Chimeric antigen receptor against glypican 3 and its use

Abstract

The present invention relates to a chimeric antigen receptor, which comprises genes encoding scFv from targeting GPC3, hinge region, transmembrane region, CD3ζ signal domain, co-stimulatory signal domain, and CD40L protein; wherein, the scFv has the amino acid sequence shown in SEQ ID NO: 1; the co-stimulatory signal domain is from CD28; CD40L has the amino acid sequence shown in SEQ ID NO: 5; the CD3ζ signal domain is one of the following variants of the natural CD3ζ signal domain sequence: 1) the membrane-proximal amino acid V2 in the natural CD3ζ signal domain sequence is replaced by L, D9 is replaced by E, and Q15 is replaced by K; 2) the tyrosine phosphorylation site Y90 in the natural CD3ζ signal domain sequence is replaced by F; 3) the membrane-proximal amino acid V2 in the natural CD3ζ signal domain sequence is replaced by L, D9 is replaced by E, Q15 is replaced by K, and the tyrosine phosphorylation site Y90 is replaced by F.

Description

Technical Field

[0001] 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

[0002] Tumor immune cell therapy, especially chimeric antigen receptor T cells (CAR-T), has achieved remarkable results in the treatment of malignant blood tumors. It has made great breakthroughs in many clinical studies on CD19 and BCMA-positive malignant blood tumors, and has also shown good prospects in preclinical and exploratory clinical studies of solid tumors such as liver cancer, bringing new hope to cancer patients. There are 7 CAR-T drugs for malignant blood tumors on the market worldwide.

[0003] CAR-T technology combines the specificity of antibodies and the killing effect of T cells, thus forming an effective way of adoptive immunity. CAR is usually composed of an extracellular antigen binding domain, a hinge region, a transmembrane region, a co-stimulatory signaling domain, and an intracellular CD3ζ signaling domain. CAR-T usually faces multiple obstacles such as limited efficacy and off-target effects in the treatment of solid tumors. At present, the global research and development of CAR-T drugs for malignant solid tumors such as liver cancer is still in the clinical trial stage, and no related products have been launched at home and abroad.

[0004] GPC3 is highly expressed in hepatocellular carcinoma (HCC) (expression rate is about 74.8%), and is also highly expressed in a variety of malignant solid tumors such as lung squamous cell carcinoma, yolk sac tumor, melanoma, osteosarcoma, etc., and is not expressed in normal tissues such as liver, kidney, stomach, etc. of healthy people (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 cell therapy for advanced liver cancer patients (Shi et al., ClinCancer Res 19: 3259, 2020) showed that CAR-T therapy has good safety in hepatocellular carcinoma, and 2 partial responses were observed among 13 patients, showing certain clinical application potential, but there is still much room for improvement in efficacy. Therefore, how to improve the efficacy of CAR-T in treating solid tumors such as liver cancer through reasonable drug design is an important technical problem that needs to be solved.

[0005] Compared with the treatment of hematological tumors, the probability of CAR-T contacting the target antigen in patients with solid tumors is low, and the solid tumor microenvironment is highly immunosuppressive. The peak expansion of CAR-T in patients with solid tumors is much lower than that in patients with hematological tumors. This is an important reason why CAR-T is not effective in treating solid tumors. Therefore, the treatment of solid tumors requires CAR-T to have stronger killing activity, in vivo expansion ability and sustainability. Summary of the invention

[0006] Currently known CAR molecules targeting GPC3 usually adopt a simple second-generation CAR design, and both the co-stimulatory signal domain and the intracellular CD3ζ signal domain adopt wild-type molecules. The inventors of the present application have found that these molecular designs may cause CAR-T to be unable to achieve the optimal binding state of immune synapse formation when binding to the target molecule, or the signal transmission to the cell after the immune synapse is formed is too strong or too weak, thereby affecting the survival and proliferation of CAR-T cells in the body, the persistence of tumor cell killing performance and the regulation of the entire immune system function, which is ultimately reflected in the difference in clinical efficacy and safety.

[0007] The intracellular signaling domain is the basic element for CAR-T activation and killing, and its structural design is crucial for the full activation and sustained survival of CAR-T. Each CD3ζ signaling domain has three immunoreceptor tyrosine 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 of the CD3ζ signaling domain, the spatial conformation of its juxtamembrane region also plays an important role in the formation of immune synapses (Guy et al., Immunol Rev 232: 7, 2009).

[0008] Therefore, the applicant of the present application mutated the amino acids of the membrane-proximal end of the CD3ζ signaling domain and the immune receptor tyrosine activation motif to improve the signal activation intensity and persistence of the CD3ζ signaling domain (refer to patent PCT / CN2021 / 076247).

[0009] 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, thereby ultimately obtaining CAR-T cells with better therapeutic effects.

[0010] 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.

[0011] Specifically, the present invention provides:

[0012] (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:

[0013] The scFv has the amino acid sequence shown in SEQ ID NO: 1;

[0014] The co-stimulatory signal domain is from CD28;

[0015] The CD40L has an amino acid sequence as shown in SEQ ID NO: 5;

[0016] The CD3ζ signaling domain is one of the following variants of the native CD3ζ signaling domain sequence:

[0017] 1) Replace the membrane-proximal amino acid V2 with L, D9 with E, and Q15 with K in the native CD3ζ signal domain sequence;

[0018] 2) Replace the tyrosine phosphorylation site Y90 in the native CD3ζ signal domain sequence with F;

[0019] 3) Substitute the proximal membrane amino acid V2 to L, D9 to E, Q15 to K, and the tyrosine phosphorylation site Y90 to F in the native CD3ζ signaling domain sequence;

[0020] The natural CD3ζ signal domain sequence corresponds to positions 52-163 of the amino acid sequence listed in NP_000725.1.

[0021] (2) The chimeric antigen receptor according to (1) above, wherein the hinge region or the transmembrane region is derived from CD8α, CD28, IgG1 or IgG4.

[0022] (3) The chimeric antigen receptor according to (1) or (2) above, further comprising a gene encoding a signal peptide.

[0023] (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.

[0024] (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.

[0025] (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.

[0026] (7) An isolated nucleic acid encoding the chimeric antigen antibody according to any one of (1) to (6) above.

[0027] (8) A vector comprising the nucleic acid described in (7) above.

[0028] (9) An isolated cell comprising the chimeric antigen antibody described in any one of (1) to (6) above or the vector described in (8) above, wherein the cell is not a germ cell or a fertilized egg.

[0029] (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.

[0030] (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.

[0031] 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

[0032] Figure 1 Shown is the transduction efficiency of the GPC3 CAR lentivirus.

[0033] Figure 2 It is the proliferation curve of total T cells and CAR-T cells during the CAR-T preparation process.

[0034] Figure 3 Shows the expression of GPC3 and CD40 on the surface of HepG2 and genetically engineered HepG2 cells.

[0035] Figure 4 This is the killing curve of CAR-T-0 and CAR-T-1 on HepG2 cells.

[0036] Figure 5 The killing effects of CAR-T-1 and CAR-T-2 on target cells with different GPC3 and CD40 expression levels are shown.

[0037] Figure 6 Showing the expansion of GPC3 CAR-T under repeated stimulation of HepG2 cells.

[0038] Figure 7 Showing cytokine secretion by GPC3 CAR-T upon stimulation of Huh7 and HepG2 cells.

[0039] Figure 8 Show the effects of CAR-T-1 and CAR-T-2 on the expression of proteins related to antigen presentation on the surface of Daudi cells.

[0040] Fig. 9 Displays the total T cells, CAR expression rate, and CAR-T cell proliferation curve during CAR-T preparation.

[0041] Fig.10 The results of the differentiation and exhaustion level tests of CAR-T cells in the resting state are shown.

[0042] Fig.11 Shows the number of tumor cells and CAR-T cells after repeated stimulation of target cells.

[0043] Fig.12 Showing the tumor inhibitory effect (A) and in vivo expansion (B) of CAR-T-2 in tumor-bearing mice. DETAILED DESCRIPTION

[0044] 1. scFv of GPC3 CAR molecule

[0045] 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.

[0046] 2. Design of the GPC3 CAR molecular signaling domain

[0047] Based on the natural CD3ζ signal domain sequence (corresponding to the amino acid sequence 52-163 listed in NP_000725.1), the CD3ζ signal domain of the CAR molecule was designed, including:

[0048] 1) Substitute the membrane-proximal amino acid V2 with L, D9 with E, and Q15 with K in the native CD3ζ signal domain sequence;

[0049] 2) The tyrosine phosphorylation site Y90 in the native CD3ζ signal domain sequence was replaced with F.

[0050] In one embodiment, the optimized CD3 zeta 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.

[0051] 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.

[0052] The amino acid sequence of CD40L is shown in SEQ ID NO: 5, and the nucleotide sequence is shown in SEQ ID NO: 6.

[0053] 3. Composition of the GPC3 CAR molecule

[0054] The present invention relates to a chimeric antigen receptor targeting GPC3, which comprises genes encoding a signal peptide, a scFv specifically recognizing GPC3, a hinge region, a transmembrane region, a CD28 costimulatory signal domain, a CD3ζ signal domain and CD40L.

[0055] In some embodiments, the signal peptide is colony stimulating factor 2 receptor alpha signal peptide.

[0056] In some embodiments, the scFv comprises, consists essentially of, or consists of the amino acid sequence shown in SEQ ID NO: 1.

[0057] In some embodiments, the hinge region or the transmembrane region is derived from CD8α, CD28, IgG1 or IgG4, etc.

[0058] In some embodiments, the CD3ζ signal domain is the following variant of the natural CD3ζ signal domain sequence (corresponding to positions 52-163 of the amino acid sequence listed in NP_000725.1):

[0059] 1) Substitute the membrane-proximal amino acid V2 with L, D9 with E, and Q15 with K in the native CD3ζ signal domain sequence;

[0060] 2) The tyrosine phosphorylation site Y90 in the native CD3ζ signal domain sequence was replaced with F.

[0061] 3) The membrane-proximal amino acid V2 in the natural CD3ζ signal domain sequence was replaced by L, D9 by E, Q15 by K, and the tyrosine phosphorylation site Y90 by F.

[0062] 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.

[0063] In one embodiment, the present invention provides a chimeric antigen receptor (CAR-1) comprising GPC3 scFv, a natural costimulatory signal domain from CD28, and an optimized CD3ζ intracellular signal 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.

[0064] In one embodiment, the present invention provides a chimeric antigen receptor (CAR-2) comprising GPC3 scFv, a natural co-stimulatory signal domain from CD28, an optimized CD3ζ intracellular signal 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.

[0065] In one embodiment, the present invention uses a chimeric antigen receptor (CAR-0) comprising GPC3 scFv, a natural costimulatory signal domain from CD28, and a natural CD3ζ intracellular signal domain as a comparative example, which has an amino acid sequence as shown in SEQ ID NO: 11.

[0066] 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; The chimeric antigen receptor (CAR-6) of scFv, natural CD28 co-stimulatory signal domain, optimized CD3ζ intracellular signal domain and 4-1BBL protein is a comparative example, which has an amino acid sequence as shown in SEQ ID NO: 15.

[0067] 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.

[0068] Sequence Description

[0069] SEQ ID NO illustrate sequence 1 scFv amino acid sequence DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS 2 scFv nucleotide sequence gatgtcgtgatgacccagtccccactgtccctgccagtgacaccaggagagcctgcatccatctcttgccggagctcccagtctctggtgcacagcaacggcaatacctacctgcactggtatctgcagaagccaggccagagcccccagctgctgatctacaaggtgtccaaccggttctctggagtgccagaccggttcagcggctccggctctggcaccgatttcacactgaagatcagcagggtggaggcagaggacgtgggcgtgtactattgctcccagaatacccacgtgccccctacatttggccagggcaccaagctggagatcaagGGAGGAGGAGGCAGCGGCGGAGGAGGCTCCGGCGGCGGCGGCTCTcaggtgcagctggtgcagtccggagcagaggtgaagaagcctggagccagcgtgaaggtgtcctgtaaggcctctggctacaccttcacagattatgagatgcactgggtgcggcaggcacctggacagggactggagtggatgggcgccctggacccaaagaccggcgatacagcctactctcagaagtttaagggcagggtgaccctgacagccgacgagagcacctccacagcctatatggagctgtctagcctgcgcagcgaggataccgccgtgtactattgcacccgcttctacagttacacttattgggggcagggcactctggtcacagtctcttca 3 Optimized amino acid sequence of the CD3ζ intracellular signaling domain RLKFSRSAEAPAYQKGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLFQGLSTATKDTYDALHMQALPPR 4 Optimized nucleotide sequence of the CD3ζ intracellular signaling domain CGCCTGAAGTTCAGCCGCAGCGCCGAGGCCCCCGCCTACCAGAAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCCGCCGCGAGGAGTACGACGTGCTGGACAAGCGCCGCGGCCGCGACCCCGAGATGGGCGGCAAGCCCCGCCGCAAGAACCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGCCGCCGCGGCAAGGGCCACGACGGCCTGTTCCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCCGC 5 CD40L amino acid sequence IETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL 6 CD40L nucleotide sequence atcgaaacatacaaccaaacttctccccgatctgcggccactggactgcccatcagcatgaaaatttttatgtatttacttactgtttttcttatcacccagatgattgggtcagcactttttgctgtgtatcttcatagaaggttggacaagatagaagatgaaaggaatcttcatgaagattttgtattcatgaaaacgatacagagatgcaacacaggagaaagatccttatccttactgaactgtgaggagattaaaagccagtttgaaggctttgtgaaggatataatgttaaacaaagaggagacgaagaaagaaaacagctttgaaatgcaaaaaggtgatcagaatcctcaaattgcggcacatgtcataagtgaggccagcagtaaaacaacatctgtgttacagtgggctgaaaaaggatactacaccatgagcaacaacttggtaaccctggaaaatgggaaacagctgaccgttaaaagacaaggactctattatatctatgcccaagtcaccttctgttccaatcgggaagcttcgagtcaagctccatttatagccagcctctgcctaaagtcccccggtagattcgagagaatcttactcagagctgcaaatacccacagttccgccaaaccttgcgggcaacaatccattcacttgggaggagtatttgaattgcaaccaggtgcttcggtgtttgtcaatgtgactgatccaagccaagtgagccatggcactggcttcacgtcctttggcttactcaaactc 7 CAR-1 Amino Acid Sequence DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRLKFSRSAEAPAYQKGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLFQGLSTATKDTYDALHMQALPPR 8 CAR-1 nucleotide sequence gatgtcgtgatgacccagtccccactgtccctgccagtgacaccaggagagcctgcatccatctcttgccggagctcccagtctctggtgcacagcaacggcaatacctacctgcactggtatctgcagaagccaggccagagcccccagctgctgatctacaaggtgtccaaccggttctctggagtgccagaccggttcagcggctccggctctggcaccgatttcacactgaagatcagcagggtggaggcagaggacgtgggcgtgtactattgctcccagaatacccacgtgccccctacatttggccagggcaccaagctggagatcaagGGAGGAGGAGGCAGCGGCGGAGGAGGCTCCGGCGGCGGCGGCTCTcaggtgcagctggtgcagtccggagcagaggtgaagaagcctggagccagcgtgaaggtgtcctgtaaggcctctggctacaccttcacagattatgagatgcactgggtgcggcaggcacctggacagggactggagtggatgggcgccctggacccaaagaccggcgatacagcctactctcagaagtttaagggcagggtgaccctgacagccgacgagagcacctccacagcctatatggagctgtctagcctgcgcagcgaggataccgccgtgtactattgcacccgcttctacagttacacttattgggggcagggcactctggtcacagtctcttcaATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGGGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCGCCTGAAGTTCAGCCGCAGCGCCGAGGCCCCCGCCTACCAGAAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCCGCCGCGAGGAGTACGACGTGCTGGACAAGCGCCGCGGCCGCGACCCCGAGATGGGCGGCAAGCCCCGCCGCAAGAACCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGCCGCCGCGGCAAGGGCCACGACGGCCTGTTCCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCCGC 9 CAR-2 amino acid sequence DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRLKFSRSAEAPAYQKGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLFQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL 10 CAR-2 nucleotide sequence gatgtcgtgatgacccagtccccactgtccctgccagtgacaccaggagagcctgcatccatctcttgccggagctcccagtctctggtgcacagcaacggcaatacctacctgcactggtatctgcagaagccaggccagagcccccagctgctgatctacaaggtgtccaaccggttctctggagtgccagaccggttcagcggctccggctctggcaccgatttcacactgaagatcagcagggtggaggcagaggacgtgggcgtgtactattgctcccagaatacccacgtgccccctacatttggccagggcaccaagctggagatcaagGGAGGAGGAGGCAGCGGCGGAGGAGGCTCCGGCGGCGGCGGCTCTcaggtgcagctggtgcagtccggagcagaggtgaagaagcctggagccagcgtgaaggtgtcctgtaaggcctctggctacaccttcacagattatgagatgcactgggtgcggcaggcacctggacagggactggagtggatgggcgccctggacccaaagaccggcgatacagcctactctcagaagtttaagggcagggtgaccctgacagccgacgagagcacctccacagcctatatggagctgtctagcctgcgcagcgaggataccgccgtgtactattgcacccgcttctacagttacacttattgggggcagggcactctggtcacagtctcttcaATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGGGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCGCCTGAAGTTCAGCCGCAGCGCCGAGGCCCCCGCCTACCAGAAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCCGCCGCGAGGAGTACGACGTGCTGGACAAGCGCCGCGGCCGCGACCCCGAGATGGGCGGCAAGCCCCGCCGCAAGAACCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGCCGCCGCGGCAAGGGCCACGACGGCCTGTTCCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCCGCGGCAGCGGCGCCACAAACTTCTCTCTGCTAAAGCAAGCAGGTGATGTTGAAGAAAACCCCGGGCCTatgatcgaaacatacaaccaaacttctccccgatctgcggccactggactgcccatcagcatgaaaatttttatgtatttacttactgtttttcttatcacccagatgattgggtcagcactttttgctgtgtatcttcatagaaggttggacaagatagaagatgaaaggaatcttcatgaagattttgtattcatgaaaacgatacagagatgcaacacaggagaaagatccttatccttactgaactgtgaggagattaaaagccagtttgaaggctttgtgaaggatataatgttaaacaaagaggagacgaagaaagaaaacagctttgaaatgcaaaaaggtgatcagaatcctcaaattgcggcacatgtcataagtgaggccagcagtaaaacaacatctgtgttacagtgggctgaaaaaggatactacaccatgagcaacaacttggtaaccctggaaaatgggaaacagctgaccgttaaaagacaaggactctattatatctatgcccaagtcaccttctgttccaatcgggaagcttcgagtcaagctccatttatagccagcctctgcctaaagtcccccggtagattcgagagaatcttactcagagctgcaaatacccacagttccgccaaaccttgcgggcaacaatccattcacttgggaggagtatttgaattgcaaccaggtgcttcggtgtttgtcaatgtgactgatccaagccaagtgagccatggcactggcttcacgtcctttggcttactcaaactc 11 CAR-0 amino acid sequence DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 12 CAR-3 Amino Acid Sequence DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRLKFSRSAEAPAYQKGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLFQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMRLGSPGLLFLLFSSLRADTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERDKITENPVSTGEKNAATWSILAVLCLLVVVAVAIGWVCRDRCLQHSYAGAWAVSPETELTGHV 13 CAR-4 Amino Acid Sequence DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRLKFSRSAEAPAYQKGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLFQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMERVQPLEENVGNAARPRFERNKLLLVASVIQGLGLLLCFTYICLHFSALQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL 14 Amino acid sequence of CAR-5 DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRLKFSRSAEAPAYQKGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLFQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP 15 CAR-6 amino acid sequence DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRLKFSRSAEAPAYQKGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLFQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 16 CAR-7 amino acid sequence DVVMTQSPLSLPVTLGENASISCRSSQSLVHSNGNTYLHWFIQRPGQSPRILIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARFYSYTYWGQGTLVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRLKFSRSAEAPAYQKGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLFQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL Example

[0070] Example 1: Design of GPC3 CAR molecules

[0071] 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.

[0072] Design principles of 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 proliferation and sustainability of CAR-T cells can be enhanced, and the anti-tumor performance of CAR-T cells can be improved.

[0073] 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.

[0074] 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 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 an amino acid sequence as shown in SEQ ID NO: 7 and a nucleotide sequence as shown in SEQ ID NO: 8. The above 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 an amino acid sequence as shown in SEQ ID NO: 9 and a nucleotide sequence as shown in SEQ ID NO: 10.

[0075] Example 2: Construction of GPC3 CAR lentiviral transfer plasmid and lentiviral preparation

[0076] 1) A chimeric gene encoding 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 method, and then double-stranded DNA was obtained by PCR amplification method, and then the PCR product was transformed and cloned into a cloning vector or expression vector (reference ProteinEng, 1992, 5: 8277-829).

[0077] 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 the gene fragment encoding GPC3 CAR with Strep tag II at the N-terminus.

[0078] 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.

[0079] 4) The lentiviral packaging plasmids pLP / VSVG, pLP1 / MDK, 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, centrifuged at 300 g to remove cell debris, and then centrifuged at 25,000 rpm in an ultracentrifuge for 3 hours. The precipitate was dissolved in 1 mL of saline to obtain the desired lentiviral vector.

[0080] Example 3: CAR-T cell preparation

[0081] In this example, a chimeric antigen receptor CAR (CAR-0) containing the above scFv, a natural costimulatory signal domain from CD28, and a natural intracellular signal domain from CD3ζ was used as a control.

[0082] T cells were isolated from peripheral blood mononuclear cells of healthy volunteers (Miaotong (Shanghai) Biotechnology Co., Ltd., China) using CD3 / CD28 Dynabeads (Thermo Fisher, Waltham, MA, USA) (marked as Day 0 time point), and the isolated T cells were cultured in fresh X-VIVO 15 culture system containing IL-2 (500 IU / mL, Beijing Shuanglu Pharmaceutical Co., Ltd.) for 48 hours, and then infected with the above lentiviral vector. After 24 hours of virus infection, the cells were centrifuged and the medium was replaced, and the culture was continued in the above culture system. At different time points of cell culture, 1×10^6 cells were taken, Dynabeads were removed using a magnetic rack, and the cells were 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 within 7 days after infection. Figure 1 B shows that 3 days after T cells were infected with CAR-2 lentivirus, about 72% of T cells were double-positive for CAR and CD40L, and among the CAR-2-positive cells, more than 95% of the cells stably expressed CD40L.

[0083] 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, and the number of CAR-T cells in the culture system was calculated based on the CAR positive rate detected above, and the T cell and CAR-T cell expansion curves were drawn, such as Figure 2 A and Figure 2 As shown in B.

[0084] Example 4: Efficiency of GPC3 CAR-T in killing target cells

[0085] 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 co-stimulatory signal domain, different CD3ζ signal domains, and co-expression or non-co-expression of CD40L on target cells expressing GPC3 as an example.

[0086] After incubation of HepG2 cells, which are positive for GPC3 expression, with APC-labeled CD40L antibody (Biolegend) at room temperature for 20 min, CD40 expression was detected by flow cytometry (NovoCyte 2060R, ACEA Biosciences, San Diego, CA, USA). It was found that HepG2 cells were double positive for GPC3 and CD40 (named HepG2 GPC3+CD40+ ), HepG2 cells were transduced with a complex of sgRNA targeting GPC3 (purchased from GenScript Biotech) and Cas9 protein (purchased from Kaixia Biotech) by electroporation, and HepG2 cells with GPC3 knockout were obtained by monoclonal screening (HepG2 GPC3-CD40+ HepG2 cells were electroporated with a complex of sgRNA targeting CD40 (purchased from GenScript Biotech) and Cas9 protein, and CD40 knockout HepG2 cells were obtained by monoclonal screening (HepG2 GPC3+CD40- HepG2 cells were infected with a lentiviral vector carrying the full CD40 gene, and HepG2 cells overexpressing CD40 were obtained through monoclonal screening (HepG2 GPC3 +CD40++ ); HepG2GPC3-CD40+ The complex formed by sgRNA targeting CD40 and Cas9 protein was transduced by electroporation, and CD40 knockout cells (HepG2 GPC3-CD40- ); HepG2 GPC3-CD40+ Infected with a lentiviral vector carrying the full CD40 gene, cells overexpressing CD40 (HepG2 GPC3-CD40++ ), the expression of GPC3 and CD40 on the surface of various target cells Figure 3 .

[0087] Add 50 μL of tumor cell culture medium to the wells of the 96-well plate E-Plate equipped with the cell label-free real-time killing detector (Agilent xCELLigence RTCA SP), place it on the RTCA Station, and perform instrument self-test; take out the E-plate 96, add 100 μL (10^4 cells / well) of mixed tumor cell suspension to the wells, and place it at room temperature for 30 min; place the E-plate 96 on the RTCA Station in the incubator and detect the cell proliferation curve overnight. Collect the CAR-T cells cultured in the culture flask, centrifuge at 400×g for 5 min, resuspend the cells in X-VIVO 15 medium, prepare the cells to a density of 1×10^6 cells / mL, and detect the CAR expression rate by flow cytometry; take out the E-plate 96, place it in a biosafety cabinet, and use a pipette to aspirate 50 μL of supernatant from each well; calculate the required volume of CAR-T cells for each group according to the required E: T ratio and CAR expression rate, add each group of CAR-T cells to the corresponding wells in two parallels, and then add culture medium to 100 μL, and add culture medium to 100 μL for the negative control group (untransduced T cells). Place the E-Plate 96 detection plate on the real-time killing instrument to start monitoring and observe the killing effect of CAR-T on tumor cells.

[0088] Figure 4 The results showed that when the effect-target ratio (CAR-T: tumor cells) was 1:1, both CAR-T-0 and CAR-T-1 could effectively kill HepG2 cells (HepG2 GPC3+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.

[0089] Further, Figure 5 The killing effects of CAR-T-1 and CAR-T-2 on HepG2 cells with different expression levels of GPC3 and CD40 were compared, including HepG2 GPC3+CD40+ 、HepG2 GPC3+CD40- 、HepG2GPC3+CD40++ 、HepG2 GPC3-CD40+ 、HepG2 GPC3-CD40- 、HepG2 GPC3-CD40++ , and found that GPC3-positive HepG2 cells (HepG2 GPC3+CD40++ 、HepG2 GPC3 +CD40+ 、HepG2 GPC3+CD40- ), compared with negative control T cells, both CAR-T cells can effectively kill target cells at all effector-target ratios; for HepG2 cells that are negative for both GPC3 and CD40, compared with negative control T cells, both CAR-T cells cannot kill HepG2 GPC3-CD40- This indicates that CAR-T-1 and CAR-T-2 only specifically recognize the GPC3 target. When the target cell surface lacks GPC3, 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. Compared with negative control T cells, CAR-T-1 cannot promote the activation of HepG2 cells with GPC3 negative and CD40 positive expression. GPC3-CD40+ and HepG2 GPC3-CD40++ Target cell death further illustrates the specificity of CAR-T-1 in recognizing GPC3, but CAR-T-2 can slightly enhance the CD40 weakly expressed target cell HepG2 at an effector-target ratio of 5:1. GPC3-CD40+ When the effector-target ratio was 1:1 and 5:1, it could significantly enhance the CD40-expressing target cell HepG2 GPC3-CD40++ This indicates 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.

[0090] Example 5: Proliferation of GPC3 CAR-T cells under stimulation of target cells

[0091] 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 co-stimulatory signal domain, different CD3ζ signal domains, and co-expression or non-co-expression of CD40L under the stimulation of HepG2 target cells expressing GPC3 as an example.

[0092] 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 at an E: T = 1: 1 ratio for stimulation and cultured in a cell culture incubator at 37°C and 5% CO2. After repeated stimulation three times, the cells were stained with trypan blue to count the total number of live cells, and the cells were incubated with Strep tag II antibodies coupled to 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 (NovoCyte2060R, ACEA Biosciences, San Diego, CA, USA), and the number of CAR-T cells in each group was calculated.

[0093] Figure 6 The results showed 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.

[0094] Example 6: Cytokine secretion by GPC3 CAR-T under target cell stimulation

[0095] 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 co-stimulatory signal domain, different CD3ζ signal domains, and co-expression or non-co-expression of CD40L under the stimulation of Huh7 or HepG2 target cells expressing GPC3 as an example.

[0096] 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 = 1: 1, with a total culture volume of 500 μL. The cells were then cultured in a 37°C, 5% CO2 cell culture incubator for 24 hours, and IFN-γ, TNF, and IL-2 in the supernatant of each group were labeled with a Cytometric Bead Array (CBA) kit (BDBiosciences), and the secretion of cytokines in the supernatant of each group was detected by flow cytometry (NovoCyte 2060R, ACEA Biosciences, San Diego, CA, USA).

[0097] Figure 7The results showed that after stimulation with Huh7 or HepG2 cells, CAR-T-0, CAR-T-1 and CAR-T-2 CAR-T cells could 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 antigen and activate T cells to express cytokines.

[0098] Example 7: Activation function of CD40L in CAR-T-2 cells on antigen presenting cells

[0099] This example is to detect the activation effect of CAR-T (CAR-T-1 and CAR-T-2) containing the above-mentioned scFv, CD28 co-stimulatory signal domain, optimized CD3ζ signal domain, and co-expression or non-co-expression of CD40L on B cell-derived Daudi cells.

[0100] Take the T, CAR-T-1 and CAR-T-2 prepared above, and Daudi cells (National Laboratory Cell Resource Sharing Service Platform), resuspend them with appropriate amount of X-VIVO in a 15mL centrifuge tube, centrifuge at 400g for 5min, resuspend them with appropriate amount of X-VIVO, centrifuge them at 400g for 5min, resuspend them with appropriate amount of X-VIVO for counting, and adjust the concentration of T, CAR-T-1, CAR-T-2, and Daudi cells to 1*10^6 / mL with X-VIVO according to the counting results, and inoculate Daudi cells at a number of 2*10^5 / well (i.e. 200μL / well) in a 24-well plate. Inoculate CAR-T cells according to the ratio of effector-target ratio CAR-T: Daudi=1:3, and finally adjust the total number of T cells in each group to the same, to a total volume of 500μL, and finally add 300 μL of X-VIVO to each well to a final volume of 1mL / well, with 2 replicates for each group. After the cells were inoculated, they were gently blown to mix and co-cultured in a carbon dioxide incubator for 48 h. After the co-culture, each well was blown evenly with a pipette, 500 μL of cell suspension was pipetted into a 1.5 mL microcentrifuge tube, centrifuged at 400 g for 5 min, the supernatant was discarded, the cell pellet was resuspended in 0.5 mL DPBS, centrifuged at 400 g for 5 min, and the supernatant was discarded. All groups were resuspended in 50 μL of FCR blocker (Biolegend) prepared in DPBS and Zombie NIR™ Fixable Viability Kit (Biolegend), and incubated at room temperature in the dark for 10 min. Six antibodies were prepared using 1% BSA (Yishen Bio) dissolved in DPBS, including Alexa Fluor 532 CD3 Monoclonal Antibody (UCHT1) (Thermo Fisher), CD19-APC (Novusbio), FITC anti-humanCD86 (Biolegend), Alexa Fluor 647 anti-human CD80 Antibody (Biolegend), BrilliantViolet 421 TM anti-human CD40 Antibody (Biolegend), and PE anti-human HLA-DR (Biolegend). 50 μL / tube was added to each group and incubated at room temperature in the dark for 20 min. 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 in each group. Samples were loaded and detected on a full-spectrum multicolor flow cytometer (NL-3000, Cytek), and 50,000 Daudi cells were collected in each group for analysis.

[0101] Figure 8The results showed 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.

[0102] Example 8: Construction of GPC3 CAR-T cells overexpressing different co-stimulatory ligands, expression rates of CAR and co-stimulatory ligands, and CAR-T cell proliferation efficiency

[0103] Referring to the method of Example 2, genes of CAR-3, CAR-4, CAR-5, CAR-6 and CAR-7 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, respectively, and then ligated with T4 DNA ligase to obtain pLenti6.3 / V5 plasmids carrying different CAR genes.

[0104] T cells were isolated from peripheral blood mononuclear cells of healthy volunteers (Miaotong (Shanghai) Biotechnology Co., Ltd., China) using CD3 / CD28 Dynabeads (Thermo Fisher, Waltham, MA, USA) (marked as Day 0 time point). After the isolated T cells were cultured in fresh X-VIVO 15 culture system containing IL-2 (500 IU / mL, Beijing Shuanglu Pharmaceutical Co., Ltd.) for 24 hours, the T cell density was adjusted to 2×10^6 cells / mL, and 1 mL of T cell suspension was taken in a 24-well plate and infected with different lentiviruses of CAR-2, CAR-3, CAR-4, CAR-5, CAR-6 and CAR-7 at an MOI of 1. After 24 hours of virus infection, the cells were centrifuged and the medium was replaced, and the cells were cultured in the above culture system. 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, 1×10^6 cells were taken, Dynabeads were removed using a magnetic stand, and the cells were incubated with PE-labeled Strep tag II antibody and APC-labeled CD40L antibody (Biolegend) or APC-labeled ICOSL antibody (Biolegend) or APC-labeled OX40L antibody (Biolegend) or APC-labeled CD70 antibody (Biolegend) or APC-labeled 4-1BBL antibody (Biolegend). After incubation at room temperature for 20 min, 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 in each group. Samples were loaded and tested on a full-spectrum multicolor flow cytometer (NL-3000, Cytek), and 50,000 T cells were collected from each group to analyze the CAR content of each group, as well as the CD40L content on the surface of CAR-T-2 and CAR-T-7 cells, the ICOSL content on the surface of CAR-T-3, the OX40L content on the surface of CAR-T-4, the CD70 content on the surface of CAR-T-5, and the 4-1BBL content on the surface of CAR-T-6. The number of CAR-T cells in the culture system was calculated, and the T cell and CAR-T cell expansion curves were drawn.

[0105] like Figure 9 As shown in the figure, the total number of T cells in each CAR-T group was higher than that of the control T cell group without virus transfection. In each CAR-T group, before Day 8, the number of cells between the groups was not much different; after Day 8, the number of CAR-T-5 cells was the largest, and the number of CAR-T-7 cells was the smallest. With the cultivation time, the CAR expression of each group was stable, and the CAR-T cells continued to grow. On Day 12, the number of CAR-T cells of CAR-T-4 and CAR-T-5 was the largest.

[0106] Example 9: Differentiation and depletion of GPC3 CAR-T cells overexpressing different co-stimulatory ligands

[0107] On Day 8 of CAR-T cell culture in each group, 1×10 6 T cells were centrifuged at 400×g for 5 min, the supernatant was discarded, and the cell pellet was resuspended in 0.5 mL DPBS, centrifuged at 400×g for 5 min, and the supernatant was discarded. All groups were resuspended in 50 μL of Zombie NIR™ Fixable Viability Kit (Biolegend) prepared with DPBS and incubated at room temperature in the dark for 10 min. Nine antibodies were prepared using 1% BSA (Yishen Biotechnology) 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 min. All samples were washed twice with 1% BSA dissolved in DPBS, 0.5 mL each time, and the cells were resuspended in 200 μL of 1% BSA solution in each group. The samples were loaded and tested 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.

[0108] like Figure 10 As shown in the figure, on the 8th day of CAR-T culture, each CAR-T group showed different degrees of differentiation, which were mainly composed of naïve and memory stem cells (TSCM). Among them, CAR-T-4 had the highest proportion of naïve and TSCM, while CAR-T-7 had the highest proportion of central memory T cells (TCM) in each group of CAR-T. - 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.

[0109] Example 10: Killing effect and expansion capacity of GPC3 CAR-T cells overexpressing different co-stimulatory ligands under repeated stimulation of GPC3-positive target cells

[0110] 50,000 tumor cells were plated per well in a 48-well plate and cultured overnight. On the second day, 1×10^6 T cells were taken, PE-labeled Strep tag II antibody was added, and the CAR content of each group was detected by flow cytometry. According to the effect-target ratio CAR-T: tumor cells = 3:1, the number of cells required for each group of CAR-T was calculated, and the total amount of CAR-T cells in each group was adjusted to the same using untransduced T cells. Four replicates were added to the 48-well plate with 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 plated per well in a 48-well plate again. After the tumor cells adhered to the wall, each group of CAR-T was transferred to the corresponding well for repeated stimulation of target cells. After 3 times, the cells in each well were collected into a 1.5 mL microcentrifuge tube, centrifuged at 400 g for 5 min, and the supernatant was discarded. The cell pellet was resuspended with 0.5 mL DPBS, centrifuged at 400 g for 5 min, and the supernatant was discarded. All groups were first resuspended with 100 μL of Zombie NIR™ Fixable Viability Kit (Biolegend) prepared in DPBS and incubated at room temperature in the dark for 10 min. Then, two flow cytometry antibodies, CD3-APC (BD Biosciences) and PE-labeled CAR antibody (prepared by Beijing Yimiao Shenzhou Pharmaceutical Technology Co., Ltd.), were added to each well and incubated at room temperature for 30 min. 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 in each group. 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 the figure. Figure 11 shown.

[0111] The results showed that under repeated stimulation of target cells, CAR-T-2 and CAR-T-3 had the strongest ability to kill tumors compared with other CAR-Ts, and were significantly better than CAR-T-4, CAR-T-5, CAR-T-6 and CAR-T-7. In terms of CAR-T persistence, CAR-T-2 had the strongest ability to continuously expand, significantly better than CAR-T-3, CAR-T-4 and CAR-T-5. The above results show that CAR-T-2 containing the above-mentioned GPC3 scFv, CD28 costimulatory signal domain, optimized CD3ζ signal domain, and co-expression of CD40L has a strong anti-tumor effect and persistence in vitro.

[0112] Example 11: Anti-tumor ability, expansion ability and sustained performance of CAR-T-2 cells in tumor-bearing mice

[0113] This example takes the detection of the anti-tumor ability, in vivo expansion ability and sustainability of CAR-T (CAR-T-2) containing the above-mentioned scFv, CD28 co-stimulatory signal domain, optimized CD3ζ signal domain, and co-expression of CD40L in tumor-bearing mice as an example.

[0114] CAR-T-2 cells were prepared according to the above CAR-T cell preparation method. When the cells were cultured to a sufficient number, they were resuspended in cryopreservation solution and stored in liquid nitrogen for later 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 / group. Each mouse was subcutaneously inoculated with 2.0×10 6 After 7 days of inoculation with Huh7-LAE cells (ATCC, USA), mice were analyzed by luciferase live imaging (Lumina II Small Animal Live Imaging System, PerkinElmer, USA) to verify whether the mouse xenogeneic inoculation liver cancer model was successful. On the 10th day after tumor inoculation, each group of mice was injected with normal saline, control T cells, and different doses of CAR-2 cells (1×10^6, 3×10^6, 5×10^6, 1×10^7 CAR-T cells / mouse) through the tail vein. The mice were analyzed by live imaging 1 day before CAR-T cell injection and on days 5, 12, 17, 22, 27, and 32 after injection, and peripheral blood CAR-T was detected on days 5, 12, 17, 22, 27, and 32 after CAR-T cell injection.

[0115] Figure 12 A 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.

[0116] Figure 12 B 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.

[0117] The above results indicate that CAR-T containing the above-mentioned GPC3 scFv, CD28 co-stimulatory signal domain, optimized CD3ζ signal domain, and co-expressing CD40L has a strong anti-tumor effect and sustainability in vivo.

[0118] 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 amplification efficiency and duration.

Claims

1. A chimeric antigen receptor comprising genes encoding a scFv targeting GPC3, a CD28 hinge region, a CD28 transmembrane region, a CD3ζ signaling domain, a co-stimulatory signaling domain, and a CD40L protein; wherein, The amino acid sequence of the scFv is shown in SEQ ID NO: 1; The co-stimulatory signal domain is from CD28; The amino acid sequence of CD40L is shown in SEQ ID NO: 5; The amino acid sequence of the CD3ζ signaling domain is shown in SEQ ID NO:

3.

2. The chimeric antigen receptor of claim 1, further comprising a gene encoding a signal peptide.

3. The chimeric antigen receptor according to 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.

4. The chimeric antigen receptor according to claim 1 or 2, whose amino acid sequence is shown in SEQ ID NO:

9.

5. An isolated nucleic acid encoding the chimeric antigen receptor according to any one of claims 1 to 4. A vector comprising the nucleic acid according to claim 5 .

7. An isolated cell comprising the chimeric antigen receptor according to any one of claims 1 to 4 or the vector according to claim 6, wherein the cell is not a germ cell or a fertilized egg.

8. Use of the chimeric antigen receptor according to any one of claims 1 to 4, the nucleic acid according to claim 5, the vector according to claim 6, or the cell according to claim 7 in the preparation of a drug for treating liver cancer.

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