WO2022151959A1 - 靶向b7-h3的car-t细胞及其在急性髓系白血病治疗中的应用 - Google Patents

靶向b7-h3的car-t细胞及其在急性髓系白血病治疗中的应用 Download PDF

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WO2022151959A1
WO2022151959A1 PCT/CN2021/141338 CN2021141338W WO2022151959A1 WO 2022151959 A1 WO2022151959 A1 WO 2022151959A1 CN 2021141338 W CN2021141338 W CN 2021141338W WO 2022151959 A1 WO2022151959 A1 WO 2022151959A1
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cells
car
cell
present
tumor
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杨林
游凤涛
范双双
李亚芬
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博生吉医药科技(苏州)有限公司
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Definitions

  • the present invention relates to the field of biotechnology, and more particularly to a CAR-T cell targeting B7-H3 and its application in the treatment of acute myeloid leukemia.
  • AML Acute myeloid leukemia
  • hematopoietic tissue a highly heterogeneous malignant clonal disease originating from hematopoietic tissue, with high mortality and low long-term survival. It is easy to relapse and seriously endangers human health and life.
  • the treatment of AML has remained largely unchanged for decades, with chemotherapy and hematopoietic stem cell transplantation being the mainstays.
  • the classic chemotherapy regimen is still anthracycline combined with cytarabine. Although this regimen can achieve a relatively high remission rate, patients have a higher risk of drug resistance and recurrence.
  • AML can be divided into eight types, M0-M7.
  • the prognosis of patients with M5 subtype is significantly worse than that of other AML. Therefore, there is an urgent need to develop better treatments for AML, especially for the M5 subtype of AML.
  • Chimeric antigen receptor T (chimeric antigen receptor T, CAR T) cellular immunotherapy is to avoid the restriction of MHC and induce specific activation of T cells by giving T cells antibodies that can recognize each other with tumor cell surface antigens.
  • the T cells were better able to recognize and kill tumors than natural T cells.
  • CAR T therapy has made great progress in the invention of cancer treatment and is considered to be one of the most promising tumor treatment methods.
  • CAR T therapy has the best curative effect and is the most widely used in the treatment of ALL.
  • CAR targeting CD19 has achieved significant efficacy in the treatment of adult and pediatric ALL, with a remission rate as high as 90%.
  • CD123 and CD33 have also achieved good results in the CAR-T treatment of AML, but these two antigens are expressed in normal myeloid cells, which will inevitably cause toxic side effects to patients, which is still a problem that cannot be ignored at present. Therefore, it is very important to find a relatively safe and effective target, and the field needs to develop new therapeutic targets and therapeutic methods.
  • the object of the present invention is to provide a CAR-T cell targeting B7-H3 and its application in the treatment of acute myeloid leukemia.
  • a chimeric antigen receptor whose antigen binding domain (ie, scFv) comprises a heavy chain variable region and a light chain variable region ,
  • the light chain variable region includes the following complementarity determining region CDRs:
  • the antigen binding domain of the chimeric antigen receptor includes the antibody heavy chain variable region shown in SEQ ID NO:1, and the antibody light chain variable region shown in SEQ ID NO:2.
  • variable region of the antibody heavy chain and the variable region of the antibody light chain are linked by a linking peptide.
  • the structure of the antigen binding domain is shown in the following formula I or II:
  • V H is the variable region of the antibody heavy chain
  • VL is the variable region of the antibody light chain
  • "-" is the connecting peptide or peptide bond.
  • the structure of the antigen binding domain is shown in formula II ( VH - VL ).
  • amino acid sequence of VH is shown in SEQ ID NO:1
  • amino acid sequence of VL is shown in SEQ ID NO:2.
  • the connecting peptide is 1-4 consecutive sequences shown in SEQ ID NO: 4 (GGGGS), preferably 2-4, more preferably 3.
  • the antigen binding domain binds to B7-H3, preferably human B7-H3.
  • variable region of the heavy chain and the variable region of the light chain of the antigen binding domain are derived from a humanized antibody.
  • the structure of the chimeric antigen receptor is shown in the following formula III:
  • L is none or a signal peptide sequence
  • scFv is an scFv targeting B7-H3;
  • H is the hinge region
  • TM is the transmembrane domain
  • C is a costimulatory signal molecule
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇ .
  • 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 macrophage colony stimulating factor-derived signal peptide.
  • the H is a hinge region selected from the following histones: CD8, CD28, CD137, Fc, 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 CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.
  • the TM is a CD8-derived transmembrane region.
  • the C is a costimulatory signal molecule of a protein selected from the group consisting of 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.
  • C is a costimulatory signal molecule derived from 4-1BB.
  • the CAR (preferably C-terminal) further comprises a cell suicide element.
  • the CAR is linked to the cell suicide element through a self-cleaving element.
  • the cell suicide element is linked to the CD3 ⁇ of the CAR through T2A.
  • the cell suicide element is selected from the group consisting of HSV-TK, iCasp9, ⁇ CD20, mTMPK, ⁇ CD19, RQR8, EGFRt, or a combination thereof.
  • the cell suicide element is tEGFR.
  • amino acid sequence of the tEGFR is shown in SEQ ID NO:5.
  • amino acid sequence of the CAR is shown in SEQ ID NO:3.
  • nucleic acid molecule encoding the chimeric antigen receptor (CAR) of the first aspect of the present invention.
  • nucleic acid molecules are isolated.
  • 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 Express the CAR described in the first aspect of the present invention.
  • the host cells include eukaryotic cells and prokaryotic cells.
  • the host cell includes Escherichia coli.
  • an engineered immune cell is provided, and the immune cell expresses the CAR described in the first aspect of the present invention.
  • 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 immune cells express exogenous cell suicide elements.
  • the CAR and the cell suicide element are co-expressed in the immune cells.
  • the engineered immune cells include T cells or NK cells, preferably (i) chimeric antigen receptor T cells (CAR-T cells); or (ii) chimeric antigen Recipient NK cells (CAR-NK cells).
  • T cells or NK cells preferably (i) chimeric antigen receptor T cells (CAR-T cells); or (ii) chimeric antigen Recipient NK cells (CAR-NK cells).
  • a preparation comprising the chimeric antigen receptor according to the first aspect of the present invention, the nucleic acid molecule according to the second aspect of the present invention, and the third aspect of the present invention.
  • the carrier, or the immune cell according to the fifth aspect of the present invention, and a pharmaceutically acceptable carrier are provided.
  • 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 chimeric antigen receptor according to the first aspect of the present invention, a nucleic acid molecule according to the second aspect of the present invention, a vector according to the third aspect of the present invention, or the present invention
  • the use of the immune cells described in the fifth aspect or the preparation described in the sixth aspect of the present invention is for preparing 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 B7-H3 positive tumor, preferably a tumor with high expression of B7-H3.
  • the medicament or preparation is used for the prevention and/or treatment of acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • 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.
  • a ninth aspect of the present invention there is provided a method for preparing engineered immune cells, the immune cells expressing the CAR described in the first aspect of the present invention, the method comprising the following steps:
  • the engineered immune cells are CAR-T cells or CAR-NK cells.
  • the method further includes the step of testing the function and effectiveness of the obtained engineered immune cells.
  • a method for treating a disease comprising administering an appropriate amount of the carrier of the third aspect of the present invention, the immune cells 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 cells 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.
  • Figure 1 shows the expression rate of B7-H3 antigen on the surface of AML tumor cells. Among them, the expression of B7-H3 on the surface of six AML tumor cells was detected by flow cytometry.
  • Figure 2 shows the structure of B7-H3-CAR and its functional verification.
  • Figure 2A shows a schematic diagram of the structure of B7-H3-CAR, which contains the EF1 promoter, B7-H3 single-chain antibody, CD8 transmembrane domain, co-stimulatory domain 41-BB, and intracellular signaling domain CD3 ⁇ .
  • Figure 2B shows the expression of B7-H3-CAR on the surface of Jurkat cells after transfection analyzed by flow cytometry.
  • Figure 2C shows the expression of CD25 and CD69 on the cell surface.
  • CD25-APC and CD69-APC antibodies were used to detect Jurkat cells and B7-H3-CAR Jurakt, respectively. Expression of CD25 and CD69 on the cell surface.
  • Figure 3 shows the specific killing of B7-H3-CAR T cells against the B7-H3 + AML cell line.
  • Figure 3A shows the positive rate of B7-H3-CAR T cells. Among them, by flow detection analysis, the positive rate of B7-H3-CAR T cells was about 51.84%.
  • Figure 3B shows the expression of B7-H3-CAR in T cells analyzed by Western bolt. Among them, T (lane 1) and B7-H3-CAR T (lane 2) cells were extracted for protein and then separated by SDS-PAGE, incubated with CD3 ⁇ chain-specific mAb and HRP-goat anti-mouse IgG, and detected by chemiluminescence immunoblotting .
  • Figure 3D shows the secretion of cytokines (IL-2, TNF, INF- ⁇ and granzyme B) in the supernatant after co-incubation. ****P ⁇ 0.0001, ***P ⁇ 0.001, **P ⁇ 0.01.
  • Figure 4 shows that B7-H3-CAR T cells have antitumor activity in AML-transplanted mice.
  • Figure 4A shows a schematic diagram of an AML transplantation model in NSG mice. Among them, 6-8 weeks old NSG female mice, 4 mice in each group, a total of three groups (PBS group, T cell group, B7-H3-CAR T cell group), were injected with 5 ⁇ 105 luciferase-GFP via tail vein -HEL cells, followed by tail vein injection of 1 ⁇ 10 7 equal volumes of effector cells (PBS is an equal volume of cell-free liquid) on the fifth day, respectively.
  • Figure 4B shows an in vivo image of mice showing leukemia progression in mice.
  • Figure 4C shows a statistical graph of the average body weight of mice.
  • Figure 4D shows a graph of mouse survival curves.
  • Figure 4E shows a graph of mouse survival time statistics. **P ⁇ 0.01, *P ⁇ 0.1.
  • Figure 5 shows that B7-H3-CAR T cells can clear tumor cells in AML-transplanted mice.
  • Figure 5A shows a schematic diagram of an AML engraftment model in NSG mice. Among them, 6-8 weeks old NSG female mice, 3 mice in each group, a total of 3 groups (PBS group, T cell group, B7-H3-CAR T cell group), 5 *105 HEL-luciferase was injected into the tail vein -GFP cells, followed by tail vein injection of 2*10 7 equal volumes of effector cells (PBS is an equal volume of cell-free liquid) on day 2, respectively.
  • Figure 5B shows an in vivo image of a mouse showing disease progression in vivo.
  • Figure 5C shows the proportion of tumor cells.
  • FIG. 5D shows tumor cell infiltration.
  • Figure 6 shows the safety validation of B7-H3-CAR T cells.
  • Figure 6A shows the statistical graph of RBC, WBC, PLT and HGB after five different classification detections in peripheral blood of mice.
  • Figure 7 shows AML mouse model modeling.
  • Figure 7A shows the positive rate of luciferase-GFP-HEL cells by flow cytometry.
  • Figure 7B shows a schematic diagram of the establishment of a mouse AML model. Among them, 6-8 week old NSG female mice were observed after 5*10 5 luciferase-GFP-HEL cells were injected into the tail vein.
  • Figure 7C shows tumor cells in bone marrow, peripheral blood, spleen, liver and kidney in tumor-bearing mice by flow cytometry.
  • Figure 8 shows the positive rate of B7-H3-CAR T cells.
  • Figure 8A shows the positive rate of B7-H3-CAR T cells in the first mouse experiment by flow cytometry.
  • Figure 8B shows the positive rate of B7-H3-CAR T cells in the second mouse experiment by flow cytometry.
  • B7-H3-CAR-T cells can significantly inhibit the growth of AML tumor cells in mice, significantly prolong the survival period of mice, and have significant anti-tumor effects in vivo.
  • the B7-H3-CAR-T cells constructed by the invention can be used as a new therapeutic method for targeted treatment of AML, and have great clinical application prospects. The present invention is completed on this basis.
  • 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).
  • B7-H3 also known as CD276, was discovered in 2001 and is mostly expressed on the cell surface, such as activated dendritic cells, monocytes, T cells, B cells and NK cells. Studies have shown that B7-H3 can stimulate T cells expansion and killing, and may selectively stimulate T-cell signaling receptors. At the same time, B7-H3 is a tumor-associated antigen that plays an important role in tumor progression and metastasis. The survival rate of patients with negative B7-H3 protein expression is higher than that of patients with positive B7-H3 protein expression. Many studies have shown that B7-H3 is abnormally expressed in human malignant tumors, including melanoma, leukemia, breast cancer, prostate, etc.
  • B7-H3 is one of the most strongly expressed B7 family molecules in AML, with the highest expression in M3 and M5 subtypes.
  • B7-H3 is not expressed in immune cells.
  • most of the literatures related to B7-H3 have proved that B7-H3 is related to tumor progression and metastasis.
  • B7-H3 has been found to be highly expressed on tumor-associated stromal cells, fibroblasts, and tumor angiogenesis. Therefore, treatment targeting B7-H3 is expected to break the inhibition of drugs by the tumor microenvironment. Novel targets that are receiving increasing attention.
  • B7-H3-CAR T cells have shown encouraging tumor-killing activity in preclinical studies of various solid tumors such as pancreatic cancer, childhood neuroblastoma, and osteosarcoma.
  • the present invention constructs a B7-H3-CAR-T cell with 4-1BB as a costimulatory domain and carrying a safety switch-truncated EGFR molecule (EGFRt), which is used in vitro and mouse models for AML tumor cell lines. The test showed good cytotoxicity.
  • EGFRt safety switch-truncated EGFR molecule
  • 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.
  • 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 B7-H3.
  • 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.
  • the antigen binding domain is fused to the intracellular domain in combination with the 4-1BB signaling domain, and the CD3 ⁇ signaling domain.
  • antigen-binding domain and “single-chain antibody fragment” each 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.
  • 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 the size of a complete antibody.
  • Single chain antibodies are preferably one amino acid chain sequence encoded by one nucleotide chain. As a preferred mode of the present invention, the scFv specifically recognizes B7-H3.
  • 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.
  • the intracellular domains in the CAR of the present invention include the signaling domain of 4-1BB and the signaling domain of CD3 ⁇ .
  • 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 techniques are well known in the art and are described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.
  • 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 cell 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 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 tumor cell marker B7-H3, 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-B7-H3 CAR-T cells elicited specific immune responses against B7-H3-expressing 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 CAR-T cells of the present invention can be used to treat acute myeloid leukemia.
  • 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 noted 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 106 to 1 x 1010 modified T cells of the invention can be administered to a patient, eg, by intravenous infusion, per treatment or per course of treatment.
  • the B7-H3-CAR-T cells constructed in the present invention have a significant in vitro anti-tumor effect on AML cell lines.
  • the B7-H3-CAR-T cells constructed in the present invention have a significant in vivo anti-tumor effect on the AML cell line HEL tumor-bearing mice.
  • KG-1, MOLM-16 and Jurkat cells were purchased from the American Type Culture Collection (ATCC; Manassas, Virginia, USA), and HEL, THP-1, HL-60 and AML5 cell lines were purchased from Soochow University Gift from the laboratory of Mr. Zhao Yun from Tang Zhongying Hematology Research Center.
  • the medium used for HEL cells, Jurkat cells and AML5 cells was RP-MI1640 supplemented with 10% FBS.
  • the medium used for KG-1 cells was IMDM supplemented with 10% FBS.
  • Normal human peripheral blood was taken, and peripheral blood mononuclear cells (Peripheral Blood Mononuclear Cell, PBMC) were extracted by Ficoll method, T cells were activated by Transact, and cultured in TexMACSTMGMP medium supplemented with IL-7 and IL-15. All cells were cultured in a 37°C cell incubator with 5% CO 2 .
  • the extracted T cells were activated in vitro for 48 hours and then transfected, and the toxicity test was performed when the cell proliferation was sufficient.
  • This application constructs a second-generation CAR.
  • the B7-H3-scFv sequence is constructed into a lentiviral vector, including B7-H3-scFv, CD8 hinge region and transmembrane region, 41BB intracellular region sequence and CD3 ⁇ sequence, human EGFR sequence.
  • Lentiviruses were prepared according to conventional methods in the prior art. After 48 hours of T cell activation, Jurkat cells were placed in a 48-well plate (5 ⁇ 10 5 cells/well), 50 ⁇ l of virus was added, and the final system was 100 ⁇ l, and 1000 ⁇ l of culture medium was added 15 hours after transfection.
  • AML cells were collected, namely AML5, KG-1, HEL, HL-60, THP-1 and MOLM-16.
  • B7-H3-CAR Jurkat cells About 2 ⁇ 10 6 cells were taken out of the cultured B7-H3-CAR Jurkat cells, Jurkat cells, B7-H3-CAR T cells and T cells, and 100ul of EGFR antibody diluted 1:1000 (final concentration of antibody: 1ug/ml) were incubated at 37°C for 20min, washed twice with PBS, and used Jurkat cells or T cells as control cells to analyze and detect B7-H3-CAR Jurkat and B7-H3-CAR T cells by flow cytometry The positive rate of B7-H3-CAR.
  • KG-1, HEL and AML5 cells were incubated with CD25-APC and CD69-APC antibodies for 20 min, and flow cytometry was used to detect the levels of CD25 and CD69.
  • T cells and B7-H3-CAR T cells were taken each, and the lysed products were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane (microwell).
  • PVDF polyvinylidene fluoride
  • CBA flow bead array
  • Model construction was carried out using 6-8 week NOD-Prkdcscidll2rgtm1/Bcgen (B-NSG) female mice purchased from Biositu. Mice were injected with 5 ⁇ 10 5 HEL-luciferase-GFP cells through the tail vein. After 2 or 5 days, the tumor burden was detected using the IVIS imaging system (IVIS-spectrum). The intraperitoneal injection of 150 mg/kg D-luciferin substrate was recorded for 10 min. After the bioluminescence of the mice, 1 ⁇ 10 7 equal volume of effector cells (PBS is an equal volume of cell-free liquid) were injected through the tail vein of the mice respectively. 100 ⁇ l of mouse peripheral blood, femoral bone marrow, ground organs, and red blood cells were lysed, and the cells were incubated with anti-human CD45 antibody for flow cytometry analysis to detect tumor cells.
  • PBS is an equal volume of effector cells
  • mice After the mice were sacrificed, the spleen and ovarian tissues were taken out, embedded in paraffin, and sliced (thickness 3 ⁇ m) for immunohistochemical analysis.
  • the paraffin sections were dewaxed and rehydrated, immersed in 0.01M citrate buffer, microwaved for 5 min, and then blocked with methanol solution containing 0.3% hydrogen peroxide.
  • the paraffin sections were dewaxed and then stained with hematoxylin-and-eosin to observe the pathological changes of tumor tissue.
  • the flow cytometry results are shown in Figure 1.
  • the expression rate of B7-H3 in HEL cells was 96.4%
  • the expression rate of B7-H3 in AML5 cells was 52.69%
  • the expression rate of B7-H3 in THP-1 cells was 40.67%
  • the expression rate of B7-H3 in MOLM-16 cells was 40.67%.
  • the expression rate of H3 was 25.35%
  • the expression rate of B7-H3 in HL-60 cells was 16.43%
  • the expression rate of B7-H3 in KG-1 cells was 11.74%.
  • HEL, AML5, and KG-1 cells were selected for later experiments.
  • a CAR vector targeting B7-H3 was constructed. It is a second-generation CAR containing B7-H3 single-chain antibody and 41BB co-stimulatory domain. It co-expresses EGFR as the detection tag of the CAR.
  • the specific structure is shown in Figure 2A. The amino acids are as follows SEQ ID NO:3. After packaging the lentivirus, it was transfected into Jurkat cells to construct B7-H3-CAR-Jurkat cells that can stably express B7-H3-CAR. .
  • B7-H3-CAR-T cells were prepared, and the positive rate of flow detection was about 51.84% (Figure 3A).
  • Western blotting revealed expression of B7-H3-CAR fusion protein in B7-H3-CAR T cells ( Figure 3B), including two endogenous CD3 ⁇ of 16kD and 28kD expressed by all T cells (lane 1). , lane 2), and one CAR-T cell expressed exogenous CD3 ⁇ (56kD of lane 2).
  • B7-H3-CAR T cells After co-incubating T cells and prepared B7-H3-CAR T cells with HEL, AML5 and KG-1 cells at an effect-to-target ratio of 1:1 for 48 h, the apoptosis of tumor cells was detected.
  • the results showed that, compared with T cells, B7-H3-CAR T cells had a good toxic effect on tumor cells with high and moderate expression of B7-H3, but not on tumor cells with low expression of B7-H3. significant difference. This indicated that B7-H3-CAR T cells had a strong toxic effect on B7-H3-expressing AML cells (Fig. 3C).
  • Example 4 B7-H3-CAR T cells can prolong the survival of tumor-bearing mice
  • luciferase-GFP-HEL cells were successfully constructed (Fig. 7A).
  • an AML mouse tumor model was established. 6-8 week old NSG female mice were taken, and 5 ⁇ 10 5 luciferase-GFP-HEL cells were injected into the tail vein, and the mice were observed. About 20 days later, depression, paralysis of hind limbs, body weight loss and weight loss occurred successively (Fig. 7B). After natural death, the bone marrow, peripheral blood, spleen, liver and kidney of the mice were taken to analyze the proportion of tumor cells by flow cytometry (Fig. 7C). Experiments show that AML model mice can be successfully established by injecting 5 ⁇ 10 5 tumor cells into the tail vein of mice.
  • the luciferase-GFP-HEL cells were tail vein injected into 12 (divided into 3 groups of 4) B-NSG mice ( Figure 4A), each mouse was injected with 5 ⁇ 10 5 cells, the first Five-day in vivo imaging of mice detected a high proportion of tumor cells in mice, which is equivalent to a middle-advanced AML case model. After that, the same amount of PBS/T/B7-H3-CAR T (positive rate) was injected into the tail vein. About 58.19%, as shown in Figure 8A) cells. The survival period of the mice was observed, and the tumor burden in the mice was detected by intravital imaging every few days.
  • the intravital imaging showed that the tumor progression in the mice in the B7-H3-CAR T group was significantly weaker than that in the other two groups ( Figure 4B). ).
  • the average body weight of the mice in the PBS group and the T group had plummeted at about 30 days, and the mice died one after another, while the weight of the mice in the B7-H3 group was still close to flat (Fig. 4C). After all the mice died, the survival time was counted.
  • the results showed that the survival time of the mice in the B7-H3-CAR T group was significantly longer than that in the PBS group and the T cell group, and there was a statistical difference (Figure 4D, Figure 4E).
  • B7-H3-CAR T cells can effectively inhibit the growth of tumor cells in tumor-bearing mice, and B7-H3-CAR T cells have a significant anti-tumor effect on B7-H3-positive AML cells in vivo.
  • Example 5 B7-H3-CAR T cells can reduce tumor burden in peripheral blood and bone marrow of tumor-bearing mice
  • FIG. 5A To further demonstrate the in vivo antitumor effect of B7-H3-CAR T cells, another batch of mouse models was established (Fig. 5A). The day after tumor cell injection, intravital imaging showed tumor cell infiltration into the bone marrow, indicating successful modeling. Then 2 ⁇ 10 7 T cells and B7-H3-CAR T cells (CAR positive rate of 52.82%, Figure 8B) were injected into mice, respectively, followed by in vivo imaging every 4-5 days, the results showed that, Compared with the mice in the PBS group and the T group, the tumor growth of the mice in the B7-H3-CAR T group was significantly inhibited (Fig. 5B).
  • mice in the PBS group developed symptoms such as weight loss, lethargy, and quadriplegia, so all mice were sacrificed uniformly, peripheral blood and bone marrow cells were collected, and the proportion of tumor cells was detected by flow cytometry (Figure 5C). .
  • the results showed that the tumor burden of the mice in the B7-H3-CAR T group was significantly lower than that of the other two groups of mice.
  • Example 6 B7-H3-CAR T does not produce toxicity to normal cells
  • the scFv sequence used in the present invention has high affinity, faster dissociation constant, higher killing activity and safety. This sequence can recognize mouse B7-H3 molecules at the same time, so the safety of this sequence can be proved by detecting the activity of normal cells in mice.
  • the peripheral blood routine of the mice in the blank group, PBS group, T cell group and B7-H3-CAR T cell group (the mouse model of Example 5) was detected respectively.
  • the results showed that there was no significant difference between the B7-H3-CAR T group and the blank group in terms of white blood cells, red blood cells, platelets or hemoglobin ( Figure 5D).
  • the HE staining results also showed that compared with healthy mice, the heart, liver, kidney, and intestine of the B7-H3-CAR T cell group mice did not suffer from lesions. Therefore, the above experiments prove that the B7-H3-CAR T developed by the present invention is a highly safe CAR-T product.
  • AML is a very refractory aggressive hematopoietic stem cell malignancy.
  • AML patients have limited treatment options and poor prognosis. They cannot be cured after chemotherapy, and are prone to relapse. New treatment methods are urgently needed.
  • CAR T therapy has made great progress in both hematological and solid tumors, and is considered to be one of the most promising tumor treatments.
  • B7-H3 transcripts are widely expressed in human solid tumors and normal tissues, B7-H3 The protein is preferentially expressed only in tumor tissues. Some scholars also pointed out that B7-H3 protein is only expressed in a few tissues and cells, including activated lymphocytes and tumor cells. Therefore, although B7-H3 is only expressed in some AMLs, it is an excellent therapeutic target for AML because it does not cause toxicity to the hematopoietic system.
  • B7-H3-CAR T cells of the present invention can effectively and specifically kill AML cells expressing B7-H3 (Figure 3C).
  • B7-H3-CAR T cells also had obvious tumor clearance effects in mice.
  • In vivo imaging detection showed that the tumor cells in the B7-H3-CAR T group were significantly lower than those in the PBS group and the T cell group ( Figure 4B, 5B).
  • B7-H3-CAR T cells can significantly prolong the survival of mice (Fig. 4D, 4E) and reduce tumors in peripheral blood, bone marrow and tissues of mice. Cell ratio (Fig. 5C, 5D). Therefore, the B7-H3-CAR T of the present invention can be used for the treatment of AML patients who are refractory and relapsed and express the B7-H3 antigen.
  • the present invention not only proves that B7-H3-CAR T can effectively and specifically remove AML tumor cells expressing B7-H3 antigen in vitro and in vivo, but also proves that it has good safety, which is a good candidate for clinical use of B7-H3-CAR T.
  • H3-CAR T cells laid the foundation for the treatment of AML. Compared with the most used CD33-CAR T and CD123-CAR T cells, which require prior myeloablation before use, the use of B7-H3-CAR T not only has the potential to prolong the survival of patients with relapsed and refractory AML, but also simplifies It reduces the difficulty of practical use of CAR T therapy in clinical practice and reduces the risk of patients.

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Abstract

本发明提供了靶向B7-H3的CAR-T细胞及其在急性髓系白血病治疗中的应用。具体地，本发明提供了一种靶向B7-H3的CAR-T细胞，其包含靶向B7-H3的scFv，41BB共刺激信号分子和CD3ζ结构域。本发明的B7-H3-CAR-T细胞对B7-H3阳性的AML肿瘤细胞具有显著的特异性杀伤，动物实验结果显示，B7-H3-CAR-T细胞可以显著抑制小鼠体内AML肿瘤细胞的生长，明显延长小鼠的生存期，具有显著的体内抗肿瘤作用。本发明的B7-H3-CAR-T细胞可以作为一种靶向治疗AML的新型治疗方法，具有巨大的临床应用前景。

Description

靶向B7-H3的CAR-T细胞及其在急性髓系白血病治疗中的应用 技术领域
本发明涉及生物技术领域，更具体地涉及一种靶向B7-H3的CAR-T细胞及其在急性髓系白血病治疗中的应用。
背景技术
急性髓系白血病(Acute M yeloid Leukemia，AML)是急性白血病中常见的一种类型，是种起源于造血组织、高度异质性的恶性克隆性疾病，患者病死率高，长期生存率低，而且易复发，严重危害人类的健康和生命。几十年来，对AML的治疗方法基本没有变化，主要手段为化疗和造血干细胞移植。经典的化疗方案仍然是蒽环类药物联合阿糖胞苷，该方案治疗后虽然可以获得相对较高的缓解率，但患者发生耐药而复发的风险较高。
根据French-American-British(FAB)的AML形态学分型，可以将AML分为M0－M7八种类型。有研究表明，M3和M4亚型的患者预后优于其他亚型，而AML中最常见的(8；12)易位与良好的预后有关，但是其在M5亚型中却很少见。同时，也有报道显示M5亚型的患者预后明显差于其他AML。因此，开发更好的AML治疗方法尤为迫切，尤其是针对M5亚型的AML。
嵌合抗原受体T(chimeric antigen receptor T，CAR T)细胞免疫疗法是通过赋予T细胞能与肿瘤细胞表面抗原相互识别的抗体，从而避开MHC的限制，诱导T细胞特异性活化，改造过的T细胞相较于天然T细胞能够更好的识别肿瘤并杀死肿瘤。近几年，CAR T疗法在癌症治疗发明取得了巨大的进展，被认为是最有前景的肿瘤治疗方式之一。
目前，CAR T疗法在治疗ALL中的疗效最好，应用也最广泛。其中靶向CD19的CAR在治疗成人和儿童ALL中都取得显著的疗效，缓解率可高达90％。在AML的CAR-T治疗中CD123和CD33也取得了不错的成绩，但是这两个抗原在正常髓细胞中都有表达，难免会对患者产生毒副作用，仍然是目前不可忽视的一个问题。因此找到一个相对安全且疗效较佳的靶点至关重要，本领域需要开发新的治疗靶点和治疗方法。
发明内容
本发明的目的在于提供一种靶向B7-H3的CAR-T细胞及其在急性髓系白血病治 疗中的应用。
在本发明的第一方面，提供了一种嵌合抗原受体(CAR)，所述嵌合抗原受体的抗原结合结构域(即，scFv)包含重链可变区和轻链可变区，
SEQ ID NO:6所示的CDR1，
SEQ ID NO:7所示的CDR2，和
SEQ ID NO:8所示的CDR3；
且所述的轻链可变区包括以下互补决定区CDR：
SEQ ID NO:9所示的CDR1’，
SEQ ID NO:10所示的CDR2’，和
SEQ ID NO:11所示的CDR3’。
在另一优选例中。所述嵌合抗原受体的抗原结合结构域包括SEQ ID NO:1所示的抗体重链可变区，和SEQ ID NO:2所示的抗体轻链可变区。
在另一优选例中，所述抗体重链可变区和抗体轻链可变区通过连接肽相连。
在另一优选例中，所述的抗原结合结构域的结构如下式I或II所示：
V L-V H             (I)；V H-V L             (II)
其中，V H为抗体重链可变区；V L为抗体轻链可变区；“-”为连接肽或肽键。
在另一优选例中，所述的抗原结合结构域的结构如式II(V H-V L)所示。
在另一优选例中，所述V H的氨基酸序列如SEQ ID NO:1所示，V L的氨基酸序列如SEQ ID NO:2所示。
在另一优选例中，所述连接肽为1-4个连续的SEQ ID NO:4(GGGGS)所示的序列，较佳地2-4个，更佳地为3个。
在另一优选例中，所述抗原结合结构域结合于B7-H3，较佳地为人B7-H3。
在另一优选例中，所述抗原结合结构域的重链可变区和轻链可变区来源于人源化抗体。
在另一优选例中，所述嵌合抗原受体的结构如下式III所示：
L-scFv-H-TM-C-CD3ζ     (III)
其中，
L为无或信号肽序列；
scFv为靶向B7-H3的scFv；
H为绞链区；
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为巨噬细胞集落刺激因子来源的信号肽。
在另一优选例中，所述的H为选自下组蛋白的铰链区：CD8、CD28、CD137、Fc、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：CD28、CD3epsi lon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、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：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，C为4-1BB来源的共刺激信号分子。
在另一优选例中，所述CAR(优选地为C端)还包括细胞自杀元件。
在另一优选例中，所述的CAR与细胞自杀元件通过自剪切元件相连接。
在另一优选例中，所述细胞自杀元件与所述CAR的CD3ζ通过T2A连接。
在另一优选例中，所述的细胞自杀元件选自下组：HSV-TK、iCasp9、ΔCD20、mTMPK、ΔCD19、RQR8、EGFRt、或其组合。
在另一优选例中，所述细胞自杀元件为tEGFR。
在另一优选例中，所述tEGFR的氨基酸序列如SEQ ID NO:5所示。
在另一优选例中，所述CAR的氨基酸序列如SEQ ID NO:3所示。
在本发明的第二方面，提供了一种核酸分子，所述核酸分子编码本发明第一方面所述的嵌合抗原受体(CAR)。
在另一优选例中，所述核酸分子为分离的。
在本发明的第三方面，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。
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。
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，所述免疫细胞表达外源性细胞自杀元件。
在另一优选例中，所述的免疫细胞中CAR与细胞自杀元件共表达。
在另一优选例中，所述的工程化的免疫细胞包括T细胞或NK细胞，较佳地为(i)嵌合抗原受体T细胞(CAR-T细胞)；或(ii)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嵌合抗原受体、本发明第二方面所述的核酸分子、本发明第三方面所述的载体、或本发明第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嵌合抗原受体、本发明第二方面所述的核酸分子、本发明第三方面所述的载体、或本发明第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B7-H3阳性肿瘤，较佳地为B7-H3高表达的肿瘤。
在另一优选例中，所述的药物或制剂用于预防和/或治疗急性髓细胞白血病(AML)。
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，提供了一种制备工程化的免疫细胞的方法，所述的免疫细胞表达本发明第一方面所述的CAR，所述方法包括以下步骤：
(a)提供待改造的免疫细胞；和
(b)将本发明第二方面所述的核酸分子或本发明第三方面所述的载体转导入所述免疫细胞内，从而获得所述工程化的免疫细胞。
在另一优选例中，所述工程化的免疫细胞为CAR-T细胞或CAR-NK细胞。
在另一优选例中，所述的方法还包括对获得的工程化免疫细胞进行功能和有效性检测的步骤。
在本发明的第十方面，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。
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AML肿瘤细胞表面B7-H3抗原的表达率。其中，通过流式细胞术检测了六株AML肿瘤细胞表面的B7-H3表达。
图2显示了B7-H3-CAR的结构及其功能性验证。图2A显示了B7-H3-CAR的结构示意图，其包含EF1启动子、B7-H3单链抗体、CD8跨膜结构域、共刺激域41-BB和胞内信号域CD3ζ。图2B显示了流式检测分析的转染后Jurkat细胞表面B7-H3-CAR的表达。图2C显示了细胞表面CD25和CD69的表达情况。其中，将Jurkat和B7-H3-CAR Jurkat细胞分别与HEL、AML5和KG-1细胞1:1共孵育48h后，使用CD25-APC和CD69-APC抗体分别检测Jurkat细胞和B7-H3-CAR Jurakt细胞表面CD25和CD69的表达。
图3显示了B7-H3-CAR T细胞对B7-H3 +的AML细胞系具有特异性的杀伤。图3A显示了B7-H3-CAR T细胞的阳性率。其中，通过流式检测分析，B7-H3-CAR T细胞的阳性率大概为51.84％。图3B显示了Western bolt分析的T细胞中B7-H3-CAR的表达。其中，将T(泳道1)，B7-H3-CAR T(泳道2)细胞提取蛋白后再SDS-PAGE分离，孵育CD3ζ链-特异性mAb和HRP-羊抗鼠IgG后通过化学发光检测免疫印迹。图3C显示了在效靶比1:1的情况下共孵育48h后靶细胞的凋亡比例。****P＜0.0001，n＝3。图3D显示了共孵育后的上清液中细胞因子(IL-2、TNF、INF-γ和颗粒酶B)的分泌情况。****P＜0.0001，***P＜0.001，**P＜0.01。
图4显示B7-H3-CAR T细胞对AML移植小鼠有抗肿瘤活性。图4A显示了NSG小鼠的AML移植模型示意图。其中，6-8周的NSG雌鼠，每组4只，一共三组(PBS组，T细胞组，B7-H3-CAR T细胞组)，尾静脉注射5×10 5个荧光素酶-GFP-HEL细胞，之后在第五天分别尾静脉注射1×10 7个等体积效应细胞(PBS为等体积无细胞液体)。图4B显示了小鼠活体成像图，显示了小鼠体内白血病进展。图4C显示了小鼠平均 体重的统计图。图4D显示了小鼠生存曲线图。图4E显示了小鼠生存期统计图。**P＜0.01，*P＜0.1。
图5显示B7-H3-CAR T细胞可以清除AML移植小鼠体内的肿瘤细胞。图5A显示了NSG小鼠的AML移植模型示意图。其中，6-8周的NSG雌鼠，每组3只，一共三组(PBS组，T细胞组，B7-H3-CAR T细胞组)，尾静脉注射5*10 5个HEL-荧光素酶-GFP细胞，之后在第2天分别尾静脉注射2*10 7个等体积效应细胞(PBS为等体积无细胞液体)。图5B显示了小鼠活体成像图，显示了小鼠体内疾病进展。图5C显示了肿瘤细胞的比例。其中，小鼠外周血和骨髓细胞裂解红细胞后，使用CD45和CD3抗体共孵育，流式检测肿瘤细胞的比例。****P＜0.0001，*P＜0.1。图5D显示了肿瘤细胞浸润情况。其中，取小鼠的脾脏和卵巢组织，B7-H3抗体免疫组化后显示，在400倍与100倍视野下，相较于B7-H3-CAR T细胞组，PBS组和T细胞小鼠内有明显的肿瘤细胞浸润。n＝3。
图6显示了B7-H3-CAR T细胞安全性验证。图6A显示了小鼠外周血五分类检测后，RBC、WBC、PLT和HGB统计图。图6B显示了小鼠组织的HE染色结果。其中，取小鼠的心脏、肝脏、肾脏和肠组织，HE染色后在显微镜下，观察100倍和400倍视野下小鼠的组织是否有病变。n＝3。
图7显示了AML小鼠模型建模。图7A显示了流式检测的荧光素酶-GFP-HEL细胞阳性率。图7B显示了小鼠AML模型建立示意图。其中，6-8周NSG雌鼠，尾静脉注射5*10 5个荧光素酶-GFP-HEL细胞后，观察小鼠。图7C显示了流式检测的荷瘤小鼠中骨髓、外周血、脾脏、肝脏和肾脏中的肿瘤细胞。
图8显示了B7-H3-CAR T细胞的阳性率。图8A显示了流式检测的第一次小鼠实验中B7-H3-CAR T细胞阳性率。图8B显示了流式检测的第二次小鼠实验中B7-H3-CAR T细胞阳性率。
具体实施方式
本发明人经过广泛而深入地研究，首次意外地发现一种靶向B7-H3的CAR-T细胞，体外功能实验结果显示，B7-H3-CAR-T细胞对B7-H3阳性的AML肿瘤细胞具有显著的特异性杀伤，动物实验结果显示，B7-H3-CAR-T细胞可以显著抑制小鼠体内AML肿瘤细胞的生长，明显延长小鼠的生存期，具有显著的体内抗肿瘤作用，说明本发明构建的B7-H3-CAR-T细胞可以作为一种靶向治疗AML的新型治疗方法，具有巨大的临床应用前景。在此基础上完全了本发明。
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)。
B7-H3
B7-H3又称CD276，于2001年被发现，多表达于细胞表面，例如活化的树突状细胞、单核细胞、T细胞、B细胞和NK细胞，有研究表明B7-H3可以刺激T细胞的扩增和杀伤，并且可能选择性刺激T细胞的信号受体。同时，B7-H3又是一种肿瘤相关抗原，在肿瘤进展和转移中起重要作用，B7-H3蛋白表达阴性的患者存活率高于B7-H3蛋白表达阳性的患者。许多研究表明B7-H3在人类恶性肿瘤中有异常表达，包括黑色素瘤、白血病、乳腺癌、前列腺等。研究发现，绝大多数癌症患者肿瘤组织中B7-H3异常高表达60％-93％，而在正常健康组织中B7-H3表达却非常有限。B7-H3是AML中表达最强的B7家族分子之一，其中在M3和M5亚型中表达最高。
有文献报道显示，B7-H3在免疫细胞中不表达，此外绝大部分B7-H3的相关文 献证明了B7-H3与肿瘤进展、转移有关。更重要的是，B7-H3被发现高表达于肿瘤相关的基质细胞、成纤维细胞、以及肿瘤新生血管上，因此靶向B7-H3的治疗有望打破肿瘤微环境对药物的抑制，是近年来受到越来越多关注的新型靶点。目前已有多个研究小组构建了
B7-H3-CAR T细胞，在胰腺癌、儿童神经母细胞瘤、骨肉瘤等多种实体肿瘤的临床前研究中表现出令人鼓舞的肿瘤杀伤活性。本发明构建了一种以4-1BB为共刺激域、携带安全开关——截短型EGFR分子(EGFRt)的B7-H3-CAR-T细胞，在针对AML肿瘤细胞株的体外和小鼠模型试验中展现出良好的细胞毒性。
嵌合抗原受体
本发明的嵌合抗原受体(CAR)包括细胞外结构域、跨膜结构域、和细胞内结构域。胞外结构域包括靶-特异性结合元件(也称为抗原结合结构域)。细胞内结构域包括共刺激信号传导区和ζ链部分。共刺激信号传导区指包括共刺激分子的细胞内结构域的一部分。共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子，而不是抗原受体或它们的配体。
在CAR的胞外结构域和跨膜结构域之间，或在CAR的胞浆结构域和跨膜结构域之间，可并入接头。如本文所用的，术语“接头”通常指起到将跨膜结构域连接至多肽链的胞外结构域或胞浆结构域作用的任何寡肽或多肽。接头可包括0-300个氨基酸，优选地2至100个氨基酸和最优选地3至50个氨基酸。
在本发明的一个较佳的实施方式中，本发明提供的CAR的胞外结构域包括靶向B7-H3的抗原结合结构域。本发明的CAR当在T细胞中表达时，能够基于抗原结合特异性进行抗原识别。当其结合其关联抗原时，影响肿瘤细胞，导致肿瘤细胞不生长、被促使死亡或以其他方式被影响，并导致患者的肿瘤负荷缩小或消除。抗原结合结构域优选与来自共刺激分子和ζ链中的一个或多个的细胞内结构域融合。优选地，抗原结合结构域与4-1BB信号传导结构域、和CD3ζ信号结构域组合的细胞内结构域融合。
如本文所用，“抗原结合结构域”“单链抗体片段”均指具有抗原结合活性的Fab片段，Fab’片段，F(ab’) 2片段，或单一Fv片段。Fv抗体含有抗体重链可变区、轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般的，Fv抗体还包含VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。抗原结合结构域通常是scFv(single-chain variable fragment)。scFv 的大小一般是一个完整抗体的1/6。单链抗体优选是由一条核苷酸链编码的一条氨基酸链序列。作为本发明的优选方式，所述scFv特异性识别B7-H3。
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。
本发明的CAR中的胞内结构域包括4-1BB的信号传导结构域和CD3ζ的信号传导结构域。
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。
CART的治疗性应用
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B7-H3，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B7-H3的CAR-T细胞引起抗表达B7-H3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-B7-H3scFv、铰链和跨膜区、和4-1BB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，本发明的CAR-T细胞可以用于治疗急性髓系白血病。
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，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)本发明构建的B7-H3-CAR-T细胞对AML细胞株具有显著的体外抗肿瘤作用。
(b)本发明构建的B7-H3-CAR-T细胞对AML细胞株HEL荷瘤小鼠具有显著的体内抗肿瘤作用。
(c)小鼠实验结果表明，本发明构建的B7-H3-CAR-T细胞的安全性较好。
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。
通用材料和方法：
1.细胞
KG-1、MOLM-16和Jurkat细胞购自美国模式培养物保藏所(ATCC；美国维吉尼亚州马纳萨斯市)，HEL、THP-1、HL-60和AML5细胞系由苏州大学唐仲英血液学研究中心赵昀老师实验室馈赠。
HEL细胞、Jurkat细胞和AML5细胞使用的培养基是添加10％FBS的RP-MI1640。KG-1细胞使用的培养基是添加10％FBS的IMDM。取正常人外周血，用Ficoll法提取外周血单核细胞(Peripheral Blood Mononuclear Cell，PBMC)，用Transact激活T细胞，应用添加IL-7和IL-15的TexMACSTMGMP培养基培养。所有细胞均在含有5％CO 2、37℃细胞培养箱中培养。提取的T细胞在体外激活48h后进行转染，待细胞增殖数量足够时进行毒性实验。
2.CAR的构建
本申请构建的是二代CAR，将B7-H3-scFv序列构建到慢病毒载体上，依次包括B7-H3-scFv，CD8铰链区和跨膜区，41BB胞内区序列和CD3ζ序列，人EGFR序列。
3.病毒包装和T/Jurkat细胞转染
按照现有技术的常规方法制备慢病毒。T细胞激活48h后，将Jurkat细胞置于48孔板(5×10 5个/孔)中，加入50μl病毒，终体系为100μl，转染15h后补加1000ul培养基进行培养。
4.流式分析
收集到六株AML细胞，分别是AML5，KG-1，HEL，HL-60，THP-1和MOLM-16，将细胞离心弃上清，PH＝7.4的PBS洗一遍，与带FITC荧光的B7-H3抗体(1:1000)孵育30分钟后，PBS洗两遍上流式细胞仪检测细胞表面B7-H3的表达。
将培养起来的B7-H3-CAR Jurkat细胞，Jurkat细胞，B7-H3-CAR T细胞，T细胞分别取出大概2×10 6个细胞，使用1:1000稀释后的EGFR抗体100ul(抗体终浓度：1ug/ml)在37℃下共孵育20min，用PBS洗涤两次，用Jurkat细胞或T细胞作为对照细胞，利用流式细胞仪，分析检测B7-H3-CAR Jurkat和B7-H3-CAR T细胞的B7-H3-CAR阳性率。
将分别与KG-1，HEL和AML5细胞共孵育后的Jurkat细胞和B7-H3-CAR Jurkat细胞分别与CD25-APC和CD69-APC抗体孵育20min后，利用流式细胞仪，检测CD25和CD69的表达。
5.WESTERN检测
各取大概1×10 6个T细胞和B7-H3-CAR T细胞，裂解后产物经SDS-PAGE分离后转移至聚偏氟乙烯(PVDF)膜(微孔)。用5％的脱脂牛奶封闭1h后在鼠抗人CD3ζ抗体中4℃孵育过夜。随后，与HRP-羊抗鼠IgG室温孵育2h后，膜上加显影液，暗室内显影曝光。
6.B7-H3 CAR T细胞对肿瘤细胞的毒性作用的检测
取1×10 5个KG-1、HEL和AML5肿瘤细胞离心后，与100μl CFSE(1:1000)于37℃孵育15分钟，PBS洗两遍后与效应细胞以1：1的比例加入24孔板中，补液至2ml。48h后取出离心，取上清进行细胞因子的检测，将沉淀用PBS洗涤一遍，使用Annexin V-APC/7-AAD凋亡检测试剂盒(LK-AP105-100；联科生物，中国杭州，http；//www.liankebio.com)，检测凋亡的肿瘤细胞比例。
7.细胞因子检测
使用流式微珠阵列(CBA)检测杀伤后的上清液中T细胞和B7-H3-CAR T细胞产 生的细胞因子量，使用购自BD公司的人颗粒酶B CBA Flex Set D7试剂盒(目录号#560304)，人TNF Flex Set D9试剂盒(目录号#558273)，人IL-2 Flex Set A4试剂盒(目录号#558270)和人IFN-γCBA Flex Set E7试剂盒(目录号#558269)进行检测。
8.AML小鼠模型
利用购自百奥赛图公司的6-8周NOD-Prkdcscidll2rgtm1/Bcgen(B-NSG)雌鼠进行模型构建。小鼠尾静脉注射5×10 5个HEL-荧光素酶-GFP细胞，2天或5天后使用IVIS成像系统(IVIS-spectrum)检测肿瘤负荷，记录腹腔注射150mg/kg D-荧光素底物10min后的小鼠的生物荧光，通过小鼠尾静脉分别注射1×10 7个等体积效应细胞(PBS为等体积无细胞液体)。取100μl小鼠外周血，股骨骨髓，研磨后的脏器，红细胞裂解后，用抗人CD45抗体孵育细胞进行流式分析，检测肿瘤细胞。
9.免疫组化分析
将小鼠处死后，取脾脏与卵巢组织，用石蜡包埋好后切片(厚度3μm)进行免疫组化分析。将石蜡切片脱蜡复水后用浸于0.01M柠檬酸盐缓冲液中，微波修复5min，再用含有0.3％过氧化氢的甲醇溶液进行封闭。用B7-H3抗体(1：300稀释)和生物素标记的二抗进行染色，最后滴加新配的DAB显色液。观察组织中肿瘤细胞的浸润情况。将石蜡切片脱蜡复水后用苏木精-伊红(hematoxylin-and-eosin)染色，观察肿瘤组织的病理变化。
10.统计学处理
采用GraphPad Prism5软件，两组间比较用t-tests检验。以****P＜0.0001，***P＜0.001，**P＜0.01和*P＜0.05表示差异有统计学意义。
实施例1 B7-H3在AML细胞系中的表达
挑选了六株AML细胞系，流式检测其B7-H3表达情况。
流式结果如图1所示，HEL细胞B7-H3表达率为96.4％，AML5细胞B7-H3表达率为52.69％，THP-1细胞B7-H3表达率为40.67％，MOLM-16细胞B7-H3表达率为25.35％，HL-60细胞B7-H3表达率为16.43％，KG-1细胞B7-H3表达率为11.74％。最终选定了HEL，AML5，KG-1细胞(B7-H3高、中、低表达)进行后期实验。
实施例2 B7-H3-CAR的构建及特异性验证
首先构建了靶向B7-H3的CAR载体，是一个包含B7-H3单链抗体和41BB共刺激域的二代CAR，共表达EGFR作为CAR的检测标签，具体结构如图2A所示，氨基酸如SEQ  ID NO:3所示。慢病毒包装后转染Jurkat细胞，构建可以稳定高表达B7-H3-CAR的B7-H3-CAR-Jurkat细胞，流式检测B7-H3-CAR-Jurkat细胞CAR阳性率高达97.19％(图2B)。
将转染前的Jurkat细胞和转染后的B7-H3-CAR Jurkat细胞分别与HEL，AML5和KG-1细胞按1:1效靶比共孵育48小时，检测发现，与高表达B7-H3抗原的肿瘤细胞共孵育后，B7-H3-CAR Jurkat细胞表面CD25和CD69的表达均高于未转染CAR的Jurkat细胞，而在与低表达B7-H3的肿瘤细胞共孵育后，B7-H3-CAR Jurkat细胞和Jurkat细胞表面CD25和CD69的表达无明显差异(图2C)。
以上结果表明，本发明构建的B7-H3-CAR-Jurkat细胞可以被B7-H3阳性肿瘤细胞特异性激活。
实施例3 B7-H3-CAR T细胞对B7-H3阳性肿瘤细胞的体外杀伤
为了进一步证明实施例2构建的B7-H3-CAR的体外生物学功能，制备了B7-H3-CAR-T细胞，流式检测其阳性率大概为51.84％(图3A)。蛋白免疫印迹检测显示，B7-H3-CAR T细胞中有B7-H3-CAR融合蛋白的表达(图3B)，其中包括两个所有T细胞都表达的16kD和28kD的内源性CD3ζ(泳道1，泳道2)，和一个CAR-T细胞表达的外源性CD3ζ(泳道2的56kD)。
将T细胞和制备好的B7-H3-CAR T细胞分别与HEL、AML5和KG-1细胞按1:1的效靶比共孵育48h后，检测肿瘤细胞的凋亡。结果显示，与T细胞相比，B7-H3-CAR T细胞对高表达和中度表达B7-H3的肿瘤细胞都有很好的毒性作用，而对于低表达B7-H3的肿瘤细胞，并没有显著的差异。这说明B7-H3-CAR T细胞对表达B7-H3的AML细胞具有很强的毒性作用(图3C)。
随后，检测了上清中细胞因子的分泌情况，与杀伤结果一致，与高表达和中度表达B7-H3的肿瘤细胞共孵育的B7-H3-CAR T细胞的细胞因子分泌量明显高于其它组(图3D)。进一步说明本发明构建的B7-H3-CAR T细胞可以很好的特异性杀伤表达B7-H3的AML肿瘤细胞。
实施例4 B7-H3-CAR T细胞可以延长荷瘤小鼠的生存期
为了进一步评估B7-H3-CAR T细胞在体内的抗肿瘤活性，成功构建了荧光素酶-GFP-HEL细胞(图7A)。
首先，建立AML小鼠肿瘤模型。取6-8周的NSG雌鼠，尾静脉注射5×10 5个荧光素酶-GFP-HEL细胞，观察小鼠，大概20天左右先后出现意志消沉，后肢瘫痪，体 型消瘦，体重下降(图7B)。待自然死亡后，取小鼠的骨髓、外周血、脾脏、，肝脏、肾脏流式分析肿瘤细胞所占比例(图7C)。实验证明，小鼠尾静脉注射5×10 5个肿瘤细胞就可以成功建立AML模型小鼠。
将荧光素酶-GFP-HEL细胞尾静脉注射到12只(分为3组，每组4只)B-NSG小鼠体内(图4A)，每只小鼠注射5×10 5个细胞，第五天小鼠活体成像检测到小鼠体内肿瘤细胞的比例很高，相当于一个中晚期的AML病例模型，之后再分别尾静脉注射等量的PBS/T/B7-H3-CAR T(阳性率大概58.19％，如图8A)细胞。观察小鼠的生存期，期间每隔几天使用活体成像检测小鼠体内的肿瘤负荷，活体成像图显示，B7-H3-CAR T组小鼠体内的肿瘤进展明显弱于另外两组(图4B)。PBS组和T组小鼠的平均体重在30天左右的时候就已经骤降，小鼠接连死亡，而B7-H3组小鼠体重仍接近平缓(图4C)。待小鼠全部死亡后统计其生存期，结果显示，B7-H3-CAR T组小鼠生存期明显比PBS组和T细胞组长，并有统计学差异(图4D，图4E)。说明B7-H3-CAR T细胞可以有效抑制荷瘤小鼠体内肿瘤细胞的增长，B7-H3-CAR T细胞对B7-H3阳性的AML细胞具有显著的体内抗肿瘤作用。
实施例5 B7-H3-CAR T细胞可以降低荷瘤小鼠外周血和骨髓的肿瘤负荷
为了进一步证明B7-H3-CAR T细胞的体内抗肿瘤作用，又建立了一批小鼠模型(图5A)。注射肿瘤细胞后的第二天，活体成像显示肿瘤细胞浸润到骨髓，说明建模成功。然后将2×10 7个T细胞和B7-H3-CAR T细胞(CAR阳性率为52.82％，图8B)分别注射到小鼠体内，之后每隔4-5天进行一次活体成像，结果显示，与PBS组和T组小鼠相比，B7-H3-CAR T组小鼠的肿瘤生长受到明显抑制(图5B)。
在20天时，PBS组小鼠出现体重下降，精神萎靡，四肢瘫痪等症状，于是将所有的小鼠统一处死，取外周血和骨髓细胞，使用流式分析检测其中肿瘤细胞的比例(图5C)。结果显示，B7-H3-CAR T组小鼠肿瘤负荷明显低于另外两组小鼠。
将小鼠的脾脏和卵巢组织进行免疫组化分析，结果显示，PBS组和T细胞组小鼠组织中有明显的肿瘤细胞浸润，而B7-H3组小鼠的组织中几乎没有或仅有少数肿瘤细胞浸润。因此，证明B7-H3-CAR T细胞可以有效的降低小鼠体内肿瘤细胞的发展。
实施例6 B7-H3-CAR T不会对正常细胞产生毒性
本发明使用的scFv序列亲和力高，具有更快的解离常数和更高的杀伤活性和安全性。该序列可同时识别小鼠B7-H3分子，因此可以通过检测小鼠体内正常细胞 的活性，来证明本序列的安全性。
分别检测了(实施例5的小鼠模型)空白组、PBS组、T细胞组和B7-H3-CAR T细胞组小鼠的外周血血常规。结果显示，无论从是白细胞，红细胞，血小板还是血红蛋白来看，B7-H3-CAR T组小鼠和空白组小鼠相比，均没有明显的差异性(图5D)。而且，HE染色结果也显示，与健康小鼠相比，B7-H3-CAR T细胞组小鼠的心脏、肝脏、肾脏和肠等重要脏器也都没有发生病变。因此，上述实验证明本发明开发的B7-H3-CAR T是一个安全性很高的CAR-T产品。
讨论
AML是一种非常难治的侵袭性造血干细胞恶性肿瘤，近十几年来，随着一些新的治疗方法的出现，AML患者的预期寿命虽然有所改善，但由于耐药和难治性，AML仍然是最致命最难治愈的恶性癌症之一。而且AML患者的治疗选择很有限，预后较差，在化疗之后并不能治愈，极易复发，非常需要新的治疗方法的出现。CAR T疗法无论是在血液瘤还是实体瘤方面都取得了巨大的进展，被认为是最有前景的肿瘤治疗方式之一。
目前针对AML的CAR T治疗因为大部分靶点在正常的髓细胞中也有很高的表达，所以在治疗前大多需要给患者进行骨髓清除，大大复杂化了治疗方法，并且会增加很多不必要的风险。因此找到一个只在恶性细胞中选择性表达，在正常细胞中缺乏的抗原很重要。曾有文章统计过，大概有40％左右的AML患者会有B7-H3的表达，并且亦有研究表明在31个胰腺肿瘤标本中>50％的细胞中检测到B7-H3的表达，而在正常胰腺组织标本中未检测到B7-H3的表达。B7-H3的表达受RNA转录的调控，是一种表面免疫调节糖蛋白，可抑制自然杀伤细胞和T细胞，尽管B7-H3转录产物在人类实体瘤和正常组织中广泛表达，但是B7-H3蛋白仅在肿瘤组织中优先表达。也有学者指出B7-H3蛋白仅在少数组织和细胞中表达，包括活化的淋巴细胞和肿瘤细胞。所以，虽然B7-H3只表达在部分AML上，但是由于它不会对造血系统产生毒性，因此是一个AML的优良治疗靶点。
已有研究表明，仅仅依靠单纯的T细胞并不足以对抗狡猾的肿瘤细胞，但是本发明的B7-H3-CAR T细胞却可以有效的特异性杀伤表达B7-H3的AML细胞(图3C)。B7-H3-CAR T细胞在小鼠体内也有很明显的清除肿瘤的作用。活体成像检测显示，B7-H3-CAR T组小鼠体内的肿瘤细胞明显低于PBS组和T细胞组(图4B，5B)。并且，即使是AML这种病程进展特别迅速的疾病，B7-H3-CAR T细胞也可以明显的延长小鼠的生存期(图4D，4E)，降低小鼠外周血、骨髓和组织中的肿瘤细胞比例(图 5C，5D)。因此，本发明的B7-H3-CAR T可用于难治复发并且表达B7-H3抗原的AML患者的治疗。
另外，还通过检测B7-H3-CAR T是否会对小鼠体内正常细胞产生脱靶毒性，来进行安全性验证。外周血血常规检测显示，B7-H3-CAR T并不会对小鼠的正常细胞产生毒性作用(图6A)。而且，HE染色结果也显示，B7-H3-CAR T细胞并不会损伤小鼠的重要脏器(图6B)。因此，本发明的B7-H3-CAR T是一个安全的CAR-T产品。
综上，本发明不但证明了B7-H3-CAR T可以有效的在体内外特异性清除表达B7-H3抗原的AML肿瘤细胞，而且还证明了其具有良好的安全性，为临床中使用B7-H3-CAR T细胞治疗AML奠定了基础。与目前使用最多的CD33-CAR T和CD123-CAR T细胞使用前需要事先给患者清髓相比，使用B7-H3-CAR T不但有延长复发难治性AML患者生存期的可能，更是简化了CAR T治疗在临床的实际使用难度，减低患者风险。
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。
本申请序列表中涉及的各序列如下：
Figure PCTCN2021141338-appb-000001

Claims (10)

  1. 一种嵌合抗原受体(CAR)，其特征在于，所述嵌合抗原受体的抗原结合结构域包含重链可变区和轻链可变区，
    所述的重链可变区包括以下互补决定区CDR：
    SEQ ID NO:6所示的CDR1，
    SEQ ID NO:7所示的CDR2，和
    SEQ ID NO:8所示的CDR3；
    且所述的轻链可变区包括以下互补决定区CDR：
    SEQ ID NO:9所示的CDR1’，
    SEQ ID NO:10所示的CDR2’，和
    SEQ ID NO:11所示的CDR3’；
    较佳地，所述的抗原结合结构域包括SEQ ID NO:1所示的抗体重链可变区，和SEQ ID NO:2所示的抗体轻链可变区。
  2. 如权利要求1所述的CAR，其特征在于，所述嵌合抗原受体的结构如下式III所示：
    L-scFv-H-TM-C-CD3ζ  (III)
    其中，
    L为无或信号肽序列；
    scFv为靶向B7-H3的scFv；
    H为绞链区；
    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。
  3. 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)。
  4. 一种载体，其特征在于，所述的载体含有权利要求3所述的核酸分子。
  5. 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。
  6. 如权利要求5所述的免疫细胞，其特征在于，所述细胞包括T细胞、NK细胞。
  7. 一种制剂，其特征在于，所述制剂含有权利要求1所述的嵌合抗原受体、权利要求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。
  8. 一种权利要求1所述的嵌合抗原受体、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、或权利要求7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。
  9. 如权利要求8所述的用途，其特征在于，所述的药物或制剂用于预防和/或治疗急性髓细胞白血病(AML)。
  10. 一种制备工程化的免疫细胞的方法，其特征在于，所述的免疫细胞表达权利要求1所述的嵌合抗原受体，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3所述的核酸分子或权利要求4所述的载体转导入所述免疫细胞内，从而获得所述工程化的免疫细胞。
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