WO2023016524A1 - 联合her2和meso双靶点car-t载体及其构建方法和在癌症中的应用 - Google Patents

联合her2和meso双靶点car-t载体及其构建方法和在癌症中的应用 Download PDF

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WO2023016524A1
WO2023016524A1 PCT/CN2022/111866 CN2022111866W WO2023016524A1 WO 2023016524 A1 WO2023016524 A1 WO 2023016524A1 CN 2022111866 W CN2022111866 W CN 2022111866W WO 2023016524 A1 WO2023016524 A1 WO 2023016524A1
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cells
car
her2
meso
vector
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French (fr)
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姜凤婷
罗剑
郑眉
丁亚红
熊斐斐
刘雪颖
周旭
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上海生物制品研究所有限责任公司
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Definitions

  • the present invention relates to the field of biotechnology, and more specifically relates to a combined HER2 and MESO dual-target CAR-T vector, its construction method and its application in cancer.
  • pancreatic cancer is the abnormal proliferation caused by the loss of normal growth regulation of normal cells in the body. It may be accompanied by distant metastasis. It has a high degree of malignancy and is a major killer that threatens human life and health in today's society.
  • pancreatic cancer is a highly malignant tumor of the digestive tract and one of the malignant tumors with the worst prognosis, with a 5-year survival rate of only 9%.
  • Primary tumors of the pancreas mainly include three tissue sources: exocrine, endocrine, and mesenchymal tissues, among which pancreatic exocrine tumors account for more than 90% of pancreatic tumors, and 80% originate from pancreatic ductal epithelium.
  • CAR-T chimeric antigen receptor-modified T cells
  • CAR-T As one of the emerging means of tumor immunotherapy, CAR-T has become the focus and hotspot in the field of tumor treatment research in recent years.
  • CAR-T uses genetic engineering technology to isolate the patient's own T cells in vitro, and expresses a single-chain antibody (ScFv) region on its surface that can specifically recognize tumor surface antigens, so as to achieve the purpose of precisely targeting and killing cancer cells .
  • the gene-edited T cells express a CAR molecule, which is mainly composed of three parts: extracellular single-chain antibody region, transmembrane region and intracellular signal transduction region, which jointly participate in signal transmission and cascade amplification.
  • CAR-T therapy still faces many challenges in the treatment of solid tumors, including: lack of ideal therapeutic targets, homing barriers, and poor persistence of CAR-T cells caused by the immunosuppressive microenvironment.
  • the object of the present invention is to provide a kind of CAR-T cell and treatment method for solid tumor, especially pancreatic cancer.
  • the CAR structure includes HER2 single-chain antibody and MESO single-chain antibody, referred to as HM CAR.
  • amino acid sequence of the signal peptide derived from CD8 is shown in SEQ ID NO: 6;
  • the HER2 single-chain antibody structure is a specific antigen-binding domain designed for the tumor-associated antigen HER2 on the surface of pancreatic cancer, and the amino acid sequence of the HER2 single-chain antibody is shown in SEQ ID NO.12.
  • the MESO single-chain antibody structure is a specific antigen-binding domain designed for the pancreatic cancer surface tumor-associated antigen MESO, and the amino acid sequence of the MESO single-chain antibody is shown in SEQ ID NO.13.
  • the HER2 single-chain antibody and the MESO single-chain antibody are connected by the hinge Inner-Linker between the single-chain antibodies, and its amino acid sequence is shown in SEQ ID NO.1.
  • CD8 ⁇ is a transmembrane region that connects the extracellular antigen-binding domain and the intracellular signaling domain, and can anchor the CAR structure on the T cell membrane. Its amino acid sequence is shown in SEQ ID NO.7.
  • 4-1BB is a co-stimulatory domain, which transduces proliferation signals and induces the production of cytokines. Its amino acid sequence is shown in SEQ ID NO.8.
  • CD3 ⁇ is an intracellular signal transduction domain. When the extracellular domain binds to the target antigen, it will transmit a TCR-like signal to the intracellular area, activate T cells to play a role in targeting and killing tumor cells.
  • the amino acid sequence is shown in SEQ ID NO.9 Show.
  • CAR is Signal Peptide-HER2V L -(G4S) 3 -HER2V H -(G4S) 5 -MESOV H -(G4S) 3 -MESOV L -CD8 ⁇ -4-1BB-CD3 ⁇ .
  • a dual-target CAR-T therapeutic vector for pancreatic cancer which consists of two parts, the lentiviral expression vector pLenti6.3/V5 and the HM CAR structure.
  • FIG. 1 the schematic diagram of the pLenti6.3/V5 structure is shown in Figure 1: as a carrier plasmid carrying the CAR gene, cPPT (Polypurine Tract) from the HIV-1 integrase gene increases the copy number of the lentivirus integrated into the host genome.
  • cPPT Polypurine Tract
  • This vector can be used for packaging, transduction and stable integration of lentiviral expression genes into the host genome.
  • the third purpose of the present invention is to provide a third purpose of the present invention.
  • a method for constructing a dual-target CAR-T therapeutic vector for pancreatic cancer is provided.
  • the above CAR structure was synthesized by conventional biosynthesis methods according to the gene sequence, and the synthesized CAR existed on the pUC57 plasmid vector; the lentiviral expression vector pLenti6.3/V5 was purchased from invitrogen. Both the PUC57 plasmid vector and the lentiviral expression vector were digested with BamHI and XhoI, and the digested products were separated by agarose gel electrophoresis, and the target band was recovered to obtain the concentration of the vector and the target fragment.
  • the fourth object of the present invention is a
  • the application of the dual-target CAR-T therapeutic vector for pancreatic cancer is provided, that is, a CAR-T cell is provided.
  • helper plasmids pLP1, pLP2 plasmid, pLP/VSVG plasmid
  • a bispecific chimeric antigen receptor (CAR) is provided, the structure of the chimeric antigen receptor is shown in Formula I below:
  • Each "-" is independently a connecting peptide or a peptide bond
  • L is an optional signal peptide sequence
  • I is a flexible joint
  • H is an optional hinge region
  • TM is the transmembrane domain
  • C is costimulatory signal molecule
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇
  • One of scFv1 and scFv2 is an antigen-binding domain targeting HER2, and the other is an antigen-binding domain targeting MESO.
  • the scFv1 is an antigen-binding domain targeting HER2
  • the scFv2 is an antigen-binding domain targeting MESO.
  • the structure of the antigen-binding domain targeting HER2 is shown in formula A or formula B below:
  • V H1 is the variable region of the heavy chain of the anti-HER2 antibody
  • V L1 is the variable region of the light chain of the anti-HER2 antibody
  • "-" is the connecting peptide or a peptide bond.
  • the structure of the HER2-targeting antigen-binding domain is shown in Formula B.
  • V H1 and V L1 are connected by a flexible linker (or connecting peptide), and the flexible linker (or connecting peptide) is 1-4 consecutive sequences shown by GGGGS, preferably 2-4, more preferably 3-4.
  • amino acid sequence of the heavy chain variable region of the anti-HER2 antibody is shown in SEQ ID NO: 2
  • amino acid sequence of the light chain variable region of the anti-HER2 antibody is shown in SEQ ID NO: 3 .
  • the structure of the MESO-targeted antigen-binding domain is shown in Formula C or Formula D below:
  • V L2 is the variable region of the light chain of the anti-MESO antibody
  • V H2 is the variable region of the heavy chain of the anti-MESO antibody
  • "-" is the connecting peptide or a peptide bond.
  • the structure of the MESO-targeting antigen-binding domain is shown in Formula D.
  • V L2 and V H2 are linked by a flexible linker (or connecting peptide), and the flexible linker (or connecting peptide) is 1-4 consecutive sequences shown by GGGGS, preferably 2-4, more preferably 3-4.
  • amino acid sequence of the heavy chain variable region of the anti-MESO antibody is shown in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region of the anti-MESO antibody is shown in SEQ ID NO: 5 .
  • the scFv1 and/or scFv2 are murine, human, human and murine chimeric, or fully humanized single-chain antibody variable region fragments.
  • the sequence of the flexible linker I includes 2-6, preferably 2-4, more preferably 3-4 consecutive sequences represented by GGGGS.
  • said L is a signal peptide of a protein selected from the following group: CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.
  • said L is a signal peptide derived from CD8.
  • amino acid sequence of L is shown in SEQ ID NO:6.
  • the TM is a transmembrane region of a protein selected from the group consisting of CD8 ⁇ , CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, GD2, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.
  • the TM is a transmembrane region derived from CD8 ⁇ .
  • sequence of TM is shown in SEQ ID NO:7.
  • said C is a co-stimulatory signal molecule selected from the following group of proteins: 4-1BB (CD137), OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, PD1 , Dap10, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof.
  • said C is a co-stimulatory signal molecule derived from 4-1BB.
  • amino acid sequence of the co-stimulatory signal molecule derived from 4-1BB is shown in SEQ ID NO:8.
  • amino acid sequence of CD3 ⁇ is shown in SEQ ID NO:9.
  • amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO:10.
  • the second aspect of the present invention provides a nucleic acid molecule encoding the chimeric antigen receptor (CAR) described in the first aspect of the present invention.
  • CAR chimeric antigen receptor
  • the nucleic acid molecule is isolated.
  • nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 11.
  • the third aspect of the present invention provides a vector containing the nucleic acid molecule described in the second aspect of the present invention.
  • the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, adeno-associated viral vector (AAV), retroviral vector, transposon, or a combination thereof .
  • the vector is selected from the group consisting of plasmids and viral vectors.
  • the vector is in the form of virus particles.
  • the vector is a lentiviral vector.
  • the fourth aspect of the present invention provides a host cell containing the vector of the third aspect of the present invention or the exogenous nucleic acid molecule of the second aspect of the present invention integrated in the chromosome or expressing the vector of the present invention The CAR described in the first aspect.
  • the cells are isolated cells.
  • the cells are genetically engineered cells.
  • the cells are mammalian cells.
  • the cells are from human or non-human mammals (such as mice).
  • the cells include T cells and NK cells.
  • the cells are CAR-T cells or CAR-NK cells, preferably CAR-T cells.
  • the cells target both HER2 and MESO.
  • the fifth invention of the present invention provides a preparation, which contains the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the carrier described in the third aspect of the present invention, Or the host cell described in the fourth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the formulation is a liquid formulation.
  • the dosage form of the preparation is injection.
  • the concentration of the cells in the preparation is 1 ⁇ 10 5 -1 ⁇ 10 8 cells/ml, preferably 1 ⁇ 10 7 -1 ⁇ 10 8 cells/ml.
  • the sixth aspect of the present invention provides a chimeric antigen receptor according to the first aspect of the present invention, the nucleic acid molecule according to the second aspect of the present invention, the vector according to the third aspect of the present invention, or the fourth aspect of the present invention
  • the use of the host cell or the preparation according to the fifth invention of the present invention is used to prepare a drug or preparation for preventing and/or treating cancer or tumor.
  • the tumor includes a solid tumor.
  • the solid tumor is selected from the group consisting of pancreatic cancer, gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, and colorectal cancer , cervical cancer, ovarian cancer, adrenal gland tumors, bladder tumors, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or combinations thereof.
  • pancreatic cancer gastric cancer
  • peritoneal metastasis of gastric cancer liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, and colorectal cancer
  • cervical cancer ovarian cancer
  • adrenal gland tumors bladder tumors
  • NSCLC non-small cell lung cancer
  • glioma endometrial cancer
  • the tumor includes HER2 and/or MESO positive solid tumors.
  • the seventh aspect of the present invention provides a kit for preparing the host cell according to the fourth aspect of the present invention, the kit comprising a container, and the nucleic acid molecule according to the second aspect of the present invention located in the container, or The carrier described in the third aspect of the present invention.
  • the eighth aspect of the present invention provides a method for preparing engineered immune cells expressing the CAR described in the first aspect of the present invention, the method comprising the following steps:
  • the engineered immune cells are CAR-T cells or CAR-NK cells.
  • the method further includes the step of testing the function and effectiveness of the obtained engineered immune cells.
  • the immune cells include T cells, NK cells, and macrophages.
  • the ninth aspect of the present invention provides a use of the host cell described in the fourth aspect of the present invention, or the preparation described in the fifth aspect of the present invention, for preventing and/or treating cancer or tumor.
  • the tenth aspect of the present invention provides a method for treating diseases, comprising administering an appropriate amount of the vector of the third aspect of the present invention, the host cell of the fourth aspect of the present invention, or the fifth aspect of the present invention to a subject in need of treatment the formulation.
  • the disease is cancer or tumor.
  • Figure 1 is a schematic diagram of the pLenti6.3/V5 vector plasmid structure
  • Fig. 2 is a flowchart of the present invention
  • Figure 3 is the result of agarose gel electrophoresis after the HM CAR gene constructed in the pLenti6.3/V5 vector was identified by double digestion with BamHI and XhoI;
  • Fig. 4 is the flow cytometric analysis detection result figure of pancreatic cancer cell SW-1990 (4-1) and ASPC-1 (4-2) HER2 and MESO antigen expression;
  • Figure 5 shows the results of transduction efficiency detection of HM CAR-T cells
  • Figure 6 is a graph showing the results of RTCA monitoring HM CAR-T cells killing pancreatic cancer cells
  • Figure 7 shows a schematic diagram of the CAR structure
  • Figure 8 shows that the therapeutic effect of HM CAR-T cells is better than that of single-target CAR-T cells.
  • the present inventors unexpectedly discovered for the first time a bispecific CAR targeting HER2 and MESO, which contains HER2 scFv and MESO scFv, as well as 4-1BB co-stimulatory domain and CD3 activation area.
  • the bispecific CAR-T cells of the present invention improve the tumor-killing effect of T cells, and the cytokines produced have a super-additive effect. Compared with single-target CAR-T, it can better clear tumor cells and alleviate The antigen escape phenomenon caused by tumor heterogeneity further strengthens the ability of CAR-T cells to kill tumors.
  • the present inventors have completed the present invention.
  • administration refers to the physical introduction of a product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or Other parenteral routes of administration, for example by injection or infusion.
  • antibody shall include, but not be limited to, an immunoglobulin that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigenic Combined part.
  • Each H chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three constant domains CH1, CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region comprises one constant domain, CL.
  • VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL contains three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • amino acids in this article are identified by international single English letters, and the corresponding three-letter abbreviations of amino acid names are: Ala(A), Arg(R), Asn(N), Asp(D), Cys (C), Gln(Q), Glu(E), Gly(G), His(H), I1e(I), Leu(L), Lys(K), Met(M), Phe(F), Pro (P), Ser(S), Thr(T), Trp(W), Tyr(Y), Val(V).
  • HER2 also known as ErbB-2/neu, is located on the long arm of chromosome 17 (17q12-21.32), and can encode a 185kDa transmembrane receptor protein with tyrosine kinase activity. Under physiological conditions, the combination of receptors and ligands promotes HER2 dimerization, activates downstream MAPK, PI3K and other signaling pathways, promotes cell proliferation and prevents cell apoptosis. When HER2 is overexpressed on the cell surface, the cells will undergo excessive proliferation and malignant metastasis. Therefore, by blocking the activated HER2 signaling pathway, it inhibits tumor cell proliferation, initiates the apoptosis pathway, and promotes the programmed death of tumor cells.
  • HER2 can be used as an ideal target to treat tumors with high expression positive rate.
  • MSLN Mesothelin
  • GPI glycosylphosphatidylinositol
  • MSLN binds with the receptor CA125/MUC16 with high affinity to promote heterotypic adhesion between cells, leading to the proliferation and metastasis of tumor cells. Therefore, according to the relatively high expression of MSLN, it can be selected as a more specific target for CAR-T cell therapy.
  • Cellular immunotherapy is an emerging tumor treatment mode with significant curative effect, and it is a new type of autoimmune anti-cancer treatment. It is a method of using biotechnology and biological agents to culture and expand immune cells collected from patients in vitro and then infuse them back into the patient's body to stimulate and enhance the body's autoimmune function, so as to achieve the purpose of treating tumors.
  • biotechnology and biological agents to culture and expand immune cells collected from patients in vitro and then infuse them back into the patient's body to stimulate and enhance the body's autoimmune function, so as to achieve the purpose of treating tumors.
  • Those skilled in the art have been devoting themselves to developing new cellular immunotherapy in order to improve the effect of cellular immunotherapy and reduce its side effects.
  • the present invention proposes a rationally optimized single-chain design and system that combines bispecific CARs that can be efficiently integrated into primary human T cells and can simultaneously target HER2 and MESO when T cells are activated.
  • the CAR-T cells of the present invention can recognize two antigens (HER2 and MESO), which is a very effective and potential method for preventing antigen escape.
  • the present invention uses bidirectionally targeted HER2 and MESO CARs, which can better clear tumor cells and reduce antigen escape caused by tumor heterogeneity compared with single-antigen-targeted CARs, and further enhance the killing of CAR-T cells Tumor capacity, with cytokine synergistic effects.
  • HER2 and MESO bidirectionally targeted HER2 and MESO CARs
  • the scope of dual-targeted CAR-T therapy is wider.
  • CAR-T targeting both HER2 and MESO on the surface of tumor cells can reduce the possibility of antigen escape caused by the downregulation or deletion of a single surface antigen.
  • Bispecificity means that the same CAR can specifically bind and immune recognize two different antigens, and CAR can generate an immune response when combined with any antigen.
  • the HER2 and MESO bispecific CAR of the present invention has a single structure, including anti-HER2 and MESO scFv.
  • CAR contains HER2 scFv and MESO scFv, and the amino acid sequence, sorting and hinge of HER2 scFv and MESO scFv are the main factors affecting its function.
  • the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes target-specific binding elements (also known as antigen binding domains).
  • the intracellular domain includes the co-stimulatory signaling region and the zeta chain portion.
  • a co-stimulatory signaling region refers to a portion of an intracellular domain that includes co-stimulatory molecules.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient response of lymphocytes to antigens.
  • a linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR.
  • the term "linker” generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to the extracellular or cytoplasmic domain of a polypeptide chain.
  • Linkers may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.
  • the extracellular domain of the CAR provided by the present invention includes an antigen-binding domain targeting the combination of HER2 and MESO.
  • the CAR of the present invention when expressed in T cells, is capable of antigen recognition based on antigen binding specificity. When it binds its cognate antigen, it affects tumor cells, causing them not to grow, being induced to die, or otherwise affected, and resulting in a reduction or elimination of the patient's tumor burden.
  • the antigen binding domain is preferably fused to an intracellular domain from one or more of the co-stimulatory molecule and the zeta chain.
  • the antigen binding domain is fused to the intracellular domain of the combination of the 4-1BB signaling domain, and the CD3 ⁇ signaling domain.
  • antigen-binding domain and “single-chain antibody fragment” all refer to a Fab fragment, Fab' fragment, F(ab') 2 fragment, or a single Fv fragment having antigen-binding activity.
  • Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant region, and the smallest antibody fragment with all antigen-binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding.
  • the antigen-binding domain is usually a scFv (single-chain variable fragment).
  • the size of scFv is generally 1/6 of a whole antibody.
  • a single-chain antibody is preferably a sequence of one amino acid chain encoded by one nucleotide chain.
  • the scFv comprises antibodies that specifically recognize HER2 and MESO.
  • the CAR can be designed to include the transmembrane domain fused to the extracellular domain of the CAR.
  • a transmembrane domain naturally associated with one of the domains in the CAR is used.
  • transmembrane domains may be selected, or modified by amino acid substitutions, to avoid binding such domains to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with the receptor complex. interactions with other members.
  • the intracellular domain in the CAR of the present invention includes the signaling domain of 4-1BB and the signaling domain of CD3 ⁇ .
  • the structure of the CAR of the present invention includes a signal peptide sequence (also known as a leader sequence), an antigen recognition sequence (antigen binding domain), an optional hinge region, a transmembrane region, a co-stimulatory factor signal region, and a CD3zeta signal transduction sequence.
  • region ( ⁇ chain part) the carrier plasmid containing the CAR gene as shown in Figure 1, as the carrier plasmid carrying the CAR gene, cPPT (Polypurine Tract) from the HIV-1 integrase gene increases the lentivirus that is integrated into the host genome.
  • Copy number the CAR structure of the present invention is shown in Figure 7.
  • the CAR of the present invention is HM CAR.
  • the antigen-binding domain targeting HER2 includes a single-chain variable region heavy chain sequence (SEQ ID NO: 2) and a single-chain variable region light chain (VL) sequence (SEQ ID NO: 3) derived from a HER2 antibody.
  • VH single-chain variable heavy chain
  • VL variable region light chain
  • HER2 single chain antibody HER2-ScFv sequence (HER2V L -(G4S)3-HER2V H )
  • the antigen-binding domain targeting MESO comprises a single-chain variable region heavy chain sequence (SEQ ID NO:4) and a single-chain variable region light chain sequence (SEQ ID NO:5) derived from a MESO antibody.
  • VH variable heavy chain
  • VL variable region light chain
  • MESO single chain antibody MESO-ScFv sequence (MESOV H -(G4S) 3 -MESOV L )
  • sequences of other elements in the CAR of the present invention are as follows:
  • the signal peptide is a signal peptide derived from CD8:
  • the connecting sequence (i.e. flexible linker I) between the heavy chain and the light chain of the single-chain variable region is:
  • the transmembrane region is the transmembrane region sequence derived from CD8 ⁇ :
  • the co-stimulatory factor signaling domain is derived from the sequence of the intracellular signaling motif of 4-1BB:
  • nucleic acid sequence and amino acid sequence of the CAR constructed in the present invention are as follows:
  • CAR-T cell As used herein, the terms “CAR-T cell”, “CAR-T”, “CART”, and “CAR-T cell of the present invention” all refer to the CAR-T targeting both HER2 and MESO described in the fourth aspect of the present invention cell.
  • the CAR structure of the CAR-T cell includes an anti-HER2 scFv, an anti-MESO scFv, a transmembrane region, and an intracellular T cell signal region, and the anti-HER2 scFv and MESO scFv pass multiple repeating structure (G4S) peptides connect.
  • G4S multiple repeating structure
  • a nucleic acid sequence encoding a desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by using standard technology, directly isolated from cells and tissues containing the gene.
  • the gene of interest can be produced synthetically.
  • the present invention also provides a vector into which an expression cassette of the present invention is inserted.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools to achieve long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in daughter cells.
  • Lentiviral vectors have an advantage over vectors derived from oncogenic retroviruses, such as murine leukemia virus, because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.
  • an expression cassette or nucleic acid sequence of the invention is typically operably linked to a promoter and incorporated into an expression vector.
  • This vector is suitable for replication and integration in eukaryotic cells.
  • a typical cloning vector contains transcriptional and translational terminators, an initial sequence and a promoter useful for regulating the expression of the desired nucleic acid sequence.
  • the expression constructs of the invention can also be used in nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, eg, US Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby incorporated by reference in their entirety.
  • the present invention provides gene therapy vectors.
  • the nucleic acid can be cloned into many types of vectors.
  • the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • vectors of interest include expression vectors, replication vectors, probe production vectors and sequencing vectors.
  • expression vectors can be provided to cells in the form of viral vectors.
  • Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks of virology and molecular biology.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO01/96584; WO01/29058; and US Patent No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the gene of choice can be inserted into a vector and packaged into retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to subject cells in vivo or ex vivo.
  • retroviral systems are known in the art.
  • an adenoviral vector is used.
  • Many adenoviral vectors are known in the art.
  • lentiviral vectors are used.
  • promoter elements can regulate the frequency of transcription initiation. Typically these are located in a region of 30-110 bp upstream of the initiation site, although it has recently been shown that many promoters also contain functional elements downstream of the initiation site.
  • the spacing between promoter elements is often flexible in order to preserve promoter function when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can act cooperatively or independently to initiate transcription.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto.
  • Another example of a suitable promoter is elongation growth factor-1 alpha (EF-1 alpha).
  • constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Ruth's sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter , myosin promoter, heme promoter and creatine kinase promoter.
  • the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention.
  • an inducible promoter provides a molecular switch capable of turning on expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turning off expression when expression is not desired.
  • inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.
  • the expression vector introduced into the cell may also contain either or both of a selectable marker gene or a reporter gene, so as to seek transfected or infected cell populations from viral vectors. Identification and selection of expressing cells.
  • selectable markers can be carried on a single piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell.
  • Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.
  • Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences.
  • a reporter gene is a gene that is absent from or expressed by a recipient organism or tissue and that encodes a polypeptide whose expression is clearly indicated by some readily detectable property, such as enzymatic activity. Expression of the reporter gene is measured at an appropriate time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, ⁇ -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79 -82).
  • Suitable expression systems are known and can be prepared using known techniques or obtained commercially.
  • the construct with the minimum of 5 flanking regions showing the highest level of reporter gene expression was identified as a promoter.
  • Such a promoter region can be linked to a reporter gene and used to assess the ability of the agent to regulate promoter-driven transcription.
  • the vector can be easily introduced into host cells, eg, mammalian, bacterial, yeast or insect cells, by any method in the art.
  • expression vectors can be transferred into host cells by physical, chemical or biological means.
  • Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, eg, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.
  • Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors.
  • Viral vectors especially retroviral vectors, have become the most widely used method of inserting genes into mammalian, eg human, cells.
  • Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, eg, US Patent Nos. 5,350,674 and 5,585,362.
  • colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipid-based systems.
  • lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and lipid-based systems.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (eg, an artificial membrane vesicle).
  • an exemplary delivery vehicle is liposomes.
  • lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo).
  • the nucleic acid can be associated with a lipid.
  • Lipid-associated nucleic acids can be encapsulated into the aqueous interior of liposomes, interspersed within the lipid bilayer of liposomes, attached via linker molecules associated with both liposomes and oligonucleotides
  • linker molecules associated with both liposomes and oligonucleotides
  • entrapped in liposomes complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, associated with lipids, contained in lipids as a suspension, contained in micelles or Complexes with micelles, or otherwise associated with lipids.
  • the lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution.
  • Lipids are fatty substances, which may be naturally occurring or synthetic lipids.
  • lipids include fat droplets, which occur naturally in the cytoplasm as well as compounds comprising long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.
  • the vector is a lentiviral vector.
  • the present invention provides a CAR-T cell containing the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the formulation is a liquid formulation.
  • the preparation is an injection.
  • the concentration of the CAR-T cells in the preparation is 1 ⁇ 10 5 -1 ⁇ 10 8 cells/ml, preferably 1 ⁇ 10 7 -1 ⁇ 10 8 cells/ml.
  • the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine ; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, sulfate buffered saline, etc.
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids
  • antioxidants such as glycine
  • chelating agents such as EDTA or glutathione
  • adjuvants eg, aluminum hydroxide
  • preservatives e.g, aluminum hydroxide
  • the invention includes therapeutic applications of cells (eg, T cells) transduced with a lentiviral vector (LV) encoding an expression cassette of the invention.
  • the transduced T cells can target tumor cell markers HER2 and MESO, activate T cells synergistically, and cause T cell immune responses, thereby significantly improving their killing efficiency against tumor cells.
  • the present invention also provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal, comprising the following steps: administering the CAR-T cells of the present invention to the mammal.
  • the present invention includes a type of cell therapy, in which a patient's own T cells (or a heterologous donor) are isolated, activated and genetically modified to produce CAR-T cells, and then injected into the same patient.
  • a patient's own T cells or a heterologous donor
  • CAR-T can treat all cancers that express that antigen.
  • CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.
  • the CAR-T cells of the invention can undergo robust in vivo T cell expansion for extended amounts of time.
  • the CAR-mediated immune response can be part of an adoptive immunotherapy step in which CAR-modified T cells induce an immune response specific for the antigen-binding domain in the CAR.
  • CAR-T cells against HER2 and MESO elicit a specific immune response against cells positive for HER2 and/or MESO.
  • Hematological cancers are cancers of the blood or bone marrow.
  • hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic , monocytic and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.
  • acute leukemias such as
  • Solid tumors are abnormal masses of tissue that usually do not contain cysts or areas of fluid. Solid tumors can be benign or malignant. The different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer and ovarian cancer.
  • the treatable cancer is a HER2 and/or MESO positive tumor, such as pancreatic cancer.
  • the CAR-modified T cells of the present invention can also be used as a type of vaccine for ex vivo immunization and/or in vivo therapy of mammals.
  • the mammal is a human.
  • cells are isolated from a mammal (preferably a human) and genetically modified (ie, transduced or transfected in vitro) with a vector expressing a CAR disclosed herein.
  • CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefit.
  • the mammalian recipient can be a human, and the CAR-modified cells can be autologous to the recipient.
  • the cells may be allogeneic, syngeneic or xenogeneic with respect to the recipient.
  • the invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.
  • the present invention provides a method of treating a tumor comprising administering to a subject in need thereof a therapeutically effective amount of the CAR-modified T cells of the present invention.
  • the CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition with a diluent and/or in combination with other components such as IL-2, IL-17 or other cytokines or cell populations.
  • the pharmaceutical compositions of the present invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; Agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, sulfate buffered saline, and the like
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids such as glycine
  • antioxidants such as EDTA or glutathione
  • adjuvants eg, aluminum hydroxide
  • preservatives eg, aluminum hydroxide
  • the pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented).
  • the amount and frequency of administration will be determined by such factors as the patient's condition, and the type and severity of the patient's disease - although appropriate dosages may be determined by clinical trials.
  • compositions of the invention to be administered can be determined by a physician, taking into account the patient (subject ) with individual differences in age, weight, tumor size, degree of infection or metastasis, and disease. It may generally be stated that pharmaceutical compositions comprising T cells as described herein may be dosed at a dose of 104 to 109 cells/kg body weight, preferably at a dose of 105 to 106 cells/kg body weight (including all integers within those ranges value) applied. T cell compositions can also be administered multiple times at these doses.
  • Cells can be administered using infusion techniques well known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).
  • the optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical art by monitoring the patient for signs of disease and adjusting treatment accordingly.
  • compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection or intraperitoneally.
  • the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection.
  • the T cell composition of the invention is preferably administered by i.v. injection.
  • Compositions of T cells can be injected directly into tumors, lymph nodes or sites of infection.
  • cells activated and expanded using the methods described herein, or other methods known in the art to expand T cells to therapeutic levels are combined with any number of relevant treatment modalities (e.g., previously , simultaneously or subsequently) to the patient in a form of treatment including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or erfatizumab treatment for psoriasis patients or other treatments for PML patients.
  • agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or erfatizumab treatment for psoriasis patients or other treatments for PML patients.
  • the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunotherapeutic agents.
  • the cell composition of the invention is administered in conjunction with (eg, before, simultaneously with, or after) bone marrow transplantation, the use of chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • a subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • the subject receives an infusion of expanded immune cells of the invention.
  • the expanded cells are administered before or after surgery.
  • Dosages administered to a patient for the above treatments will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration can be implemented according to practice accepted in the art. Usually, 1 ⁇ 10 6 to 1 ⁇ 10 10 modified T cells (CAR-T cells) of the present invention can be administered to the patient for each treatment or each course of treatment, for example, through intravenous infusion.
  • CAR-T cells modified T cells
  • the bispecific CAR-T cells of the present invention have significant killing effects on HER2-positive target cells and MESO-positive target cells.
  • the bispecific CAR-T cells of the present invention improve the tumor-killing effect of T cells, and the cytokines produced have a super-additive effect. Compared with single-target CAR-T, it can better clear tumor cells and alleviate The antigen escape phenomenon caused by tumor heterogeneity further strengthens the ability of CAR-T cells to kill tumors.
  • the present invention found for the first time that since the T cells can simultaneously express single-chain antibodies against tumor-associated antigens HER2 and MESO on the surface of pancreatic cancer cells in tandem, the range of tumor cell recognition is greatly increased, and single-target CAR- T cells recognize cancer cells below the threshold, which promotes the widespread application of tandem CAR-T cells in heterogeneous tumor subpopulations, so the killing range of pancreatic cancer cells is wider; at the same time, tandem CAR-T cells can improve T cell The effect of killing tumors, the cytokines produced have a super-additive effect, compared with single-target CAR-T, it can better clear tumor cells, reduce the antigen escape phenomenon caused by tumor heterogeneity, and further strengthen CAR-T The ability of cells to kill tumors.
  • HM CAR plasmids HM CAR-T cells, construction methods and applications capable of simultaneously expressing in series targeting HER2 and MESO.
  • Fig. 2 is a flowchart of the present invention, which is specifically shown in the following embodiments.
  • FIG. 3 is the result of agarose gel electrophoresis after the HM CAR gene constructed in the pLenti6.3/V5 vector was identified by double digestion with BamHI and XhoI.
  • Tube 1 Labeled "DNA"
  • Test Tube 2 Labeled "TfxR"
  • test tube 1 After 1 minute, pour test tube 1 into test tube 2 or combine the two solutions in the opposite way, and vortex briefly;
  • HM CAR-LV lentiviral stock solution
  • HM CAR-LV stock solution was higher than 10 7 TU/mL, and the titer could be as high as 10 9 TU/mL after concentrating the lentivirus with a 100KD ultrafiltration tube.
  • T cell CD3CD28 stimulator (note: add 10 ⁇ L stimulator for every 10 cells)
  • T cells were transfected with lentivirus at MOI 15, and mixed by pipetting;
  • the CAR gene in CAR-T cells was detected by real-time fluorescent quantitative PCR, which further proved the successful transfection of T cells with lentivirus at the gene level.
  • 1 ⁇ 10 6 HM CAR-T cells and non-transfected T cells were collected respectively, the cell DNA was extracted, and the CAR gene copy number was detected by qPCR.
  • RTCA was used to detect the killing of two pancreatic cancer cell lines by HM CAR-T cells, and the effect-to-target ratio was set to 4:1 for killing. 8 ⁇ 10 4 ASPC-1 cells and 1 ⁇ 10 4 SW-1990 pancreatic cancer cells were plated, cultured for 24 hours, and then CAR-T cells and T cells with an effect-to-target ratio of 4:1 were added to compare the effects of the two. Differential killing of tumor cells. The results are shown in Figure 6: 6-1 is HM CAR-T cells killing ASPC-1, 6-2 is HM CAR-T cells killing SW-1990.
  • the experimental results prove that compared with the control group, the killing effect of HM CAR-T cells is more superior, indicating that the tandem HM CAR-T cells expressing both HER2 and MESO can specifically target HER2- and MESO-positive pancreatic cancer cells. To lethal effect.

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Abstract

提供了联合HER2和MESO双靶点CAR-T载体及其构建方法和在癌症中的应用。具体地，提供了一种双特异性嵌合抗原受体(CAR)，其包含HER2 scFv和MESO scFv，以及4-1BB共刺激域和CD3激活域。双特异性CAR-T细胞对HER2阳性靶细胞和MESO阳性靶细胞有显著的杀伤作用，可提高T细胞杀伤肿瘤的作用，产生的细胞因子具有超级加和效应，与单靶点CAR-T相比，可以更好地清除肿瘤细胞，减轻肿瘤异质性引起的抗原逃逸现象，进一步加强了CAR-T细胞杀伤肿瘤的能力。

Description

联合HER2和MESO双靶点CAR-T载体及其构建方法和在癌症中的应用 技术领域
本发明涉及生物技术领域，更具体地涉及联合HER2和MESO双靶点CAR-T载体及其构建方法和在癌症中的应用。
背景技术
癌症，是由于机体的正常细胞失去正常生长调控而发生的异常增殖，可伴有远处转移，其恶性程度较高，是当今社会威胁人类生命健康的一大杀手。其中，胰腺癌是一种恶性程度很高的消化道肿瘤，是预后最差的恶性肿瘤之一，5年生存率仅9％。胰腺的原发性肿瘤主要包括三种组织源：外分泌、内分泌和间叶组织，其中胰腺外分泌部肿瘤占胰腺肿瘤的90％以上，且80％来源于胰腺导管上皮。由于其生理解剖位置隐匿，早期不易被发现而丧失了诊断及手术的最佳时机，绝大部分患者被确诊时已经是晚期或转移性肿瘤，这也导致其成为了癌症导致死亡的第四大常见肿瘤。预计到2030年，胰腺癌将成为美国第二大癌症死亡原因。临床治疗仍以传统的手术切除为主结合放、化疗。由于其致病因素复杂多样，目前并没有找到攻克癌症的最佳手段。近年来，以嵌合抗原受体修饰T细胞(Chimeric antigen receptor-T cell，CAR-T)为代表的肿瘤细胞免疫疗法为治疗肿瘤带来新的希望。
CAR-T作为肿瘤免疫治疗的新兴手段之一，成为近年来肿瘤治疗研究领域的焦点和热点。CAR-T是利用基因工程技术，将患者自身的T细胞分离体外，并在其表面表达一个能够特异性识别肿瘤表面抗原的单链抗体(ScFv)区域，实现精准靶向癌细胞并杀伤的目的。基因编辑后的T细胞表达一个CAR分子，主要由胞外单链抗体区、跨膜区及胞内信号传导区三部分组成，共同参与信号的传递和级联放大。
目前，CAR-T疗法对于实体肿瘤的治疗还存在诸多挑战，包括：缺乏理想的治疗靶点、归巢障碍、以及免疫抑制微环境导致的CAR-T细胞持久性表现不良等。
因此，本领域还需要开发新的用于实体瘤，尤其是胰腺癌的CAR-T细胞和治疗方法。
发明内容
本发明的目的在于提供一种用于实体瘤，尤其是胰腺癌的CAR-T细胞和治 疗方法。
本发明第一个目的：
提供一种串联的嵌合抗原受体，CAR结构包括HER2单链抗体及MESO单链抗体，简称HM CAR。
其中，CD8来源的信号肽氨基酸序列如SEQ ID NO:6所示；
其中，HER2单链抗体结构是针对胰腺癌表面肿瘤相关抗原HER2设计的特异性抗原结合域，HER2单链抗体氨基酸序列如SEQ ID NO.12所示。
其中，MESO单链抗体结构是针对胰腺癌表面肿瘤相关抗原MESO设计的特异性抗原结合域，MESO单链抗体氨基酸序列如SEQ ID NO.13所示。
HER2单链抗体和MESO单链抗体由单链抗体间铰链Inner-Linker相连接，其氨基酸序列如SEQ ID NO.1所示。
CD8α为跨膜区，连接胞外抗原结合域和胞内信号域，能将CAR结构锚定于T细胞膜上，其氨基酸序列如SEQ ID NO.7所示。
4-1BB为共刺激结构域，转导增殖信号并诱导细胞因子的产生，其氨基酸序列如SEQ ID NO.8所示。
CD3ζ为胞内信号转导域，当胞外区和目标抗原结合时，会向胞内传导TCR样信号，激活T细胞发挥靶向性杀伤肿瘤细胞的作用，氨基酸序列如SEQ ID NO.9所示。
进一步地，CAR结构组成为Signal Peptide-HER2V L-(G4S) 3-HER2V H-(G4S) 5-MESOV H-(G4S) 3-MESOV L-CD8α-4-1BB-CD3ζ。
本发明的第二个目的：
提供一种针对胰腺癌的双靶点CAR-T治疗载体，包括慢病毒表达载体pLenti6.3/V5和HM CAR结构两部分构成。
其中，pLenti6.3/V5结构示意图如图1所示：作为载体质粒携带CAR基因，来自HIV-1整合酶基因的cPPT(Polypurine Tract)，增加了整合入宿主基因组的慢病毒的拷贝数。该载体可用于包装、转导和稳定地将慢病毒表达基因整合到宿主的基因组中。
本发明的第三个目的：
提供一种针对胰腺癌的双靶点CAR-T治疗载体的构建方法。
将上述CAR结构按照基因序列，采用常规生物合成方法进行合成，合成后的CAR存在于PUC57质粒载体上；慢病毒表达载体pLenti6.3/V5购自invitrogen。 将PUC57质粒载体和慢病毒表达载体均采用BamHI、XhoI双酶切，将酶切产物经琼脂糖凝胶电泳分离，回收目的条带，得出载体和目的片段的浓度，将两者按照摩尔比为1:5进行连接转化，质粒提取，最终获得含有嵌合抗原受体结构的重组质粒，最终获得含有特定CAR结构的重组质粒。
本发明第四个目的：
提供针对胰腺癌的双靶点CAR-T治疗载体的应用，即提供一种CAR-T细胞。
采用四质粒包装系统进行慢病毒包装，三个辅助质粒(pLP1、pLP2质粒、pLP/VSVG质粒)与慢病毒表达载体共转染HEK293细胞，收集培养48h-55h的病毒液，将此病毒液浓缩，并测病毒滴度，最终以MOI＝15感染T细胞，最终获得CAR-T细胞。
在本发明的第一方面，提供了一种双特异性嵌合抗原受体(CAR)，所述嵌合抗原受体的结构如下式I所示：
L-scFv1-I-scFv2-H-TM-C-CD3ζ     (I)
式中，
各“-”独立地为连接肽或肽键；
L为任选的信号肽序列；
I为柔性接头；
H为任选的铰链区；
TM为跨膜结构域；
C为共刺激信号分子；
CD3ζ为源于CD3ζ的胞浆信号传导序列；
scFv1和scFv2两者中一个为靶向HER2的抗原结合结构域，另一个为靶向MESO的抗原结合结构域。
在另一优选例中，所述scFv1为靶向HER2的抗原结合结构域，所述scFv2为靶向MESO的抗原结合结构域。
在另一优选例中，所述靶向HER2的抗原结合结构域的结构如下式A或式B所示：
V H1-V L1   (A)；V L1-V H1   (B)
式中，V H1为抗HER2抗体重链可变区；V L1为抗HER2抗体轻链可变区；“-”为连接肽或肽键。
在另一优选例中，所述靶向HER2的抗原结合结构域的结构如式B所示。
在另一优选例中，所述的V H1和V L1通过柔性接头(或连接肽)相连，所述的柔性接头(或连接肽)为1-4个连续的GGGGS所示的序列，较佳地2-4个，更佳地3-4个。
在另一优选例中，所述抗HER2抗体重链可变区的氨基酸序列如SEQ ID NO:2所示，所述抗HER2抗体轻链可变区的氨基酸序列如SEQ ID NO:3所示。
在另一优选例中，所述靶向MESO的抗原结合结构域的结构如下式C或式D所示：
V L2-V H2   (C)；V H2-V L2   (D)
式中，V L2为抗MESO抗体轻链可变区；V H2为抗MESO抗体重链可变区；“-”为连接肽或肽键。
在另一优选例中，所述靶向MESO的抗原结合结构域的结构如式D所示。
在另一优选例中，所述的V L2和V H2通过柔性接头(或连接肽)相连，所述的柔性接头(或连接肽)为1-4个连续的GGGGS所示的序列，较佳地2-4个，更佳地3-4个。
在另一优选例中，所述抗MESO抗体重链可变区的氨基酸序列如SEQ ID NO:4所示，所述抗MESO抗体轻链可变区的氨基酸序列如SEQ ID NO:5所示。
在另一优选例中，所述的scFv1和/或scFv2为鼠源、人源、人源和鼠源嵌合、或者全人源化的单链抗体可变区片段。
在另一优选例中，所述柔性接头I的序列包含2-6个，较佳地为2－4个，更佳地3-4个连续的GGGGS所示的序列。
在另一优选例中，所述的L为选自下组的蛋白的信号肽：CD8、CD28、GM-CSF、CD4、CD137、或其组合。
在另一优选例中，所述的L为CD8来源的信号肽。
在另一优选例中，L的氨基酸序列如SEQ ID NO:6所示。
在另一优选例中，所述的TM为选自下组的蛋白的跨膜区：CD8α、CD28、CD3 epsilon、CD45、CD4、CD5、CD8、CD9、CD16、GD2、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、或其组合。
在另一优选例中，所述的TM为CD8α来源的跨膜区。
在另一优选例中，TM的序列如SEQ ID NO:7所示。
在另一优选例中，所述的C为选自下组的蛋白的共刺激信号分子：4-1BB(CD137)、OX40、CD2、CD7、CD27、CD28、CD30、CD40、CD70、CD134、PD1、Dap10、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)、NKG2D、GITR、 TLR2、或其组合。
在另一优选例中，所述的C为4-1BB来源的共刺激信号分子。
在另一优选例中，所述的4-1BB来源的共刺激信号分子的氨基酸序列如SEQ ID NO:8所示。
在另一优选例中，CD3ζ的氨基酸序列如SEQ ID NO:9所示。
在另一优选例中，所述嵌合抗原受体的氨基酸序列如SEQ ID NO:10所示。
本发明第二方面提供了一种核酸分子，所述核酸分子编码本发明第一方面所述的嵌合抗原受体(CAR)。
在另一优选例中，所述核酸分子为分离的。
在另一优选例中，所述核酸分子的核苷酸序列如SEQ ID NO:11所示。
本发明第三方面提供了一种载体，所述的载体含有本发明第二方面所述的核酸分子。
在另一优选例中，所述的载体选自下组：DNA、RNA、质粒、慢病毒载体、腺病毒载体、腺相关病毒载体(AAV)、逆转录病毒载体、转座子、或其组合。
在另一优选例中，所述的载体选自下组：质粒、病毒载体。
在另一优选例中，所述载体为病毒颗粒的形式。
在另一优选例中，所述载体为慢病毒载体。
本发明第四方面提供了一种宿主细胞，所述的宿主细胞中含有本发明第三方面所述的载体或染色体中整合有外源的本发明第二方面所述的核酸分子或表达本发明第一方面所述的CAR。
在另一优选例中，所述细胞为分离的细胞。
在另一优选例中，所述细胞为基因工程化的细胞。
在另一优选例中，所述细胞为哺乳动物细胞。
在另一优选例中，所述的细胞来自人或非人哺乳动物(如鼠)。
在另一优选例中，所述细胞包括T细胞、NK细胞。
在另一优选例中，所述细胞为CAR-T细胞或CAR-NK细胞，较佳地为CAR-T细胞。
在另一优选例中，所述细胞同时靶向HER2和MESO。
本发明第五发明提供了一种制剂，所述制剂含有本发明第一方面所述的嵌合抗原受体、本发明第二方面所述的核酸分子、本发明第三方面所述的载体、或本发明第四方面所述的宿主细胞，以及药学上可接受的载体、稀释剂或赋形剂。
在另一优选例中，所述制剂为液态制剂。
在另一优选例中，所述制剂的剂型为注射剂。
在另一优选例中，所述制剂中所述细胞的浓度为1×10 5-1×10 8个细胞/ml，较佳地1×10 7-1×10 8个细胞/ml。
本发明第六方面提供了一种本发明第一方面所述的嵌合抗原受体、本发明第二方面所述的核酸分子、本发明第三方面所述的载体、或本发明第四方面所述的宿主细胞、或本发明第五发明所述的制剂的用途，用于制备预防和/或治疗癌症或肿瘤的药物或制剂。
在另一优选例中，所述肿瘤包括实体瘤。
在另一优选例中，所述实体瘤选自下组：胰腺癌、胃癌、胃癌腹膜转移、肝癌、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、宫颈癌、卵巢癌、肾上腺肿瘤、膀胱肿瘤、非小细胞肺癌(NSCLC)、脑胶质瘤、子宫内膜癌、或其组合。
在另一优选例中，所述的肿瘤包括HER2和/或MESO阳性实体肿瘤。
本发明第七方面提供了一种用于制备本发明第四方面所述的宿主细胞的试剂盒，所述试剂盒含有容器，以及位于容器内的本发明第二方面所述的核酸分子、或本发明第三方面所述的载体。
本发明第八方面提供了一种制备工程化的免疫细胞的方法，所述的免疫细胞表达本发明第一方面所述的CAR，所述方法包括以下步骤：
(a)提供待改造的免疫细胞；和
(b)将本发明第二方面所述的核酸分子或本发明第三方面所述的载体转导入所述免疫细胞内，从而获得所述工程化的免疫细胞。
在另一优选例中，所述工程化的免疫细胞为CAR-T细胞或CAR-NK细胞。
在另一优选例中，所述的方法还包括对获得的工程化免疫细胞进行功能和有效性检测的步骤。
在另一优选例中，所述免疫细胞包括T细胞、NK细胞、巨噬细胞。
本发明第九方面提供了一种本发明第四方面所述宿主细胞、或本发明第五方面所述的制剂的用途，用于预防和/或治疗癌症或肿瘤。
本发明第十方面提供了一种治疗疾病的方法，包括给需要治疗的对象施用适量的本发明第三方面所述的载体、本发明第四方面所述的宿主细胞、或本发明第五方面所述的制剂。
在另一优选例中，所述疾病为癌症或肿瘤。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施 例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为pLenti6.3/V5载体质粒结构示意图；
图2为本发明流程图；
图3为构建在pLenti6.3/V5载体的HM CAR基因经BamHI和XhoI双酶切鉴定后，琼脂糖凝胶电泳结果；
图4为胰腺癌细胞SW-1990(4-1)和ASPC-1(4-2)HER2和MESO抗原表达情况的流式细胞分析检测结果图；
图5为HM CAR-T细胞的转导效率检测结果；
图6为RTCA监测HM CAR-T细胞杀伤胰腺癌细胞的结果图；
图7显示为CAR结构示意图；
图8显示了HM CAR-T细胞治疗效果优于单靶点CAR-T细胞。
具体实施方式
本发明人经过广泛而深入地研究，首次意外地发现一种靶向HER2和MESO双特异性CAR，所述的双特异性CAR包含HER2 scFv和MESO scFv，以及4-1BB共刺激域和CD3激活域。实验表明，本发明的双特异性CAR-T细胞提高T细胞杀伤肿瘤的作用，产生的细胞因子具有超级加和效应，与单靶点CAR-T相比，可以更好地清除肿瘤细胞，减轻肿瘤异质性引起的抗原逃逸现象，进一步加强了CAR-T细胞杀伤肿瘤的能力。在此基础上，本发明人完成了本发明。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。
术语“给予”是指使用本领域技术人员已知的各种方法和递送系统中的任一种将本发明的产品物理引入受试者，包括静脉内，肌内，皮下，腹膜内，脊髓或其它肠胃外给药途径，例如通过注射或输注。
术语“抗体”(Ab)应包括但不限于免疫球蛋白，其特异性结合抗原并包含通过二硫键互连的至少两条重(H)链和两条轻(L)链，或其抗原结合部分。每条H链包含重链可变区(本文缩写为VH)和重链恒定区。重链恒定区包含三个恒定结构域CH1、CH2和CH3。每条轻链包含轻链可变区(本文缩写为VL)和轻链恒定区。轻链恒定区包含一个恒定结构域CL。VH和VL区可以进一步细分为称为互补决定区(CDR)的高变区，其散布有更保守的称为框架区(FR)的区域。每个VH和VL包含三个CDR和四个FR，从氨基末端到羧基末端按照以下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。
应理解，本文中氨基酸名称采用国际通用的单英文字母标识，与其相对应的氨基酸名称三英文字母简写分别是：Ala(A)、Arg(R)、Asn(N)、Asp(D)、Cys(C)、Gln(Q)、Glu(E)、Gly(G)、His(H)、I1e(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser(S)、Thr(T)、Trp(W)、Tyr(Y)、Val(V)。
HER2和MESO抗原
HER2又称ErbB-2/neu，位于17号染色体长臂(17q12-21.32)，可编码185kDa的具有酪氨酸激酶活性的跨膜受体蛋白。生理情况下，通过受体与配体相结合，促使HER2二聚化，激活下游MAPK、PI3K等信号通路，促进细胞增殖、防止细胞凋亡。当细胞表面HER2过表达，细胞会发生过度增殖及恶性转移。因此，通过阻断活化的HER2信号通路，抑制肿瘤细胞增殖、启动凋亡途径，促进肿瘤细胞程序性死亡。约20％的原发性乳腺浸润性导管癌有HER2过表达；雄激素非依赖性前列腺癌(AIPC)组患者HER2表达阳性率高于良性前列腺增生组和雄激素依赖性前列腺癌组；约20％～60％的胰腺导管腺癌(PDAC)患者中也有阳性表达。因此，HER2可以作为一个理想的靶点来治疗表达阳性率高的肿瘤。
间皮素(MESO)是由糖基磷脂酰肌醇(GPI)锚定的糖蛋白，分子量约40KD。在正常组织细胞中不表达，或微量表达于间皮细胞。几乎100％的胰腺癌MSLN均阳性，其他肿瘤如肝外胆管癌(95％)，子宫内膜癌(89％)，三阴性乳腺癌(66％)，食道癌(46％)，结直肠癌(30％)和宫颈癌(25％)等均有不同程度表达。异常MSLN高表达使肿瘤进一步恶化，发生途径主要有两种：一是通过GPI激活胞内信号通路，并持续活化NF-κB、MAPK和PI3-激酶信号通路，促进肿瘤细胞增殖并增强其抗凋亡能力；二是MSLN与受体CA125/MUC16发生高亲和力结合，促进细胞 间异型粘附，导致肿瘤细胞发生扩散及转移。因此，根据MSLN相对高表达，可以选作为CAR-T细胞治疗较特异性的靶点。
靶向HER2和MESO的双特异性嵌合抗原受体
细胞免疫治疗是一种新兴的、具有显著疗效的肿瘤治疗模式，是一种自身免疫抗癌的新型治疗方法。它是运用生物技术和生物制剂对从病人体内采集的免疫细胞进行体外培养和扩增后回输到病人体内的方法，来激发、增强机体自身免疫功能，从而达到治疗肿瘤的目的。本领域技术人员一直致力于开发新的细胞免疫疗法，以提高细胞免疫疗法的效果，并降低其副作用。
本发明提出了一种合理的优化的单链设计和系统，即联合双特异性CAR，所述CAR可以有效地整合到初级人类T细胞，当T细胞激活时可同时靶向HER2和MESO。本发明CAR-T细胞能够识别两种抗原(HER2和MESO)，是一种很有效的潜在的预防抗原逃逸的方法。
本发明使用双向靶向的HER2和MESO的CAR，与靶向单抗原的CAR相比，可以更好地清除肿瘤细胞，减轻肿瘤异质性引起的抗原逃逸现象,进一步加强了CAR-T细胞杀伤肿瘤的能力，具有细胞因子协同效应。此外，由于HER2和MESO在肿瘤细胞中的表达水平不均一，双靶向CAR-T治疗范围更广泛。同时靶向肿瘤细胞表面的HER2和MESO的CAR-T可以减少因单一表面抗原下调或者缺失造成的抗原逃逸的可能性。
双特异性是指同一个CAR可以特异结合、免疫识别两个不同的抗原，CAR结合任意一个抗原都能产生免疫反应。
本发明的HER2和MESO双特异性的CAR为单一结构，包含抗HER2和MESO的scFv。其中CAR包含HER2 scFv和MESO scFv，HER2 scFv和MESO scFv的氨基酸序列、排序和铰链是其功能的主要影响因素。
具体地，本发明的嵌合抗原受体(CAR)包括细胞外结构域、跨膜结构域、和细胞内结构域。胞外结构域包括靶-特异性结合元件(也称为抗原结合结构域)。细胞内结构域包括共刺激信号传导区和ζ链部分。共刺激信号传导区指包括共刺激分子的细胞内结构域的一部分。共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子，而不是抗原受体或它们的配体。
在CAR的胞外结构域和跨膜结构域之间，或在CAR的胞浆结构域和跨膜结构域之间，可并入接头。如本文所用的，术语“接头”通常指起到将跨膜结构域连接至多肽链的胞外结构域或胞浆结构域作用的任何寡肽或多肽。接头可包括0-300个氨基酸，优选地2至100个氨基酸和最优选地3至50个氨基酸。
在本发明的一个较佳的实施方式中，本发明提供的CAR的胞外结构域包括靶向HER2和MESO联合的抗原结合结构域。本发明的CAR当在T细胞中表达时，能够基于抗原结合特异性进行抗原识别。当其结合其关联抗原时，影响肿瘤细胞，导致肿瘤细胞不生长、被促使死亡或以其他方式被影响，并导致患者的肿瘤负荷缩小或消除。抗原结合结构域优选与来自共刺激分子和ζ链中的一个或多个的细胞内结构域融合。优选地，抗原结合结构域与4-1BB信号传导结构域、和CD3ζ信号结构域 组合的细胞内结构域融合。
如本文所用，“抗原结合结构域”“单链抗体片段”均指具有抗原结合活性的Fab片段，Fab’片段，F(ab’) 2片段，或单一Fv片段。Fv抗体含有抗体重链可变区、轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般的，Fv抗体还包含VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。抗原结合结构域通常是scFv(single-chain variable fragment)。scFv的大小一般是一个完整抗体的1/6。单链抗体优选是由一条核苷酸链编码的一条氨基酸链序列。作为本发明的优选方式，所述scFv包含特异性识别HER2和MESO的抗体。
对于铰链区和跨膜区(跨膜结构域)，CAR可被设计以包括融合至CAR的胞外结构域的跨膜结构域。在一个实施方式中，使用天然与CAR中的结构域之一相关联的跨膜结构域。在一些例子中，可选择跨膜结构域，或通过氨基酸置换进行修饰，以避免将这样的结构域结合至相同或不同的表面膜蛋白的跨膜结构域，从而最小化与受体复合物的其他成员的相互作用。
本发明的CAR中的胞内结构域包括4-1BB的信号传导结构域和CD3ζ的信号传导结构域。
优选的，本发明的CAR的结构依次包括信号肽序列(又称前导序列)、抗原识别序列(抗原结合结构域)、任选的铰链区、跨膜区、共刺激因子信号区和CD3zeta信号传导区(ζ链部分)，含CAR基因的载体质粒如图1所示，作为载体质粒携带CAR基因，来自HIV-1整合酶基因的cPPT(Polypurine Tract)，增加了整合入宿主基因组的慢病毒的拷贝数，本发明的CAR结构如图7所示。
在另一优选例中，本发明CAR为HM CAR。其中，靶向HER2的抗原结合结构域包含HER2抗体来源的单链可变区重链序列(SEQ ID NO:2)和单链可变区轻链(VL)序列(SEQ ID NO:3)。
HER2抗体来源的单链可变区重链(VH)的氨基酸序列：
Figure PCTCN2022111866-appb-000001
HER2抗体来源的单链可变区轻链(VL)序列：
Figure PCTCN2022111866-appb-000002
HER2单链抗体HER2-ScFv序列(HER2V L-(G4S)3-HER2V H)
Figure PCTCN2022111866-appb-000003
Figure PCTCN2022111866-appb-000004
靶向MESO的抗原结合结构域包含MESO抗体来源的单链可变区重链序列(SEQ ID NO:4)和单链可变区轻链序列(SEQ ID NO:5)。
MESO抗体来源的单链可变区重链(VH)序列：
Figure PCTCN2022111866-appb-000005
MESO抗体来源的单链可变区轻链(VL)序列：
Figure PCTCN2022111866-appb-000006
MESO单链抗体MESO-ScFv序列(MESOV H-(G4S) 3-MESOV L)
Figure PCTCN2022111866-appb-000007
具体地，在本发明CAR中其它元件的序列如下：
信号肽为CD8来源的信号肽：
Figure PCTCN2022111866-appb-000008
单链可变区重链与轻链之间的连接序列(即柔性接头I)为：
氨基酸序列：GGGGSGGGGSGGGGS(SEQ ID NO:1)
跨膜区为CD8α来源的跨膜区序列：
Figure PCTCN2022111866-appb-000009
共刺激因子信号区来自4-1BB的胞内信号传导基序的序列：
Figure PCTCN2022111866-appb-000010
CD3ζ的信号传导域序列：
Figure PCTCN2022111866-appb-000011
在优选的实施方式中，本发明构建的CAR完整的核酸序列和氨基酸序列如下所示：
HM CAR的完整核酸序列
Figure PCTCN2022111866-appb-000012
HM CAR的完整氨基酸序列
Figure PCTCN2022111866-appb-000013
Figure PCTCN2022111866-appb-000014
嵌合抗原受体T细胞(CAR-T细胞)
如本文所用，术语“CAR-T细胞”、“CAR-T”、“CART”、“本发明CAR-T细胞”均指本发明第四方面所述的同时靶向HER2和MESO的CAR-T细胞。具体的所述CAR-T细胞的CAR结构依次包括抗HER2的scFv，抗MESO的scFv，跨膜区，和胞内T细胞信号区，并且抗HER2scFv和MESOscFv通过多个重复结构(G4S)肽段连接。与靶向单抗原的CAR-T相比，同时识别两个靶点的CAR-T细胞可以更好地清除肿瘤细胞，减轻肿瘤异质性引起的抗原逃逸现象，进一步加强了CAR-T细胞杀伤肿瘤的能力。
载体
编码期望分子的核酸序列可利用在本领域中已知的重组方法获得，诸如例如通过从表达基因的细胞中筛选文库，通过从已知包括该基因的载体中得到该基因，或通过利用标准的技术，从包含该基因的细胞和组织中直接分离。可选地，感兴趣的基因可被合成生产。
本发明也提供了其中插入本发明的表达盒的载体。源于逆转录病毒诸如慢病毒的载体是实现长期基因转移的合适工具，因为它们允许转基因长期、稳定的整合并且其在子细胞中增殖。慢病毒载体具有超过源自致癌逆转录病毒诸如鼠科白血病病毒的载体的优点，因为它们可转导非增殖的细胞，诸如肝细胞。它们也具有低免疫原性的优点。
简单概括，通常可操作地连接本发明的表达盒或核酸序列至启动子，并将其并入表达载体。该载体适合于复制和整合真核细胞。典型的克隆载体包含可用于调节期望核酸序列表达的转录和翻译终止子、初始序列和启动子。
本发明的表达构建体也可利用标准的基因传递方案，用于核酸免疫和基因疗法。基因传递的方法在本领域中是已知的。见例如美国专利号5,399,346、5,580,859、5,589,466，在此通过引用全文并入。在另一个实施方式中，本发明提供了基因疗法载体。
该核酸可被克隆入许多类型的载体。例如，该核酸可被克隆入如此载体，其包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。特定的感兴趣载体包括表达载体、复制载体、探针产生载体和测序载体。
进一步地，表达载体可以以病毒载体形式提供给细胞。病毒载体技术在本领域中是公知的并在例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)和其他病毒学和分子生物学手册中进行了描述。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记(例如，WO01/96584；WO01/29058；和美国专利号6,326,193)。
已经开发许多基于病毒的系统，用于将基因转移入哺乳动物细胞。例如，逆转录病毒提供了用于基因传递系统的方便的平台。可利用在本领域中已知的技术将选择的基因插入载体并包装入逆转录病毒颗粒。该重组病毒可随后被分离和传递至体内或离体的对象细胞。许多逆转录病毒系统在本领域中是已知的。在一些实施方式中，使用腺病毒载体。许多腺病毒载体在本领域中是已知的。在一个实施方式中，使用慢病毒载体。
额外的启动子元件，例如增强子，可以调节转录开始的频率。通常地，这些位于起始位点上游的30-110bp区域中，尽管最近已经显示许多启动子也包含起始位点下游的功能元件。启动子元件之间的间隔经常是柔性的，以便当元件相对于另一个被倒置或移动时，保持启动子功能。在胸苷激酶(tk)启动子中，启动子元件之间的间隔可被增加隔开50bp，活性才开始下降。取决于启动子，表现出单个元件可合作或独立地起作用，以起动转录。
合适的启动子的一个例子为即时早期巨细胞病毒(CMV)启动子序列。该启动子序列为能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动子序列。合适的启动子的另一个例子为延伸生长因子-1α(EF-1α)。然而，也可使用其他组成型启动子序列，包括但不限于类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、艾伯斯坦-巴尔(Epstein-Barr)病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地，本发明不应被限于组成型启动子的应用。诱导型启动子也被考虑为本发明的一部分。诱导型启动子的使用提供了分子开关，其能够当这样的表达是 期望的时，打开可操作地连接诱导型启动子的多核苷酸序列的表达，或当表达是不期望的时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
为了评估CAR多肽或其部分的表达，被引入细胞的表达载体也可包含可选择的标记基因或报道基因中的任一个或两者，以便于从通过病毒载体寻求被转染或感染的细胞群中鉴定和选择表达细胞。在其他方面，可选择的标记可被携带在单独一段DNA上并用于共转染程序。可选择的标记和报道基因两者的侧翼都可具有适当的调节序列，以便能够在宿主细胞中表达。有用的可选择标记包括例如抗生素抗性基因，诸如neo等等。
报道基因用于鉴定潜在转染的细胞并用于评价调节序列的功能性。通常地，报道基因为以下基因：其不存在于受体有机体或组织或由受体有机体或组织进行表达，并且其编码多肽，该多肽的表达由一些可容易检测的性质例如酶活性清楚表示。在DNA已经被引入受体细胞后，报道基因的表达在合适的时间下进行测定。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿色萤光蛋白的基因(例如，Ui-Tei等，2000FEBS Letters479:79-82)。合适的表达系统是公知的并可利用已知技术制备或从商业上获得。通常，显示最高水平的报道基因表达的具有最少5个侧翼区的构建体被鉴定为启动子。这样的启动子区可被连接至报道基因并用于评价试剂调节启动子-驱动转录的能力。
将基因引入细胞和将基因表达入细胞的方法在本领域中是已知的。在表达载体的内容中，载体可通过在本领域中的任何方法容易地引入宿主细胞，例如，哺乳动物、细菌、酵母或昆虫细胞。例如，表达载体可通过物理、化学或生物学手段转移入宿主细胞。
将多核苷酸引入宿主细胞的物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔等等。生产包括载体和/或外源核酸的细胞的方法在本领域中是公知的。见例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)。将多核苷酸引入宿主细胞的优选方法为磷酸钙转染。
将感兴趣的多核苷酸引入宿主细胞的生物学方法包括使用DNA和RNA载体。病毒载体，特别是逆转录病毒载体，已经成为最广泛使用的将基因插入哺乳动物例如人细胞的方法。其他病毒载体可源自慢病毒、痘病毒、单纯疱疹病毒I、腺病毒和腺伴随病毒等等。见例如美国专利号5,350,674和5,585,362。
将多核苷酸引入宿主细胞的化学手段包括胶体分散系统，诸如大分子复合物、纳米胶囊、微球、珠；和基于脂质的系统，包括水包油乳剂、胶束、混合胶束和脂质体。用作体外和体内传递工具(delivery vehicle)的示例性胶体系统为脂质体(例如，人造膜囊)。
在使用非病毒传递系统的情况下，示例性传递工具为脂质体。考虑使用脂质制剂，以将核酸引入宿主细胞(体外、离体(ex vivo)或体内)。在另一方面，该核酸可与脂质相关联。与脂质相关联的核酸可被封装入脂质体的水性内部中，散布在脂质体的脂双层内，经与脂质体和寡核苷酸两者都相关联的连接分子附接至脂质体，陷入脂质体，与脂质体复合，分散在包含脂质的溶液中，与脂质混合，与脂质联合，作为悬浮液包含在脂质中，包含在胶束中或与胶束复合，或以其他方式与脂质相关联。与组合物相关联的脂质、脂质/DNA或脂质/表达载体不限于溶液中的任何具体结构。例如，它们可存在于双分子层结构中，作为胶束或具有“坍缩的(collapsed)”结构。它们也可简单地被散布在溶液中，可能形成大小或形状不均一的聚集体。脂质为脂肪物质，其可为天然发生或合成的脂质。例如，脂质包括脂肪小滴，其天然发生在细胞质以及包含长链脂肪族烃和它们的衍生物诸如脂肪酸、醇类、胺类、氨基醇类和醛类的该类化合物中。
在本发明的一个优选地实施方式中，所述载体为慢病毒载体。
制剂
本发明提供了一种含有本发明的CAR-T细胞，以及药学上可接受的载体、稀释剂或赋形剂。在一个实施方式中，所述制剂为液态制剂。优选地，所述制剂为注射剂。优选地，所述制剂中所述CAR-T细胞的浓度为1×10 5-1×10 8个细胞/ml，较佳地1×10 7-1×10 8个细胞/ml。
在一个实施方式中，所述制剂可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的制剂优选配制用于静脉内施用。
治疗性应用
本发明包括用编码本发明表达盒的慢病毒载体(LV)转导的细胞(例如，T细胞)进行的治疗性应用。转导的T细胞可靶向肿瘤细胞的标志物HER2和MESO，协 同激活T细胞，引起T细胞免疫应答，从而显著提高其对肿瘤细胞的杀伤效率。
因此，本发明也提供了刺激对哺乳动物的靶细胞群或组织的T细胞-介导的免疫应答的方法，其包括以下步骤：给哺乳动物施用本发明的CAR-T细胞。
在一个实施方式中，本发明包括一类细胞疗法，分离病人自体T细胞(或者异源供体)，激活并进行基因改造产生CAR-T细胞，随后注入同一病人体内。这种方式患移植物抗宿主病概率极低，抗原被T细胞以无MHC限制方式识别。此外，一种CAR-T就可以治疗表达该抗原的所有癌症。不像抗体疗法，CAR-T细胞能够体内复制，产生可导致持续肿瘤控制的长期持久性。
在一个实施方式中，本发明的CAR-T细胞可经历稳固的体内T细胞扩展并可持续延长的时间量。另外，CAR介导的免疫应答可为过继免疫疗法步骤的一部分，其中CAR-修饰T细胞诱导对CAR中的抗原结合结构域特异性的免疫应答。例如，抗HER2和MESO的CAR-T细胞引起针对HER2和/或MESO阳性的细胞的特异性免疫应答。
尽管本文公开的数据具体公开了包括HER2-MESO scFv、4-1BB胞内区和CD3ζ信号传导结构域的慢病毒载体，但本发明应被解释为包括对构建体组成部分中的每一个的任何数量的变化。
血液学癌症为血液或骨髓的癌症。血液学(或血原性)癌症的例子包括白血病，包括急性白血病(诸如急性淋巴细胞白血病、急性髓细胞白血病、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病)、慢性白血病(诸如慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病)、真性红细胞增多症、淋巴瘤、霍奇金氏疾病、非霍奇金氏淋巴瘤(无痛和高等级形式)、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血症、重链疾病、骨髓增生异常综合征、多毛细胞白血病和脊髓发育不良。
实体瘤为通常不包含囊肿或液体区的组织的异常肿块。实体瘤可为良性或恶性的。不同类型的实体瘤以形成它们的细胞类型命名(诸如肉瘤、癌和淋巴瘤)。实体瘤诸如肉瘤和癌的例子包括纤维肉瘤、粘液肉瘤、脂肪肉瘤间皮瘤、淋巴恶性肿瘤、胰腺癌卵巢癌。
在优选的实施方式中，可治疗的癌症为HER2和/或MESO阳性肿瘤，如胰腺癌等。
本发明的CAR-修饰T细胞也可用作对哺乳动物离体免疫和/或体内疗法的疫苗类型。优选地，哺乳动物为人。
对于离体免疫，以下中的至少一项在将细胞施用进入哺乳动物前在体外发 生：i)扩增细胞，ii)将编码CAR的核酸引入细胞，和/或iii)冷冻保存细胞。
离体程序在本领域中是公知的，并在以下更完全地进行讨论。简单地说，细胞从哺乳动物(优选人)中分离并用表达本文公开的CAR的载体进行基因修饰(即，体外转导或转染)。CAR-修饰的细胞可被施用给哺乳动物接受者，以提供治疗益处。哺乳动物接受者可为人，和CAR-修饰的细胞可相对于接受者为自体的。可选地，细胞可相对于接受者为同种异基因的、同基因的(syngeneic)或异种的。
除了就离体免疫而言使用基于细胞的疫苗之外，本发明也提供了体内免疫以引起针对患者中抗原的免疫应答的组合物和方法。
本发明提供了治疗肿瘤的方法，其包括施用给需要其的对象治疗有效量的本发明的CAR-修饰的T细胞。
本发明的CAR-修饰的T细胞可被单独施用或作为药物组合物与稀释剂和/或与其他组分诸如IL-2、IL-17或其他细胞因子或细胞群结合施用。简单地说，本发明的药物组合物可包括如本文所述的靶细胞群，与一种或多种药学或生理学上可接受载体、稀释剂或赋形剂结合。这样的组合物可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的组合物优选配制用于静脉内施用。
本发明的药物组合物可以以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由这样的因素确定，如患者的病症、和患者疾病的类型和严重度——尽管适当的剂量可由临床试验确定。
当指出“免疫学上有效量”、“抗肿瘤有效量”、“肿瘤-抑制有效量”或“治疗量”时，待施用的本发明组合物的精确量可由医师确定，其考虑患者(对象)的年龄、重量、肿瘤大小、感染或转移程度和病症的个体差异。可通常指出：包括本文描述的T细胞的药物组合物可以以10 4至10 9个细胞/kg体重的剂量，优选10 5至10 6个细胞/kg体重的剂量(包括那些范围内的所有整数值)施用。T细胞组合物也可以以这些剂量多次施用。细胞可通过使用免疫疗法中公知的注入技术(见例如Rosenberg等，NewEng.J.of Med.319:1676，1988)施用。对于具体患者的最佳剂量和治疗方案可通过监测患者的疾病迹象并因此调节治疗由医学领域技术人员容易地确定。
对象组合物的施用可以以任何方便的方式进行，包括通过喷雾法、注射、 吞咽、输液、植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内(i.v.)注射或腹膜内施用给患者。在一个实施方式中，本发明的T细胞组合物通过皮内或皮下注射被施用给患者。在另一个实施方式中，本发明的T细胞组合物优选通过i.v.注射施用。T细胞的组合物可被直接注入肿瘤，淋巴结或感染位置。
在本发明的某些实施方式中，利用本文描述的方法或本领域已知的其他将T细胞扩展至治疗性水平的方法活化和扩展的细胞，与任何数量的有关治疗形式结合(例如，之前、同时或之后)施用给患者，所述治疗形式包括但不限于用以下试剂进行治疗：所述试剂诸如抗病毒疗法、西多福韦和白细胞介素-2、阿糖胞苷(也已知为ARA-C)或对MS患者的那他珠单抗治疗或对牛皮癣患者的厄法珠单抗治疗或对PML患者的其他治疗。在进一步的实施方式中，本发明的T细胞可与以下结合使用：化疗、辐射、免疫抑制剂，诸如，环孢菌素、硫唑嘌呤、甲氨喋呤、麦考酚酯和FK506，抗体或其他免疫治疗剂。在进一步的实施方式中，本发明的细胞组合物与骨髓移植、利用化疗剂诸如氟达拉滨、外部光束放射疗法(XRT)、环磷酰胺结合(例如，之前、同时或之后)而施用给患者。例如，在一个实施方式中，对象可经历高剂量化疗的标准治疗，之后进行外周血干细胞移植。在一些实施方式中，在移植后，对象接受本发明的扩展的免疫细胞的注入。在一个额外的实施方式中，扩展的细胞在外科手术前或外科手术后施用。
施用给患者的以上治疗的剂量将随着治疗病症的精确属性和治疗的接受者而变化。人施用的剂量比例可根据本领域接受的实践实施。通常，每次治疗或每个疗程，可将1×10 6个至1×10 10个本发明经修饰的T细胞(CAR-T细胞)，通过例如静脉回输的方式，施用于患者。
本发明的主要优点包括：
(1)本发明的双特异性CAR-T细胞对HER2阳性靶细胞和MESO阳性靶细胞有显著的杀伤作用。
(2)本发明的双特异性CAR-T细胞提高T细胞杀伤肿瘤的作用，产生的细胞因子具有超级加和效应，与单靶点CAR-T相比，可以更好地清除肿瘤细胞，减轻肿瘤异质性引起的抗原逃逸现象，进一步加强了CAR-T细胞杀伤肿瘤的能力。
(3)本发明首次发现，由于该T细胞可同时串联表达针对胰腺癌细胞表面的肿瘤相关抗原HER2和MESO的单链抗体，使其识别肿瘤细胞的范围大大增加，可识别单靶点CAR-T细胞识别阈值以下的癌细胞，促进了串联CAR-T细胞在异质性 肿瘤亚群中的广泛应用，因此对胰腺癌细胞的杀伤范围更广；同时，串联CAR-T细胞可以提高T细胞杀伤肿瘤的作用，产生的细胞因子具有超级加和效应，与单靶点CAR-T相比，可以更好地清除肿瘤细胞，减轻肿瘤异质性引起的抗原逃逸现象,进一步加强了CAR-T细胞杀伤肿瘤的能力。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
如无特别说明，则实施例中所用的试剂、材料均为市售产品。
本发明披露了能同时串联表达靶向HER2和MESO的HM CAR质粒、HM CAR-T细胞、构建方法及其应用。图2为本发明的流程图，具体如下各实施例所示。
实施例1 能同时表达HER2单链抗体及MESO单链抗体的HM CAR质粒的构建
依次连接Signal Peptide-HER2V L-(G4S) 3-HER2V H-(G4S) 5-MESOV H-(G4S) 3-MESOV L-CD8α-4-1BB-CD3ζ。序列两端分别连接BamHI和XhoI酶切位点。所有序列都是人源化的，由生工生物工程(上海)股份有限公司合成，以PUC57质粒形式保存。
将得到的基因序列片段使用酶切连接的方法连接至慢病毒表达载体pLenti6.3/V5，得到HM CAR质粒。图3为构建在pLenti6.3/V5载体的HM CAR基因经BamHI和XhoI双酶切鉴定后，琼脂糖凝胶电泳结果。
实施例2 慢病毒包装
将HEK293细胞调整至2×10 6活细胞/mL后，取25.5mL，加1.5mL LV-MAX Supplement；
准备DNA/LV-MAX转染试剂复合物：
试管1：标记为“DNA”
(1)加入1.5mL Opti-MEM无血清培养基；
(2)添加三个辅助质粒混合物(1.5μg/mL)和慢病毒表达载体pLenti6.3/V5 (1μg/mL)；
试管2：标记为“TfxR”
(1)加入1.5mL Opti-MEM无血清培养基；
(2)添加LV-MAX转染试剂180μL，短暂涡旋并在室温下孵育1分钟；
(3)1分钟后，将试管1倒至试管2或以相反方式将两种溶液合并，短暂涡旋；
(4)室温下混合溶液孵育10分钟后，直接将DNA/LV-MAX转染复合物加入HEK293细胞；
(5)转染5-6小时后，向细胞中添加1.2mL LV-MAX增强剂。
转染48-55小时后，将慢病毒原液(HM CAR-LV)移至50mL离心管，1300g离心15min，收集上清。再用0.45μm低蛋白结合过滤器过滤去除细胞碎片，100KD超滤管4℃超滤浓缩，将30mL体系浓缩至5mL，将慢病毒浓缩液等分并存储在-80℃冰箱。
实时荧光定量PCR(qPCR)检测慢病毒滴度
(1)用多聚赖氨酸处理24孔板，防止293T细胞在转染时脱落；
(2)用密度为2×10 5/ml细胞铺板1ml，37℃含5％CO2培养箱培养过夜；
(3)用无血清培养基稀释慢病毒，将慢病毒原液分别稀释10倍、100倍、1000倍。吸弃旧培养基，细胞中加入500μL慢病毒稀释液和10μL转染剂，增加转染效率，继续培养。第二天，将细胞培养基换为含10％FBS的DMEM培养基；
(4)转染72h后，消化293T细胞，并抽提细胞DNA，qPCR检测CAR基因拷贝数。得到的HM CAR-LV原液滴度高于10 7TU/mL，用100KD超滤管浓缩慢病毒后滴度可高达10 9TU/mL。结果如下表所示：
Figure PCTCN2022111866-appb-000015
实施例3 CAR-T细胞制备
PBMC细胞复苏及磁珠分选
(1)将人PBMC从液氮罐中小心拿出，放入37℃水浴，待溶解至一小冰块后，移入安全柜；
(2)移液管吸5mL HBSS(含10％人血白蛋白)，再吸入细胞至50mL离心管中，用5mL HBSS(含10％人血白蛋白)润洗冻存管；
(3)缓慢加入30mL HBSS(含10％人血白蛋白)至总体积为40mL，400g离心10min，弃上清；
(4)加1mL1640培养基(含10％FBS)和8μL DNAse，37℃静置15min；
(5)再加入29mL 1640培养基(含10％FBS)，37℃静置4～6小时；
(6)静置数小时后，进行细胞计数，确定总细胞量；
(7)300g离心10min，弃上清，加入一定量体积MACs Buffer Running Buffer(简称Buffer)重悬细胞，再加入一定体积CD3分选磁珠，4℃，离心15min；(注:每10 7个细胞悬于80μL Buffer中；每10 7个细胞加20μL CD3磁珠。)
(8)加入一定体积Buffer，300g离心10min，弃上清；(注：每10 7个细胞加2mL Buffer)
(9)再用一定体积Buffer重悬细胞；(注：每10 7个细胞加500μL Buffer)
(10)第8步离心细胞时，进行柱平衡(用3mL Buffer激活LS柱)；
(11)将第9步重悬的细胞悬液加入柱中，重力作用下流出流穿并收集；
(12)用Buffer清洗柱子，每次3mL，洗三遍，并收集流穿；
(13)将柱子从分离器中取下，并放在合适的收集管上；
(14)用5mL Buffer洗脱LS柱，并进行细胞计数；
(15)300g，离心10min，弃上清，加入TexMACs培养基(含IL-2)，调整细胞浓度为1×10 6/mL；
(16)添加T细胞CD3CD28刺激剂；(注：每10 6个细胞加10μL刺激剂)
(17)用500μL铺24孔板，37℃培养过夜，为第二天转染T细胞做准备。
CAR-T细胞制备
(1)慢病毒以MOI 15转染T细胞，吹打混匀；
(2)再加入Polybrene，增强转染效率；
(3)4h后补加500μL TexMACs培养基。(注:每两天进行一次换液)
CAR-T细胞阳性率检测
取1×10 6个转导后T细胞，与生物素标记的HER2和Meso 4℃避光孵育1h，用PBS(含2％FBS)清洗两遍；加入100μL PBS(含2％FBS)重悬细胞，加10μL PE标记的亲和素，4℃避光孵育1h，PBS(含2％FBS)清洗两遍，再以600μL PBS重悬细胞进行上机。通过流式细胞仪检测PE荧光信号，反映CAR-T细胞在总细胞中的阳性率。
检测结果如图5所示，由结果显示HM CAR-T细胞阳性率为50％左右。
通过实时荧光定量PCR检测CAR-T细胞中CAR基因，进一步从基因水平证明慢病毒成功转染T细胞。分别取1×10 6个HM CAR-T细胞和未转染的T细胞，抽提细胞DNA，qPCR检测CAR基因拷贝数。
具体实验结果见表1，实验结果证明，CAR基因成功整合到T细胞基因组中。
表1.实时荧光定量PCR检测细胞CAR基因
Figure PCTCN2022111866-appb-000016
实施例4 双靶点CAR-T细胞对胰腺癌细胞的体外杀伤作用
胰腺癌细胞SW-1990和ASPC-1表面HER2和MESO抗原表达情况的通过流式细胞分析检测，其抗原阳性率结果图4所示。
采用RTCA检测HM CAR-T细胞杀伤两株胰腺癌细胞，设置效靶比4:1进行杀伤。以8×10 4个ASPC-1细胞和1×10 4个SW-1990的胰腺癌细胞进行铺板，培养24h，再加入效靶比4:1的CAR-T细胞和T细胞，对比两者对肿瘤细胞的杀伤差异性。结果如图6所示：6-1为HM CAR-T细胞杀伤ASPC-1，6-2为HM CAR-T细胞杀伤SW-1990。实验结果证明：相较于对照组，HM CAR-T细胞杀伤效果更显优势，表明同时表达靶向HER2和MESO的串联HM CAR-T细胞对HER2和MESO阳性的胰腺癌细胞具有特异性地靶向杀伤作用。
结果显示，HM CAR-T细胞显著延迟了肿瘤的生长，与对照组相比，p<0.05(图6)。
此外，通过RTCA检测三种CAR-T细胞对ASPC-1细胞的杀伤作用。通过80K个的细胞量进行铺板，培养24h，再加入效靶比4:1的CAR-T细胞和T细胞，对比不同细胞对肿瘤细胞的杀伤差异性。实验结果表明，相较于阴性对照组，T细胞和CAR-T细胞均有不同程度的杀伤作用，但是以串联组CAR-T细胞杀伤效果更显优势。(图8)
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申 请所附权利要求书所限定的范围。

Claims (10)

  1. 一种双特异性嵌合抗原受体(CAR)，其特征在于，所述嵌合抗原受体的结构如下式I所示：
    L-scFv1-I-scFv2-H-TM-C-CD3ζ  (I)
    式中，
    各“-”独立地为连接肽或肽键；
    L为任选的信号肽序列；
    I为柔性接头；
    H为任选的铰链区；
    TM为跨膜结构域；
    C为共刺激信号分子；
    CD3ζ为源于CD3ζ的胞浆信号传导序列；
    scFv1和scFv2两者中一个为靶向HER2的抗原结合结构域，另一个为靶向MESO的抗原结合结构域。
  2. 如权利要求1所述的双特异性嵌合抗原受体，其特征在于，所述scFv1为靶向HER2的抗原结合结构域，所述scFv2为靶向MESO的抗原结合结构域。
  3. 如权利要求1所述的双特异性嵌合抗原受体，其特征在于，所述靶向HER2的抗原结合结构域的结构如下式A或式B所示：
    V H1-V L1  (A)；V L1-V H1  (B)
    式中，V H1为抗HER2抗体重链可变区；V L1为抗HER2抗体轻链可变区；“-”为连接肽或肽键。
  4. 一种核酸分子，其特征在于，所述核酸分子编码权利要求1所述的嵌合抗原受体(CAR)。
  5. 一种载体，其特征在于，所述的载体含有权利要求4所述的核酸分子。
  6. 一种宿主细胞，其特征在于，所述的宿主细胞中含有权利要求5所述的载体或染色体中整合有外源的权利要求4所述的核酸分子或表达权利要求1所述的CAR。
  7. 一种制剂，其特征在于，所述制剂含有权利要求1所述的嵌合抗原受体、权利要求4所述的核酸分子、权利要求5所述的载体、或权利要求6所述的宿主细胞，以及药学上可接受的载体、稀释剂或赋形剂。
  8. 一种权利要求1所述的嵌合抗原受体、权利要求4所述的核酸分子、权 利要求5所述的载体、或权利要求6所述的宿主细胞、或权利要求7所述的制剂的用途，其特征在于，用于制备预防和/或治疗癌症或肿瘤的药物或制剂。
  9. 一种用于制备权利要求6所述的宿主细胞的试剂盒，其特征在于，所述试剂盒含有容器，以及位于容器内的权利要求4所述的核酸分子、或权利要求5所述的载体。
  10. 一种制备工程化的免疫细胞的方法，其特征在于，所述的免疫细胞表达权利要求1所述的CAR，所述方法包括以下步骤：
    (a)提供待改造的免疫细胞；和
    (b)将权利要求4所述的核酸分子或权利要求5所述的载体转导入所述免疫细胞内，从而获得所述工程化的免疫细胞。
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