WO2022143928A1 - 膜融合蛋白及其在免疫细胞中的应用 - Google Patents

膜融合蛋白及其在免疫细胞中的应用 Download PDF

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WO2022143928A1
WO2022143928A1 PCT/CN2021/143209 CN2021143209W WO2022143928A1 WO 2022143928 A1 WO2022143928 A1 WO 2022143928A1 CN 2021143209 W CN2021143209 W CN 2021143209W WO 2022143928 A1 WO2022143928 A1 WO 2022143928A1
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cells
car
meso
cell
tumor
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PCT/CN2021/143209
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French (fr)
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董琦
江鹏斐
沈连军
曹卫
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亘喜生物科技（上海）有限公司
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Priority to CN202180088001.4A priority Critical patent/CN116648457A/zh
Publication of WO2022143928A1 publication Critical patent/WO2022143928A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464466—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K39/464468—Mesothelin [MSLN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/49—Breast

Definitions

  • the present invention relates to the field of immune cell therapy, more particularly to engineered immune cells that co-express interleukin-15 mutant and IL-15R ⁇ fusion protein and target solid tumors and hematological tumors, and applications thereof.
  • Immune cell therapy is a new mode of drug development. Significant progress has been made in the treatment of B-cell malignancies by genetically engineering human immune cells into active drugs.
  • CAR-T cell immunotherapy includes autologous and universal CAR-T therapy, in which autologous CAR-T therapy uses the patient's immune cells, while allogeneic immunotherapy uses CAR-T cells from allogeneic subjects to transfer the required immune cells.
  • the cells are cultured in vitro, gene edited and expanded and then infused back into the patient. These cells recognize tumor cells in a non-MHC-restricted manner and do not require an antigen-presenting mechanism to recognize tumor antigens, reducing tumor cells mediated by downregulation of MHC and antigen presentation immune escape.
  • the chimeric antibody receptor (CAR) molecules on the cell surface are designed to specifically recognize the antigen targets expressed by tumor cells, and directly attack and kill tumor cells by secreting cytokines such as interferon and perforin. achieve the purpose of treating or relieving the disease.
  • immunotherapy for hematological tumors has outstanding curative effect, but the curative effect in solid tumors is not ideal.
  • the main reasons include three aspects, one is safe tumor-specific antigen targets, the other is the heterogeneity of solid tumors, and the third is the existence of complex tumor cell microenvironment and other factors, including immunosuppressive cells Tregs and MDSCs, as well as immunosuppressive factors.
  • the existence of it is not conducive to the survival of immune cells, thereby inhibiting the effect of immunotherapy.
  • Mesothelin is a differentiation antigen that exists on normal mesothelial cells.
  • mesothelioma lung cancer, pancreatic cancer, breast cancer, ovarian cancer and other tumors, and in normal tissues such as normal pleura, pericardium and peritoneum.
  • mesothelin can be used as an effective immunotherapy target.
  • immunotherapy strategies are designed for MSLN, including antibody therapy, immunotoxin and chimeric antigen receptor T cell therapy.
  • Antibody drugs include Amatuximab (MORAb-009), Anetumab Ravtansine (BAY94-9343), DMOT4039A, MDX-1204, etc., but their efficacy in the treatment of solid tumors is not satisfactory, and it is necessary to improve the existing treatment technology to enhance the efficacy and reduce the treatment risks in the process.
  • the purpose of the present invention is to provide a chimeric antigen receptor immune cell expressing interleukin-15 and its application.
  • a chimeric antigen receptor (CAR) construct is provided, and the structure of the CAR construct is shown in the following formula I or II,
  • each "-" is independently a linking peptide or peptide bond
  • X is a CAR targeting tumor antigens
  • A is the self-shearing element
  • E is the IL-15/IL-15R ⁇ complex.
  • the structure of the CAR construct is shown in formula I.
  • the IL-15/IL-15R ⁇ complex comprises IL-15 and IL-15R ⁇ .
  • the IL-15 and IL-15R ⁇ are derived from human.
  • the IL-15 and IL-15R ⁇ are linked through a linking peptide.
  • the IL-15/IL-15R ⁇ complex further comprises a signal peptide element.
  • the structure of the IL-15/IL-15R ⁇ complex is shown in the following formula III,
  • each "-" is independently a linking peptide or peptide bond
  • L' is none or signal peptide
  • M is IL-15 or a mutant thereof
  • I is a flexible joint
  • R is IL-15R ⁇ .
  • the IL-15 mutant has the biological activity of IL-15.
  • amino acid sequence of the IL-15 is shown in SEQ ID NO.: 1.
  • amino acid sequence of the IL-15 mutant (IL-15N72D) is shown in SEQ ID NO.:2.
  • the IL-15R ⁇ is a complete IL-15R ⁇ element.
  • the IL-15R ⁇ comprises a transmembrane domain and an intracellular domain.
  • the IL-15R ⁇ includes an extracellular region, a transmembrane region and an intracellular region of the sushi domain (active fragment that binds to IL-15) in sequence from the N-terminus to the C-terminus.
  • amino acid sequence of the IL-15R ⁇ is shown in SEQ ID NO.:3.
  • the flexible linker is a connecting peptide, preferably, the amino acid sequence of the connecting peptide is shown in SEQ ID NO.: 4 (SGGGGSGGGGSGGGGSGGGGSGGGSLQ).
  • the L' is a signal peptide derived from IgE, IL-2.
  • amino acid sequence of the IL-15/IL-15R ⁇ complex is shown in SEQ ID NO.:5.
  • amino acid sequence of the IL-15 mutant/IL-15R ⁇ complex is shown in SEQ ID NO.:6.
  • the self-shearing element includes T2A and P2A.
  • each "-" is independently a linking peptide or peptide bond
  • L is none or signal peptide
  • scFv is an antibody single-chain variable region targeting tumor antigens
  • H is the hinge-free region
  • TM is the transmembrane domain
  • C is a costimulatory signal molecule
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇
  • the tumor antigen is selected from the group consisting of mesothelin, Claudin18.2, MUCl, GPC3, PSCA, Her2, CD19, or a combination thereof.
  • the tumor antigen is mesothelin.
  • the L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.
  • the L is a signal peptide derived from CD8.
  • the H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof.
  • the H is a hinge region derived from CD8.
  • the TM is a transmembrane region of a protein selected from the group consisting of: ICOS, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, MUCl-Tn, CD33, CD37, CD64 , CD80, CD86, CD134, CD137, CD154, or a combination thereof.
  • the TM is a transmembrane region derived from CD8 or CD28.
  • the C is a costimulatory signal molecule selected from the following group of proteins: ICOS, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137) , PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.
  • proteins ICOS, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137) , PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.
  • the C is a costimulatory signal molecule derived from CD28.
  • amino acid sequence of the X is shown in SEQ ID NO.: 8.
  • amino acid sequence of the CAR construct is shown in SEQ ID NO.:7.
  • nucleic acid molecule encoding the CAR construct described in the first aspect of the present invention, or,
  • the nucleic acid molecule comprises a first nucleic acid molecule encoding a CAR targeting a tumor antigen and a second nucleic acid molecule encoding an IL-15/IL-15R ⁇ complex, wherein the CAR targeting a tumor antigen and IL-15/IL-15R ⁇
  • the IL-15R ⁇ complex is defined as described above.
  • first nucleic acid molecule and the second nucleic acid molecule may be connected in series, or may exist independently.
  • a vector is provided, and the vector contains 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 virus vector (AAV), retroviral vector, transposon, or a combination thereof .
  • the vector is selected from the group consisting of plasmid and viral vector.
  • the vector is in the form of virus particles.
  • the vector is a lentiviral vector.
  • a host cell contains the vector of the third aspect of the present invention or the exogenous nucleic acid molecule of the second aspect of the present invention is integrated into the chromosome or The CAR construct described in the first aspect of the present invention is expressed.
  • the host cells include eukaryotic cells and prokaryotic cells.
  • the host cell includes Escherichia coli.
  • an engineered immune cell is provided, the immune cell expresses the CAR construct described in the first aspect of the present invention, or
  • the immune cells express a CAR targeting a tumor antigen and an IL-15/IL-15R ⁇ complex, wherein the definition of the CAR targeting a tumor antigen and an IL-15/IL-15R ⁇ complex is as described above.
  • the CAR targeting the tumor antigen and the IL-15/IL-15R ⁇ complex are independently expressed on the cell membrane of the immune cells.
  • the cells are isolated cells, and/or the cells are genetically engineered cells.
  • the immune cells are derived from human or non-human mammals (eg, mice).
  • the cells include T cells and NK cells.
  • the engineered immune cells can be chimeric antigen receptor T cells (CAR-T cells) or chimeric antigen receptor NK cells (CAR-NK cells).
  • CAR-T cells chimeric antigen receptor T cells
  • CAR-NK cells chimeric antigen receptor NK cells
  • a preparation in the sixth aspect of the present invention, contains the CAR construct described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, and the vector described in the third aspect of the present invention. , or the immune cells described in the fifth aspect of the present invention, and a pharmaceutically acceptable carrier.
  • the preparation is a liquid preparation.
  • the dosage form of the preparation is an injection.
  • the concentration of the CAR-T cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml .
  • the preparation further comprises a second anti-tumor active ingredient, preferably a second antibody, or a chemotherapeutic agent.
  • the chemotherapeutic agent is selected from the group consisting of docetaxel, carboplatin, or a combination thereof.
  • a CAR construct described in the first aspect of the present invention a nucleic acid molecule described in the second aspect of the present invention, a vector described in the third aspect of the present invention, or the fifth aspect of the present invention.
  • Use of the immune cells described in this aspect, or the preparation described in the sixth aspect of the present invention is used to prepare a medicament or preparation for preventing and/or treating cancer or tumor.
  • the tumor is selected from the group consisting of hematological tumors, solid tumors, or a combination thereof.
  • the hematological tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse B-cell lymphoma (DLBCL), or a combination thereof.
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • DLBCL diffuse B-cell lymphoma
  • the solid tumor is selected from the group consisting of gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, Cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or a combination thereof.
  • gastric cancer gastric cancer peritoneal metastasis
  • liver cancer leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, Cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or a combination thereof.
  • NSCLC non-small
  • the tumor is a mesothelin-positive tumor, preferably a tumor with high mesothelin expression.
  • kits for preparing the host cell according to the fourth aspect of the present invention comprising a container and the nucleic acid according to the second aspect of the present invention located in the container molecule, or the vector according to the third aspect of the present invention.
  • 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.
  • a method for treating a disease comprising administering an appropriate amount of the carrier of the third aspect of the present invention, the immune cell of the fifth aspect of the present invention, or the present invention to a subject in need of treatment
  • the preparation of the sixth aspect comprising administering an appropriate amount of the carrier of the third aspect of the present invention, the immune cell of the fifth aspect of the present invention, or the present invention to a subject in need of treatment.
  • the disease is cancer or tumor.
  • an IL-15/IL-15R ⁇ complex is provided, and the structure of the IL-15/IL-15R ⁇ complex is shown in the following formula III,
  • each "-" is independently a linking peptide or peptide bond
  • L' is none or signal peptide
  • M is IL-15 or a mutant thereof
  • I is a flexible joint
  • R is IL-15R ⁇
  • the IL-15R ⁇ comprises a transmembrane region and an intracellular region.
  • IL-15/IL-15R ⁇ complex according to the eleventh aspect of the present invention, for preparing a preparation for enhancing CAR-T Persistence of cells and/or enhanced cytotoxicity of CAR-T cells.
  • the preparation is used for adoptive immunotherapy based on CAR-T cells.
  • the enhancing the persistence of CAR-T cells refers to enhancing the sustained killing ability of CAR-T cells to tumor cells.
  • Figure 1 shows a schematic diagram of the mechanism of action of the membrane fusion protein of the present invention.
  • FIG. 2A shows a schematic diagram of the CAR structure employed in the present invention.
  • Figure 2B shows a schematic diagram of the huIL-15/IL-15 mutant complex.
  • Figure 2C shows the structure of huIL-15R ⁇ of the huIL-15/IL-15 mutant complex.
  • Figure 3A shows the amino acid sequence of the MESO-E1m1 gene.
  • Figure 3B shows the amino acid sequence of the E1m1 gene.
  • Figure 4 shows the detection chart of the positive rate of CAR-T cells.
  • Figure 5A shows the lysis rate of CAR-T cells to target cells in the in vitro co-culture system of Meso-1, Meso-E1 and Meso-E1m1 CAR-T cells and ovarian cancer cell OVCAR3.
  • Figure 5B shows the cytokine secretion of CAR-T cells after CAR-T cells were co-cultured with ovarian cancer OVCAR3 in vitro.
  • Figure 6A shows the lysis rate of CAR-T cells to target cells in the in vitro co-culture system of Meso-1, Meso-E1 and Meso-E1m1 CAR-T cells and triple-negative breast cancer MDA-MB-231-MESO cells. .
  • Figure 6B shows the cytokine secretion of CAR-T cells after in vitro co-culture of CAR-T cells with triple-negative breast cancer MDA-MB-231-MESO cells.
  • Figure 7A shows Meso-E1 and Meso-E1m1 CAR-T in vivo efficacy experiment-1, HCC70 (breast cancer cells) tumor volume change curve.
  • Figure 7B shows Meso-E1 and Meso-E1m1 CAR-T cells in vivo efficacy experiment-1, animal body weight change curve.
  • Figure 8A shows Meso-1 and Meso-E1m1 CAR-T in vivo efficacy experiment-2, HCC70 (breast cancer cells) tumor volume change curve.
  • Figure 8B shows Meso-1 and Meso-E1m1 CAR-T in vivo efficacy test-2, animal body weight changes.
  • Figure 8C shows the in vivo efficacy of Meso-1 and Meso-E1m1 CAR-T experiments-2, the tumor volume changes of a single mouse in each group.
  • Figure 9A shows the multi-round killing experiment design.
  • Figure 9B and Figure 9C show the comparison of target cell lysis rate and CAR-T cell expansion after multiple rounds of killing of the in vitro co-culture system Meso-1, Meso-E1 and Meso-E1m1 CAR-T cells and ovarian cancer cells OVCAR3, respectively.
  • Figure 9D and Figure 9E show the target cell lysis rate and CAR-T cells after multiple rounds of stimulation with triple-negative breast cancer MDA-MB-231-MESO cells in the in vitro co-culture system Meso-1, Meso-E1 and Meso-E1m1 CAR-T cells, respectively - Comparison of T cell expansion.
  • Figure 10A shows the lysis rate of target cells after multiple rounds of stimulation of Meso-1 and Meso-E1m1 CAR-T cells with ovarian cancer cells OVCAR3 after adding different concentrations of TGF- ⁇ 1 in vitro co-culture system.
  • Figure 10B shows the expansion curve of CAR-T cells after multiple rounds of stimulation of Meso-1 and Meso-E1m1 CAR-T cells with ovarian cancer cells OVCAR3 after adding different concentrations of TGF- ⁇ 1 in vitro co-culture system.
  • Figure 10C shows the detection of apoptosis of CAR-T cells after the second round of multi-round killing of Meso-1 and Meso-E1m1 CAR-T cells with ovarian cancer cells OVCAR3 after adding different concentrations of TGF- ⁇ 1 in vitro co-culture system.
  • Figure 11 shows the expansion values of PBNK cells at 48 and 72 hours after co-culture with PBNK without CAR-T.
  • 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 routes of parenteral administration, such as by injection or infusion.
  • antibody shall include, but is not 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 antigens thereof combined part.
  • Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region contains 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 contains 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 the amino terminus to the 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 the international single English letter, and the corresponding three English letter abbreviations of the 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).
  • chimeric antigen receptor chimeric membrane antigen receptor
  • membrane fusion protein membrane fusion protein
  • recombinant membrane fusion protein can be used interchangeably, and all represent the IL-15/IL- 15R ⁇ complex.
  • IL-15 is a cytokine that can stimulate the growth of immune cells.
  • IL-15-based drug development has long focused on soluble IL-15 engineering.
  • literature and patent reports focusing on the development of soluble IL-15 drugs, mainly RL1, ALT803, HRP008, P22339 four structures.
  • RL1 is a complex of IL-15 and the Sushi domain of IL-15 receptor ⁇ , which is connected by a linker.
  • ALT803 is a complex of two IL-15N72D mutants with a Sushi domain/Fc fusion protein of dimeric IL-15 receptor alpha, a complex of IL-15(N72D):IL-15RalphaSu/Fc.
  • HRP00018 is a complex of IL-15/Fc fusion protein and Sushi domain/Fc fusion protein of IL-15 receptor ⁇ linked by Knob-into-Hole.
  • P22339 is the IL-15(L52C) mutant and the IL-15R ⁇ Sushi domain(S40C)/Fc complex forming a disulfide bond at the mutation site, which is connected by Knob-into-Hole form, thereby stabilizing the structure of the complex.
  • the four structures above overcome the short half-life of recombinant IL-15, significantly prolong the half-life in vivo, and improve its biological activity, such as promoting the proliferation of CD8+ memory T cells, NKT and NK cells.
  • it still has the problems of toxic side effects and short half-life, and needs to be administered multiple times in clinical use, which limits its clinical practicability.
  • the present invention provides an "IL-15/IL-15R ⁇ complex", which comprises IL-15 or a mutant thereof, and a complete IL-15R ⁇ comprising a transmembrane region and an intracellular region, which can be used to enhance CAR- Persistence of T cells and/or enhanced cytotoxicity of CAR-T cells.
  • the IL-15/IL-15R ⁇ complex of the present invention may include IL-15N72D/IL-15R ⁇ complex, may include signal peptide (IgE, IL-2, etc.), and may share the basic structure of CAR Express.
  • the IL-15(N72D)-IL-15R ⁇ structure of the present invention is a complex expressed on the surface of immunotherapy cells.
  • IL-15 and IL-15R ⁇ are connected by linker, wherein IL-15R ⁇ structure refers to the complete IL-15R ⁇ molecule including extracellular domain (including sushi domain), transmembrane domain and intracellular domain.
  • IL-15R ⁇ can promote the proliferation of immunotherapy cells and inhibit the apoptosis of immunotherapy cells by recruiting the self-expressed IL-2R ⁇ and IL-2R ⁇ c chains on the surface of immunotherapy cells and activating downstream signaling signals such as JAK1/JAK3, Stat3/Stat5 , so as to obtain a better therapeutic effect.
  • the complex is restricted to the surface of immunotherapy cells to avoid the possible immunosuppressive effects of activating the proliferation of immunosuppressive T cells, NK and NKT cells (such as Treg), and to avoid excessive activation of T cells, NK and NKT cells. systemic or systemic side effects.
  • the present invention exemplifies the construction of CAR-T cells expressing the IL-15/IL-15R ⁇ complex by using the basic structure of Meso-CAR, that is, the CAR structure targeting mesothelin.
  • the IL-15/IL-15R ⁇ complex is linked to the C-terminus of the basic CAR structure by a self-cleaving element, and is expressed on the membrane of the CAR-T cell after cleavage.
  • the Meso-CAR is composed of a signal peptide, a single-chain variable region of Mesothelin, CD28/4-1bb/ICOS, and CD3 ⁇ in series.
  • 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 referred to as antigen binding domains).
  • the intracellular domain includes the costimulatory signaling region and the zeta chain portion.
  • a costimulatory signaling region refers to a portion of an intracellular domain that includes a costimulatory molecule.
  • Costimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that are required for an efficient lymphocyte response to an antigen.
  • the CAR of the present invention comprises a costimulatory signaling molecule derived from CD28.
  • 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 the transmembrane domain to the extracellular or cytoplasmic domain of a polypeptide chain.
  • the linker 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 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 to its cognate antigen, it affects tumor cells, causing the tumor cells to not grow, being driven to die, or otherwise being 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 costimulatory molecule and the zeta chain.
  • antigen binding domain and “single chain antibody fragment” both 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 regions, and are the smallest antibody fragment with all antigen-binding sites. Typically, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure 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 that of a complete antibody.
  • Single chain antibodies are preferably one amino acid chain sequence encoded by one nucleotide chain.
  • the scFv comprises an antibody that specifically recognizes Meso, preferably a humanized single-chain antibody.
  • the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR.
  • the 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 receptor complexes interactions with other members.
  • Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by using standard technology to isolate directly from cells and tissues that contain the gene. Alternatively, the gene of interest can be produced synthetically.
  • the present invention also provides vectors into which the expression cassettes of the present invention are 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 proliferation in daughter cells.
  • Lentiviral vectors have advantages 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.
  • the vector is suitable for replication and integration in eukaryotic cells.
  • Typical cloning vectors contain transcriptional and translational terminators, initial sequences and promoters that can be used to regulate the expression of the desired nucleic acid sequence.
  • the expression constructs of the present 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 generation 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, herpesviruses, 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 (eg, WO01/96584; WO01/29058; and U.S. Patent No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected gene 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.
  • Many retroviral systems are known in the art.
  • adenoviral vectors are 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 so that promoter function is maintained 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 before activity begins to decline.
  • individual elements appear to 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 ⁇ (EF-1 ⁇ ).
  • constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, the mouse breast cancer virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russell 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 present invention.
  • an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is not desired.
  • inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
  • the expression vector introduced into the cells may also contain either or both of a selectable marker gene or a reporter gene to facilitate the search for the transfected or infected cell population from the viral vector Identification and selection of expressing cells.
  • the selectable marker can be carried on a single piece of DNA and used in co-transfection procedures. Both the selectable marker and the reporter gene can 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 evaluate the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is clearly indicated by some readily detectable property such as enzymatic activity. After the DNA has been introduced into the recipient cells, the expression of the reporter gene is measured at an appropriate time.
  • Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (eg, Ui-Tei et al., 2000 FEBS Letters 479:79). -82).
  • Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Typically, constructs with a minimum of 5 flanking regions showing the highest levels of reporter gene expression are identified as promoters. Such promoter regions can be linked to reporter genes and used to assess the ability of an agent to modulate promoter-driven transcription.
  • an expression vector can be readily introduced into a host cell, eg, mammalian, bacterial, yeast or insect cells, by any method known in the art.
  • a host cell eg, mammalian, bacterial, yeast or insect cells
  • an expression vector can be transferred into a host cell by physical, chemical or biological means.
  • Physical methods of introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods of 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). The 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 lipids plastid.
  • lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and lipids plastid.
  • Exemplary colloidal systems for use as in vitro and in vivo delivery vehicles are liposomes (eg, artificial membrane vesicles).
  • exemplary delivery vehicles are liposomes.
  • lipid formulations is contemplated to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo).
  • nucleic acid can be associated with a lipid.
  • Nucleic acids associated with lipids 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 to liposomes, 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 Complex 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. For example, they may exist in bilayer structures, as micelles or have a "collapsed" structure.
  • Lipids are fatty substances, which can be naturally occurring or synthetic lipids.
  • lipids include lipid droplets, which occur naturally in the cytoplasm as well as in such compounds comprising long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols 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 formulation is an injection.
  • the concentration of the CAR-T cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, more preferably 1 ⁇ 10 4 -1 ⁇ 10 7 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 present invention includes therapeutic applications of cells (eg, T cells) transduced with lentiviral vectors (LVs) encoding the expression cassettes of the present invention.
  • the transduced T cells can target the tumor cell marker Meso, synergistically activate T cells, and cause T cell immune responses, thereby significantly improving their killing efficiency against tumor cells.
  • the present invention also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal, comprising the steps of: administering to the mammal a CAR-T cell of the present invention.
  • the present invention includes a type of cell therapy wherein a patient's autologous T cells (or a heterologous donor) are isolated, activated and genetically engineered to produce CAR-T cells, and subsequently infused into the same patient.
  • a patient's autologous T cells or a heterologous donor
  • CAR-T can treat all cancers that express this 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.
  • a CAR-mediated immune response can be part of an adoptive immunotherapy step in which CAR-modified T cells induce an immune response specific to the antigen binding domain in the CAR.
  • anti-Meso CAR-T cells elicit specific immune responses against Meso-positive cells.
  • Cancers that can be treated include tumors that are not vascularized or substantially not vascularized, as well as tumors that are vascularized. Cancers may include non-solid tumors (such as hematological tumors, eg, leukemias and lymphomas) or may include solid tumors. Cancer types treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas, and sarcomas, and certain leukemic or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Also includes adult tumors/cancers and pediatric tumors/cancers.
  • 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 myeloid leukemia, and myeloblastoid, promyelocytic, myelomonocytic type) , monocytic and erythroleukemia), chronic leukemia (such as chronic myeloid (myeloid) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non- Hodgkin's lymphoma (painless and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.
  • acute leukemias such
  • Solid tumors are abnormal masses of tissue that typically do not contain cysts or areas of fluid. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell type that forms 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, ovarian cancer.
  • the treatable cancer is a Meso positive tumor.
  • 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.
  • CAR-modified cells are isolated from mammals (preferably human) and genetically modified (ie, transduced or transfected in vitro) with vectors expressing the CARs disclosed herein.
  • CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefit.
  • the mammalian recipient can be 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 present 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 methods of treating tumors comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the present invention.
  • the CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations.
  • the pharmaceutical compositions of the present invention may include a target cell population as described herein in association 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; chelates Adjuvants such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • the compositions of the present invention are preferably formulated for intravenous administration.
  • compositions of the present invention can be administered in a manner appropriate to the disease to be treated (or prevented).
  • the amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease - although appropriate doses may be determined by clinical trials.
  • the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the patient (subject ) individual differences in age, weight, tumor size, degree of infection or metastasis, and condition. It may generally be indicated that the pharmaceutical compositions comprising the T cells described herein may be administered at a dose of 104 to 109 cells/kg body weight, preferably 105 to 106 cells/kg body weight (including all integers within those ranges). value) application. The T cell composition 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).
  • Optimal dosages and treatment regimens for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting treatment accordingly.
  • compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinal, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • the T cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection.
  • the T cell composition of the present invention is preferably administered by i.v. injection.
  • the composition of T cells can be injected directly into tumors, lymph nodes or the site 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 therapeutic modalities (eg, previously , concurrently or subsequently) to a 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 therapy for MS patients or elfazizumab therapy 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 therapy for MS patients or elfazizumab therapy for psoriasis patients or other treatments for PML patients.
  • the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunotherapeutics.
  • the cellular composition of the invention is administered in combination with (eg, before, concurrently or after) bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • the 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.
  • the dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration can be carried out in accordance with art-accepted practice. Typically, 1 x 10 6 to 1 x 10 10 modified T cells (eg, CAR-T cells) of the invention can be administered to a patient per treatment or per course of treatment, for example, by intravenous infusion .
  • 1 x 10 6 to 1 x 10 10 10 modified T cells (eg, CAR-T cells) of the invention can be administered to a patient per treatment or per course of treatment, for example, by intravenous infusion .
  • the CAR structure of the CAR-T cell of the present invention contains both the basic structure of the CAR and the IL-15/IL-15R ⁇ complex or the IL-15 mutant/IL-15R ⁇ complex, each of which functions without interfering with each other.
  • the immune cells of the present invention can significantly enhance NK cell expansion.
  • Example 1 Isolation of PBMC and expansion of T cells from donor blood
  • Mononuclear cells were isolated from peripheral blood, subjected to density gradient centrifugation using Ficoll, and enriched for T cells (EasySep human T cell enrichment kit, Stemcell Technologies), and activated to culture and expand T cells using coupled anti-CD3/CD28 magnetic beads , the cell culture system used x-vivo 15 (5% FBS, 300IU/mL rhIL-2), and the cells were cultured at 37°C and continuously cultured in a 5% CO 2 incubator.
  • MSLN-expressing cell lines were obtained from ATCC: OVCAR3 (human ovarian cancer cell line, ATCC HTB-161), HCC70 (human breast cancer cell line), MDA-MB-231 (human breast cancer cell line), in which MDA-MB- 231MESO transferred MSLN antigen into MDA-MB231 cells by lentivirus, and then obtained a stable cell line with high expression of MSLN after monoclonal screening.
  • OVCAR3 human ovarian cancer cell line, ATCC HTB-161
  • HCC70 human breast cancer cell line
  • MDA-MB-231 human breast cancer cell line
  • MDA-MB- 231MESO transferred MSLN antigen into MDA-MB231 cells by lentivirus
  • This example exemplarily uses the basic structure of Meso-CAR, that is, the CAR structure targeting mesothelin, to construct CAR-T cells expressing IL-15/IL-15R ⁇ complexes.
  • the core structure of CAR includes CD8 extracellular signal peptide, P4scFv (scFv specifically targeting mesothelin), CD8-derived hinge region and CD8/CD28 transmembrane region, and uses CD28 intracellular segment costimulatory signal to construct 3. kind of Meso-CAR.
  • the three Meso-CAR genes were cloned into the FUW lentiviral vector, and the lentiviral packaging plasmids pMD2.G (Addgene, Plasmid#12259) and psPAX2 (Addgene, Plasmid#12260) were transfected into 293T cells using PEI transfection reagent. , the expression vector, the virus was collected for 48 hours and 72 hours, respectively, and after ultracentrifugation and concentration, the activated T cells were infected.
  • Example 4 CAR-T cell preparation - lentivirus infection, CAR positive rate detection and in vitro proliferation
  • the isolated and purified primary T cells were added to the concentrated lentivirus in Example 3, and the CAR positive rate was detected 72 hours after cell infection.
  • Biotin-labeled MSLN antigen was used as the primary antibody, APC-Streptavidin (BD) ) as secondary antibody, and anti-IL-15R ⁇ antibody to detect scFv and IL-15R ⁇ expression.
  • T cells were continuously cultured in a 37°C, 5% CO 2 incubator, supplemented every other day, harvested on day 10, cryopreserved, counted, and calculated in vitro proliferation rate.
  • E1 CAR-T cells were compared with E1m1 CAR-T cells. There was no significant difference in the proliferation of T cells.
  • the T cells harvested in Example 4 were subjected to in vitro killing experiments.
  • RTCA method target cells were plated on 96-well RTCA plates at 1x10 4 /well, and after 18 hours of culture, CAR T and target cells OVCAR3 were co-cultured at a ratio of 1:1, 1:3, 1:6, and cultured continuously for 1- After 2 days, the growth status of target cells was recorded in real time, the survival rate of target cells was detected, and the killing efficiency of CAR-T cells was calculated. After continuous culturing for 1-2 days, the RTCA plate was taken out, and the co-culture supernatant was collected by centrifugation, and frozen at -20°C.
  • HumanTh1/Th2Cytokine kit II (BD, Cat.551809) was used to detect cytokines in the co-culture supernatant.
  • the co-culture supernatant was thawed, and mixed capture beads and Human Th1/Th2-II PE Detection were configured. Samples or standards were incubated in the dark for 3 hours, after incubation, centrifuged at 300g for 5min, and the supernatant was discarded. Add 100 ⁇ l washing buffer to resuspend, shake for 5 min, and detect by flow cytometer. Data analysis was performed with FCAP Array v.3 software.
  • mice subcutaneously inject 5E6 HCC70 cells, and continuously detect tumor burden.
  • they are divided into groups, 2-3 mice in each group, and 200uL DPBS/mouse is injected into the tail vein one day after grouping, 5E6 E1/E1m1- CAR-T/mouse, on the first day after CAR-T cell injection, a small amount of mouse blood was taken to detect the number of CAR-T cells in vivo, and then blood samples were taken once a week to detect various phenotypes of CAR-T cells. Subcutaneous tumor size was measured twice.
  • mice injected with Meso-E1 CAR-T cells and Meso-E1m1 CAR-T cells showed a significant decrease in tumor volume on D10; the tumor volume of the animals was continued to be observed on D17, D21, and D24, and the E1 group The tumor volume of animals recovered, and continued to decrease in the E1m1 group.
  • the body weight of the mice is shown in Figure 7B. After the injection of CAR-T cells, the body weight of the mice in the Meso-E1m1 group basically did not change.
  • mice subcutaneously inject 5E6 HCC70 cells, and continuously detect tumor burden.
  • the tumor When the tumor is growing rapidly, they are divided into groups, 2-3 mice in each group, and 200uL DPBS/mouse is injected into the tail vein one day after grouping, high (indicates HD)
  • mice injected with Meso-1 CAR-T cells and Meso-E1m1 CAR-T cells started to have a more obvious decrease in tumor volume at D10; on D17, D21, and D24, the tumor volume of the animals was continued to be observed, and the tumor volume was higher. .
  • the tumor volume of the low-dose Meso-1 group recovered, but the tumor volume of the high-dose E1m1 group did not increase.
  • the tumor volume of the low-dose E1m1 group started to recur at D40. Compared with the Meso-1 group, the tumor volume was significantly smaller.
  • the T cells harvested in Example 4 were subjected to multiple rounds of killing experiments in vitro.
  • the experimental method is shown in the schematic diagram (Fig. 9A).
  • the CAR-T cells and the target cells were co-incubated at a ratio of 1:3 for 2-3 days.
  • the growth of the target cells was observed under a microscope. When all the target cells are lysed, take half of the T cells to detect the phenotype and cell number of the T cells, digest the target cells with trypsin, and count them as the residual value of the target cells.
  • the formula for calculating the killing efficiency of CAR-T cells is: CAR-T cells
  • the killing efficiency (tumor-only group-experimental group)/tumor-only group*100%, the remaining half of the cells continue to co-culture with new target cells, and continue to kill continuously according to the above method.
  • the T cells harvested in Example 4 were subjected to multiple rounds of killing experiments in vitro.
  • the experimental protocol is shown in Figure 9A.
  • the CAR-T cells and target cells were co-incubated at a ratio of 1:5, and different concentrations of recombinant TGF- ⁇ 1 were added during the culture, supplemented every two days, and cultured for 2-3 days.
  • the growth status of the target cells was recorded in real time. When all the target cells were lysed by CAR-T cells, half of the T cells were taken to detect the phenotype and cell number of the T cells.
  • the T cells harvested in Example 4 were subjected to in vitro killing experiments.
  • the CAR-T cells were incubated with the target cells (MDA-MB231-Meso) at a ratio of 1:2. After 3 days of continuous culture, the CAR-T cells were moved to fresh target cells. In the cells, after culturing for 3 days, the apoptosis of CAR-T cells was detected. During the culturing process, two groups were divided into groups without or with the addition of TGF- ⁇ 1 at the indicated concentrations.
  • the T cells harvested in Example 4 were co-cultured with the autologous PBNK cells cultured in vitro at a ratio of 1:1.
  • the PBNK cells were first labeled with CFSE, and the number of PBNK cells was detected at 48 hours and 96 hours, respectively.
  • the flow detection method was used to detect FITC + cells in the culture system.
  • CFSE labeling method PBNK cells were centrifuged and resuspended to 1E6/mL with PBS, added with a final concentration of 4uM CFSE, incubated at room temperature for 2 minutes, and then added medium to stop the reaction.
  • PBNK is derived from monocytes from the same Donor as T cells, and is isolated and purified using anti-CD56 microbeads. For specific methods, see Miltenyi's protocol.
  • E1 CAR-T cells were co-cultured with E1m1 CAR-T cells and PBNK cells for 48-72 hours. Compared with NT cells, E1 and E1-m1 CAR-T cells could significantly promote the growth of PBNK cells. Amplification.

Abstract

一种表达白介素-15的嵌合抗原受体免疫细胞及其应用。具体地，提供了一种基因重组的嵌合抗原受体免疫细胞，其细胞表面表达白介素-15突变体与IL-15Ra融合蛋白,实验显示该突变体免疫细胞在抗原持续刺激情况下，明显提高免疫细胞存活，促进免疫细胞的扩增，减少TME对免疫细胞的抑制，并增强CAR-T细胞的持久性和抗肿瘤活性。

Description

膜融合蛋白及其在免疫细胞中的应用 技术领域
本发明涉及免疫细胞治疗领域，更具体地涉及共表达白介素-15突变体和IL-15Rα融合蛋白且靶向实体瘤和血液瘤的工程化免疫细胞及其应用。
背景技术
免疫细胞疗法是一种全新的药物开发模式。利用基因工程化改造人类免疫细胞使其成为活性药物，在治疗B细胞恶性肿瘤方面已经取得了显著的进展。
目前已开发了多种免疫疗法，包括LAK、DC、CIK、DC-CIK、TCR-T、CAR-T、NK、CAR-NK等等。常用的CAR-T细胞免疫疗法包括自体和通用型CAR-T疗法，其中自体CAR-T疗法采用病人的免疫细胞，而异体免疫疗法采用同种异体对象的CAR-T细胞，将所需的免疫细胞进行体外培养、基因编辑和扩增后回输病人体内，这些细胞以非MHC限制方式识别肿瘤细胞，不需要抗原递呈的机制识别肿瘤抗原，减少肿瘤细胞通过下调MHC以及抗原递呈介导的免疫逃逸。针对肿瘤相关抗原靶点(TAA)，设计细胞表面嵌合抗体受体(CAR)分子特异识别肿瘤细胞表达的抗原靶点，通过分泌干扰素、穿孔素等细胞因子直接攻击并杀伤肿瘤细胞，从而达到治疗或缓解疾病的目的。
目前，免疫治疗血液瘤已有突出的疗效，而在实体瘤中疗效并不理想。主要原因包括三方面，一是安全的肿瘤特异性抗原靶点，二是实体肿瘤的异质性，三是存在复杂的肿瘤细胞微环境等因素，包括免疫抑制细胞Tregs和MDSCs等以及免疫抑制因子的存在，不利于免疫细胞存活，从而抑制免疫治疗效果。间皮素是一种存在于正常间皮细胞上的分化抗原，在间皮瘤、肺癌、胰腺癌、乳腺癌、卵巢癌等肿瘤中均有高表达，正常组织如正常胸膜、心包和腹膜的间皮细胞中有限表达，在气管，卵巢，睾丸，扁桃体和输卵管的上皮细胞表面上表达最低。因此间皮素可作为有效的免疫治疗靶点，目前针对MSLN设计免疫治疗策略，主要包括抗体疗法、免疫毒素和嵌合抗原受体T细胞疗法。抗体类药物包括Amatuximab(MORAb-009)，Anetumab Ravtansine(BAY94-9343),DMOT4039A,MDX-1204等，但其治疗实体瘤疗效并不理想，尚需完善现有的治疗技术，增强疗效，降低治疗过程中的风险。
综上所述，本领域尚需进一步开发更有效、持久、安全靶向实体瘤的免疫疗法。
发明内容
本发明的目的在于提供一种表达白介素-15的嵌合抗原受体免疫细胞及其应 用。
在本发明的第一方面，提供了一种嵌合抗原受体(CAR)构建物，所述CAR构建物的结构如下式I或II所示，
X-A-E  (I)
E-A-X  (II)
式中，
各“-”独立地为连接肽或肽键；
X为靶向肿瘤抗原的CAR；
A为自剪切元件；
E为IL-15/IL-15Rα复合体。
在另一优选例中，所述的CAR构建物的结构如式I所示。
在另一优选例中，所述的IL-15/IL-15Rα复合体包含IL-15和IL-15Rα。
在另一优选例中，所述的IL-15和IL-15Rα来源于人。
在另一优选例中，所述的IL-15和IL-15Rα通过连接肽连接。
在另一优选例中，所述的IL-15/IL-15Rα复合体还包含信号肽元件。
另一优选例中，所述的IL-15/IL-15Rα复合体的结构如下式III所示，
L’-M-I-R   (III)
式中，
各“-”独立地为连接肽或肽键；
L’为无或信号肽；
M为IL-15或其突变体；
I为柔性接头；
R为IL-15Rα。
在另一优选例中，所述的IL-15突变体具有IL-15的生物活性。
在另一优选例中，所述的IL-15的氨基酸序列如SEQ ID NO.:1所示。
在另一优选例中，所述的IL-15突变体(IL-15N72D)的氨基酸序列如SEQ ID NO.:2所示。
在另一优选例中，所述的IL-15Rα为完整的IL-15Rα元件。
在另一优选例中，所述的IL-15Rα包含跨膜区和胞内区。
在另一优选例中，所述的IL-15Rα从N端到C端依次包括sushi结构域(与IL-15结合的活性片段)胞外区、跨膜区和胞内区。
在另一优选例中，所述的IL-15Rα的氨基酸序列如SEQ ID NO.:3所示。
在另一优选例中，所述的柔性接头为连接肽，较佳地，所述连接肽的氨基酸序列如SEQ ID NO.:4所示(SGGGSGGGGSGGGGSGGGGSGGGSLQ)。
在另一优选例中，所述的L’为来源IgE,IL-2的信号肽。
在另一优选例中，所述的IL-15/IL-15Rα复合体的氨基酸序列如SEQ ID NO.:5所示。
在另一优选例中，所述的IL-15突变体/IL-15Rα复合体的氨基酸序列如SEQ ID NO.:6所示。
在另一优选例中，所述的自剪切元件包括T2A、P2A。
在另一优选例中，所述的X(CAR)的结构如下式IV所示，
L-scFv-H-TM-C-CD3ζ   (IV)
各“-”独立地为连接肽或肽键；
L为无或信号肽；
scFv为靶向肿瘤抗原的抗体单链可变区；
H为无铰链区；
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；
在另一优选例中，所述的肿瘤抗原选自下组：间皮素、Claudin18.2、MUC1、GPC3、PSCA、Her2、CD19、或其组合。
在另一优选例中，所述的肿瘤抗原为间皮素。
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，所述的H为选自下组的蛋白的铰链区：CD8、CD28、CD137、或其组合。
在另一优选例中，所述的H为CD8来源的铰链区。
在另一优选例中，所述的TM为选自下组的蛋白的跨膜区：ICOS、CD28、CD3epsilon、CD45、CD4、CD5、CD8、CD9、CD16、MUC1-Tn、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、或其组合。
在另一优选例中，所述的TM为CD8或CD28来源的跨膜区。
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：ICOS、OX40、CD2、CD7、CD27、CD28、CD30、CD40、CD70、CD134、4-1BB(CD137)、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CD28来源的共刺激信号分子。
在另一优选例中，所述的X的氨基酸序列如SEQ ID NO.:8所示。
在另一优选例中，所述的CAR构建物的氨基酸序列如SEQ ID NO.:7所示。
在本发明的第二方面，提供了一种核酸分子，所述的核酸分子编码本发明第一方面所述的CAR构建物，或者，
所述的核酸分子包含编码靶向肿瘤抗原的CAR的第一核酸分子和编码IL-15/IL-15Rα复合体的第二核酸分子，其中所述的靶向肿瘤抗原的CAR和IL-15/IL-15Rα复合体的定义如上所述。
在另一优选例中，所述的第一核酸分子和第二核酸分子可以是串联的，也可以是独立存在的。
在本发明的第三方面，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。
在本发明的第四方面，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构建物。
在另一优选例中，所述的宿主细胞包括真核细胞和原核细胞。
在另一优选例中，所述的宿主细胞包括大肠杆菌。
在本发明的第五方面，提供了一种工程化的免疫细胞，所述的免疫细胞表达有本发明第一方面所述的CAR构建物，或者
所述的免疫细胞表达有靶向肿瘤抗原的CAR和IL-15/IL-15Rα复合体，其中所述的靶向肿瘤抗原的CAR和IL-15/IL-15Rα复合体的定义如上所述。
在另一优选例中，靶向肿瘤抗原的CAR和IL-15/IL-15Rα复合体独立地表达于所述免疫细胞的细胞膜上。
在另一优选例中，所述细胞为分离的细胞，和/或所述细胞为基因工程化的细胞。
在另一优选例中，所述的免疫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，所述的工程化的免疫细胞可以是嵌合抗原受体T细胞(CAR-T细胞)或嵌合抗原受体NK细胞(CAR-NK细胞)。
在本发明的第六方面，提供了一种制剂，所述制剂含有本发明第一方面所述的CAR构建物、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、或本发明第五方面所述的免疫细胞，以及药学上可接受的载体。
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CAR-T细胞的浓度为1×10 3-1×10 8个细胞/ml，较佳地1×10 4-1×10 7个细胞/ml。
在另一优选例中，所述的制剂还包含抗肿瘤的第二活性成分，较佳地包括第二抗体、或化疗剂。
在另一优选例中，所述的化疗剂选自下组：多西他赛、卡铂、或其组合。
在本发明的第七方面，提供了一种本发明第一方面所述的CAR构建物、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、或本发明第五方面所述的免疫细胞、或本发明第六方面所述的制剂的用途，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，所述肿瘤选自下组：血液肿瘤、实体瘤、或其组合。
在另一优选例中，所述血液肿瘤选自下组：急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、或其组合。
在另一优选例中，所述实体瘤选自下组：胃癌、胃癌腹膜转移、肝癌、白血病、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、淋巴癌、鼻咽癌、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，所述的肿瘤为间皮素阳性肿瘤，较佳地为间皮素高表达的肿瘤。
在本发明的第八方面，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。
在本发明的第九方面，提供了一种制备本发明第五方面所述的工程化的免疫 细胞的方法，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。
在本发明的第十方面，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、本发明第五方面所述的免疫细胞、或本发明第六方面所述的制剂。
在另一优选例中，所述疾病为癌症或肿瘤。
在本发明的第十一方面，提供了一种IL-15/IL-15Rα复合体，所述的IL-15/IL-15Rα复合体的结构如下式III所示，
L’-M-I-R   (III)
式中，
各“-”独立地为连接肽或肽键；
L’为无或信号肽；
M为IL-15或其突变体；
I为柔性接头；
R为IL-15Rα，
其中，所述的IL-15Rα包含跨膜区和胞内区。
在本发明的第十二方面，提供了一种本发明的第十一方面所述的IL-15/IL-15Rα复合体的用途，用于制备一制剂，所述制剂用于增强CAR-T细胞的持久性和/或增强CAR-T细胞的细胞毒性。
在另一优选例中，所述制剂用于基于CAR-T细胞的过继免疫治疗。
在另一优选例中，所述的增强CAR-T细胞的持久性是指增强CAR-T细胞对肿瘤细胞的持续杀伤能力。
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显示了本发明所述膜融合蛋白的作用机理示意图。
图2A显示本发明采用的CAR结构的示意图。
图2B显示huIL-15/IL-15突变体复合物示意图。
图2C显示huIL-15/IL-15突变体复合物的huIL-15Rα的结构。
图3A显示MESO-E1m1基因的氨基酸序列。
图3B显示E1m1基因的氨基酸序列。
图4显示CAR-T细胞的阳性率检测图。
图5A显示Meso-1，Meso-E1和Meso-E1m1 CAR-T细胞与卵巢癌细胞OVCAR3体外共培养体系中，CAR-T细胞对靶细胞裂解率。
图5B显示CAR-T细胞与卵巢癌OVCAR3体外共培养后，CAR-T细胞的细胞因子分泌。
图6A显示Meso-1，Meso-E1和Meso-E1m1 CAR-T细胞与三阴乳腺癌MDA-MB-231-MESO细胞体外共培养体系中，CAR-T细胞对靶细胞裂解率。。
图6B显示CAR-T细胞与三阴乳腺癌MDA-MB-231-MESO细胞体外共培养后,CAR-T细胞的细胞因子分泌。
图7A显示Meso-E1和Meso-E1m1 CAR-T体内药效实验-1，HCC70(乳腺癌细胞)肿瘤体积变化曲线。
图7B显示Meso-E1和Meso-E1m1 CAR-T细胞体内药效实验-1，动物体重变化曲线。
图8A显示Meso-1和Meso-E1m1 CAR-T体内药效实验-2，HCC70(乳腺癌细胞)肿瘤体积变化曲线。
图8B显示Meso-1和Meso-E1m1 CAR-T体内药效实验-2，动物体重变化。
图8C显示Meso-1和Meso-E1m1 CAR-T体内药效实验-2，每组单只小鼠的肿瘤体积变化。
图9A显示多轮杀伤实验设计方案。
图9B、图9C显示体外共培养体系Meso-1，Meso-E1和Meso-E1m1 CAR-T细胞分别与卵巢癌细胞OVCAR3多轮杀伤后,靶细胞裂解率和CAR-T细胞扩增比较。
图9D、图9E显示体外共培养体系Meso-1，Meso-E1和Meso-E1m1 CAR-T细胞分别与三阴乳腺癌MDA-MB-231-MESO细胞多轮刺激后,靶细胞裂解率和CAR-T细胞扩增比较。
图10A显示添加不同浓度TGF-β1体外共培养体系，Meso-1和Meso-E1m1 CAR-T细胞分别与卵巢癌细胞OVCAR3多轮刺激后,靶细胞的裂解率。
图10B显示添加不同浓度TGF-β1体外共培养体系，Meso-1和Meso-E1m1 CAR-T细胞分别与卵巢癌细胞OVCAR3多轮刺激后,CAR-T细胞扩增曲线。
图10C显示添加不同浓度TGF-β1体外共培养体系，Meso-1和Meso-E1m1 CAR-T细胞分别与卵巢癌细胞OVCAR3第二轮多轮杀伤后,CAR-T细胞凋亡的检测。
图11显示不CAR-T与PBNK共培养后，PBNK细胞在48和72小时扩增值。
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基因重组的嵌合抗原受体免疫细胞及其应用，所述嵌合抗原受体免疫细胞表面表达超级白介素-15，即为白介素-15突变体与IL-15Ra融合蛋白,体外实验显示该突变体能显著增强免疫细胞的增殖能力、存活能力，持续杀伤力和促进NK细胞的扩增；体内实验显示该突变体持久抗肿瘤活性。在此基础上完成了本发明。
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)。
术语“嵌合抗原受体”、“嵌合膜抗原受体”、“膜融合蛋白”、和“重组的膜融合蛋白”可互换使用，均代表本发明所构建的IL-15/IL-15Rα复合体。
IL-15
IL-15是一种可以刺激免疫细胞生长的细胞因子。基于IL-15的药物开发长期以来集中在可溶型的IL-15改造上。已有文献和专利报道，集中在开发可溶型IL-15药物，主要有RL1,ALT803,HRP008,P22339四种结构。
RL1是IL-15与IL-15受体α的Sushi domain的复合体，两者之间通过一个Linker连接。ALT803是两个IL-15N72D突变体与二聚体IL-15受体α的Sushi domain/Fc融合蛋白的复合体，即IL-15(N72D):IL-15RαSu/Fc的复合体。HRP00018是IL-15/Fc融合蛋白与IL-15受体α的Sushi domain/Fc融合蛋白通过Knob-into-Hole形式相连形成复合体。P22339是IL-15(L52C)突变体与IL-15RαSushi domain(S40C)/Fc复合体在突变位点形成二硫键，通过Knob-into-Hole形式连接，进而稳定复合物的结构。
综上所述的四种结构，克服了重组的IL-15半衰期短的缺点，都明显延长体内的半衰期，并提高其生物学活性，如促进了CD8+记忆T细胞、NKT和NK细胞增殖。但其仍旧存在毒副作用和半衰期短的问题，在临床使用中需多次给药，限制其临床实用性。
IL-15/IL-15Rα复合体
本发明提供了一种“IL-15/IL-15Rα复合体”，其包含IL-15或其突变体，以及完整的包含跨膜区和胞内区的IL-15Rα，可以用于增强CAR-T细胞的持久性和/或增强CAR-T细胞的细胞毒性。
在优选的实施方式中，本发明的IL-15/IL-15Rα复合体可以包含IL-15N72D/IL-15Rα复合体，可以包含信号肽(IgE,IL-2等)，可以与CAR基本结构共表达。
本发明的IL-15(N72D)-IL-15Rα结构是表达在免疫治疗细胞表面的复合物。IL-15和IL-15Rα通过linker相连，其中IL-15Rα结构是指完整的IL-15Rα分子包括胞外区(含sushi domain)、跨膜区和胞内区。IL-15Rα可通过招募免疫治疗细胞表面自身表达的IL-2Rβ和IL-2Rγc链，激活下游信号通路信号比如JAK1/JAK3，Stat3/Stat5达到促进免疫治疗细胞增殖，抑制免疫治疗细胞凋亡的目的，从而获得更好的治疗效果。同时将该复合物限制在免疫治疗细胞表面，避免激活免疫抑制型T细胞、NK和NKT细胞的增殖(比如Treg)可能带来的免疫抑制作用，也能够避免过度激活T细胞、NK和NKT细胞带来的系统性或者全身性的毒副作用。
嵌合抗原受体(CAR)
本发明示例性的采用Meso-CAR基本结构，即靶向间皮素的CAR结构进行表达 IL-15/IL-15Rα复合体的CAR-T细胞的构建。
在优选的实施方式中，所述IL-15/IL-15Rα复合体通过自剪切元件连接至基本CAR结构的C端，并在剪切后在CAR-T细胞的膜上表达。
在优选的实施方式中，所述Meso-CAR由信号肽、Mesothelin单链可变区、CD28/4-1bb/ICOS、CD3ζ串联而成。
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。在优选的实施方式中，本发明的CAR包含来源CD28的共刺激信号分子。
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Meso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。
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Meso的抗体，较佳地为人源化的单链抗体。
对于绞链区和跨膜区(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。
序列
本申请序列表中涉及的各序列如下：
Figure PCTCN2021143209-appb-000001
Figure PCTCN2021143209-appb-000002
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 3-1×10 8个细胞/ml，更优地1×10 4-1×10 7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Meso的CAR-T细胞引起抗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抗-Meso scFv、铰链和CD28跨膜区和胞内区和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。
可治疗的癌症包括没有被血管化或基本上还没有被血管化的肿瘤，以及血管 化的肿瘤。癌症可包括非实体瘤(诸如血液学肿瘤，例如白血病和淋巴瘤)或可包括实体瘤。用本发明的CAR治疗的癌症类型包括但不限于癌、胚细胞瘤和肉瘤，和某些白血病或淋巴恶性肿瘤、良性和恶性肿瘤、和恶性瘤，例如肉瘤、癌和黑素瘤。也包括成人肿瘤/癌症和儿童肿瘤/癌症。
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Meso阳性肿瘤。
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(如，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：
(a)本发明CAR-T细胞的CAR结构中同时包含CAR的基本结构和IL-15/IL-15Rα复合体或IL-15突变体/IL-15Rα复合体，各自发挥功能，互不干扰。
(b)体内实验结果显示本发明免疫细胞Meso-E1m1能明显增强CAR-T细胞的细胞毒性和持久性,并明显优于Meso-E1。
(c)体外实验结果显示，与Meso-1 CAR-T比较，本发明免疫细胞Meso-E1m1在靶细胞和TGF-β1持续共培养情况下，明显提高免疫细胞存活率，促进免疫细胞的扩增，减少TGF-β1对免疫细胞的抑制，同时增强CAR-T细胞的细胞毒性和持久性。
(d)与Meso-1 CAR-T比较，本发明免疫细胞Meso-E1m1在体外瞬时杀伤实验中，明显减少炎性因子TNF-α分泌。
(e)本发明的免疫细胞可明显增强NK细胞扩增。
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。
实施例1从供体血液中分离PBMC和扩增T细胞
从外周血中分离单核细胞，使用Ficoll进行密度梯度离心，并富集T细胞(EasySep human T cell enrichment kit，Stemcell Technologies)，使用偶联anti-CD3/CD28磁珠激活培养和扩增T细胞，细胞培养体系采用x-vivo 15(5％FBS，300IU/mL rhIL-2)，细胞培养在37℃，5％CO 2培养箱连续培养。
实施例2细胞培养及构建
从ATCC获得表达MSLN的细胞系：OVCAR3(人卵巢癌细胞系，ATCC HTB-161)，HCC70(人乳腺癌细胞系)，MDA-MB-231(人乳腺癌细胞系),其中MDA-MB-231MESO在MDA-MB231细胞基础上通过慢病毒将MSLN抗原转入，再经过单克隆筛选后获得高表达MSLN的稳定细胞系。以上细胞按照ATCC指引配制培养基并培养细胞。
实施例3 CAR结构设计与转导
本实施例示例性的采用Meso-CAR基本结构，即靶向间皮素的CAR结构进行表达IL-15/IL-15Rα复合物的CAR-T细胞构建。
本实施例构建了二代和四代CAR，结构如表1所示。
CAR的核心结构包括CD8胞外信号肽，P4scFv(特异性靶向间皮素的scFv)，CD8来源的铰链区和CD8/CD28跨膜区，并采用CD28胞内段共刺激信号，构建了3种Meso-CAR。
表1
命名 共刺激信号 胞内激活区 增强子
Meso-1 CD28 3ζ N/A
Meso-E1 CD28 3ζ IL-15/IL-15Rα
Meso-E1m1 CD28 3ζ IL-15突变体/IL-15Rα
将3种Meso-CAR基因分别克隆至FUW慢病毒载体中，与慢病毒包装质粒pMD2.G(Addgene,Plasmid#12259)和psPAX2(Addgene,Plasmid#12260)使用PEI转染试剂转入293T细胞中，表达载体，分别收集48小时和72小时病毒，超离浓缩后，感染激活T细胞。
结果显示，利用三种Meso-CAR基因，成功构建了慢病毒载体。
实施例4 CAR-T细胞制备-慢病毒感染、CAR阳性率检测和体外增殖
分离纯化的原代T细胞在激活48小时后，分别加入实施例3中浓缩的慢病毒，细胞感染72小时后检测CAR阳性率，分别使用生物素标记MSLN抗原作为一抗，APC-Streptavidin(BD)作为二抗，以及anti-IL-15Rα抗体检测scFv和IL-15Rα表达。
流式检测结果如图4所示
转染后T细胞置于37℃，5％CO 2培养箱中连续培养，隔天补液，在第10天收获细胞，冻存、计数，计算体外增殖率，E1 CAR-T细胞与E1m1 CAR-T细胞的增殖无明显差异。
实施例5体外杀伤实验和细胞因子检测
对实施例4中收获的T细胞进行体外杀伤实验。通过RTCA方法，以1x10 4/孔将靶细胞铺于96孔RTCA平板，培养18小时后，CAR T与靶细胞OVCAR3按比例1:1,1:3,1:6共培养，连续培养1-2天，实时记录靶细胞的生长状况，检测靶细胞的存活率，计算CAR-T细胞的杀伤效率。连续培养1-2天后，将RTCA平板取出，离心取共培养上清液，冻存-20℃。
结果如图5A所示，Meso-1,Meso-E1与Meso-E1m1 CAR-T细胞与OVCAR3靶细胞共培养下，有明显杀伤，且三者之间杀伤无明显差异，NT(Non-transduced T cell)分别与肿瘤细胞进行共培养时(E:T＝1:1,1:3,1:6)对肿瘤细胞没有明显杀伤。
按说明书推荐方法，使用HumanTh1/Th2Cytokine kit Ⅱ(BD，Cat.551809)检测共培养上清细胞因子，先将共培养上清液解冻，配置mixed capture beads和Human Th1/Th2-Ⅱ PE Detection,与样品或标准品避光孵育3小时，孵育后，300g离心5min，甩掉上清。加入100μl washing buffer重悬，震荡5min，流式细胞仪上机检测。用FCAP Array v.3软件数据分析。结果见图5B所示，Meso-1,Meso-E1与Meso-E1m1 CAR-T细胞与OVCAR3靶细胞共培养的上清中，IFN-γ含量无明显差异；Meso-E1与Meso-E1m1 CAR-T细胞与OVCAR3靶细胞共培养上清，TNF-α含量明显低于Meso-1。
结果如图6A所示，Meso-1,Meso-E1与Meso-E1m1CAR-T细胞与MDA-MB-231-MESO靶细胞共培养下，有明显杀伤，且三者之间杀伤无明显差异,NT(Non-transduced T cell)分别与肿瘤细胞进行共培养时(E:T＝3:1,1:1,1:3)对肿瘤细胞没有明显杀伤。用HumanTh1/Th2 Cytokine kit Ⅱ(BD)检测共培养上清细胞因子，结果如图6B,Meso-E1与Meso-E1m1 CAR-T细胞与MDA-MB-231-MESO靶细胞共培养上清，TNF-α含量明显低于Meso-1。
实施例6体内药效研究-1
选取NOD小鼠，皮下注射5E6 HCC70细胞，连续检测肿瘤负荷，待肿瘤处于高速生长时，分组，每组2-3只小鼠，分组后一天尾静脉注射200uL DPBS/鼠，5E6 E1/E1m1-CAR-T/鼠，CAR-T细胞注射后第1天，取少量小鼠血检测CAR-T细胞体内存活数量，其后每周取一次血样，检测CAR-T细胞各种表型，每周两次检测皮下瘤大小。
结果如图7A所示，注射Meso-E1 CAR-T细胞与Meso-E1m1 CAR-T细胞的小鼠在D10出现较为明显的肿瘤体积下降；在D17、D21、D24继续观察动物肿瘤体积，E1组动物肿瘤体积回升，E1m1组继续下降。
小鼠体重如图7B所示，注射CAR-T细胞后，Meso-E1m1组小鼠的体重基本没有变化。
实施例7体内药效研究-2
选取NOD小鼠，皮下注射5E6 HCC70细胞，连续检测肿瘤负荷，待肿瘤处于高速生长时，分组，每组2-3只小鼠，分组后一天尾静脉注射200uL DPBS/鼠，高(表示HD)低剂量(表示LD)两组CAR-T,每只小鼠注射剂量分别为5E6或2E6，CAR-T分别为Meso-1/Meso-E1m1，CAR-T细胞注射后第1天，取少量小鼠血检测 CAR-T细胞体内存活数量，其后每周取一次血样，检测CAR-T细胞各种表型，每周两次检测皮下瘤大小。
结果如图8A所示，注射Meso-1 CAR-T细胞与Meso-E1m1 CAR-T细胞的小鼠在D10开始出现较为明显的肿瘤体积下降；在D17、D21、D24继续观察动物肿瘤体积，高、低剂量Meso-1组动物肿瘤体积回升，高剂量E1m1组动物肿瘤体积没有上升，低剂量E1m1组动物在D40开始肿瘤体积有复发，与Meso-1组比较，肿瘤体积明显小。
如图8B所示，各组小鼠体重无明显变化。
如图8C所示，注射CAR-T细胞后，各组中单只小鼠的肿瘤体积变化，与Meso-1组比较，高剂量Meso-E1m1组动物肿瘤体积无复发，肿瘤体积均一并明显小于Meso-1组。
实施例8体外药效研究—多轮杀伤
对实施例4中收获的T细胞进行体外多轮杀伤实验。实验方法见示意图(图9A)，将CAR-T细胞与靶细胞按1：3共孵育，连续培养2-3天，在显微镜下观察靶细胞的生长状况，当其中一种CAR-T细胞将靶细胞全部裂解时，取一半T细胞检测T细胞的表型及细胞数，并用胰酶消化靶细胞，将其计数作为靶细胞残余值，CAR-T细胞杀伤效率的计算公式：CAR-T细胞的杀伤效率＝(肿瘤only组-实验组)/肿瘤only组*100％，余下一半细胞继续与新的靶细胞共培养，按上述方法继续连续杀伤。
结果如图9B所示，各组CAR-T与OVCAR3共培养下(E:T＝1:3)，Meso-E1/E1m1在第三轮连续培养后显示明显杀伤优势，如图9C,显示Meso-E1/E1m1在每一轮连续杀伤后，根据CAR阳性率和T细胞的计数，计算CAR-T细胞值，结果显示Meso-E1/E1m1连续扩增明显多于Meso-1 CAR-T,Meso-E1/E1m1之间无显著差异。
结果如图9D所示，各组CAR-T与MDA-MB231-Meso共培养下(E:T＝1:3)，Meso-E1/E1m1在第四轮连续培养后显示明显杀伤优势。如图9E,显示Meso-E1/E1m1在每一轮连续杀伤后，根据CAR阳性率和T细胞的计数，计算CAR-T细胞值，结果显示Meso-E1/E1m1连续扩增明显多于Meso-1 CAR-T,Meso-E1/E1m1之间无显著差异。
实施例9体外药效研究—TGF-β存在下多轮杀伤
对实施例4中收获的T细胞进行体外多轮杀伤实验。实验方案如图9A所示。将CAR-T细胞与靶细胞按1：5共孵育，培养过程中加入不同浓度重组TGF-β1，每两天补加一次，连续培养2-3天，实时记录靶细胞生长状况，当其中一种CAR-T细胞将靶细胞全部裂解时，取一半T细胞检测T细胞的表型，细胞数，胰酶消化 靶细胞后，计数即得到CAR-T细胞的杀伤效率＝(对照组-实验组)/对照组*100％，对照组为肿瘤only组，余下一半细胞继续与靶细胞共培养，按上述方法继续连续杀伤。
结果如图10A所示，各组CAR-T与OVCAR3共培养下(E:T＝1:5)，Meso-E1m1在第二(R2)、四轮(R4)连续培养后显示明显杀伤优势。表1显示每轮杀伤后根据细胞阳性率，T细胞计数值和稀释系数，计算出Meso-1、Meso-E1m1 CAR-T细胞数。图10B为根据表2和表3所绘制CAR-T细胞扩增曲线，结果显示Meso-E1m1在连续杀伤第四轮，CAR-T细胞扩增明显比Meso-1多；Meso-E1m1在添加1.25、2.5、5ng/mL TGFβ1培养条件下，在连续杀伤第二轮，CAR-T细胞扩增明显比Meso-1多17倍、19倍、15倍；Meso-E1m1在添加1.25ng/mL TGFβ1培养条件下,连续杀伤第四轮有明显杀伤靶细胞能力明显优于Meso-1，CAR-T细胞的扩增也明显多于相同培养条件的Meso-1 CAR-T，实验结果显示Meso-E1m1 CAR-T能有效减少TGFβ抑制T细胞增殖、活化能力，从而减少TGFβ的免疫抑制作用。
表2 Meso-1
Figure PCTCN2021143209-appb-000003
表3 Meso-E1m1
Figure PCTCN2021143209-appb-000004
实施例10 Annexin V染色检测细胞凋亡
对实施例4中收获的T细胞进行体外杀伤实验，CAR-T细胞与靶细胞(MDA-MB231-Meso)按1：2共孵育，连续培养3天后，将CAR-T细胞移到新鲜的靶细胞中，继续培养3天后，检测CAR-T细胞的凋亡情况，培养过程中分两组分别为不添加/添加图示浓度的TGF-β1组。
结果如图10C所示，Annexin V和7-AAD双阴细胞，Meso-E1m1 CAR-T活细胞 比例最高为60％，加入不同浓度TGF-β，其活率也没有明显变化，而Meso-1 CAR组在TGF-β1培养条件下，凋亡细胞随着TGF-β1浓度而升高，早期凋亡和晚期细胞明显增多，实验结果显示Meso-E1m1 CAR-T能有效减少TGFβ促进T细胞凋亡能力，从而减少TGFβ的免疫抑制作用。
实施例11 CAR-T细胞与PBNK细胞共孵育
对实施例4中收获的T细胞与体外培养的自体PBNK细胞按1：1比例共培养，PBNK细胞先用CFSE标记，分别检测48小时，96小时的PBNK细胞数量，采用流式检测方法，检测培养体系中FITC +细胞。
CFSE标记方法：PBNK细胞离心后用PBS重悬至1E6/mL，加入终浓度4uM CFSE,室温孵育2分钟，再加入培养基终止反应。
自体PBNK来源于T细胞同一Donor的单核细胞，使用anti-CD56microbeads分离、纯化，具体方法见美天旎的protocol。
实验结果如图11所示，E1 CAR-T细胞与E1m1 CAR-T细胞与PBNK细胞共培养48-72小时，与NT细胞相比，E1和E1-m1 CAR-T细胞能明显促进PBNK细胞的扩增。
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 (15)

  1. 一种嵌合抗原受体(CAR)构建物，其特征在于，所述CAR构建物的结构如下式I或II所示，
    X-A-E  (I)
    E-A-X  (II)
    式中，
    各“-”独立地为连接肽或肽键；
    X为靶向肿瘤抗原的CAR；
    A为自剪切元件；
    E为IL-15/IL-15Rα复合体。
  2. 如权利要求1所述的CAR构建物，其特征在于，所述的IL-15/IL-15Rα复合体的结构如下式III所示，
    L’-M-I-R  (III)
    式中，
    各“-”独立地为连接肽或肽键；
    L’为无或信号肽；
    M为IL-15或其突变体；
    I为柔性接头；
    R为IL-15Rα。
  3. 如权利要求2所述的CAR构建物，其特征在于，所述的IL-15突变体为IL-15N72D，其氨基酸序列如SEQ ID NO.:2所示。
  4. 如权利要求2所述的CAR构建物，其特征在于，所述的IL-15Rα为完整的IL-15Rα元件，其氨基酸序列如SEQ ID NO.:3所示。
  5. 如权利要求2所述的CAR构建物，其特征在于，所述的IL-15/IL-15Rα复合体的氨基酸序列如SEQ ID NO.:5或6所示。
  6. 如权利要求1所述CAR构建物，其特征在于，所述的X(CAR)的结构如下式IV所示，
    L-scFv-H-TM-C-CD3ζ  (IV)
    各“-”独立地为连接肽或肽键；
    L为无或信号肽；
    scFv为靶向肿瘤抗原的抗体单链可变区；
    H为无铰链区；
    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。
  7. 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)构建物，或者，
    所述的核酸分子包含编码靶向肿瘤抗原的CAR的第一核酸分子和编码IL-15/IL-15Rα复合体的第二核酸分子。
  8. 一种载体，其特征在于，所述的载体含有权利要求7所述的核酸分子。
  9. 一种工程化的免疫细胞，其特征在于，所述的免疫细胞表达有权利要求1所述的CAR构建物，或者
    所述的免疫细胞表达有靶向肿瘤抗原的CAR和IL-15/IL-15Rα复合体。
  10. 如权利要求9所述的免疫细胞，其特征在于，所述靶向肿瘤抗原的CAR和IL-15/IL-15Rα复合体独立地表达于所述免疫细胞的细胞膜上。
  11. 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构建物、权利要求3所述的核酸分子、权利要求4所述的载体、或权利要求5所述的免疫细胞，以及药学上可接受的载体。
  12. 一种权利要求1所述的嵌合抗原受体构建物、权利要求7所述的核酸分子、权利要求8所述的载体、或权利要求9所述的免疫细胞、或权利要求11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。
  13. 一种制备权利要求9所述的工程化的免疫细胞的方法，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7所述的核酸分子或权利要求8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。
  14. 一种IL-15/IL-15Rα复合体的用途，用于制备一制剂，所述制剂用于增强CAR-T细胞的持久性和/或增强CAR-T细胞的细胞毒性，
    其中，所述的IL-15/IL-15Rα复合体的结构如下式III所示，
    L’-M-I-R  (III)
    式中，
    各“-”独立地为连接肽或肽键；
    L’为无或信号肽；
    M为IL-15或其突变体；
    I为柔性接头；
    R为IL-15Rα，
    其中，所述的IL-15Rα包含跨膜区和胞内区。
  15. 一种治疗疾病的方法，其特征在于，所述方法包括给需要治疗的对象施用适 量的如权利要求8所述的载体、如权利要求9所述的免疫细胞、或如权利要求11所述的制剂，其中所述疾病为癌症或肿瘤。
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