 in the native CD3ζ signaling domain sequence was replaced with L, D9 with E, and Q15 with K;

[0051] 2) The tyrosine phosphorylation site Y90 in the native CD3ζ signal domain sequence was replaced with F.

[0052] In one embodiment, the optimized CD3zeta signaling domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: 4.

[0053] In one embodiment, the optimized CD3ζ signaling domain comprises V2L, D9E, Q15K, and Y90F substitutions, and a CD40L protein linked to a P2A sequence is added to the C-terminus of the CD3ζ signaling domain.

[0054] The amino acid sequence of CD40L is shown in SEQ ID NO: 5, and the nucleotide sequence is shown in SEQ ID NO: 6.

[0055] 3. Composition of the GPC3 CAR molecule

[0056] The present invention relates to a chimeric antigen receptor targeting GPC3, which comprises genes encoding a signal peptide, a scFv that specifically recognizes GPC3, a hinge region, a transmembrane region, a CD28 costimulatory signal domain, a CD3ζ signal domain and CD40L.

[0057] In some embodiments, the signal peptide is colony stimulating factor 2 receptor alpha signal peptide.

[0058] In some embodiments, the scFv comprises, consists essentially of, or consists of the amino acid sequence shown in SEQ ID NO: 1.

[0059] In some embodiments, the hinge region or transmembrane region is derived from CD8α, CD28, IgG1, or IgG4.

[0060] In some embodiments, the CD3ζ signaling domain is a variant of the native CD3ζ signaling domain sequence (corresponding to amino acid positions 52-163 of the sequence listed in NP_000725.1):

[0061] 1) The membrane-proximal amino acid V2 in the native CD3ζ signaling domain sequence was replaced with L, D9 with E, and Q15 with K;

[0062] 2) The tyrosine phosphorylation site Y90 in the native CD3ζ signal domain sequence was replaced with F.

[0063] 3) The membrane-proximal amino acid V2 in the native CD3ζ signaling domain sequence was replaced with L, D9 was replaced with E, Q15 was replaced with K, and the tyrosine phosphorylation site Y90 was replaced with F.

[0064] In some more specific embodiments, the CD3ζ signaling domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: 4.

[0065] In one embodiment, the present invention provides a chimeric antigen receptor (CAR-1) comprising GPC3 scFv, a natural costimulatory signaling domain from CD28, and an optimized CD3ζ intracellular signaling domain, which has an amino acid sequence as shown in SEQ ID NO: 7 and a nucleotide sequence as shown in SEQ ID NO: 8.

[0066] In one embodiment, the present invention provides a chimeric antigen receptor (CAR-2) comprising GPC3 scFv, a natural costimulatory signaling domain from CD28, an optimized CD3ζ intracellular signaling domain and a CD40L protein, which has an amino acid sequence as shown in SEQ ID NO: 9 and a nucleotide sequence as shown in SEQ ID NO: 10.

[0067] In one embodiment, the present invention uses a chimeric antigen receptor (CAR-0) comprising GPC3 scFv, a natural costimulatory signaling domain from CD28, and a natural CD3ζ intracellular signaling domain as a comparative example, which has an amino acid sequence as shown in SEQ ID NO: 11.

[0068] In other embodiments, the present invention uses a chimeric antigen receptor (CAR-3) comprising GPC3 scFv, a natural costimulatory signal domain from CD28, an optimized CD3ζ intracellular signal domain and ICOSL protein as a comparative example, which has an amino acid sequence as shown in SEQ ID NO: 12; a chimeric antigen receptor (CAR-4) comprising GPC3 scFv, a natural costimulatory signal domain from CD28, an optimized CD3ζ intracellular signal domain and OX40L protein as a comparative example, which has an amino acid sequence as shown in SEQ ID NO: 13; a chimeric antigen receptor (CAR-5) comprising GPC3 scFv, a natural costimulatory signal domain from CD28, an optimized CD3ζ intracellular signal domain and CD70 protein as a comparative example, which has an amino acid sequence as shown in SEQ ID NO: 14; a chimeric antigen receptor (CAR-6) comprising GPC3 scFv, a natural costimulatory signal domain from CD28, an optimized CD3ζ intracellular signal domain and CD70 protein as a comparative example, which has an amino acid sequence as shown in SEQ ID NO: 15; A chimeric antigen receptor (CAR-6) comprising scFv, a natural costimulatory signaling domain from CD28, an optimized CD3ζ intracellular signaling domain, and 4-1BBL protein is used as a comparative example and has an amino acid sequence as shown in SEQ ID NO: 15.

[0069] In other embodiments, the present invention uses a chimeric antigen receptor (CAR-7) comprising a control GPC3 scFv (reference patent CN202110764887.1), a natural co-stimulatory signal domain from CD28, an optimized CD3ζ intracellular signal domain and a CD40L protein as a comparative example, which has an amino acid sequence as shown in SEQ ID NO: 16.

[0070] Sequence Description

[0071] Example

[0072] Example 1: Design of GPC3 CAR molecules

[0073] This example takes the design of a GPC3 CAR molecule with an optimized CD3ζ signaling domain and CD40L protein, and the preparation of CAR-T cells as an example.

[0074] Design principles of the CD3ζ signaling domain: By modifying the amino acid sites related to signal transduction in the CD3ζ intracellular signaling region, the intracellular signal transduction pathway of CAR-T cells can be improved, the expansion and sustainability of CAR-T cells can be enhanced, and the anti-tumor performance of CAR-T cells can be improved.

[0075] Design principle of co-stimulatory molecule CD40L: By introducing the co-stimulatory ligand CD40L molecule, on the one hand, CD40-positive tumor cells can be identified and killed, and on the other hand, CD40-positive antigen-presenting cells can be activated, ultimately obtaining CAR-T cells with better efficacy.

[0076] The amino acid sequence of the scFv of GPC3 CAR is as shown in SEQ ID NO: 1, and the nucleotide sequence is as shown in SEQ ID NO: 2. The above-mentioned scFv, the natural costimulatory signal domain from CD28, and the optimized CD3ζ signal domain are connected to form a chimeric antigen receptor CAR (CAR-1), which has the amino acid sequence shown in SEQ ID NO: 7 and the nucleotide sequence shown in SEQ ID NO: 8. The above-mentioned scFv, the natural costimulatory signal domain from CD28, the optimized CD3ζ signal domain, and CD40L are connected to form a chimeric antigen receptor CAR (CAR-2), which has the amino acid sequence shown in SEQ ID NO: 9 and the nucleotide sequence shown in SEQ ID NO: 10.

[0077] Example 2: Construction of GPC3 CAR Lentiviral Transfer Plasmid and Lentivirus Preparation

[0078] 1) A chimeric gene encoding the GPC3 CAR with Strep tag II at the N-terminus and restriction enzyme sites XbaI and SalI at both ends was synthesized by gene synthesis (Beijing Biomed Gene Technology Co., Ltd.). That is, overlapping single-stranded oligonucleotide primers were designed and synthesized, and template DNA was formed by overlap extension. Double-stranded DNA was then obtained by PCR amplification. The PCR product was then transformed and cloned into a cloning vector or expression vector (reference: Protein Eng, 1992, 5: 8277-829).

[0079] 2) The plasmid containing the gene fragment synthesized in step 1) was double-digested with XbaI and SalI (Thermo Fisher, Waltham, MA, USA) by restriction enzyme digestion to obtain a gene fragment encoding GPC3 CAR with Strep tag II at the N-terminus.

[0080] 3) The lentiviral vector pLenti6.3 / V5 (Thermo Fisher, Waltham, MA, USA) was double-digested with XbaI and SalI by restriction enzyme digestion and ligated with the gene fragment obtained in step 2) to obtain a lentiviral transfer plasmid carrying the GPC3-targeting CAR gene.

[0081] 4) The lentiviral packaging plasmids pLP / VSVG, pLP1 / MDK, and pLP2 / RSK (Thermo Fisher, Waltham, MA, USA) and the lentiviral transfer plasmid obtained in step 3) were transfected into HEK293T cells using Lipofectamine 3000 (Thermo Fisher, Waltham, MA, USA). After 48 hours, the culture medium was collected and centrifuged at 300 g to remove cell debris. The culture medium was then ultracentrifuged at 25,000 rpm for 3 hours. The precipitate was dissolved in 1 mL of saline to obtain the desired lentiviral vector.

[0082] Example 3: CAR-T cell preparation

[0083] This example uses a chimeric antigen receptor CAR (CAR-0) formed by connecting the above-mentioned scFv, a natural costimulatory signal domain from CD28, and a natural intracellular signal domain from CD3ζ as a control.

[0084] T cells were isolated from peripheral blood mononuclear cells (PBMCs) from healthy volunteers (Miaotong (Shanghai) Biotechnology Co., Ltd., China) using CD3 / CD28 Dynabeads (Thermo Fisher, Waltham, MA, USA) (referred to as Day 0). After 48 hours of culture in fresh X-VIVO 15 medium supplemented with IL-2 (500 IU / mL, Beijing Shuanglu Pharmaceutical Co., Ltd.), the isolated T cells were infected with the aforementioned lentiviral vectors. Twenty-four hours after infection, the cells were centrifuged and the medium was exchanged, and cultured again in the aforementioned medium. At different time points in cell culture, 1×10^6 cells were taken, Dynabeads were removed using a magnetic stand, and incubated with PE-labeled Strep tag II antibody (prepared by Beijing Yimiao Shenzhou Pharmaceutical Technology Co., Ltd., for details, see patent ZL202011421882.0) and APC-labeled CD40L antibody (Biolegend) at room temperature for 20 minutes. The CAR content of CAR-T-0, CAR-T-1, and CAR-T-2 cells, as well as the CD40L content on the surface of CAR-T-2 cells, were detected by flow cytometry (NovoCyte 2060R, ACEA Biosciences, San Diego, CA, USA). Figure 1A shows that when the lentiviral infection MOI = 1, the transduction efficiency of each CAR gene was above 70% 3 days after lentiviral infection and remained stable for 7 days after infection. Figure 1B shows that 3 days after CAR-2 lentivirus infection of T cells, approximately 72% of T cells were CAR and CD40L double-positive cells, and among the CAR-2 positive cells, more than 95% of the cells stably expressed CD40L.

[0085] At different time points of CAR-T culture (Day 5, Day 9, Day 13, Day 17, Day 21), the total T cells in the culture system were counted. Based on the CAR positivity rate detected above, the number of CAR-T cells in the culture system was calculated, and the T cell and CAR-T cell expansion curves were plotted, as shown in Figures 2A and 2B.

[0086] Example 4: Efficiency of GPC3 CAR-T in killing target cells

[0087] This example takes the detection of the killing efficiency of CAR-T (CAR-T-0, CAR-T-1 and CAR-T-2) containing the above-mentioned scFv, CD28 costimulatory signal domain, different CD3ζ signal domains, and co-expression or non-co-expression of CD40L against target cells expressing GPC3 as an example.

[0088] HepG2 cells, a hepatocellular carcinoma cell line expressing GPC3, were incubated with APC-labeled CD40L antibody (Biolegend) at room temperature for 20 minutes and then analyzed by flow cytometry (NovoCyte 2060R, ACEA Biosciences, San Diego, CA). Diego, CA, USA) to detect CD40 expression and found that HepG2 cells were double positive for GPC3 and CD40 (named HepG2GPC3+CD40+). ​​Therefore, HepG2 cells were electroporated with a complex formed by sgRNA targeting GPC3 (purchased from GenScript Biotech) and Cas9 protein (purchased from Kaixia Biotech), and GPC3-knockout HepG2 cells (HepG2GPC3-CD40+) were obtained through monoclonal screening. HepG2 cells were electroporated with a complex formed by sgRNA targeting CD40 (purchased from GenScript Biotech) and Cas9 protein, and CD40-knockout HepG2 cells (HepG2GPC3+CD40+) were obtained through monoclonal screening. D40-); HepG2 cells were infected with a lentiviral vector carrying the full CD40 gene, and HepG2 cells overexpressing CD40 (HepG2GPC3+CD40++) were obtained through monoclonal screening; HepG2GPC3-CD40+ were transduced with a complex formed by sgRNA targeting CD40 and Cas9 protein by electroporation, and CD40 knockout cells (HepG2GPC3-CD40-) were obtained through monoclonal screening; HepG2GPC3-CD40+ were infected with a lentiviral vector carrying the full CD40 gene, and cells overexpressing CD40 (HepG2GPC3-CD40++) were obtained through monoclonal screening. The expression of GPC3 and CD40 on the surface of various target cells is shown in Figure 3.

[0089] Add 50 μL of tumor cell culture medium to the wells of the 96-well E-Plate equipped with a label-free real-time cell killing detector (Agilent xCELLigence RTCA SP), place it on the RTCA Station, and perform instrument self-test; remove the E-plate 96, add 100 μL (10^4 cells / well) of mixed tumor cell suspension to the wells, and let it stand at room temperature for 30 minutes; place the E-plate 96 on the RTCA Station in the incubator, and monitor the cell proliferation curve overnight. CAR-T cells cultured in the culture flask were collected and centrifuged at 400 × g for 5 minutes. The cells were resuspended in X-VIVO 15 medium and prepared to a density of 1 × 10^6 cells / mL. CAR expression rate was detected by flow cytometry. The E-Plate 96 was removed and placed in a biosafety cabinet. 50 μL of supernatant was aspirated from each well using a pipette. The required volume of CAR-T cells for each group was calculated based on the desired E:T ratio and CAR expression rate. In duplicate, each group of CAR-T cells was added to the corresponding wells, and then medium was added to 100 μL. The negative control group (untransduced T cells) was supplemented with medium to 100 μL. The E-Plate 96 detection plate was placed on a real-time killing instrument to monitor the killing effect of CAR-T on tumor cells.

[0090] Figure 4 shows that when the effector-target ratio (CAR-T: tumor cells) is 1:1, both CAR-T-0 and CAR-T-1 can effectively kill HepG2 cells (HepG2GPC3+CD40+), indicating that both the wild-type CD3ζ signaling domain and the mutated CD3ζ signaling domain can induce CAR-T to produce a killing effect on antigen-positive target cells.

[0091] Furthermore, Figure 5 compares the killing effects of CAR-T-1 and CAR-T-2 on HepG2 cells with different expression levels of GPC3 and CD40, including HepG2GPC3+CD40+, HepG2GPC3+CD40-, HepG2GPC3+CD40++, HepG2GPC3-CD40+, HepG2GPC3-CD40-, and HepG2GPC3-CD40++. Compared with negative control T cells, both CAR-Ts could effectively kill target cells at all effector-target ratios. For HepG2 cells with negative GPC3 and CD40, compared with negative control T cells, both CAR-Ts could not kill HepG2GPC3-CD40- cells, indicating that CAR-T-1 and CAR-T-2 only CAR-T-1 specifically recognizes the GPC3 target. When GPC3 is lacking on the target cell surface, CAR cannot activate T cells to exert its effect, and the overexpressed CD40L in CAR-T-2 cannot affect the viability of CD40-negative target cells. For HepG2 cells that are GPC3-negative and CD40-positive, compared with negative control T cells, CAR-T-1 cannot promote the death of HepG2GPC3-CD40+ and HepG2GPC3-CD40++ target cells, further demonstrating the specificity of CAR-T-1 in recognizing GPC3. However, CAR-T-2 can slightly enhance the death of HepG2GPC3-CD40+ target cells with weak CD40 expression at an effector-target ratio of 5:1, and significantly enhance the death of HepG2GPC3-CD40++ target cells with strong CD40 expression at effector-target ratios of 1:1 and 5:1, indicating that CD40L overexpressed on the surface of CAR-T-2 cells can help CAR-T induce apoptosis of tumor cells that are GPC3 antigen-negative but CD40-positive.

[0092] Example 5: Proliferation of GPC3 CAR-T cells under target cell stimulation

[0093] This example takes the detection of the proliferation efficiency of CAR-T (CAR-0, CAR-1 and CAR-2) containing the above-mentioned scFv, CD28 costimulatory signal domain, different CD3ζ signal domains, and co-expression or non-co-expression of CD40L under stimulation of HepG2 target cells expressing GPC3 as an example.

[0094] 2 × 10^5 CAR-T cells were added to each well of a 6-well cell culture plate (Corning Incorporated, Corning, NY, USA). HepG2 cells were added every two days for stimulation at an E:T ratio of 1:1 and cultured in a 37°C, 5% CO2 cell culture incubator. After repeated stimulation three times, the cells were stained with trypan blue to count the total number of viable cells, and the cells were incubated with Strep tag II antibodies conjugated with PE fluorescent molecules and APC-labeled CD3 antibodies (Biolegend). The proportion of CAR-T cells in each group of cells was detected by flow cytometry (NovoCyte 2060R, ACEA Biosciences, San Diego, CA, USA), and the number of CAR-T cells in each group was calculated.

[0095] Figure 6 shows that after repeated stimulation of HepG2 cells, the expansion efficiency of CAR-1 and CAR-2 was better than that of CAR-0, indicating that compared with the wild-type CD3ζ signaling domain, the mutated CD3ζ signaling domain can enhance the expansion efficiency of CAR-T after target cell stimulation.

[0096] Example 6: Cytokine secretion by GPC3 CAR-T cells under target cell stimulation

[0097] This example takes the detection of cytokine secretion by CAR-T (CAR-T-0, CAR-T-1, and CAR-T-2) containing the above-mentioned scFv, CD28 costimulatory signaling domain, different CD3ζ signaling domains, and co-expression or non-co-expression of CD40L under stimulation of Huh7 or HepG2 target cells expressing GPC3 as an example.

[0098] 1 × 10^5 Huh7 or HepG2 cells were added to each well of a 96-well cell culture plate (Corning Incorporated, Corning, NY, USA), and various CAR-T cells were added at an E:T ratio of 1:1 in a total culture volume of 500 μL. The cells were then cultured in a 37°C, 5% CO2 cell incubator for 24 hours. IFN-γ, TNF, and IL-2 were labeled in the supernatant of each group using Cytometric Bead Array (CBA) kits (BD Biosciences), and cytokine secretion in the supernatant of each group was detected using flow cytometry (NovoCyte 2060R, ACEA Biosciences, San Diego, CA, USA).

[0099] Figure 7 shows that after stimulation with Huh7 or HepG2 cells, CAR-T-0, CAR-T-1, and CAR-T-2 CAR-T cells can significantly secrete cytokines such as IFN-γ, TNF, and IL-2 compared with control T cells, indicating that these CAR-T cells can effectively recognize GPC3 antigens and activate T cells to express cytokines.

[0100] Example 7: Activation Function of CD40L in CAR-T-2 Cells on Antigen Presenting Cells

[0101] This example detects the activation effect of CAR-T (CAR-T-1 and CAR-T-2) containing the above-mentioned scFv, CD28 costimulatory signaling domain, optimized CD3ζ signaling domain, and co-expression or non-co-expression of CD40L on B cell-derived Daudi cells.

[0102] The T, CAR-T-1, and CAR-T-2 cells prepared above, as well as Daudi cells (National Laboratory Cell Resource Sharing Service Platform), were taken and resuspended in an appropriate amount of X-VIVO in a 15mL centrifuge tube, centrifuged at 400g for 5 minutes, and then resuspended in an appropriate amount of X-VIVO and centrifuged at 400g for 5 minutes. The cells were resuspended in an appropriate amount of X-VIVO and counted. Based on the count results, the concentration of T, CAR-T-1, CAR-T-2, and Daudi cells was adjusted to 1*10^6 cells / mL with X-VIVO. Daudi cells were seeded at a density of 2*10^5 / well (i.e., 200μL / well) in a 24-well plate. CAR-T cells were seeded at a ratio of CAR-T:Daudi = 1:3. Finally, the total T cell number in each group was adjusted to the same, and the total volume was 500μL. Finally, 300μL of X-VIVO was added to each well to a final volume of 1mL / well. Two replicates were set for each group. After the cells were inoculated, they were gently pipetted to mix and co-cultured in a carbon dioxide incubator for 48 hours. After the co-culture, each well was pipetted evenly, 500 μL of cell suspension was transferred to a 1.5 mL microcentrifuge tube, centrifuged at 400 g for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in 0.5 mL DPBS, centrifuged at 400 g for 5 minutes, and the supernatant was discarded. All groups were first treated with 50 μL of FCR blocker (Biolegend) prepared in DPBS and Zombie NIR TMCells were resuspended using the Fixable Viability Kit (Biolegend) and incubated at room temperature in the dark for 10 minutes. Six antibodies, including Alexa Fluor 532 CD3 Monoclonal Antibody (UCHT1) (Thermo Fisher), CD19-APC (Novusbio), FITC anti-human CD86 (Biolegend), Alexa Fluor 647 anti-human CD80 Antibody (Biolegend), Brilliant Violet 421™ anti-human CD40 Antibody (Biolegend), and PE anti-human HLA-DR (Biolegend), were prepared using 1% BSA (Yisheng Bio) dissolved in DPBS. 50 μL / tube was added to each group and incubated for another 20 minutes at room temperature in the dark. All samples were washed twice with 1% BSA dissolved in DPBS (0.5 mL / time), and cells were resuspended in 200 μL of 1% BSA solution per group. The samples were loaded and detected on a full-spectrum multicolor flow cytometer (NL-3000, Cytek), and 50,000 Daudi cells were collected from each group for analysis.

[0103] Figure 8 shows that compared with T cells and CAR-T-1, CAR-T-2 can significantly enhance the expression of CD40 and CD80 on the surface of Daudi cells and slightly increase the expression of HLA-DR, suggesting that the overexpressed CD40L in CAR-T-2 has the function of activating antigen-presenting cells.

[0104] Example 8: Construction of GPC3 CAR-T cells overexpressing different co-stimulatory ligands, CAR and co-stimulatory ligand expression rates, and CAR-T cell proliferation efficiency

[0105] Referring to the method of Example 2, CAR-3, CAR-4, CAR-5, CAR-6 and CAR-7 genes with XbaI and SalI restriction sites at the 5' and 3' ends, respectively, were synthesized (Beijing Bomade Gene Technology Co., Ltd.); the five gene plasmids synthesized above were double-digested with XbaI and SalI, and the vector pLenti6.3 / V5 (Thermo Fisher, Waltham, MA, USA) was also double-digested with XbaI and SalI. The gene fragments and plasmid fragments after double enzyme digestion were purified using a DNA gel recovery kit and then ligated with T4 DNA ligase to obtain pLenti6.3 / V5 plasmids carrying different CAR genes.

[0106] T cells were isolated from peripheral blood mononuclear cells (PBMCs) from healthy volunteers (Miaotong (Shanghai) Biotechnology Co., Ltd., China) using CD3 / CD28 Dynabeads (Thermo Fisher, Waltham, MA, USA) (designated as Day 0). After 24 hours of culture in fresh X-VIVO 15 medium supplemented with IL-2 (500 IU / mL, Beijing Shuanglu Pharmaceutical Co., Ltd.), the T cell density was adjusted to 2 × 10^6 cells / mL. 1 mL of this T cell suspension was plated in a 24-well plate and infected with CAR-2, CAR-3, CAR-4, CAR-5, CAR-6, and CAR-7 lentiviruses at an MOI of 1. Twenty-four hours after infection, the cells were centrifuged and the medium was replaced, and cultured again in the above-described medium. At different time points of CAR-T culture (Day 6, Day 8, Day 10, Day 12), the total T cells in the culture system were counted, and 1×10^6 cells were taken. The Dynabeads were removed using a magnetic stand and incubated with PE-labeled Strep tag II antibody and APC-labeled CD40L antibody (Biolegend), APC-labeled ICOSL antibody (Biolegend), APC-labeled OX40L antibody (Biolegend), APC-labeled CD70 antibody (Biolegend), or APC-labeled 4-1BBL antibody (Biolegend). After incubation at room temperature for 20 minutes, all samples were washed twice with 1% BSA dissolved in DPBS, 0.5 mL each time, and the cells in each group were resuspended in 200 μL of 1% BSA solution. Samples were loaded and analyzed on a full-spectrum multicolor flow cytometer (NL-3000, Cytek). 50,000 T cells were collected from each group to analyze the CAR content, CD40L content on the cell surfaces of CAR-T-2 and CAR-T-7, ICOSL content on the surface of CAR-T-3, OX40L content on the surface of CAR-T-4, CD70 content on the surface of CAR-T-5, and 4-1BBL content on the surface of CAR-T-6. The number of CAR-T cells in the culture system was calculated, and T cell and CAR-T cell expansion curves were plotted.

[0107] As shown in Figure 9, the total number of T cells in each CAR-T group was higher than that in the untransfected control T cell group. Before Day 8, there was little difference in cell number between the CAR-T groups. After Day 8, CAR-T-5 cells were the most numerous, while CAR-T-7 cells were the least numerous. Over time, CAR expression stabilized in each group, and CAR-T cells continued to grow. On Day 12, CAR-T-4 and CAR-T-5 cells had the largest number of CAR-T cells.

[0108] Example 9: Differentiation and depletion of GPC3 CAR-T cells overexpressing different co-stimulatory ligands

[0109] On Day 8 of CAR-T culture in each group, 1×10 6 T cells, centrifuged at 400×g for 5 min, discarded the supernatant, resuspended the cell pellet in 0.5 mL DPBS, centrifuged at 400×g for 5 min, discarded the supernatant. TM The cells were resuspended using Fixable Viability Kit (Biolegend) and incubated at room temperature in the dark for 10 min. Nine antibodies were prepared using 1% BSA (Yishen Bio) dissolved in DPBS, including anti-CD3-AF488 (Biolegend), anti-CAR-PE (manufactured by Yimiao Shenzhou), anti-CD4-BV750 (Biolegend), anti-CD8 PerCP / eFlour710 (Biolegend), anti-CD45RA-BV510 (Biolegend), anti-CD62L-APC / Fire750 (Biolegend), anti-PD-1-BV650 (Biolegend), anti-LAG-3-PE / Cy7 (Biolegend), and anti-CD39-BV421 (Biolegend). These antibodies were added to each well and incubated at room temperature for 20 minutes. All samples were washed twice with 1% BSA dissolved in DPBS, 0.5 mL each time, and the cells in each group were resuspended in 200 μL of 1% BSA solution. The samples were loaded and detected on a full-spectrum multicolor flow cytometer (NL-3000, Cytek), and 50,000 T cells were collected from each group to analyze the differentiation and exhaustion of CAR-T cells in each group.

[0110] As shown in Figure 10, on the 8th day of CAR-T culture, each CAR-T group showed different degrees of differentiation, all with the initial and memory stem cells (TSCM), among which CAR-T-4 The proportion of PD-1 and TSCM was the highest, while the level of central memory T cells (TCM) of CAR-T-7 was the highest among all CAR-T groups. - LAG-3 +The proportion was the largest, but there was no significant difference. Studies have shown that CD39 can be used as an indicator of T cell exhaustion (Moesta, Achim K et al. Nature reviews. Immunology vol. 20, 12 (2020): 739-755). Among the CAR-T groups, CAR-T-2 had the lowest CD39 positive ratio, while CAR-T-7 had the highest positive ratio.

[0111] Example 10: Killing Effect and Expansion Ability of GPC3 CAR-T Overexpressing Different Costimulatory Ligands Upon Repeated Stimulation of GPC3-Positive Target Cells

[0112] 50,000 tumor cells were plated per well in a 48-well plate and cultured overnight. The next day, 1×10^6 T cells were harvested and treated with PE-labeled Strep Tag II antibody. CAR expression in each group was determined by flow cytometry. The required number of CAR-T cells for each group was calculated, using a target-to-target ratio of 3:1. Untransduced T cells were used to adjust the total number of CAR-T cells in each group to the same level. Four replicates of each group were added to the 48-well plate containing the tumor cells. A negative control group (untransduced T cells) and a tumor cell group were set up. On the third day, 50,000 tumor cells were re-plated per well in the 48-well plate. After the tumor cells adhered, the CAR-T cells from each group were transferred to the corresponding well for target cell stimulation. After three replicates, cells from each well were collected into a 1.5 mL microcentrifuge tube and centrifuged at 400 g for 5 minutes. The supernatant was discarded. The cell pellet was resuspended in 0.5 mL of DPBS and centrifuged at 400 g for 5 minutes. The supernatant was discarded. All groups were first treated with 100 μL of Zombie NIR prepared in DPBS. TM The cells were resuspended with Fixable Viability Kit (Biolegend) and incubated at room temperature in the dark for 10 minutes. Two flow cytometry antibodies, CD3-APC (BD Biosciences) and PE-labeled CAR antibody (prepared by Beijing Yimiao Shenzhou Pharmaceutical Technology Co., Ltd.), were then added to each well and incubated at room temperature for 30 minutes. All samples were washed twice with 1% BSA dissolved in DPBS, 0.5 mL / time, and the cells were resuspended in 200 μL of 1% BSA solution for each group. The samples were loaded and tested on a full-spectrum multicolor flow cytometer (NL-3000, Cytek), and 100 μL of cell suspension was collected from each group to detect the residual amount of tumor cells and the number of CAR-T after repeated stimulation. The specific results are shown in Figure 11.

[0113] Results showed that under repeated stimulation of target cells, CAR-T-2 and CAR-T-3 demonstrated the strongest tumor-killing ability compared to other CAR-Ts, significantly outperforming CAR-T-4, CAR-T-5, CAR-T-6, and CAR-T-7. Regarding CAR-T persistence, CAR-T-2 demonstrated the strongest sustained expansion, significantly outperforming CAR-T-3, CAR-T-4, and CAR-T-5. These results demonstrate that CAR-T-2, containing the aforementioned GPC3 scFv, CD28 costimulatory signaling domain, optimized CD3ζ signaling domain, and co-expressing CD40L, exhibits potent and sustained anti-tumor efficacy in vitro.

[0114] Example 11: Anti-tumor ability, expansion ability and sustained performance of CAR-T-2 cells in tumor-bearing mice

[0115] This example uses the detection of the anti-tumor ability, in vivo expansion ability and persistence ability of CAR-T (CAR-T-2) containing the above-mentioned scFv, CD28 costimulatory signal domain, optimized CD3ζ signal domain, and co-expression of CD40L in tumor-bearing mice as an example.

[0116] CAR-T-2 cells were prepared according to the above CAR-T cell preparation method. When sufficient cells were cultured, the cells were resuspended in freezing solution and stored in liquid nitrogen for use. A total of 36 6-8 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 2.0×10 6 Seven days after inoculation with Huh7-LAE cells (ATCC, USA), mice were analyzed using luciferase live imaging (Lumina II Small Animal Live Imaging System, PerkinElmer, USA) to verify the success of the mouse xenograft HCC model. Ten days after tumor inoculation, mice in each group were injected via the tail vein with saline, control T cells, or different doses of CAR-2 cells (1×10^6, 3×10^6, 5×10^6, and 1×10^7 CAR-T cells per mouse). Live imaging analysis was performed on the day before CAR-T cell injection and on days 5, 12, 17, 22, 27, and 32 after injection. CAR-T cells were detected in peripheral blood on days 5, 12, 17, 22, 27, and 32 after CAR-T cell injection.

[0117] Figure 12A shows that compared with the tumor burden of mice in the saline group and the control T cell group, different doses of CAR-T-2 cells can effectively reduce the tumor burden within 32 days, indicating that GPC3 CAR-T containing an optimized CD3ζ signaling domain and co-expressing CD40L has significant anti-GPC3 positive tumor activity.

[0118] Figure 12B shows that GPC3 CAR-T cells were injected into the tail vein of mice and the CAR-T cell content in the peripheral blood of mice was detected at different times. After CAR-T transfusion, significantly higher levels of CAR-T cells were detected in the peripheral blood, and after 12 days, CAR-T further expanded in the body and maintained a certain level.

[0119] The above results indicate that CAR-T containing the above-mentioned GPC3 scFv, CD28 co-stimulatory signaling domain, optimized CD3ζ signaling domain, and co-expressing CD40L has a strong and sustained anti-tumor effect in vivo.

[0120] From the above in vitro and in vivo experimental results, it can be seen that the GPC3 CAR-T designed by the inventors of the present application has good anti-tumor activity, in vivo expansion efficiency and duration.

Claims

1. A chimeric antigen receptor comprising genes encoding a scFv targeting GPC3, a hinge region, a transmembrane region, a CD3ζ signaling domain, a co-stimulatory signaling domain, and a CD40L protein; wherein, The scFv has the amino acid sequence shown in SEQ ID NO: 1; The co-stimulatory signal domain is from CD28; The CD40L has an amino acid sequence shown in SEQ ID NO: 5; The CD3ζ signaling domain is one of the following variants of the native CD3ζ signaling domain sequence: 1) The proximal amino acid V2 in the natural CD3ζ signal domain sequence was replaced with L, D9 with E, and Q15 with K; 2) replacing the tyrosine phosphorylation site Y90 in the native CD3ζ signal domain sequence with F; 3) replacing the membrane-proximal amino acid V2 with L, D9 with E, Q15 with K, and the tyrosine phosphorylation site Y90 with F in the natural CD3ζ signal domain sequence; The natural CD3ζ signal domain sequence corresponds to positions 52-163 of the amino acid sequence listed in NP_000725.

1.

2. The chimeric antigen receptor of claim 1, wherein the hinge region or the transmembrane region is derived from CD8α, CD28, IgG1 or IgG4.

3. The chimeric antigen receptor according to claim 1 or 2, further comprising a gene encoding a signal peptide.

4. The chimeric antigen receptor of claim 1 or 2, wherein the gene encoding the CD40L protein is connected to the C-terminus of the CD3ζ signaling domain via a linker peptide.

5. The chimeric antigen receptor of claim 1 or 2, wherein the CD3ζ signaling domain has the amino acid sequence shown in SEQ ID NO:

3.

6. The chimeric antigen receptor according to claim 1 or 2, which comprises or has the amino acid sequence shown in SEQ ID NO:

9.

7. An isolated nucleic acid encoding the chimeric antigen antibody according to any one of claims 1 to 6. A vector comprising the nucleic acid according to claim 7.

9. An isolated cell comprising the chimeric antigen antibody according to any one of claims 1 to 6 or the vector according to claim 8, wherein the cell is not a germ cell or a fertilized egg.

10. Use of the chimeric antigen receptor according to any one of claims 1 to 6, the nucleic acid according to claim 7, the vector according to claim 8 or the cell according to claim 9 in the preparation of a medicament for treating GPC3-positive malignant tumors.

11. The use according to claim 10, wherein the drug is used to prepare a drug for treating liver cancer, lung cancer, osteosarcoma or melanoma.

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