WO2023116637A1 - 转基因免疫细胞及其构建方法和应用 - Google Patents

转基因免疫细胞及其构建方法和应用 Download PDF

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WO2023116637A1
WO2023116637A1 PCT/CN2022/140094 CN2022140094W WO2023116637A1 WO 2023116637 A1 WO2023116637 A1 WO 2023116637A1 CN 2022140094 W CN2022140094 W CN 2022140094W WO 2023116637 A1 WO2023116637 A1 WO 2023116637A1
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chain variable
seq
variable region
acid sequence
amino acid
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French (fr)
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王先进
彭亮
叶立军
黄倩
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深圳市菲鹏生物治疗股份有限公司
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Definitions

  • the present invention relates to the technical field of CART, in particular to transgenic immune cells and their construction method and application.
  • Chimeric antigen receptor T cell immunotherapy is a method of transforming the patient's T cells in vitro, so that the patient's T cells have the ability to recognize tumor cells, and then expand and culture them in vitro and return them to the patient for treatment.
  • CD19-targeted CART has achieved great results in the treatment of B-cell hematological tumors, but according to clinical research results, the efficacy of CD19 CART in the treatment of B-cell lymphoma is far inferior to that in the treatment of B-cell acute lymphoma. This may be because B-cell lymphoma is a solid tumor, and it is difficult for CART cells to reach and infiltrate the tumor in large numbers.
  • solid tumor cells can overexpress a large number of PD-L1 molecules on the surface, thus evading the killing of CART cells.
  • insufficient contact between CART cells and solid tumor cells also significantly affects the ability of CART cells Proliferation and persistence accelerated the rate of depletion of CART cells in vivo.
  • the treatment of solid tumors puts higher requirements on CART cells, which require CART cells to have the ability to resist or even destroy the microenvironment of solid tumors, and continue to penetrate deep into the interior of solid tumors to play a therapeutic role.
  • Cytokines are synthesized and secreted by immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.)
  • immune cells such as monocytes, macrophages, T cells, B cells, NK cells, etc.
  • non-immune cells endothelial cells, epidermal cells, fibroblasts, etc.
  • Interleukin is one of the important categories. It has multiple functions such as immune regulation, hematopoiesis and inflammation regulation. So far, more than 30 kinds of interleukins have been reported, among which IL-21 is produced by CD4 T cells and NKT cells.
  • IL-21 stimulate the maturation of CD8 T cells and NK cells and enhance their cytotoxicity, and at the same time have the functions of promoting the differentiation of memory CD8 T cells.
  • the many potencies of IL-21 make it a potential target for immunotherapy, but IL-21R is widely expressed including T cells, B cells, NK cells and myeloid cells.
  • Chemokines are a class of small cytokines or signaling proteins secreted by cells. They are named chemotactic cytokines because of their ability to induce directed chemotaxis of nearby responding cells. Chemotactic cytokines can be divided into four subfamilies, CXC, CC, C, and CX3C, according to the arrangement of their amino-terminal (N-terminal) cysteines. Among them, CCL19 and CCL21 of the CC chemokine subfamily are CCR7. Ligand, T cells express CCR7 protein, so CCL19 and CCL21 chemokines can chemoattract T cell migration.
  • cytokines and chemokines are beneficial to fight against and improve the microenvironment of solid tumors, and provide a persistent killing environment for immune cells.
  • the purpose of the present invention is to provide a multifunctional immune cell for the microenvironment of solid tumors, which is expected to not only resist the microenvironment that is gradually formed during the development of solid tumors and is beneficial to its stability and spread, but also be able to chemoattract autoimmunity.
  • Cells activate the body's own immune system to a greater extent, and improve the body's own strength against solid tumors.
  • the present invention provides the following technical solutions:
  • the present invention provides a gene comprising three coding regions, wherein the coding region (I) encodes a chimeric antigen receptor, and the chimeric antigen receptor contains an extracellular region that specifically recognizes a tumor antigen;
  • the coding region (II) encodes a fusion protein, and the fusion protein includes immune checkpoint antibodies and cytokines;
  • Coding region (III) encodes chemokines.
  • the tumor antigen is selected from at least one of MSLN, GD2, GPC3, CD19, EGFR VIII, GUCY2C, HER2, MUC16 or Claudin 18.2.
  • the extracellular region contains anti-MSLN antibodies.
  • the extracellular region contains an anti-GUCY2C antibody; in an optional embodiment, the chimeric antigen receptor contains an anti-GUCY2C single-chain antibody;
  • the amino acid sequence of the anti-MSLN antibody is (a) the amino acid sequence shown in SEQ ID NO: 1, or (b) one or several amino acids are substituted, deleted or added in the amino acid sequence defined in (a) and
  • the encoded protein has a derived amino acid sequence that specifically recognizes MSLN antigenic activity.
  • the immune checkpoint is selected from at least one of PD1, PD-L1, TIGIT, LAG3, CTLA4, BTLA or TIM3.
  • the immune checkpoint antibody is an anti-PD1 antibody.
  • the amino acid sequence of the anti-PD1 antibody is (c) the amino acid sequence shown in SEQ ID NO: 2, or (d) one or several amino acids are substituted, deleted or added in the amino acid sequence defined in (c) and
  • the edited protein has a derived amino acid sequence targeting PD1 function.
  • the cytokine is (A) or (B);
  • (B) is a protein derived from (A) with regulatory immune cell activity.
  • the cytokine is IL-21.
  • IL-21 has the amino acid sequence shown in SEQ ID NO: 3.
  • the chemokine is (c) or (d);
  • (d) is a protein derived from (c) capable of inducing directional migration of immune cells.
  • the CXC chemokine is at least one of CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16 or CXCL17.
  • the CC chemokine is CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, At least one of CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, or CCL28.
  • the CX3C chemokine is CX3CL1.
  • the XC chemokine is XCL1.
  • the chemokine is CCL19 or CCL21.
  • CCL19 has the amino acid sequence shown in SEQ ID NO: 4;
  • CCL21 has the amino acid sequence shown in SEQ ID NO: 5.
  • the present invention provides a recombinant nucleic acid.
  • the recombinant nucleic acid includes a first nucleic acid molecule, a second nucleic acid molecule, and a third nucleic acid molecule.
  • the first nucleic acid molecule contains the coding region (I) of any of the foregoing embodiments
  • the second nucleic acid molecule Containing the coding region (II) of any of the foregoing embodiments
  • the third nucleic acid molecule contains the coding region (III) of any of the foregoing embodiments.
  • the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are linked by the nucleic acid sequence of the 2A peptide.
  • the 2A peptide is selected from at least one of P2A, T2A, F2A or E2A.
  • the first nucleic acid molecule is connected to the second nucleic acid molecule through the nucleic acid sequence of the 2A peptide, and the second nucleic acid molecule is connected to the third nucleic acid molecule through the nucleic acid sequence of the 2A peptide.
  • the present invention provides biological materials, which include any of the following:
  • a recombinant vector contains the gene of any of the foregoing embodiments, or contains the recombinant nucleic acid of any of the foregoing embodiments;
  • constructs include non-pathogenic viruses, and non-pathogenic viruses contain the genes, recombinant nucleic acids or (i) recombinant vectors of any of the foregoing embodiments.
  • non-pathogenic viruses include retroviruses, lentiviruses or adenoviruses.
  • the present invention provides transgenic immune effector cells, which contain the gene, recombinant nucleic acid or biological material according to any of the aforementioned embodiments.
  • the method for constructing transgenic immune effector cells includes introducing genes, recombinant nucleic acids or biological materials in any of the aforementioned embodiments into immune effector cells to obtain transgenic immune effector cells;
  • the immune effector cells are selected from at least one of T cells, NK cells, NKT cells, macrophages or CIK cells.
  • the immune effector cells are T cells.
  • the present invention provides the application of the gene, recombinant nucleic acid, biological material, transgenic immune effector cell or transgenic immune effector cell constructed by the construction method in the preparation of anti-tumor products according to any of the foregoing embodiments.
  • the tumor comprises a solid tumor.
  • the present invention provides anti-tumor drugs, which include at least one of the genes, recombinant nucleic acids, biological materials or transgenic immune effector cells in any of the foregoing embodiments.
  • the tumor comprises a solid tumor.
  • the present invention provides a method for treating tumors, the method comprising administering to a subject a therapeutically effective amount of at least one of the aforementioned genes, recombinant nucleic acids, biological materials or transgenic immune effector cells.
  • the tumor comprises a solid tumor.
  • the present invention provides a pharmaceutical composition, which includes at least one of the aforementioned genes, recombinant nucleic acids, biological materials or transgenic immune effector cells, and a pharmaceutically acceptable carrier.
  • the genes, recombinant nucleic acids, biological materials and transgenic immune effector cells provided by the present invention can all encode three proteins, and the expression of the three proteins enables the immune effector cells to have multiple functions at the same time, including specific recognition of tumor antigens and targeted immune detection point, as well as chemotaxis and regulation of immune cell activity, thereby reducing the inhibitory effect of the solid tumor microenvironment on immune effector cells and prolonging the killing time of immune effector cells; at the same time, the expression of chemokines can recruit and chemoattract the body itself
  • the T cells in the tumor reach the tumor site, and under the regulation of cytokines, they jointly kill tumor cells and enhance the anti-tumor efficacy of immune effector cells.
  • Example 1 is a schematic diagram of the composition of the recombinant nucleic acid sequence used in Example 1, Example 2 and Comparative Example 1 of the present invention (Schema graph of lentiviral vector coding gene, that is, a schematic diagram of lentiviral vector coding gene);
  • Fig. 2 provides CART cell positive rate detection result for embodiment 3 of the present invention, embodiment 4 and comparative example 1;
  • Figure 3 provides the comparison results of the in vitro tumoricidal ability of CART cells in Example 3, Example 4 and Comparative Example 1 of the present invention
  • Figure 4 provides the comparative results of CART cell chemotactic ability for Example 3, Example 4 and Comparative Example 1 of the present invention
  • Example 5 is a schematic diagram of the composition of the recombinant nucleic acid used in Example 5 and Comparative Example 2 of the present invention (Schema graph of lentiviral vector coding gene, that is, a schematic diagram of lentiviral vector coding gene);
  • Fig. 6 is the detection result figure of the CART cell positive rate of Experimental Example 5 of the present invention.
  • Fig. 7 is a comparison result diagram of the in vitro tumor killing ability of CART cells in Experimental Example 6 of the present invention.
  • Fig. 8 is a graph showing the results of an in vivo tumor inhibition experiment in Experimental Example 7 of the present invention.
  • first and second are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features.
  • the features defined as “first” and “second” may explicitly or implicitly include at least one of these features.
  • “plurality” means at least two, such as two, three, etc., unless otherwise specifically defined.
  • the present invention provides a gene containing three coding regions, wherein, the coding region (I) encodes a chimeric antigen receptor, and the chimeric antigen receptor contains a gene that specifically recognizes a tumor antigen. extracellular region;
  • the coding region (II) encodes a fusion protein, and the fusion protein includes immune checkpoint antibodies and cytokines;
  • Coding region (III) encodes chemokines.
  • genes of the present invention should be understood as all genes that include the above three coding regions. As for whether other coding regions are included in addition to the above three coding regions, the present invention does not limit, and those skilled in the art can choose to add other genes according to actual needs. coding region.
  • connection sequence of the three coding regions in the above-mentioned genes of the present invention can be selected according to actual needs, and no special limitation is required.
  • the gene of the present invention may also contain appropriate introns without affecting the independent expression of the three coding regions.
  • the above-mentioned chimeric antigen receptor refers to the coupling of chimeric antigen receptor T cells into a chimeric protein in vitro through the antigen-binding part of an antibody that recognizes a certain tumor antigen, and the transmembrane region and intracellular region.
  • the method transfects the patient's T cells to express chimeric antigen receptors, so that the patient's T cells can be "recoded” to generate a large number of tumor-specific CART cells.
  • the chimeric antigen receptor can specifically track and recognize and guide the T cells to kill tumor cells.
  • the present invention extends the scope of application of chimeric antigen receptors to other immune effector cells, such as T cells, NK cells, NKT cells, macrophages or CIK cells, etc., the antigen of the antibody
  • the binding part is the extracellular region that recognizes tumor antigens in the present invention, while the transmembrane region and intracellular region can be obtained through conventional selection according to actual needs.
  • the transmembrane region is the transmembrane segment of CD8.
  • the intracellular region is the intracellular segment of the immune co-stimulatory molecule and the CD3 Zeta chain.
  • the immune co-stimulatory molecule is selected from any one or more of 4-1BB, CD28, CD3, OX-40, CD40L, CD27, CD30, or their derivatives.
  • the immune co-stimulatory molecule is selected from 4-1BB.
  • the tumor antigen of the present invention is selected from at least one of MSLN, GD2, GPC3, CD19, EGFRVIII, GUCY2C, HER2, MUC16 or Claudin 18.2.
  • the extracellular region contains anti-MSLN antibodies.
  • the amino acid sequence of the anti-MSLN antibody is (a) the amino acid sequence shown in SEQ ID NO: 1, or (b) one or several amino acids are substituted, deleted or added in the amino acid sequence defined in (a) and
  • the encoded protein has a derived amino acid sequence that specifically recognizes MSLN antigenic activity.
  • Amino acid sequence (SEQ ID NO: 1) of anti-MSLN antibody is N-(SEQ ID NO: 1) of anti-MSLN antibody:
  • antibody is used in the broadest sense encompassing various antibody structures including but not limited to monoclonal/polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies, trispecific antibodies, etc.), murine antibodies /chimeric antibodies, full-length antibodies or antigen-binding fragments thereof (eg scFv), as long as they exhibit the desired antigen-binding activity.
  • complementarity determining regions refer to the hypervariable regions of the heavy and light chains of immunoglobulins, which are the major contributors within the variable domains of antibodies to antigen specificity. area of sexual union.
  • the complementarity determining region of the heavy chain is represented by HCDR, and the three CDR regions contained in the variable region of the heavy chain are: HCDR1, HCDR2 and HCDR3;
  • the complementarity determining region of the light chain is represented by LCDR, and the three CDR regions contained in the variable region of the light chain are: LCDR1, LCDR2, and LCDR3.
  • the amino acid sequence boundaries of CDRs can be determined by various known schemes, for example: “Kabat” numbering convention (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health , Bethesda, MD), "Chothia” numbering sequence, "ABM” numbering sequence, "contact” numbering sequence (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and ImMunoGenTics (IMGT) numbering Rules (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)), etc.; the correspondence between various numbering systems is well known to those skilled in the art.
  • Kabat numbering convention
  • IMGT ImMunoGenTics
  • the chimeric antigen receptor comprises an anti-MSLN antibody.
  • the anti-MSLN antibody is a single-chain antibody (scFv), which is formed by linking the antibody light chain variable region (VL) and heavy chain variable region (VH) directly or through a peptide linker (L), such as N-terminal To the C-terminus: scFv of VH-L-VL or VL-L-VH, the linker L can be selected from (GxS)y linker, wherein, x is selected from an integer of 1-5, and y is selected from an integer of 0-6 , for example x is 4 and y is 3).
  • the anti-MSLN antibody comprises a heavy chain variable region and a light chain variable region;
  • the heavy chain variable region of the anti-MSLN antibody comprises the heavy chain variable region in SEQ ID NO: 1 (SEQ ID NO: 9 ),
  • the light chain variable region of the anti-MSLN antibody comprises LCDR1, LCDR2 and LCDR3 of the light chain variable region (SEQ ID NO: 10) in SEQ ID NO: 1; in some embodiments, HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system.
  • HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 are defined by the Kabat numbering system.
  • the amino acid sequence of the CDRs (defined by the Kabat numbering system) of the anti-MSLN antibody is as follows: HCDR1: GYTMN (SEQ ID NO: 11); HCDR2: LITPYNGASSYNQKFRG (SEQ ID NO: 12); HCDR3: GGYDGRGFDY (SEQ ID NO: 13); LCDR1: SASSSVSYMH (SEQ ID NO: 14); LCDR2: DTSKLAS (SEQ ID NO: 15); LCDR3: QQWSKHPLT (SEQ ID NO: 16).
  • the heavy chain variable region of the anti-MSLN antibody comprises the heavy chain variable region of SEQ ID NO: 1, and the light chain variable region of the anti-MSLN antibody comprises the light chain variable region of SEQ ID NO: 1 district.
  • an anti-MSLN antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and the light chain can The variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10; in some embodiments, the heavy chain variable region and the light chain variable region comprise HCDR1, HCDR2, HCDR3 of any of the preceding anti-MSLN antibodies , LCDR1, LCDR2 and LCDR3; In some embodiments, the anti-MSLN antibody comprises a heavy chain variable region shown in SEQ ID NO: 9 and a light chain variable region shown in SEQ ID NO: 10.
  • the anti-MSLN antibody is a single chain antibody comprising SEQ ID NO: 1.
  • the anti-MSLN antibody is a single-chain antibody, which includes an amino acid sequence derived from SEQ ID NO: 1, obtained by substituting, deleting or adding one or several amino acids to the amino acid sequence derived from SEQ ID NO: 1, and the edited protein It has the function of targeting MSLN.
  • amino acid sequence of the heavy chain variable region of the anti-MSLN antibody (SEQ ID NO: 9):
  • Anti-MSLN antibody light chain variable region amino acid sequence (SEQ ID NO: 10):
  • the tumor antigen of the present invention is selected from GUCY2C.
  • the chimeric antigen receptor comprises an anti-GUCY2C antibody.
  • the anti-GUCY2C antibody is a single chain antibody (scFv).
  • an anti-GUCY2C antibody comprises a heavy chain variable region and a light chain variable region.
  • the heavy chain variable region of the anti-GUCY2C antibody includes HCDR1, HCDR2, and HCDR3 in SEQ ID NO:17
  • the light chain variable region of the anti-GUCY2C antibody includes LCDR1, LCDR2, and LCDR3
  • HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system system definition.
  • the amino acid sequence of the CDRs (defined by the IMGT numbering system) of the anti-GUCY2C antibody is as follows: HCDR1: GYTFTEYT (SEQ ID NO: 20); HCDR2: INPNNGGA (SEQ ID NO: 21); HCDR3: ARAPYYYGSSYYAMDY (SEQ ID NO: 22); LCDR1: ESVDNYGISF (SEQ ID NO: 23); LCDR2: AAS; LCDR3: QQSKEVPFT (SEQ ID NO: 24).
  • an anti-GUCY2C antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 17, and the light chain can be The variable region comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 18; in some embodiments, the heavy chain variable region and the light chain variable region comprise HCDR1, HCDR2 of any of the aforementioned anti-GUCY2C antibodies , HCDR3, LCDR1, LCDR2, and LCDR3. In some embodiments, the heavy chain variable region of the anti-GUCY2C antibody comprises SEQ ID NO: 17, and the light chain variable region of the anti-GUCY2C antibody comprises SEQ ID NO: 18.
  • the anti-GUCY2C antibody comprises the heavy chain variable region shown in SEQ ID NO: 17 and the light chain variable region shown in SEQ ID NO: 18.
  • the anti-GUCY2C antibody is a single chain antibody comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 19; in some embodiments, the antibody comprises an anti-GUCY2C antibody as described in any of the foregoing The heavy chain variable region and the light chain variable region.
  • the anti-GUCY2C single chain antibody comprises SEQ ID NO: 19.
  • Anti-GUCY2C antibody heavy chain variable region amino acid sequence (SEQ ID NO: 17):
  • Anti-GUCY2C antibody light chain variable region amino acid sequence (SEQ ID NO: 18):
  • Anti-GUCY2C single-chain antibody amino acid sequence (SEQ ID NO: 19):
  • Immune checkpoints are inhibitory pathways in the immune system that are regulated by ligand/receptor interactions. It plays an important role in maintaining autoimmune tolerance and regulating the duration and magnitude of physiological immune responses, thereby preventing the immune system from causing damage and destruction to normal tissues.
  • tumor cells in the microenvironment of solid tumors bind to immune checkpoint receptors (such as PD1 receptors) expressed by immune cells (such as T cells) by overexpressing immune checkpoint ligands (such as PD-L1 ligands) to suppress immunity. Cell activity, so as to avoid being killed by immune cells.
  • the present invention provides the above-mentioned immune checkpoint antibody in the fusion protein.
  • the combination of immune checkpoints expressed by cells can shield the immune checkpoint ligands expressed by tumor cells, so that immune cells can maintain continuous killing activity.
  • the immune checkpoint of the fusion protein of the present invention is selected from at least one of PD1, PD-L1, TIGIT, LAG3, CTLA4, BTLA or TIM3. In an optional embodiment, the immune checkpoint of the fusion protein of the present invention is selected from PD1.
  • the immune checkpoint antibody is an anti-PD1 antibody.
  • the amino acid sequence of the anti-PD1 antibody is (c) the amino acid sequence shown in SEQ ID NO: 2, or (d) one or several amino acids are substituted, deleted or added in the amino acid sequence defined in (c) and
  • the edited protein has a derived amino acid sequence targeting PD1 function.
  • Amino acid sequence of anti-PD1 antibody (SEQ ID NO: 2):
  • the anti-PD1 antibody is a single chain antibody (scFv).
  • the anti-PD1 antibody comprises a heavy chain variable region and a light chain variable region; in some embodiments, the heavy chain variable region of an anti-PD1 antibody comprises a heavy chain variable region in SEQ ID NO: 2 ( HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 25), the light chain variable region of the anti-PD1 antibody comprises LCDR1, LCDR2 and LCDR3 of the light chain variable region (SEQ ID NO: 26) in SEQ ID NO: 2; In some embodiments, HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system.
  • the amino acid sequence of the CDRs (defined by the Kabat numbering system) of the anti-PD1 antibody is as follows: HCDR1: NSGMH (SEQ ID NO: 27); HCDR2: VIWYDGSKRYYADSVKG (SEQ ID NO: 28); HCDR3: NDDY (SEQ ID NO: 28); NO: 29); LCDR1: RASQSVSSYLA (SEQ ID NO: 30); LCDR2: DASNRAT (SEQ ID NO: 31); LCDR3: QQSSNWPRT (SEQ ID NO: 32).
  • the heavy chain variable region of the anti-PD1 antibody comprises the heavy chain variable region in SEQ ID NO: 2
  • the light chain variable region of the anti-PD1 antibody comprises the light chain variable region in SEQ ID NO: 2 district.
  • an anti-PD1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 25, and the light chain can be The variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 26; in some embodiments, the heavy chain variable region and the light chain variable region comprise HCDR1, HCDR2 of any of the preceding anti-PD1 antibodies , HCDR3, LCDR1, LCDR2, and LCDR3.
  • the heavy chain variable region of the anti-PD1 antibody comprises the amino acid sequence of SEQ ID NO:25, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:26.
  • the anti-PD1 antibody comprises a heavy chain variable region set forth in SEQ ID NO:25 and a light chain variable region set forth in SEQ ID NO:26.
  • the anti-PD1 antibody is a single chain antibody comprising SEQ ID NO:2.
  • the anti-PD1 antibody is a single-chain antibody, which includes the amino acid sequence derived from SEQ ID NO: 2, the amino acid sequence derived from SEQ ID NO: 2 is obtained through substitution, deletion or addition of one or several amino acids, and the edited protein It has the function of targeting PD1.
  • amino acid sequence of the heavy chain variable region of the anti-PD1 antibody (SEQ ID NO: 25):
  • Anti-PD1 antibody light chain variable region amino acid sequence (SEQ ID NO: 26):
  • cytokines are low-molecular-weight soluble proteins produced by various cells induced by immunogens, mitogens or other stimulators, and have the ability to regulate innate and adaptive immunity, hematopoiesis, Various functions such as cell growth and damaged tissue repair. According to different functions, cytokines can be divided into interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, and growth factors.
  • cytokines play a role in the body through paracrine, autocrine or endocrine, etc., and have various physiological characteristics such as pleiotropy, overlap, antagonism, and synergy, forming a very complex cytokine regulatory network, participating in many aspects of the human body. an important physiological function. Different cytokines have different functions and degrees in the regulation of different immune cells.
  • the cytokines in the fusion protein provided by the invention can be selected according to the selected immune effector cells. For example, interleukins can activate T lymphocytes to produce active mediators. , Therefore, interleukin can be selected as the cytokine in the fusion protein when T cells are transgenic.
  • the cytokine of the present invention is (A) or (B);
  • (B) is a protein derived from (A) with regulatory immune cell activity.
  • the cytokine is IL-21.
  • IL-21 has the amino acid sequence shown in SEQ ID NO: 3.
  • the main function of the above chemokines is to manage the migration (homing) of leukocytes to their respective locations during inflammation and homeostasis, including (1) basal homing chemokines: basal ones produced in thymus and lymphoid tissues Steady-state chemokine.
  • basal homing chemokines basal ones produced in thymus and lymphoid tissues
  • the chemokines CCL19 and CCL21 expressed in lymph nodes and lymphatic endothelial cells
  • their receptor CCR7 play a homeostatic function in cell homing.
  • Inflammatory homing chemokines Inflammatory chemokines are produced in high concentrations during infection or injury and determine the migration of inflammatory leukocytes to damaged areas.
  • Typical inflammatory chemokines include: CCL2, CCL3 and CCL5, CXCL1, CXCL2 and CXCL8.
  • the chemokine of the present invention is (c) or (d);
  • (d) is a protein derived from (c) capable of inducing directional migration of immune cells.
  • the CXC chemokine is at least one of CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16 or CXCL17.
  • the CC chemokine is CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, At least one of CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, or CCL28.
  • the CX3C chemokine is CX3CL1.
  • the XC chemokine is XCL1.
  • the chemokine is CCL19 or CCL21.
  • CCL19 has the amino acid sequence shown in SEQ ID NO: 4;
  • CCL21 has the amino acid sequence shown in SEQ ID NO: 5.
  • the present invention provides a recombinant nucleic acid.
  • the recombinant nucleic acid includes a first nucleic acid molecule, a second nucleic acid molecule, and a third nucleic acid molecule.
  • the first nucleic acid molecule contains the coding region (I) of any of the foregoing embodiments
  • the second nucleic acid molecule Containing the coding region (II) of any of the foregoing embodiments
  • the third nucleic acid molecule contains the coding region (III) of any of the foregoing embodiments.
  • the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are linked by the nucleic acid sequence of the 2A peptide.
  • the 2A peptide is selected from at least one of P2A, T2A, F2A or E2A.
  • the first nucleic acid molecule is connected to the second nucleic acid molecule through the nucleic acid sequence of the 2A peptide, and the second nucleic acid molecule is connected to the third nucleic acid molecule through the nucleic acid sequence of the 2A peptide.
  • the structure of the recombinant nucleic acid is as shown in CAR-Mesothelin&PD1-IL21&CCL19 or CAR-Mesothelin&PD1-IL21&CCL21 in accompanying drawing 1, or as shown in CAR-GUCY2C&PD1-IL21&CCL19 or CAR-GUCY2C&PD1-IL21&CCL21 in accompanying drawing 5 .
  • the present invention provides biological materials, which include any of the following:
  • Recombinant vector contains the gene of any of the aforementioned embodiments, or contains the recombinant nucleic acid of any of the aforementioned embodiments; the original plasmid vector used by the recombinant vector can be routinely selected by those skilled in the art according to actual needs.
  • constructs include non-pathogenic viruses, and non-pathogenic viruses contain the genes, recombinant nucleic acids or (i) recombinant vectors of any of the foregoing embodiments.
  • non-pathogenic viruses include retroviruses, lentiviruses or adenoviruses.
  • the present invention provides transgenic immune effector cells, which contain the gene, recombinant nucleic acid or biological material according to any of the aforementioned embodiments.
  • the construction method includes introducing the gene, recombinant nucleic acid or biological material in any of the foregoing embodiments into immune effector cells to obtain transgenic immune effector cells;
  • the immune effector cells are selected from at least one of T cells, NK cells, NKT cells, macrophages or CIK cells.
  • the immune effector cells are T cells.
  • the present invention provides the application of the gene, recombinant nucleic acid, biological material, transgenic immune effector cell or transgenic immune effector cell constructed by the construction method in the preparation of anti-tumor products according to any of the foregoing embodiments.
  • the tumor comprises a solid tumor.
  • the present invention provides anti-tumor drugs, which include at least one of the genes, recombinant nucleic acids, biological materials or transgenic immune effector cells in any of the foregoing embodiments.
  • the tumor comprises a solid tumor.
  • the present invention provides a method for treating tumors, the method comprising administering to a subject a therapeutically effective amount of at least one of the aforementioned genes, recombinant nucleic acids, biological materials or transgenic immune effector cells.
  • the tumor comprises a solid tumor.
  • the present invention provides a pharmaceutical composition, which includes at least one of the aforementioned genes, recombinant nucleic acids, biological materials or transgenic immune effector cells, and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier refers to an inactive substance used to deliver drugs such as antibodies in a preparation.
  • Pharmaceutically acceptable carriers can be anti-adherents, binders, coatings, disintegrants, fillers, diluents, preservatives, sweeteners, absorption delaying agents, wetting agents, emulsifiers, buffers etc.
  • the pharmaceutical composition is used to treat tumors.
  • a lentivirus targeting Mesothelin expressing Anti PD1-IL-21 fusion protein and CCL19 was constructed, as follows:
  • Nucleotide sequence of recombinant nucleic acid CAR-Mesothelin&PD1-IL21&CCL19 SEQ ID NO: 6
  • Example 1 The difference between this example and Example 1 is that CCL19 is replaced by CCL21, as shown in the third Schema Graph in Figure 1, and the rest are consistent with Example 1, and the expression of Anti PD1-IL-21 fusion protein and CCL21 targeting Mesothelin Lentivirus.
  • This example uses the lentivirus provided in Example 1 to produce CART cells, including the following steps:
  • T cells were separated with Stemcell T cell sorting kit (Cat. No.: 19051).
  • Example 2 the lentivirus provided in Example 2 was used to produce CART cells according to the method of Example 3, which was named CAR-Mesothelin&PD1-IL21&CCL21 group.
  • Example 5 Construction of Lentivirus Targeting GUCY2C Expressing Anti PD1-IL-21 Fusion Protein and CCL19 or CCL21
  • a lentivirus targeting GUCY2C expressing Anti PD1-IL-21 fusion protein and CCL19 or CCL21 was constructed, as follows:
  • Nucleotide sequence of recombinant nucleic acid CAR-GUCY2C&PD1-IL21&CCL19 SEQ ID NO: 7
  • Nucleotide sequence of recombinant nucleic acid CAR-GUCY2C&PD1-IL21&CCL21 SEQ ID NO: 8
  • Example 5 Using the GUCY2C-targeting lentivirus expressing Anti PD1-IL-21 fusion protein and CCL19 constructed in Example 5, and the GUCY2C-targeting lentivirus expressing Anti PD1-IL-21 fusion protein and CCL21, respectively according to Example 3
  • the method to produce CART cells was named CAR-GUCY2C&PD1-IL21&CCL19 and CAR-GUCY2C&PD1-IL21&CCL21 respectively.
  • This comparative example constructs a kind of lentivirus, and the difference from Example 1 is that the CCL19 coding region is omitted, and the rest is consistent with Example 1, as shown in the first Schema Graph in Figure 1. Then, using the lentivirus, according to The method of Example 3 produces CART cells, named CAR-Mesothelin&PD1-IL21 group.
  • CART cells were constructed: CAR-GUCY2C&PD1-IL21.
  • First construct the GUCY2C-targeting lentivirus expressing the Anti PD1-IL-21 fusion protein (the difference between the construction method and the construction method of the GUCY2C-targeting lentivirus expressing the Anti PD1-IL-21 fusion protein and CCL19 in Example 5 is only In that, the nucleic acid omits the CCL19 coding region, and the rest is consistent with Example 5), the structure of the lentivirus is shown in the first Schema Graph in Figure 5; then use the lentivirus to produce CART cells according to the method of Example 3, and obtain The CART cells were named CAR-GUCY2C&PD1-IL21.
  • the CAR positive rate detection results on D6 (day 6) after T cell infection showed that the CAR positive rate in the CAR-Mesothelin&PD1-IL21 group was 47.00%, and that in the CAR-Mesothelin&PD1-IL21&CCL19 group was 47.00%. 35.50%, CAR-Mesothelin&PD1-IL21&CCL21 group, the CAR-positive rate was 29.87%. From the results, it can be seen that the present invention successfully prepared CAR-Mesothelin&PD1-IL21&CCL19 and CAR-Mesothelin&PD1-IL21&CCL21.
  • each group of cells on D6 after infection was detected by flow cytometry.
  • T, B, and NK cells are grouped, and the groups of CD4, CD8, PD1, and PD-L1 in T cells are detected at the same time.
  • T cells Using T cells as a control, the in vitro killing function of the cells in the CAR-Mesothelin&PD1-IL21&CCL19 group in Example 3 and the CAR-Mesothelin&PD1-IL21&CCL21 group in Example 4 on D6 after infection was evaluated, using xCELLigence real-time, cell-mediated The cytotoxicity system (Acea Biosciences Inc.) of CART cell-mediated cytotoxicity was evaluated.
  • the cytotoxicity system Acea Biosciences Inc.
  • OVCAR3 cells 1 ⁇ 10 4 OVCAR3 cells were cultured in 150 ⁇ L of growth medium per well of E-Plate 16 (Acea Biosciences) and incubated overnight in a 37 °C incubator to quantify electrical resistance every 15 min using the RTCA DP Analyzer system Antibody and RTCA software version 2.0 (Acea Biosciences Inc.). After approximately 24 hours, 50 ⁇ L of CART cells (E:T ratio 3:1) or 50 ⁇ L of medium were added as negative, and cell-mediated killing was quantified over the next 24 hours, with electrical impedance readings taken every 15 minutes.
  • CAR-Mesothelin&PD1-IL21 group cells CAR-Mesothelin&PD1-IL21&CCL19 group cells and CAR-Mesothelin&PD1-IL21&CCL21 group cells were cultured on the 8th day, each cell culture supernatant 2mL, 300g, 5min, RT centrifugation, and then take the supernatant Add them to the lower chamber of a transwell plate (Corning, 3422), set up three duplicate wells for each group, and add 0.6 mL of supernatant to each well.
  • CAR-GUCY2C&PD1-IL21&CCL19 In the production of CAR-GUCY2C&PD1-IL21&CCL19, CAR-GUCY2C&PD1-IL21&CCL21 in Example 6 and in the production of CAR-GUCY2C&PD1-IL21 in Comparative Example 2, after 72 hours of virus infection, use flow cytometry to analyze the cell positive rate of the CART cells obtained respectively .
  • the experimental results are shown in Figure 6.
  • the CAR positive rate detection results on D6 after T cell infection showed that the CAR positive rate in the CAR-GUCY2C&PD1-IL21 group was 59.77%, and that in the CAR-GUCY2C&PD1-IL21&CCL19 group was 66.62%.
  • the positive rate of CAR in the CAR-GUCY2C&PD1-IL21&CCL21 group was 50.10%. It can be seen from the results that the present invention successfully prepared CAR-GUCY2C&PD1-IL21&CCL19 and CAR-GUCY2C&PD1-IL21&CCL21.
  • OVCAR3 cells 1 ⁇ 10 4 OVCAR3 cells were cultured in 150 ⁇ L of growth medium per well of E-Plate 16 (Acea Biosciences) and incubated overnight in a 37 °C incubator to quantify electrical resistance every 15 min using the RTCA DP Analyzer system Antibody and RTCA software version 2.0 (Acea Biosciences Inc.). After approximately 24 hours, add 50 ⁇ L of T cells or 50 ⁇ L of CART cells (E:T ratio 3:1) or 50 ⁇ L of media as negative, and quantify cell-mediated killing over the next 24 hours, reading every 15 minutes electrical impedance.
  • the experimental results are shown in Figure 7.
  • the experimental results showed that when co-cultured with target cells in vitro, compared with T cells and CAR-GUCY2C&PD1-IL21 cells, cells in CAR-GUCY2C&PD1-IL21&CCL19 group and CAR-GUCY2C&PD1-IL21&CCL21 group had Very obvious tumor killing ability.
  • mice In vivo tumor suppression ability experiment in mice:
  • LS1034 human colorectal cancer cell tumor model
  • NPG mice Beijing Weitongda Biotechnology Co., Ltd.
  • a total of 25 mice divided into 5 groups, 5 mice in each group, when the tumor grew for 12 days , by tail vein administration, each mouse was injected with 5.0E+06 CAR+ cells or T cells or PBS once, and then the tumor volume was measured twice a week.
  • the experimental results are shown in Figure 8.
  • PD1 is expressed on the surface of T cells, mainly CD8+ T cells, and the formed PD1 antibody is compatible with IL-21.
  • the -21 fusion protein will selectively bind to the surface of T cells and CART cells to exercise its dual functions; at the same time, the fusion protein will greatly increase the half-life of the drug due to its larger molecular weight.
  • the present invention builds CCL19 or CCL21 into the CAR structure through molecular construction, so that CAR T cells can express CAR and CCL19 or CCL21 chemokines at the same time.
  • CAR T cells can express CAR and CCL19 or CCL21 chemokines at the same time.
  • the secreted CCL19 or CCL21 can recruit and chemotaxis the body's natural
  • the T cells from the T cells reach the tumor site and help the CART cells to kill the tumor.

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Abstract

涉及CART技术领域，尤其是转基因免疫细胞及其构建方法和应用。提供了含有三个编码区的基因、含有该基因的重组核酸、生物材料和转基因免疫效应细胞，能够编码三种功能蛋白，三种蛋白的表达使得免疫效应细胞具有多种功能，从而降低实体瘤微环境对免疫效应细胞抑制作用，延长免疫效应细胞的杀伤作用时间；同时增强免疫效应细胞抗肿瘤疗效。

Description

转基因免疫细胞及其构建方法和应用
本申请要求申请日为2021年12月21日的中国专利申请(申请号：202111572804.5，发明名称：转基因免疫细胞及其构建方法和应用)的优先权，该中国专利申请的全文通过引用方式整体并入到本申请。
技术领域
本发明涉及CART技术领域，尤其是涉及转基因免疫细胞及其构建方法和应用。
背景技术
嵌合抗原受体T细胞免疫疗法，简称CART技术，是通过体外改造病人的T细胞，使病人的T细胞具备识别肿瘤细胞的能力，体外扩大培养后回输到病人体内进行治疗的一种方法。目前，以CD19为靶点的CART在治疗B细胞血液肿瘤方面取得了巨大的成果，但根据临床研究结果来看，CD19 CART在治疗B细胞淋巴瘤方面的疗效远远不及在治疗B细胞急性淋巴细胞白血病方面的疗效，这可能是因为B细胞淋巴瘤是实体肿瘤，CART细胞难以大量到达并浸润到肿瘤内部。对于浸润到肿瘤内部的CART细胞，实体瘤细胞能够通过表面过表达大量的PD-L1分子，从而逃避CART细胞的杀伤，同时，CART细胞与实体瘤细胞的不充分接触还显著影响了CART细胞的增殖和持久性，加快了CART细胞在体内的耗竭速度。
因此，与血液瘤的治疗相比，使用实体瘤的治疗对CART细胞提出了更高的要求，需要CART细胞具有抵抗，甚至破坏实体瘤微环境的能力，持续深入实体瘤内部发挥治疗作用。
细胞因子是由免疫细胞(如单核、巨噬细胞、T细胞、B细胞、NK细胞等)和某些非免疫细胞(内皮细胞、表皮细胞、纤维母细胞等)经刺激而合成、分泌的一类具有广泛生物学活性的小分子蛋白质，通过结合相应受体调节细胞生长、分化和效应，调控免疫应答。白细胞介素是其中重要分类之一，具有免疫调节、造血以及炎症调控等多种作用，目前已报道的白细胞介素已多达三十余种，其中IL-21由CD4 T细胞和NKT细胞产生，刺激CD8 T细胞和NK细胞的成熟并增强其细胞毒性，同时具备促进记忆性CD8 T细胞的分化等功能。IL-21的诸多效能使其成为免疫治疗的潜在靶点，但由于IL-21R广泛表达包括T细胞、B细胞、NK细胞和骨髓细胞。
趋化因子(chemokines)，是一类由细胞分泌的小细胞因子或信号蛋白。由于它们具有诱导附近反应细胞定向趋化的能力，因而命名为趋化细胞因子。趋化性细胞因子根据其氨基端(N端)半胱氨酸的排列方式，可分为CXC、CC、C和CX3C四个亚族，其 中CC趋化因子亚族的CCL19和CCL21是CCR7的配体，T细胞表达CCR7蛋白，所以CCL19和CCL21趋化因子能够趋化T细胞迁移。
上述细胞因子和趋化因子均有利于对抗和改善实体瘤微环境，为免疫细胞提供持久的杀伤环境。
发明内容
如何对在现有免疫细胞的基础上，整合多种功能分子共同对抗实体瘤微环境，显著有效地改善免疫细胞的治疗效果是目前亟需解决的问题。
本发明的目的在于，针对实体瘤微环境提供一种多功能免疫细胞，期望不仅能够对抗实体瘤在发展过程中逐渐形成的、对其稳定和扩散有利的微环境，同时还能够趋化自身免疫细胞，更大限度激活机体自身免疫系统，提高机体自身与实体瘤的对抗强度。
为了解决上述问题，实现上述目的，本发明提供了以下技术方案：
第一方面，本发明提供了含有三个编码区的基因，其中，编码区(I)编码嵌合抗原受体，嵌合抗原受体含有特异性识别肿瘤抗原的胞外区；
编码区(II)编码融合蛋白，融合蛋白包括免疫检查点抗体和细胞因子；
编码区(III)编码趋化因子。
可选实施方式中，肿瘤抗原选自MSLN、GD2、GPC3、CD19、EGFR VIII、GUCY2C、HER2、MUC16或Claudin 18.2中的至少一种。
可选实施方式中，胞外区含有抗MSLN抗体。
可选实施方式中，胞外区含抗GUCY2C抗体；可选实施方式中，嵌合抗原受体包含抗GUCY2C单链抗体；
优选地，抗MSLN抗体的氨基酸序列为(a)如SEQ ID NO：1所示的氨基酸序列，或者(b)在(a)限定的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且编码的蛋白质具有特异性识别MSLN抗原活性的衍生氨基酸序列。
可选实施方式中，免疫检查点选自PD1、PD-L1、TIGIT、LAG3、CTLA4、BTLA或TIM3中至少一种。
可选实施方式中，免疫检查点抗体为抗PD1抗体。
优选地，抗PD1抗体的氨基酸序列为(c)如SEQ ID NO：2所示的氨基酸序列，或者(d)在(c)限定的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且编辑的蛋白质具有靶向PD1功能的衍生氨基酸序列。
可选实施方式中，细胞因子为(A)或(B)；
(A)选自IL-21、IL-23、IL-2、IL-7、IL-9、IL-12、IL-15或IL-18中至少一种；
(B)为具有调节免疫细胞活性的由(A)衍生的蛋白质。
可选实施方式中，细胞因子为IL-21。
优选地，IL-21具有如SEQ ID NO：3所示的氨基酸序列。
可选实施方式中，趋化因子为(c)或(d)；
(c)选自CXC趋化因子、CC趋化因子、CX3C趋化因子或XC趋化因子中的至少一种；
(d)为具有诱导免疫细胞定向迁移功能的由(c)衍生的蛋白质。
优选地，CXC趋化因子为CXCL1、CXCL2、CXCL3、CXCL4、CXCL5、CXCL6、CXCL7、CXCL8、CXCL9、CXCL10、CXCL11、CXCL12、CXCL13、CXCL14、CXCL15、CXCL16或CXCL17中的至少一种。
优选地，CC趋化因子为CCL1、CCL2、CCL3、CCL4、CCL5、CCL6、CCL7、CCL8、CCL9、CCL10、CCL11、CCL12、CCL13、CCL14、CCL15、CCL16、CCL17、CCL18、CCL19、CCL20、CCL21、CCL22、CCL23、CCL24、CCL25、CCL26、CCL27或CCL28中的至少一种。
优选地，CX3C趋化因子为CX3CL1。
优选地，XC趋化因子为XCL1。
可选实施方式中，趋化因子为CCL19或CCL21。
优选地，CCL19具有如SEQ ID NO：4所示的氨基酸序列；
优选地，CCL21具有如SEQ ID NO：5所示的氨基酸序列。
第二方面，本发明提供了重组核酸，重组核酸包括第一核酸分子、第二核酸分子和第三核酸分子，第一核酸分子含有前述任一实施方式的编码区(I)，第二核酸分子含有前述任一实施方式的编码区(II)，第三核酸分子含有前述任一实施方式的编码区(III)。
可选实施方式中，第一核酸分子、第二核酸分子和第三核酸分子之间通过2A肽的核酸序列相连。
优选地，2A肽选自P2A、T2A、F2A或E2A中的至少一种。
可选实施方式中，第一核酸分子通过2A肽的核酸序列与第二核酸分子相连，第二核酸分子通过2A肽的核酸序列和第三核酸分子相连。
第三方面，本发明提供了生物材料，生物材料包括以下任一项：
(i)重组载体，重组载体含有前述任一实施方式基因，或者含有前述任一实施方式的重组核酸；
(ii)构建体，构建体包括非致病性病毒，非致病性病毒含有前述任一实施方式的基因、重组核酸或(i)重组载体。
可选实施方式中，非致病性病毒包括反转录病毒、慢病毒或腺病毒。
第四方面，本发明提供了转基因免疫效应细胞，转基因免疫效应细胞含有前述任一实施方式基因、重组核酸或生物材料。
可选实施方式中，转基因免疫效应细胞的构建方法包括，将前述任一实施方式基因、重组核酸或生物材料导入免疫效应细胞中，获得转基因免疫效应细胞；
免疫效应细胞选自T细胞、NK细胞、NKT细胞、巨噬细胞或CIK细胞中的至少一种。
优选地，免疫效应细胞为T细胞。
第五方面，本发明提供了前述任一实施方式基因、重组核酸、生物材料、转基因免疫效应细胞或构建方法构建得到的转基因免疫效应细胞在制备抗肿瘤产品中的应用。
优选地，肿瘤包括实体瘤。
第六方面，本发明提供了抗肿瘤药物，药物包括前述任一实施方式基因、重组核酸、生物材料或转基因免疫效应细胞中的至少一种。
优选地，肿瘤包括实体瘤。
第七方面，本发明提供了治疗肿瘤的方法，方法包括包括向受试者施用治疗有效量的如前任一基因、重组核酸、生物材料或转基因免疫效应细胞中的至少一种。优选地，肿瘤包括实体瘤。
第八方面，本发明提供了一种药物组合物，其包括如前任一基因、重组核酸、生物材料或转基因免疫效应细胞中的至少一种，以及药学上可接受的载体。
本发明提供的基因、重组核酸、生物材料和转基因免疫效应细胞均能够编码三种蛋白，三种蛋白的表达使得免疫效应细胞同时具有了多种功能，包括特异性识别肿瘤抗原和靶向免疫检查点，以及趋化并调节免疫细胞活性，从而在实现降低实体瘤微环境对免疫效应细胞抑制作用的同时，延长免疫效应细胞的杀伤作用时间；同时趋化因子的表达能够招募和趋化机体自身的T细胞到达肿瘤部位，在细胞因子的调节作用下共同杀伤肿瘤细胞，增强免疫效应细胞抗肿瘤疗效。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例1、实施例2和对比例1采用的重组核酸序列构成示意图(Schema graph of lentiviral vector coding gene，也即慢病毒载体编码基因示意图)；
图2为本发明实施例3、实施例4和对比例1提供CART细胞阳性率检测结果；
图3为本发明实施例3、实施例4和对比例1提供CART细胞体外杀瘤能力对比结果；
图4为本发明实施例3、实施例4和对比例1提供CART细胞趋化能力对比结果；
图5为本发明实施例5和对比例2采用的重组核酸构成示意图(Schema graph of lentiviral vector coding gene，也即慢病毒载体编码基因示意图)；
图6为本发明实验例5的CART细胞阳性率检测结果图；
图7为本发明实验例6的CART细胞体外杀瘤能力对比结果图；
图8为本发明实验例7的体内抑制肿瘤实验结果图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。通常在此处附图中描述和示出的本发明实施例的组件可以以各种不同的配置来布置和设计。
因此，以下对在附图中提供的本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
应注意到：相似的标号和字母在下面的附图中表示类似项，因此，一旦某一项在一个附图中被定义，则在随后的附图中不需要对其进行进一步定义和解释。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
在某个具体实施方式中，第一方面，本发明提供了含有三个编码区的基因，其中，编码区(I)编码嵌合抗原受体，嵌合抗原受体含有特异性识别肿瘤抗原的胞外区；
编码区(II)编码融合蛋白，融合蛋白包括免疫检查点抗体和细胞因子；
编码区(III)编码趋化因子。
本发明基因应当理解为，包含上述三个编码区的所有基因，至于除上述三个编码区以外，是否还含有其他编码区本发明并未进行限定，本领域技术人员可以根据实际需求选择添加其他编码区。
本发明上述三个编码区以独立表达为目的，因此，本发明上述基因中三个编码区的连接顺序可以根据实际需求进行选择，并不需特殊限定。
本发明基因在不影响三个编码区独立表达的情况下，还可以包含适当的内含子。
上述嵌合抗原受体指的是嵌合抗原受体T细胞通过识别某种肿瘤抗原的抗体的抗原结合部与跨膜区和胞内区在体外偶联为一个嵌合蛋白，通过基因转导的方法转染患者的T细胞，使其表达嵌合抗原受体，使得患者的T细胞被“重编码”后，能够生成大量肿瘤特异性的CART细胞。重新编码后的嵌合抗原受体T细胞加进患者身体后，所述嵌合抗原受体可以特异性地追踪和识别并引导T细胞杀伤肿瘤细胞。本发明在现有CART细胞基础上，将嵌合抗原受体的应用范围扩展至其他免疫效应细胞，例如T细胞、NK细胞、NKT细胞、巨噬细胞或CIK细胞等，所述的抗体的抗原结合部即为本发明所述的识别肿瘤抗原的胞外区，而跨膜区和胞内区则可根据实际需求通过常规选择获得。
优选地，跨膜区为CD8的跨膜段。
优选地，胞内区为免疫共刺激分子的胞内段和CD3Zeta链。
优选地，免疫共刺激分子选自4-1BB、CD28、CD3、OX-40、CD40L、CD27、CD30、或他们的衍生物中的任一种或几种。可选实施方式中，免疫共刺激分子选自4-1BB。
可选实施方式中，本发明肿瘤抗原选自MSLN、GD2、GPC3、CD19、EGFRVIII、GUCY2C、HER2、MUC16或Claudin 18.2中的至少一种。
可选实施方式中，胞外区含有抗MSLN抗体。
优选地，抗MSLN抗体的氨基酸序列为(a)如SEQ ID NO：1所示的氨基酸序列，或者(b)在(a)限定的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且编码的蛋白质具有特异性识别MSLN抗原活性的衍生氨基酸序列。
抗MSLN抗体的氨基酸序列(SEQ ID NO：1)：
Figure PCTCN2022140094-appb-000001
术语“抗体”在最广义上使用，其涵盖各种抗体结构，包括但不限于单克隆抗体/多克隆抗体，多特异性抗体(例如双特异性抗体、三特异性抗体等)，鼠源抗体/嵌合抗体，全长抗体或其抗原结合片段(例如scFv)，只要它们展示出所期望的抗原结合活性。
术语“互补性决定区”、“CDR”或“CDRs”或“互补决定区”是指免疫球蛋白的重链和轻链的高度可变区，其是抗体可变结构域内主要促成与抗原特异性结合的区域。重链互补决定区用HCDR表示，重链可变区中含有的3个CDR区：HCDR1、HCDR2和HCDR3；轻链互补决定区用LCDR表示，轻链可变区中含有的3个CDR区：LCDR1、LCDR2和LCDR3。可以通过各种公知方案来确定CDR的氨基酸序列边界，例如：“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则、“ABM”编号规则、“contact”编号规则(参见Martin，ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001)和ImMunoGenTics(IMGT)编号规则(Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003))等；各种编号系统之间的对应关系是本领域技术人员熟知的。
可选实施方式中，嵌合抗原受体包含抗MSLN抗体。可选实施方式中，抗MSLN抗体为单链抗体(scFv，由抗体轻链可变区(VL)与重链可变区(VH)直接或通过肽接头(L)连接而成，例如N端到C端：VH-L-VL或者VL-L-VH的scFv，连接子L可以选自(GxS)y连接子，其中，x选自1-5的整数，y选自0-6的整数，例如x为4，y为3)。可选实施方式中，抗MSLN抗体包含重链可变区和轻链可变区；抗MSLN抗体的重链可变区包括SEQ ID NO：1中的重链可变区(SEQ ID NO：9)的HCDR1、HCDR2和HCDR3，抗MSLN抗体的轻链可变区包括SEQ ID NO：1中的轻链可变区(SEQ ID NO：10)的LCDR1、LCDR2和LCDR3；在一些实施方式中，HCDR1、HCDR2和HCDR3以及LCDR1、LCDR2和LCDR3由IMGT编号系统定义，或由Kabat编号系统定义，或由Chothia编号系统定义，或由Contact编号系统定义，或由AbM编号系统定义。在一些实施方式中，HCDR1、HCDR2和HCDR3以及LCDR1、LCDR2和LCDR3 由Kabat编号系统定义。可选实施方式中，抗MSLN抗体的CDRs(由Kabat编号系统定义)氨基酸序列如下：HCDR1：GYTMN(SEQ ID NO：11)；HCDR2：LITPYNGASSYNQKFRG(SEQ ID NO：12)；HCDR3：GGYDGRGFDY(SEQ ID NO：13)；LCDR1：SASSSVSYMH(SEQ ID NO：14)；LCDR2：DTSKLAS(SEQ ID NO：15)；LCDR3：QQWSKHPLT(SEQ ID NO：16)。在一些实施方式中，抗MSLN抗体的重链可变区包括SEQ ID NO：1中的重链可变区，抗MSLN抗体的轻链可变区包括SEQ ID NO：1中的轻链可变区。在一些实施方式中，抗MSLN抗体包含重链可变区和轻链可变区，其中，重链可变区包含与SEQ ID NO：9具有至少85％序列同一性的氨基酸序列，轻链可变区包含SEQ ID NO：10具有至少85％序列同一性的氨基酸序列；在一些实施方式中，重链可变区和轻链可变区包括前面任一的抗MSLN抗体的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3；在一些实施方式中，抗MSLN抗体包含SEQ ID NO：9所示的重链可变区和SEQ ID NO：10所示的轻链可变区。在一些实施方式中，抗MSLN抗体为单链抗体，其包括SEQ ID NO：1。在一些实施方式中，抗MSLN抗体为单链抗体，其包括SEQ ID NO：1衍生氨基酸序列，SEQ ID NO：1衍生氨基酸序列经过取代、缺失或添加一个或几个氨基酸获得，其编辑的蛋白质具有靶向MSLN功能。
可选实施方式中，抗MSLN抗体重链可变区氨基酸序列(SEQ ID NO：9)：
Figure PCTCN2022140094-appb-000002
抗MSLN抗体轻链可变区氨基酸序列(SEQ ID NO：10)：
Figure PCTCN2022140094-appb-000003
可选实施方式中，本发明肿瘤抗原选自GUCY2C。可选实施方式中，嵌合抗原受体包含抗GUCY2C抗体。可选实施方式中，抗GUCY2C抗体为单链抗体(scFv)。可选实施方式中，抗GUCY2C抗体包含重链可变区和轻链可变区。在一些实施方式中，抗GUCY2C抗体的重链可变区包括SEQ ID NO：17中的HCDR1、HCDR2和HCDR3，抗GUCY2C抗体的轻链可变区包括SEQ ID NO：18中的LCDR1、LCDR2和LCDR3；在一些实施方式中，HCDR1、HCDR2和HCDR3以及LCDR1、LCDR2和LCDR3由IMGT编号系统定义，或由Kabat编号系统定义，或由Chothia编号系统定义，或由Contact编号系统定义，或由AbM编号系统定义。可选实施方式中，抗GUCY2C抗体的CDRs(由IMGT编号系统定义)氨基酸序列如下：HCDR1：GYTFTEYT(SEQ ID NO：20)；HCDR2：INPNNGGA(SEQ ID NO：21)；HCDR3：ARAPYYYGSSYYAMDY(SEQ ID NO：22)；LCDR1：ESVDNYGISF(SEQ ID NO：23)；LCDR2：AAS；LCDR3：QQSKEVPFT(SEQ ID NO：24)。在一些实施方式中，抗GUCY2C抗体包含重链可变区和轻链可变区，其中，重链可变区包含与SEQ ID NO：17具有至少85％序列同一性的氨基酸序列，轻链可变区包含与SEQ ID NO：18具有至少85％序列同一性的氨 基酸序列；在一些实施方式中，重链可变区和轻链可变区包括前面任一述的抗GUCY2C抗体的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3。在一些实施方式中，抗GUCY2C抗体的重链可变区包括SEQ ID NO：17，抗GUCY2C抗体的轻链可变区包括SEQ ID NO：18。在一些实施方式中，抗GUCY2C抗体包含SEQ ID NO：17所示的重链可变区和SEQ ID NO：18所示的轻链可变区。在一些实施方式中，抗GUCY2C抗体为单链抗体，其包括与SEQ ID NO：19具有至少85％序列同一性的氨基酸序列；在一些实施方式中，抗体包含前面任一项述的抗GUCY2C抗体的重链可变区和轻链可变区。在一些实施方式中，抗GUCY2C单链抗体包括SEQ ID NO：19。
抗GUCY2C抗体重链可变区氨基酸序列(SEQ ID NO：17)：
Figure PCTCN2022140094-appb-000004
抗GUCY2C抗体轻链可变区氨基酸序列(SEQ ID NO：18)：
Figure PCTCN2022140094-appb-000005
抗GUCY2C单链抗体氨基酸序列(SEQ ID NO：19)：
Figure PCTCN2022140094-appb-000006
免疫检查点(免疫检查点分子)是免疫系统中的抑制性通路，由配体/受体的相互作用所调控。它对于维持自身免疫耐受、调节生理性免疫应答的持续时间和幅度起重要作用，从而避免免疫系统对正常组织造成损伤和破坏。而实体瘤微环境中的肿瘤细胞通过过表达免疫检查点配体(例如PD-L1配体)与免疫细胞(例如T细胞)表达的免疫检查站受体(例如PD1受体)结合，抑制免疫细胞活性，从而免于被免疫细胞杀伤，本发明为了阻止免疫细胞和肿瘤细胞表达的免疫检查点配受体之间的结合，在融合蛋白中提供了上述免疫检查点抗体，该抗体提前与免疫细胞表达的免疫检查点相结合，从而屏蔽肿瘤细胞表达的免疫检查点配体，使免疫细胞保持持续杀伤活性。
可选实施方式中，本发明融合蛋白的免疫检查点选自PD1、PD-L1、TIGIT、LAG3、CTLA4、BTLA或TIM3中至少一种。可选实施方式中，本发明融合蛋白的免疫检查点选自PD1。
可选实施方式中，免疫检查点抗体为抗PD1抗体。
优选地，抗PD1抗体的氨基酸序列为(c)如SEQ ID NO：2所示的氨基酸序列，或者(d)在(c)限定的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且编辑的蛋白质具有靶向PD1功能的衍生氨基酸序列。
抗PD1抗体的氨基酸序列(SEQ ID NO：2)：
Figure PCTCN2022140094-appb-000007
可选实施方式中，抗PD1抗体为单链抗体(scFv)。可选实施方式中，抗PD1抗体包含重链可变区和轻链可变区；在一些实施方式中，抗PD1抗体的重链可变区包括SEQ ID NO：2中重链可变区(SEQ ID NO：25)的HCDR1、HCDR2和HCDR3，抗PD1抗体的轻链可变区包括SEQ ID NO：2中的轻链可变区(SEQ ID NO：26)的LCDR1、LCDR2和LCDR3；在一些实施方式中，HCDR1、HCDR2和HCDR3以及LCDR1、LCDR2和LCDR3由IMGT编号系统定义，或由Kabat编号系统定义，或由Chothia编号系统定义，或由Contact编号系统定义，或由AbM编号系统定义。可选实施方式中，抗PD1抗体的CDRs(由Kabat编号系统定义)氨基酸序列如下：HCDR1：NSGMH(SEQ ID NO：27)；HCDR2：VIWYDGSKRYYADSVKG(SEQ ID NO：28)；HCDR3：NDDY(SEQ ID NO：29)；LCDR1：RASQSVSSYLA(SEQ ID NO：30)；LCDR2：DASNRAT(SEQ ID NO：31)；LCDR3：QQSSNWPRT(SEQ ID NO：32)。在一些实施方式中，抗PD1抗体的重链可变区包括SEQ ID NO：2中的重链可变区，抗PD1抗体的轻链可变区包括SEQ ID NO：2中的轻链可变区。在一些实施方式中，抗PD1抗体包含重链可变区和轻链可变区，其中，重链可变区包含与SEQ ID NO：25具有至少85％序列同一性的氨基酸序列，轻链可变区包含与SEQ ID NO：26具有至少85％序列同一性的氨基酸序列；在一些实施方式中，重链可变区和轻链可变区包括前面任一项的抗PD1抗体的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3。在一些实施方式中，抗PD1抗体的重链可变区包括SEQ ID NO：25的氨基酸序列，轻链可变区包括SEQ ID NO：26的氨基酸序列。在一些实施方式中，抗PD1抗体包含SEQ ID NO：25所示的重链可变区和SEQ ID NO：26所示的轻链可变区。在一些实施方式中，抗PD1抗体为单链抗体，其包括SEQ ID NO：2。在一些实施方式中，抗PD1抗体为单链抗体，其包括SEQ ID NO：2衍生氨基酸序列，SEQ ID NO：2衍生氨基酸序列经过取代、缺失或添加一个或几个氨基酸获得，其编辑的蛋白质具有靶向PD1功能。
可选实施方式中，抗PD1抗体重链可变区氨基酸序列(SEQ ID NO：25)：
Figure PCTCN2022140094-appb-000008
抗PD1抗体轻链可变区氨基酸序列(SEQ ID NO：26)：
MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK；上述细胞因子是免疫原、丝裂原或其他刺激剂诱导多种细胞产生的低分 子量可溶性蛋白质，具有调节固有免疫和适应性免疫、血细胞生成、细胞生长以及损伤组织修复等多种功能。按照功能不同，细胞因子可被分为白细胞介素、干扰素、肿瘤坏死因子超家族、集落刺激因子、以及生长因子等。众多细胞因子在体内通过旁分泌、自分泌或内分泌等方式发挥作用，具有多效性、重叠性、拮抗性、协同性等多种生理特性，形成了十分复杂的细胞因子调节网络，参与人体多种重要的生理功能。不同细胞因子对不同免疫细胞的调节存在功能和程度上的差异，本发明提供的融合蛋白中的细胞因子可以根据选用的免疫效应细胞进行选择，例如，白细胞介素能够活化T淋巴细胞产生活性介质，因此，在对T细胞进行转基因改造时，可以选择白细胞介素作为融合蛋白中的细胞因子。
可选实施方式中，本发明细胞因子为(A)或(B)；
(A)选自IL-21、IL-23、IL-2、IL-7、IL-9、IL-12、IL-15或IL-18中至少一种；
(B)为具有调节免疫细胞活性的由(A)衍生的蛋白质。
可选实施方式中，细胞因子为IL-21。
优选地，IL-21具有如SEQ ID NO：3所示的氨基酸序列。
IL-21的氨基酸序列(SEQ ID NO：3)：
Figure PCTCN2022140094-appb-000009
上述趋化因子的主要功能是在炎症和体内平衡过程中管理白细胞向各自位置的迁移(归巢)，具体包括(1)基础归巢作用趋化因子：在胸腺和淋巴组织中产生的基础的稳态趋化因子。例如，趋化因子CCL19和CCL21(在淋巴结和淋巴管内皮细胞中表达)及其受体CCR7在细胞归巢中起到稳态功能。(2)炎症归巢作用趋化因子：炎症趋化因子在感染或损伤过程中产生高浓度，并决定炎症性白细胞向受损区域的迁移。典型的炎性趋化因子包括：CCL2、CCL3和CCL5、CXCL1、CXCL2和CXCL8。
可选实施方式中，本发明趋化因子为(c)或(d)；
(c)选自CXC趋化因子、CC趋化因子、CX3C趋化因子或XC趋化因子中的至少一种；
(d)为具有诱导免疫细胞定向迁移功能的由(c)衍生的蛋白质。
优选地，CXC趋化因子为CXCL1、CXCL2、CXCL3、CXCL4、CXCL5、CXCL6、CXCL7、CXCL8、CXCL9、CXCL10、CXCL11、CXCL12、CXCL13、CXCL14、CXCL15、CXCL16或CXCL17中的至少一种。
优选地，CC趋化因子为CCL1、CCL2、CCL3、CCL4、CCL5、CCL6、CCL7、CCL8、CCL9、CCL10、CCL11、CCL12、CCL13、CCL14、CCL15、CCL16、CCL17、CCL18、CCL19、CCL20、CCL21、CCL22、CCL23、CCL24、CCL25、CCL26、CCL27或CCL28中的至少一种。
优选地，CX3C趋化因子为CX3CL1。
优选地，XC趋化因子为XCL1。
可选实施方式中，趋化因子为CCL19或CCL21。
优选地，CCL19具有如SEQ ID NO：4所示的氨基酸序列；
CCL19的氨基酸序列(SEQ ID NO：4)：
Figure PCTCN2022140094-appb-000010
优选地，CCL21具有如SEQ ID NO：5所示的氨基酸序列。
CCL21的氨基酸序列(SEQ ID NO：5)：
Figure PCTCN2022140094-appb-000011
第二方面，本发明提供了重组核酸，重组核酸包括第一核酸分子、第二核酸分子和第三核酸分子，第一核酸分子含有前述任一实施方式的编码区(I)，第二核酸分子含有前述任一实施方式的编码区(II)，第三核酸分子含有前述任一实施方式的编码区(III)。
可选实施方式中，第一核酸分子、第二核酸分子和第三核酸分子之间通过2A肽的核酸序列相连。
优选地，2A肽选自P2A、T2A、F2A或E2A中的至少一种。
可选实施方式中，第一核酸分子通过2A肽的核酸序列与第二核酸分子相连，第二核酸分子通过2A肽的核酸序列和第三核酸分子相连。
可选实施方式中，重组核酸的结构如附图1中的CAR-Mesothelin&PD1-IL21&CCL19或CAR-Mesothelin&PD1-IL21&CCL21所示，或者如附图5中的CAR-GUCY2C&PD1-IL21&CCL19或CAR-GUCY2C&PD1-IL21&CCL21所示。第三方面，本发明提供了生物材料，生物材料包括以下任一项：
(i)重组载体，重组载体含有前述任一实施方式基因，或者含有前述任一实施方式的重组核酸；重组载体使用的原始质粒载体，本领域技术人员可以根据实际需求进行常规选择。
(ii)构建体，构建体包括非致病性病毒，非致病性病毒含有前述任一实施方式的基因、重组核酸或(i)重组载体。
可选实施方式中，非致病性病毒包括反转录病毒、慢病毒或腺病毒。第四方面，本发明提供了转基因免疫效应细胞，转基因免疫效应细胞含有前述任一实施方式基因、重组核酸或生物材料。
可选实施方式中，构建方法包括，将前述任一实施方式基因、重组核酸或生物材料导入免疫效应细胞中，获得转基因免疫效应细胞；
免疫效应细胞选自T细胞、NK细胞、NKT细胞、巨噬细胞或CIK细胞中的至少一种。
优选地，免疫效应细胞为T细胞。
第五方面，本发明提供了前述任一实施方式基因、重组核酸、生物材料、转基因免疫效应细胞或构建方法构建得到的转基因免疫效应细胞在制备抗肿瘤产品中的应用。
优选地，肿瘤包括实体瘤。
第六方面，本发明提供了抗肿瘤药物，药物包括前述任一实施方式基因、重组核酸、生物材料或转基因免疫效应细胞中的至少一种。
优选地，肿瘤包括实体瘤。
第七方面，本发明提供了治疗肿瘤的方法，方法包括包括向受试者施用治疗有效量的如前任一基因、重组核酸、生物材料或转基因免疫效应细胞中的至少一种。优选地，肿瘤包括实体瘤。
第八方面，本发明提供了一种药物组合物，其包括如前任一基因、重组核酸、生物材料或转基因免疫效应细胞中的至少一种，以及药学上可接受的载体。药学上可接受的载体是指制剂中用于递送抗体等药物的无活性物质。药学上可接受的载体可以是抗粘附剂、粘合剂、包衣、崩解剂、充填剂、稀释剂、防腐剂、增甜剂、吸收延迟剂、润湿剂、乳化剂、缓冲剂等等。在一些实施方式中，药物组合物用于治疗肿瘤。
下面结合附图，对本发明的一些实施方式作详细说明。在不冲突的情况下，下述的实施例及实施例中的特征可以相互组合。
实施例1 构建表达Anti PD1-IL-21融合蛋白和CCL19的靶向Mesothelin的慢病毒
本实施例构建了表达Anti PD1-IL-21融合蛋白和CCL19的靶向Mesothelin的慢病毒，如下步骤：
(1)重组病毒载体制备
通过人工基因合成重组核酸CAR-Mesothelin&PD1-IL21&CCL19，如图1第二条Schema Graph所示，重组核酸的核苷酸序列如SEQ ID NO：6所示，按照慢病毒载体酶切位点，将核苷酸片段构建至慢病毒载体上，设计引物，通过测序结果验证载体构建的正确性。
重组核酸CAR-Mesothelin&PD1-IL21&CCL19的核苷酸序列(SEQ ID NO：6)：
Figure PCTCN2022140094-appb-000012
Figure PCTCN2022140094-appb-000013
Figure PCTCN2022140094-appb-000014
(2)包装并浓缩慢病毒
将293t细胞按照8×10 6cells/150mm 2培养皿的密度接种，次日观察细胞的状态，用PEI转染的方法将3代慢病毒包装载体共转至293t细胞，转染完6小时后换液，按照15mL/150mm 2培养皿添加含有10％胎牛血清的DMEM培养基，转染完48小时与72小时收集病毒上清，2000rpm 4℃ 10min离心，去除细胞碎片，之后0.45微米的滤器过滤杂质，过滤后的病毒悬液用25000rpm 4℃ 2小时浓缩慢病毒，浓缩后的病毒加入适量的培养基重悬，置于-80℃保存。
实施例2 构建表达Anti PD1-IL-21融合蛋白和CCL21的靶向Mesothelin的慢病毒
本实施例与实施例1的区别在于，将CCL19替换为CCL21，如图1第三条Schema Graph所示，其余与实施例1一致，获得表达Anti PD1-IL-21融合蛋白和CCL21的靶向Mesothelin的慢病毒。
实施例3 生产CAR-Mesothelin&PD1-IL21&CCL19细胞
本实施例使用实施例1提供的慢病毒生产CART细胞，包括如下步骤：
抽血20mL，Ficall梯度离心分离PBMC，用Stemcell公司T细胞分选试剂盒(货号：19051)分离T细胞，分离后的T细胞用添加5％人AB血清及300单位/mL IL-2X-VIVO 15培养基重悬T细胞至1×10 6cells/mL，用含1％FBS X-VIVO 15清洗beads，按照磁珠∶T细胞＝2∶1体积比例加入预先清洗过的磁珠(Cat#40203D，10mL，Life technology)，2～3天后用新鲜的培养基重悬T细胞至3～5×10 6cells/mL，按照MOI＝10值加入慢病毒，同时加入8ug/mL的Polybrene，4～6小时之后，补加培养基稀释细胞至1×10 6cells/mL，次日更换新鲜培养基，使细胞浓度维持在0.2～0.3×10 6PBMC/mL，之后每隔2～3天更换一次培养基，得到的CART细胞命名为CAR-Mesothelin&PD1-IL21&CCL19组。
实施例4 生产CAR-Mesothelin&PD1-IL21&CCL21细胞
本实施例使用实施例2提供的慢病毒，按照实施例3的方法生产CART细胞，命名为CAR-Mesothelin&PD1-IL21&CCL21组。
实施例5：构建表达Anti PD1-IL-21融合蛋白和CCL19或CCL21的靶向GUCY2C的慢病毒
本实施例构建了表达Anti PD1-IL-21融合蛋白和CCL19或CCL21的靶向GUCY2C的慢病毒，如下步骤：
(1)重组病毒载体制备
通过人工基因合成重组核酸CAR-GUCY2C&PD1-IL21&CCL19，如图5第二条Schema Graph所示，重组核酸的核苷酸序列如SEQ ID NO：7所示；通过人工基因合成重组核酸CAR-GUCY2C&PD1-IL21&CCL21，如图5第三条Schema Graph所示，重组核酸的核苷酸序列如SEQ ID NO：8所示，按照慢病毒载体酶切位点，将核苷酸片段构建至慢病毒载体上，设计引物，通过测序结果验证载体构建的正确性。
重组核酸CAR-GUCY2C&PD1-IL21&CCL19的核苷酸序列(SEQ ID NO：7)：
Figure PCTCN2022140094-appb-000015
Figure PCTCN2022140094-appb-000016
重组核酸CAR-GUCY2C&PD1-IL21&CCL21的核苷酸序列(SEQ ID NO：8)：
Figure PCTCN2022140094-appb-000017
Figure PCTCN2022140094-appb-000018
Figure PCTCN2022140094-appb-000019
(2)包装并浓缩慢病毒
将293t细胞按照8×10 6cells/150mm 2培养皿的密度接种，次日观察细胞的状态，用PEI转染的方法将3代慢病毒包装载体共转至293t细胞，转染完6小时后换液，按照15mL/150mm 2培养皿添加含有10％胎牛血清的DMEM培养基，转染完48小时与72小时收集病毒上清，2000rpm 4℃ 10min离心，去除细胞碎片，之后0.45微米的滤器过滤杂质，过滤后的病毒悬液用25000rpm 4℃ 2小时浓缩慢病毒，浓缩后的病毒加入适量的培养基重悬，置于-80℃保存。
实施例6.生产CART细胞：CAR-GUCY2C&PD1-IL21&CCL19和CAR-GUCY2C&PD1-IL21&CCL21
使用实施例5构建的表达Anti PD1-IL-21融合蛋白和CCL19的靶向GUCY2C的慢病毒、以及表达Anti PD1-IL-21融合蛋白和CCL21的靶向GUCY2C的慢病毒，分别按照实施例3的方法生产CART细胞，分别命名为CAR-GUCY2C&PD1-IL21&CCL19和CAR-GUCY2C&PD1-IL21&CCL21。
对比例1
本对比例构建了一种慢病毒，与实施例1的区别在于，省略了CCL19编码区，其余与实施例1一致，如图1第一条Schema Graph所示，然后，使用该慢病毒，按照实施例3的方法生产CART细胞，命名为CAR-Mesothelin&PD1-IL21组。
对比例2
本对比例构建了CART细胞：CAR-GUCY2C&PD1-IL21。首先构建表达Anti PD1-IL-21融合蛋白的靶向GUCY2C的慢病毒(构建方法与实施例5中表达Anti PD1-IL-21融合蛋白和CCL19的靶向GUCY2C的慢病毒的构建方法的区别仅在于，核酸省略了CCL19编码区，其余与实施例5一致)，该慢病毒结构如图5中第一条Schema Graph所示；然后使用该慢病毒，按照实施例3的方法生产CART细胞，获得的CART细胞命名为CAR-GUCY2C&PD1-IL21。
实验例1
在实施例3的CAR-Mesothelin&PD1-IL21&CCL19组、实施例4的CAR-Mesothelin&PD1-IL21&CCL21组和对比例1的CAR-Mesothelin&PD1-IL21组中，病毒侵染完72小时后，使用流式分析各自得到的CART细胞的细胞阳性率。
如图2所示，T细胞侵染后D6(第6天)的CAR阳性率检测结果显示，CAR-Mesothelin&PD1-IL21组的CAR阳性率为47.00％、CAR-Mesothelin&PD1-IL21&CCL19组的CAR阳性率为35.50％、CAR-Mesothelin&PD1-IL21&CCL21组的CAR阳性率为29.87％；由结果可以看出：本发明成功制备得到了CAR-Mesothelin&PD1-IL21&CCL19和CAR-Mesothelin&PD1-IL21&CCL21。
实验例2
对实施例3的CAR-Mesothelin&PD1-IL21&CCL19组、实施例4的CAR-Mesothelin&PD1-IL21&CCL21组和对比例1的CAR-Mesothelin&PD1-IL21组中侵染后D6的各组细胞，使用流式细胞术检测其T、B、NK细胞分群，同时检测T细胞中CD4、CD8、PD1、PD-L1的分群。
结果如表1所示，可以看出：表达PD1-IL21组的PD1分子表达水平降低，提示PD1-IL21融合蛋白可能会调控T细胞表面分子PD1的表达，大大降低了T细胞表面PD1表达的比例。
表1流式细胞术对三组CART细胞的表征结果
Figure PCTCN2022140094-appb-000020
实验例3
以T细胞为对照，实施例3的CAR-Mesothelin&PD1-IL21&CCL19组、实施例4的CAR-Mesothelin&PD1-IL21&CCL21组中侵染后D6的各组细胞的体外杀伤功能进行评估，采用xCELLigence实时、细胞介导的细胞毒性系统(Acea Biosciences Inc.)评估CART细胞介导的细胞毒性。将1×10 4OVCAR3细胞培养于E-Plate 16(Acea Biosciences)的每个孔中的150μL生长培养基中，并在37℃培养箱中培养过夜，使用RTCA DP Analyzer系统每15分钟量化一次电阻抗和RTCA软件版本2.0(Acea Biosciences Inc.)。大约24小时后，加入50μL CART细胞(E∶T比例为3∶1)或50μL培养基作为阴性，在接下来的24小时内量化细胞介导的杀伤，每15分钟读取一次电阻抗。
结果如图3所示，可以看出，在体外与靶细胞共培养时，与T细胞相比，CAR-Mesothelin&PD1-IL21&CCL19组和CAR-Mesothelin&PD1-IL21&CCL21组细胞都具有非常明显的杀瘤能力。
实验例4
以T细胞为对照，对实施例3的CAR-Mesothelin&PD1-IL21&CCL19组、实施例4的CAR-Mesothelin&PD1-IL21&CCL21组和对比例1的CAR-Mesothelin&PD1-IL21组共3组细胞开展趋化实验。T Cell、CAR-Mesothelin&PD1-IL21组细胞、CAR-Mesothelin&PD1-IL21&CCL19组细胞和CAR-Mesothelin&PD1-IL21&CCL21组细胞培养第8天，各取细胞培养上清2mL，300g，5min，RT离心，再取上清分别加入transwell孔板(Corning，3422)的下室中，每组设置三个复孔，每孔加入0.6mL上清。取T-Cell，计数离心，用X-vivo培养基重悬至2E6/mL，上室加入100uL细胞悬液，放置培养箱中培养6h后，收集下室中的细胞悬液并计数，计算迁移到下室中的细胞数。
实验结果如图4所示，可以看出，与T细胞和对比例1相比，CAR-Mesothelin&PD1-IL21&CCL19组细胞和CAR-Mesothelin&PD1-IL21&CCL21组细胞上清中分泌的CCL19和CCL21对T细胞具有非常显著的趋化作用。
实验例5
在实施例6生产CAR-GUCY2C&PD1-IL21&CCL19、CAR-GUCY2C&PD1-IL21&CCL21以及在对比例2生产CAR-GUCY2C&PD1-IL21中，病毒侵染完72小时后，使用流式分析各自得到的CART细胞的细胞阳性率。
实验结果如图6所示，T细胞侵染后D6的CAR阳性率检测结果显示，CAR-GUCY2C&PD1-IL21组的CAR阳性率为59.77％、CAR-GUCY2C&PD1-IL21&CCL19组的CAR阳性率为66.62％、CAR-GUCY2C&PD1-IL21&CCL21组的CAR阳性率为50.10％；由结果可以看出：本发明成功制备得到了CAR-GUCY2C&PD1-IL21&CCL19和CAR-GUCY2C&PD1-IL21&CCL21。
实验例6
以T细胞和CART细胞CAR-GUCY2C&PD1-IL21为对照，对实施例6的CAR-GUCY2C&PD1-IL21&CCL19和CAR-GUCY2C&PD1-IL21&CCL21侵染后D6的各组细胞的体外杀伤功能进行评估，采用xCELLigence实时、细胞介导的细胞毒性系统(Acea Biosciences Inc.)评估CART细胞介导的细胞毒性。将1×10 4OVCAR3细胞培养于E-Plate 16(Acea Biosciences)的每个孔中的150μL生长培养基中，并在37℃培养箱中培养过夜，使用RTCA DP Analyzer系统每15分钟量化一次电阻抗和RTCA软件版本2.0(Acea Biosciences Inc.)。大约24小时后，加入50μL T细胞或50μL CART细胞(E∶T比例为3∶1)或50μL培养基作为阴性，在接下来的24小时内量化细胞介导的杀伤，每15分钟读取一次电阻抗。
实验结果如图7所示，实验结果表明，在体外与靶细胞共培养时，与T细胞和CAR-GUCY2C&PD1-IL21细胞相比，CAR-GUCY2C&PD1-IL21&CCL19组和CAR-GUCY2C&PD1-IL21&CCL21组细胞都具有非常明显的杀瘤能力。
实验例7
小鼠体内抑制肿瘤能力实验：
采用LS1034(人结直肠癌细胞)肿瘤模型，皮下接种至NPG小鼠(北京维通达生物科技有限公司)，一共25只小鼠，分5组，每组5只小鼠，待肿瘤生长12天时，采用尾静脉给药的方式，每只小鼠注射5.0E+06个CAR+细胞或者T细胞或者PBS，给药一次，随后每周测量两次肿瘤体积。实验结果见附图8，结果显示，与T细胞和CAR-GUCY2C&PD1-IL21细胞相比，CAR-GUCY2C&PD1-IL21&CCL19组和CAR-GUCY2C&PD1-IL21&CCL21组细胞在NPG小鼠体内对LS1034肿瘤都具有非常明显的抑制肿瘤生长的能力。
由上述实施例和实验例可以看出，在CART细胞中将PD1抗体与IL-21融合是一种理想途径，PD1表达在T细胞表面，主要是CD8+T细胞，这样形成的PD1抗体与IL-21融合蛋白会选择性的结合在T细胞和CART细胞表面，行使其双重功能；与此同时，融合蛋白由于分子量变大，也大大的提高了药物的半衰期。
本发明通过分子构建，将CCL19或者CCL21构建到CAR结构中，让CART细胞同时表达CAR和CCL19或者CCL21趋化因子，当CART杀伤肿瘤时，同时表达分泌的CCL19或者CCL21能够招募和趋化机体天然的T细胞到达肿瘤部位，帮助CART细胞杀伤肿瘤。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (16)

  1. 含有三个编码区的基因，其特征在于，
    编码区(I)编码嵌合抗原受体，所述嵌合抗原受体含有特异性识别肿瘤抗原的胞外区；
    编码区(II)编码融合蛋白，所述融合蛋白包括免疫检查点抗体和细胞因子；
    编码区(III)编码趋化因子。
  2. 根据权利要求1所述的基因，其特征在于，所述肿瘤抗原选自MSLN、GD2、GPC3、CD19、EGFR VIII、GUCY2C、HER2、MUC16或Claudin 18.2中的至少一种；可选地，所述肿瘤抗原为MSLN或GUCY2C。
  3. 根据权利要求2所述的基因，其特征在于，所述胞外区含有抗MSLN抗体；可选地，所述抗MSLN抗体为单链抗体；
    可选地，
    (a)所述抗MSLN抗体的氨基酸序列如SEQ ID NO：1所示；
    (b)所述抗MSLN抗体的氨基酸序列为在(a)限定的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且编码的蛋白质具有特异性识别MSLN抗原活性的SEQ ID NO：1的衍生氨基酸序列；
    (c)所述抗MSLN抗体包含重链可变区和轻链可变区，所述重链可变区包括SEQ ID NO：9中的HCDR1、HCDR2和HCDR3，所述轻链可变区包括SEQ ID NO：10中的LCDR1、LCDR2和LCDR3；可选地，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由Kabat、IMGT、Chothia、Contact或AbM编号系统定义；
    (d)所述抗MSLN抗体包含重链可变区和轻链可变区，所述重链可变区的HCDR1包含SEQ ID NO：11的氨基酸序列，HCDR2包含SEQ ID NO：12的氨基酸序列，和HCDR3包含SEQ ID NO：13的氨基酸序列；所述轻链可变区的LCDR1包含SEQ ID NO：14的氨基酸序列，LCDR2包含SEQ ID NO：15的氨基酸序列，和LCDR3包含SEQ ID NO：16的氨基酸序列；或者
    (e)所述抗MSLN抗体包含重链可变区和轻链可变区，其中，重链可变区包含与SEQ ID NO：9具有至少85％序列同一性的氨基酸序列，轻链可变区包含SEQ ID NO：10具有至少85％序列同一性的氨基酸序列；可选地，所述重链可变区和轻链可变区包括(c)或(d)所述的抗MSLN抗体的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3；可选地，所述抗MSLN抗体包含SEQ ID NO：17所示的重链可变区和SEQ ID NO：18所示的轻链可变区。
  4. 根据权利要求2所述的基因，其特征在于，所述嵌合抗原受体包含抗GUCY2C抗体；可选地，所述抗GUCY2C抗体为单链抗体；
    可选地，
    (a)所述抗GUCY2C抗体包含重链可变区和轻链可变区，所述重链可变区包括SEQ ID NO：17中的HCDR1、HCDR2和HCDR3，所述轻链可变区包括SEQ ID NO：18中的LCDR1、LCDR2和LCDR3；可选地，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由Kabat、IMGT、Chothia、Contact或AbM编号系统定义；
    (b)所述抗GUCY2C抗体包含重链可变区和轻链可变区，所述重链可变区的HCDR1包含SEQ ID NO：20的氨基酸序列，HCDR2包含SEQ ID NO：21的氨基酸序列，和HCDR3包含SEQ ID NO：22的氨基酸序列；所述轻链可变区的LCDR1包含SEQ ID NO：23的氨基酸序列，LCDR2包含氨基酸序列：AAS，和LCDR3包含SEQ ID NO：24的氨基酸序列；
    (c)所述抗GUCY2C抗体包含重链可变区和轻链可变区，其中，重链可变区包含与SEQ ID NO：17具有至少85％序列同一性的氨基酸序列，轻链可变区包含与SEQ ID NO：18具有至少85％序列同一性的氨基酸序列；可选地，所述重链可变区和轻链可变区包括(a)或(b)所述的抗GUCY2C抗体的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3；可选地，所述抗GUCY2C抗体包含SEQ ID NO：17所示的重链可变区和SEQ ID NO：18所示的轻链可变区；或
    (d)所述抗GUCY2C抗体为单链抗体，其包括与SEQ ID NO：19具有至少85％序列同一性的氨基酸序列；可选地，所述抗体包含(c)所述的抗GUCY2C抗体的重链可变区和轻链可变区；可选地，所述抗GUCY2C单链抗体包括SEQ ID NO：19。
  5. 根据权利要求1～4任一项所述的基因，其特征在于，所述免疫检查点选自PD1、PD-L1、TIGIT、LAG3、CTLA4、BTLA或TIM3中至少一种；可选地，所述免疫检查点选自PD1。
  6. 根据权利要求5所述的基因，其特征在于，所述免疫检查点抗体为抗PD1抗体；可选地，所述抗PD1抗体为单链抗体；
    可选地，
    (a)所述抗PD1抗体包含重链可变区和轻链可变区，所述重链可变区包括SEQ ID NO：25中的HCDR1、HCDR2和HCDR3，所述轻链可变区包括SEQ IDNO：26中的LCDR1、LCDR2和LCDR3；可选地，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由Kabat、IMGT、Chothia、Contact或AbM编号系统定义；
    (b)所述抗PD1抗体包含重链可变区和轻链可变区，所述重链可变区的HCDR1包含SEQ ID NO：27的氨基酸序列，HCDR2包含SEQ ID NO：28的氨基酸序列，和HCDR3包含SEQ ID NO：29的氨基酸序列；所述轻链可变区的LCDR1包含SEQ ID NO：30的氨基酸序列，LCDR2包含SEQ ID NO：31的氨基酸序列，和LCDR3包含SEQ ID NO：32的氨基酸序列；
    (c)所述抗PD1抗体的氨基酸序列如SEQ ID NO：2所示；
    (d)在(c)限定的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且编辑的蛋白质具有靶向PD1功能的SEQ ID NO：2的衍生氨基酸序列；或者
    (e)所述抗PD1抗体包含重链可变区和轻链可变区，其中，重链可变区包含与SEQ ID NO：25具有至少85％序列同一性的氨基酸序列，轻链可变区包含与SEQ ID NO：26具有至少85％序列同一性的氨基酸序列；可选地，所述重链可变区和轻链可变区包括(a)或(b)所述的抗PD1抗体的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3；可选地，所述抗PD1抗体包含SEQ ID NO：17所示的重链可变区和SEQ ID NO：18所示的轻链可变区。
  7. 根据权利要求1～6任一项所述的基因，其特征在于，所述细胞因子为(A)或(B)；
    所述(A)选自IL-21、IL-23、IL-2、IL-7、IL-9、IL-12、IL-15或IL-18中至少一种；
    所述(B)为具有调节免疫细胞活性的由(A)衍生的蛋白质；
    可选地，所述细胞因子为IL-21；
    可选地，所述IL-21具有如SEQ ID NO：3所示的氨基酸序列。
  8. 根据权利要求1～7任一项所述的基因，其特征在于，所述趋化因子为(c)或(d)；
    所述(c)选自CXC趋化因子、CC趋化因子、CX3C趋化因子或XC趋化因子中的至少一种；
    所述(d)为具有诱导免疫细胞定向迁移功能的由(c)衍生的蛋白质；
    优选地，所述CXC趋化因子为选自CXCL1、CXCL2、CXCL3、CXCL4、CXCL5、CXCL6、CXCL7、CXCL8、CXCL9、CXCL10、CXCL11、CXCL12、CXCL13、CXCL14、CXCL15、CXCL16或CXCL17中的至少一种；
    优选地，所述CC趋化因子为选自CCL1、CCL2、CCL3、CCL4、CCL5、CCL6、CCL7、CCL8、CCL9、CCL10、CCL11、CCL12、CCL13、CCL14、CCL15、CCL16、CCL17、CCL18、CCL19、CCL20、CCL21、CCL22、CCL23、CCL24、CCL25、CCL26、CCL27或CCL28中的至少一种；
    优选地，所述CX3C趋化因子为CX3CL1；
    优选地，所述XC趋化因子为XCL1；
    可选地，所述趋化因子为CCL19或CCL21；优选地，所述CCL19具有如SEQ ID NO：4所示的氨基酸序列；优选地，所述CCL21具有如SEQ ID NO：5所示的氨基酸序列。
  9. 一种重组核酸，其特征在于，所述重组核酸包括第一核酸分子、第二核酸分子和第三核酸分子，所述第一核酸分子含有权利要求1～8任一项所述的编码区(I)，所述第二核酸分子含有权利要求1～8任一项所述的编码区(II)，所述第三核酸分子含有权利要求1～8任一项所述的编码区(III)。
  10. 根据权利要求9所述的重组核酸，其特征在于，所述第一核酸分子、第二核酸分子和第三核酸分子之间通过2A肽的核酸序列相连；优选地，所述2A肽选自P2A、T2A、F2A或E2A中的至少一种；
    可选地，所述第一核酸分子通过2A肽的核酸序列与第二核酸分子相连，所述第二核酸分子通过2A肽的核酸序列和第三核酸分子相连。
  11. 一种生物材料，其特征在于，所述生物材料包括以下(i)或(ii)：
    (i)重组载体，所述重组载体含有权利要求1～8任一项所述基因，或者含有权利要求9～10任一项所述的重组核酸；
    (ii)构建体，所述构建体包括非致病性病毒，所述非致病性病毒含有权利要求1～8任一项所述基因、权利要求9或10所述的重组核酸或所述(i)重组载体；
    可选地，所述非致病性病毒包括反转录病毒、慢病毒或腺病毒。
  12. 一种转基因免疫效应细胞，其特征在于，所述转基因免疫效应细胞含有权利要求1～8任一项所述基因、权利要求9或10所述的重组核酸或权利要求11所述生物材料；
    可选地，所述免疫效应细胞选自T细胞、NK细胞、NKT细胞、巨噬细胞或CIK细胞中的至少一种；
    可选地，所述免疫效应细胞为T细胞。
  13. 根据权利要求12所述的转基因免疫效应细胞，其特征在于，所述转基因免疫效应细胞含有跨膜区和/或胞内区；
    可选地，所述跨膜区含CD8的跨膜段；
    可选地，所述胞内区含免疫共刺激分子的胞内段和/或CD3 Zeta链；
    可选地，所述免疫共刺激分子选自4-1BB、CD28、CD3、OX-40、CD40L、CD27、CD30、或他们的衍生物中的任一种或几种；可选地，所述免疫共刺激分子选自4-1BB。
  14. 一种权利要求12或13所述转基因免疫效应细胞的构建方法，其特征在于，所述构建方法包括，将权利要求1～8任一项所述基因、权利要求9或10所述的重组核酸或权利要求11所述生物材料导入免疫效应细胞中，获得所述转基因免疫效应细胞；
    可选地，所述免疫效应细胞选自T细胞、NK细胞、NKT细胞、巨噬细胞或CIK细胞中的至少一种；
    可选地，所述免疫效应细胞为T细胞。
  15. 一种药物组合物，其特征在于，包括：权利要求1～8任一项所述基因、权利要求9或10所述重组核酸、权利要求11所述生物材料、权利要求12或13所述的转基因免疫效应细胞或权利要求14所述的构建方法构建得到的转基因免疫效应细胞，以及药学上可接受的载体。
  16. 权利要求1～8任一项所述基因、权利要求9或10所述重组核酸、权利要求11所述生物材料、权利要求12或13所述的转基因免疫效应细胞、权利要求14所述的构建方法构建得到的转基因免疫效应细胞或权利要求15所述的药物组合物在制备抗肿瘤产品中的应用；可选地，所述肿瘤包括实体瘤。
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Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016122738A1 (en) * 2015-01-31 2016-08-04 The Trustees Of The University Of Pennsylvania Compositions and methods for t cell delivery of therapeutic molecules
CN106467573A (zh) * 2015-08-21 2017-03-01 科济生物医药（上海）有限公司 抗间皮素全人抗体以及靶向间皮素的免疫效应细胞
CN109971714A (zh) * 2017-12-28 2019-07-05 上海细胞治疗研究院 自表达pd-1抗体并靶向间皮素的嵌合抗原受体修饰t细胞及其用途
CN110894241A (zh) * 2018-09-13 2020-03-20 中国科学院生物物理研究所 一种靶向抗原特异性t细胞诱导其向记忆干性细胞分化的融合蛋白
WO2020057641A1 (zh) * 2018-09-20 2020-03-26 科济生物医药（上海）有限公司 表达有趋化因子的细胞及用途
WO2020150402A2 (en) * 2019-01-15 2020-07-23 Caerus Therapeutics, Corp. Advanced chimeric antigen receptor vectors for targeting solid tumors
CN111548420A (zh) * 2020-05-14 2020-08-18 深圳普瑞金生物药业有限公司 一种抗间皮素嵌合抗原受体、表达基因、表达载体、t细胞及其用途
CN111849910A (zh) * 2020-05-27 2020-10-30 南京北恒生物科技有限公司 工程化免疫细胞及其用途
CN113106068A (zh) * 2021-03-26 2021-07-13 深圳市先康达生命科学有限公司 一种自分泌IL-15与anti-PD1融合蛋白的免疫细胞
CN113286874A (zh) * 2018-10-12 2021-08-20 美商生物细胞基因治疗有限公司 嵌合抗原受体(CARs)组合物及使用方法
CN113286634A (zh) * 2018-05-23 2021-08-20 辉瑞公司 对gucy2c特异性的抗体及其用途

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016122738A1 (en) * 2015-01-31 2016-08-04 The Trustees Of The University Of Pennsylvania Compositions and methods for t cell delivery of therapeutic molecules
CN106467573A (zh) * 2015-08-21 2017-03-01 科济生物医药（上海）有限公司 抗间皮素全人抗体以及靶向间皮素的免疫效应细胞
CN109971714A (zh) * 2017-12-28 2019-07-05 上海细胞治疗研究院 自表达pd-1抗体并靶向间皮素的嵌合抗原受体修饰t细胞及其用途
CN113286634A (zh) * 2018-05-23 2021-08-20 辉瑞公司 对gucy2c特异性的抗体及其用途
CN110894241A (zh) * 2018-09-13 2020-03-20 中国科学院生物物理研究所 一种靶向抗原特异性t细胞诱导其向记忆干性细胞分化的融合蛋白
WO2020057641A1 (zh) * 2018-09-20 2020-03-26 科济生物医药（上海）有限公司 表达有趋化因子的细胞及用途
CN113286874A (zh) * 2018-10-12 2021-08-20 美商生物细胞基因治疗有限公司 嵌合抗原受体(CARs)组合物及使用方法
WO2020150402A2 (en) * 2019-01-15 2020-07-23 Caerus Therapeutics, Corp. Advanced chimeric antigen receptor vectors for targeting solid tumors
CN111548420A (zh) * 2020-05-14 2020-08-18 深圳普瑞金生物药业有限公司 一种抗间皮素嵌合抗原受体、表达基因、表达载体、t细胞及其用途
CN111849910A (zh) * 2020-05-27 2020-10-30 南京北恒生物科技有限公司 工程化免疫细胞及其用途
CN113106068A (zh) * 2021-03-26 2021-07-13 深圳市先康达生命科学有限公司 一种自分泌IL-15与anti-PD1融合蛋白的免疫细胞

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHEN, SHANLING ET AL.: "Engineered IL-21 Cytokine Muteins Fused to Anti-PD-1 Antibodies Can Improve CD8+ T Cell Function and Anti-tumor Immunity", FRONTIERS IN IMMUNOLOGY, vol. 11, 31 May 2020 (2020-05-31), XP055854614, DOI: 10.3389/fimmu.2020.00832 *

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