WO2018068766A1 - Cd19靶向性的嵌合抗原受体及其制法和应用 - Google Patents

Cd19靶向性的嵌合抗原受体及其制法和应用 Download PDF

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WO2018068766A1
WO2018068766A1 PCT/CN2017/106135 CN2017106135W WO2018068766A1 WO 2018068766 A1 WO2018068766 A1 WO 2018068766A1 CN 2017106135 W CN2017106135 W CN 2017106135W WO 2018068766 A1 WO2018068766 A1 WO 2018068766A1
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car
seq
cell
cells
sequence
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姚意弘
朱侍贵
姚昕
李志远
何佳平
朱蔚
朱琳
王庆霞
林南静
张莉
黄家琪
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西比曼生物科技（上海）有限公司
西比曼生物科技（无锡）有限公司
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Definitions

  • the present invention belongs to the field of biomedicine, and in particular, the present invention relates to a CD19-targeting chimeric antigen receptor, a preparation method and application thereof.
  • the CD19 molecule is a receptor for specific signal transduction on the surface of B lymphocytes, which exists in various stages of B cell maturation, appears in the B progenitor cell stage and is stably and continuously expressed.
  • CD19 is expressed only on the surface of pre-B cells and mature B cells, but not in hematopoietic stem cells, plasma cells and other normal tissue cells.
  • CD19 molecules are relatively exposed on the membrane, are easily accessible, have no significant internalization and shedding after binding with monoclonal antibodies, and do not undergo antigen modulation due to binding to antibodies, so CD19 is the most reliable surface of B cells. Biomarker one.
  • CD19 regulates signaling through B cell receptors, which play an important role in B cell development, proliferation and differentiation, and malignant transformation.
  • CD19 is absolutely necessary for stabilizing Myc protein.
  • Myc gene is stable and at a high level, Myc Genes contribute to cancer development, and patients with high levels of Myc protein are more likely to die from lymphoma.
  • CD19 may be a switch on the Myc gene pathway, and controlling the on/off switch may represent a powerful tool for treating lymphocytic tumors. Based on the above phenomena, CD19 is an important target for the treatment of cells that develop and develop CD19-positive B-cell malignancies.
  • CD19 has high levels of expression in most B cell lymphomas (such as DLBCL, FL, and mantle cell lymphoma), acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and a subset of acute myeloid leukemia.
  • B cell lymphomas such as DLBCL, FL, and mantle cell lymphoma
  • acute lymphocytic leukemia chronic lymphocytic leukemia
  • hairy cell leukemia and a subset of acute myeloid leukemia.
  • CD19 is not expressed in hematopoietic stem cells, plasma cells and other normal human tissues. Therefore, the targeted treatment of CD19 does not cross-react with other normal tissues.
  • the main side effect is B cell deficiency.
  • the anti-CD19-specific antibody Blinatumomab has been approved by the US FDA for the treatment of relapsed or refractory Philadelphia chromosome-negative B-cell acute lymphoblastic leuk
  • Cellular immunotherapy is an emerging and highly effective tumor treatment model, and is a new type of treatment for autoimmune and anti-cancer. It is a method for in vitro culture and amplification of immune cells collected from a patient using biotechnology and biological agents to be returned to a patient to stimulate and enhance the body's own immune function, thereby achieving the purpose of treating tumors.
  • biotechnology and biological agents to be returned to a patient to stimulate and enhance the body's own immune function, thereby achieving the purpose of treating tumors.
  • Those skilled in the art have been working to develop new cellular immunotherapies to increase the effectiveness of cellular immunotherapy and reduce its side effects.
  • the object of the present invention is to optimize the CD19-targeting chimeric antigen receptor and its preparation and use.
  • a chimeric antigen receptor (sequence)
  • the antigen binding domain ie, scFv
  • the chimeric antigen receptor is as follows:
  • V H antibody heavy chain variable region V L is an antibody light chain variable region; and "-" connecting peptide or a peptide bond.
  • the antibody heavy chain variable region comprises the following three complementarity determining region CDRs:
  • the antibody heavy chain variable region has the amino acid sequence set forth in SEQ ID NO.
  • the antibody light chain variable region comprises the following three complementarity determining region CDRs:
  • the antibody light chain variable region has the amino acid sequence set forth in SEQ ID NO.
  • the structure of the chimeric antigen receptor is as follows:
  • L is an optional leader sequence (signal peptide sequence);
  • H is a hinge region
  • TM is a transmembrane domain
  • 4-1BB is a costimulatory molecule derived from 4-1BB;
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇
  • V H and V L are as described above, respectively.
  • sequence of L is as shown in SEQ ID NO. 22 or SEQ ID NO.
  • sequence of H comprises the amino acid sequence set forth in SEQ ID NO. 14; preferably, the sequence of H is set forth in SEQ ID NO.
  • sequence of TM is as shown in SEQ ID NO.
  • sequence of 4-1BB is as shown in SEQ ID NO. 18 or 27.
  • sequence of CD3 ⁇ is as shown in SEQ ID NO. 20 or SEQ ID NO.
  • the antigen binding domain of the chimeric antigen receptor is set forth in SEQ ID NO.
  • sequence of the chimeric antigen receptor is as shown in SEQ ID NO. 3 or SEQ ID NO.
  • a nucleic acid molecule encoding the chimeric antigen receptor (CAR) of the first aspect of the invention.
  • the nucleic acid molecule comprises a hinge region nucleic acid sequence encoding the CD8a selected from the group consisting of:
  • the nucleic acid molecule comprises a nucleic acid sequence encoding a transmembrane region of the CD8a selected from the group consisting of:
  • the nucleic acid molecule comprises a nucleic acid sequence encoding an intracellular signal domain encoding the 4-1BB (CD137) selected from the group consisting of:
  • the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of an intracellular signal domain encoding the CD3 ⁇ :
  • the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of:
  • the nucleotide sequence has a homology of SEQ ID NO. 4 or SEQ ID NO. 2 of ⁇ 95% (preferably ⁇ 98%) and encodes SEQ ID NO. 3 or SEQ ID NO. a polynucleotide of the amino acid sequence shown in 1;
  • the nucleic acid molecule is isolated.
  • the nucleic acid molecule further comprises a polynucleotide encoding a leader sequence (a leader sequence, a signal peptide), the amino acid sequence of which is represented by SEQ ID NO. 22 or SEQ ID NO.
  • a leader sequence a leader sequence, a signal peptide
  • the polynucleotide encoding the leader sequence is set forth in SEQ ID NO. 23 or 25.
  • sequence of the nucleic acid molecule is as shown in SEQ ID NO. 4 or SEQ ID NO.
  • a vector comprising the nucleic acid molecule of the first aspect of the invention is provided.
  • the vector is a lentiviral vector.
  • a host cell comprising the vector of the third aspect of the present invention or the nucleic acid according to the second aspect of the present invention in which the exogenous source is integrated molecule.
  • the cell is an isolated cell, and/or the cell is a genetically engineered cell.
  • the cell is a mammalian cell.
  • the cell is a T cell.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the chimeric antigen receptor of the first aspect of the invention, the nucleic acid of the second aspect of the invention, is provided A molecule, the vector of the third aspect of the invention, or the cell of the fourth aspect of the invention.
  • the chimeric antigen receptor of the first aspect of the invention the nucleic acid molecule of the second aspect of the invention, the vector of the third aspect of the invention, or the fourth aspect of the invention.
  • the tumor comprises a CD19 positive B cell malignancy.
  • a seventh aspect of the invention provides a method for treating a disease comprising administering an appropriate amount of the chimeric antigen receptor of the first aspect of the invention, the nucleic acid molecule of the second aspect of the invention, to a subject in need of treatment,
  • the vector of the third aspect of the invention, or the cell of the fourth aspect of the invention, or the pharmaceutical composition of the fifth aspect of the invention is administered to a subject in need of treatment.
  • the disease is a tumor.
  • a method for producing a CAR-T cell (CAR-modified T cell) expressing the chimeric antigen receptor of the first aspect of the invention a method for producing a CAR-T cell (CAR-modified T cell) expressing the chimeric antigen receptor of the first aspect of the invention
  • the method comprises the steps of: transducing the nucleic acid molecule of the second aspect of the invention or the vector of the third aspect of the invention into a T cell, thereby obtaining the CAR-T cell.
  • Figure 1 is a schematic view showing the structure of CAR-19.1, 19.2CAR-19.3, and CAR-19.4.
  • Figure 2 is an ELISA assay for IFN- ⁇ release of CAR-T19.1 and 19.2 cells co-cultured with three tumor cells.
  • Ramos is a CD19 positive cell and Molt4 and SEM are CD19 negative cells.
  • Figure 3 shows the flow profiles of CAR-T19.1, CAR-T19.3, and CAR-T19.4 prepared by lentiviral transfection. Protein L staining was used to label CAR positive cells, and the donor of PBMC was healthy.
  • Figure 4 is an ELISA assay for IFN-[gamma] release of CAR-T19.1, 19.3 and 19.4 cells co-cultured with four tumor cells.
  • Raji and Ramos are CD19 positive cells
  • Molt4 and Karpas620 are CD19 negative cells.
  • Figure 5 is a flow cytometric analysis of upregulation of CD137 expression by CAR-T19 cells co-cultured with four tumor cells.
  • Figure 6 is a cell killing experiment of four tumor cells using CAR-T19 cells using an LDH kit.
  • Figure 7 shows the flow cytometry of degranulation of CAR-T19 cells stimulated by four tumor cells, and CD107a is a degranulated marker molecule.
  • Figure 8 is a flow analysis of CAR-T19.1, CAR-T19.3, and CAR-T19.4 prepared using lentiviral transfection, and the donor of PBMC is a B-ALL patient.
  • Figure 9 is an ELISA assay for IFN- ⁇ release co-cultured with CAR-T19 cells and tumor cells.
  • P, CD19 positive cells, N, CD19 negative cells, and donors of PBMC are B-ALL patients.
  • Figure 10 is a flow cytometric analysis of up-regulation of CD137 expression in co-cultured with CAR-T19 cells and tumor cells.
  • the donor of PBMC is a B-ALL patient.
  • Figure 11 is a schematic illustration of the structure of a chimeric antigen receptor and vector according to the present invention.
  • the present inventors have obtained an optimized CD19-targeting chimeric antigen receptor and its preparation and application by extensive and intensive research, the extracellular antigen binding domain of the chimeric antigen receptor N-terminal to C
  • the ends are the antibody light chain variable region and the antibody heavy chain variable region.
  • the experimental results show that the present invention also provides a chimeric antigen receptor, the specific light chain variable region-heavy chain variable region sequence arrangement, the regulation of the extracellular hinge region length, and the adjustment of the signal transduction domain. It can significantly increase the activity of antigen-binding receptors and greatly improve the killing ability of tumor cells.
  • the invention provides chimeric antigen receptors (CARs) comprising an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes a target-specific binding element (also known as an antigen binding domain).
  • the intracellular domain includes a costimulatory signaling region and a purine 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 required for efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands.
  • a linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR.
  • the term "linker” generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain.
  • the linker may comprise from 0 to 300 amino acids, preferably from 2 to 100 amino acids and most preferably from 3 to 50 amino acids.
  • the invention provides cells (e.g., T cells) that have been genetically engineered to express CAR, which exhibit significant anti-tumor properties.
  • the CAR of the invention may also include an extracellular domain having an antigen binding domain fused to an intracellular signaling domain of a T cell antigen receptor complex ⁇ chain (eg, CD3 ⁇ ).
  • the CAR of the present invention when expressed in T cells, is capable of altering antigen recognition based on antigen binding specificity.
  • An exemplary antigen is CD19 because the antigen is expressed on malignant B cells.
  • the invention is not limited to targeting CD19.
  • the invention encompasses any antigen binding domain that, when bound to its associated antigen, affects the tumor cells, results in the tumor cells not growing, being promoted to death or otherwise affected, and causing the patient's tumor burden to shrink or eliminate.
  • the antigen binding domain is preferably The intracellular domain from one or more of the costimulatory molecule and the ⁇ chain is fused.
  • the antigen binding domain is fused to an intracellular domain in combination with a CD137 (4-1BB) signaling domain, and a CD3 ⁇ signal domain.
  • a CD19-targeting CAR of the invention comprises a specific signaling domain comprising the invention (the transmembrane region of CD8, the intracellular signal domains of CD137 and CD3 ⁇ are made in series).
  • the signaling domain of the invention significantly increases anti-tumor activity and in vivo persistence of CAR-T cells compared to other modes of CD19-targeted CAR.
  • the amino acid sequence of the chimeric antigen receptor (CAR) provided by the present invention is as follows:
  • the coding polynucleotide sequence is as follows:
  • the amino acid sequence of the chimeric antigen receptor (CAR) provided by the present invention is as follows:
  • the coding polynucleotide sequence is as follows:
  • a CAR of the invention comprises a target-specific binding element referred to as an antigen binding domain.
  • the choice of antigen binding domain depends on the type and number of ligands that define the surface of the target cell.
  • an antigen binding domain can be selected to recognize a ligand that acts as a cell surface marker on a target cell associated with a particular disease state.
  • cell surface markers include those associated with viral, bacterial and parasitic infections, autoimmune diseases and cancer cells.
  • the antigen is CD19.
  • the antigen binding domain of a CAR of the invention targets CD19.
  • the antigen binding domain in the CAR of the invention is as follows (N-terminal to C-terminal):
  • V H antibody heavy chain variable region V L is an antibody light chain variable region; and "-" connecting peptide or a peptide bond.
  • the antibody heavy chain variable region comprises the following three complementarity determining region CDRs:
  • CDR1 GVSLPDYG, SEQ ID NO. 5,
  • CDR2 IWGSETT, SEQ ID NO. 6, and
  • CDR3 AKHYYYGGSYAMDY, SEQ ID NO.
  • amino acid sequence of the antibody heavy chain variable region is as follows:
  • the antibody light chain variable region comprises the following three complementarity determining region CDRs:
  • amino acid sequence of the variable region of the antibody light chain is as follows:
  • amino acid sequence of the antigen binding domain in the CAR of the invention is as follows:
  • the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR.
  • a transmembrane domain that is 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 the transmembrane domain of the same or different surface membrane proteins, thereby minimizing complexes with receptors. The interaction of other members.
  • Transmembrane domains can be derived from natural or synthetic sources. In natural sources, this domain can be derived from any membrane-bound protein or transmembrane protein.
  • the transmembrane region specifically used in the present invention may be derived from the ⁇ , ⁇ or ⁇ chain of the T-cell receptor, CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80. , CD86, CD134, CD137, CD154 (ie, including at least the transmembrane region(s) in the above).
  • the hinge region and the transmembrane region in the CAR of the invention are the hinge region and the transmembrane region of CD8a.
  • the hinge region of CD8a comprises the following amino acid sequence:
  • TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO. 14); its coding polynucleotide sequence is as follows:
  • amino acid sequence of the hinge region in the CAR of the invention is as follows:
  • TRLSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SEQ ID NO. 15; its coding polynucleotide sequence is as follows:
  • the transmembrane region of CD8a comprises the following amino acid sequence
  • the coding polynucleotide sequence is as follows:
  • the intracellular domain of the CAR of the invention or the additional intracellular signaling domain is responsible for the activation of at least one normal effector function of the immune cells in which the CAR has been placed.
  • effector function refers to the proprietary function of a cell.
  • the effector function of a T cell can be a cytolytic activity or a helper activity including secretion of a cytokine.
  • intracellular signaling domain refers to a transduction effector function letter And direct the cell part of the protein to perform a proprietary function.
  • the entire intracellular signaling domain can generally be used, in many instances it is not necessary to use the entire chain.
  • intracellular signaling domain In the case of a truncated portion of an intracellular signaling domain, such a truncated portion can be used in place of the entire strand as long as it transduces an effector function signal.
  • the term intracellular signaling domain thus refers to any truncated portion comprising an intracellular signaling domain sufficient to transduce an effector function signal.
  • Preferred examples of the intracellular signaling domain of the CAR for use in the present invention include a cytoplasmic sequence of a T cell receptor (TCR) and a co-receptor that acts synergistically to initiate signal transduction after antigen receptor binding, and these sequences Any derivative or variant and any synthetic sequence that has the same functional ability.
  • TCR T cell receptor
  • co-receptor that acts synergistically to initiate signal transduction after antigen receptor binding
  • T cell activation can be thought of as mediated by two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation by TCR (primary cytoplasmic signaling sequence) and in an antigen-independent manner Those that function to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).
  • the primary cytosolic signaling sequence modulates the primary activation of the TCR complex in a stimulatory or inhibitory manner.
  • the primary cytoplasmic signaling sequence that acts in a stimulatory manner can comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.
  • the cytosolic signaling molecule in the CAR of the invention comprises a cytosolic signaling sequence derived from CD3 ⁇ .
  • the cytoplasmic domain of the CAR can be designed to include the CD3- ⁇ signaling domain itself, or any other desired cytoplasmic domain that can be useful in the context of the CAR of the invention (a Or multiple) unions.
  • the cytoplasmic domain of a CAR can include a CD3 ⁇ chain portion and a costimulatory signaling region.
  • a costimulatory signaling region refers to a portion of a CAR that includes an intracellular domain of a costimulatory molecule.
  • Costimulatory molecules are cell surface molecules required for efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, LIGHT,
  • cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the invention may be linked to each other at random or in a defined order.
  • a short oligopeptide or polypeptide linker preferably 2 and 10 amino acids in length, can form the linkage.
  • the glycine-serine doublet provides a particularly suitable linker.
  • the cytoplasmic domain of the CAR of the invention is designed to include a signaling domain of 4-1BB and a signaling domain of CD3 ⁇ , wherein the signaling domain of 4-1BB comprises SEQ ID NO:
  • the nucleic acid sequence set forth in 4 and the signaling domain of CD3- ⁇ include the nucleic acid sequence set forth in SEQ ID NO: 5.
  • the cytoplasmic domain of the CAR of the invention is designed to include a signaling domain of 4-1BB and a signaling domain of CD3 ⁇ , wherein the signaling domain of 4-1BB comprises encoding SEQ ID NO
  • the nucleic acid sequence of the amino acid sequence of 18, and the signaling domain of CD3 ⁇ comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 19.
  • the cytoplasmic domain of the CAR of the invention is designed to include a signaling domain of 4-1BB and a signaling domain of CD3 ⁇ , wherein the signaling domain of 4-1BB comprises SEQ ID NO:
  • the amino acid sequence set forth in 18, and the signaling domain of CD3 ⁇ include the one presented in SEQ ID NO: 19. Amino acid sequence.
  • the intracellular signal domain of 4-1BB comprises the following amino acid sequence:
  • the intracellular signal domain of 4-1BB comprises the following amino acid sequence:
  • the intracellular signal domain of CD3 ⁇ comprises the following amino acid sequence:
  • the intracellular signal domain of CD3 ⁇ comprises an amino acid sequence comprising a mutated amino acid residue (underlined):
  • the invention includes a DNA construct comprising a CAR sequence, wherein the sequence comprises a nucleic acid sequence of an antigen binding domain of a nucleic acid sequence operably linked to a signaling domain.
  • Exemplary signaling domains for CARs useful in the present invention include anti-CD19 scFv, CD8 hinge and transmembrane regions, and CD137 and CD3 intracellular signaling domains.
  • the CAR of the invention comprises the nucleic acid sequence set forth in SEQ ID NO: 10.
  • the CAR of the invention comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:9.
  • the CAR of the invention comprises the amino acid sequence set forth in SEQ ID NO:9.
  • a nucleic acid sequence encoding a desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a vector known to include the gene, or by utilizing standard Techniques, isolated directly from cells and tissues containing the gene.
  • interested Genes can be produced synthetically.
  • the present invention also provides a vector into which the DNA of the present invention is inserted.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of the transgene and its proliferation in daughter cells.
  • Lentiviral vectors have the advantage over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.
  • expression of a native or synthetic nucleic acid encoding a CAR is typically achieved by operably linking a nucleic acid encoding a CAR polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector.
  • This vector is suitable for replication and integration of eukaryotic cells.
  • a typical cloning vector comprises a transcriptional and translational terminator, an initial sequence and a promoter that can be used to modulate expression of a desired nucleic acid sequence.
  • the expression constructs of the invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5, 580, 859, 5, 589, 466, incorporated herein by reference.
  • the invention provides a gene therapy vector.
  • the nucleic acid can be cloned into many types of vectors.
  • the nucleic acid can be cloned into such vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • Specific vectors of interest include expression vectors, replication vectors, probe production vectors, and sequencing vectors.
  • the expression vector can be provided to the cells in the form of a viral vector.
  • 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 handbooks.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector comprises an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (eg, WO01/96584; WO01/29058; and the United States) 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 a subject cell in vivo or ex vivo.
  • retroviral systems are known in the art.
  • an adenoviral vector is used.
  • Many adenoviral vectors are known in the art.
  • a lentiviral vector is used.
  • promoter elements can regulate the frequency of transcription initiation.
  • these are located in the 30-110 bp region upstream of the start site, although it has recently been shown that many promoters also contain functional elements downstream of the start site.
  • the spacing between the promoter elements is often flexible to maintain the promoter function when the element is inverted or moved relative to the other.
  • tk thymidine kinase
  • the interval between promoter elements can be increased by 50 bp, and the activity begins to decrease.
  • a single element can function cooperatively or independently to initiate transcription.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto.
  • Another example of a suitable promoter is Elongation Growth Factor-1 alpha (EF-1 alpha).
  • constitutive promoter sequences can also be used, including but not limited to human prion 40 (SV40) early promoter, mouse breast cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Lu A sarcoma virus promoter, and a human gene promoter such as, but not limited to, an actin promoter, a myosin promoter, a heme promoter, and a creatine kinase promoter.
  • the invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered as part of the invention.
  • an inducible promoter provides a molecular switch capable of opening expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or shutting down expression when expression is undesirable.
  • 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 comprise any one or both of a selectable marker gene or reporter gene to facilitate seeking a population of cells that are transfected or infected by the viral vector. Identify and select expression cells.
  • selectable markers can be carried on a single piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene 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 were used to identify potentially transfected cells and to assess the functionality of regulatory sequences.
  • the reporter gene is a gene that is not present in or expressed by the recipient organism or tissue, and which encodes a polypeptide whose expression is clearly indicated by some readily detectable properties such as enzymatic activity. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is determined at an appropriate time.
  • Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein genes (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 commercially available.
  • a construct with a minimum of 5 flanking regions showing the highest level of reporter gene expression is identified as a promoter.
  • Such a promoter region can be ligated to a reporter gene and used to assess the ability of the agent to modulate promoter-driven transcription.
  • an expression vector can be readily introduced into a host cell, for example, a mammalian, bacterial, yeast or insect cell by any method in the art.
  • an expression vector can be transferred into a host cell by physical, chemical or biological means.
  • Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods of producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method of introducing a polynucleotide into a host cell is calcium phosphate transfection.
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors particularly retroviral vectors, have become the most widely used method of inserting genes into mammals, such as human cells.
  • Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.
  • Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipids. Platinum.
  • An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (eg, an artificial membrane sac).
  • an exemplary delivery tool is a liposome. It is contemplated to use a lipid formulation to introduce the nucleic acid into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid can be associated with a lipid.
  • the nucleic acid associated with the lipid can be encapsulated into the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached via a linker molecule associated with both the liposome and the oligonucleotide
  • a linker molecule associated with both the liposome and the oligonucleotide
  • the lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include fat droplets that occur naturally in the cytoplasm and 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, more preferably a pWPT-GFP lentiviral vector.
  • the present inventors have confirmed that the construction of the CAR of the present invention using the lentiviral vector has high transfection efficiency for T cells and is highly reproducible.
  • the vector further comprises a signal peptide coding sequence.
  • the signal peptide sequence is ligated upstream of the antigenic tuberculosis domain nucleic acid sequence.
  • the signal peptide is a rat growth hormone signal peptide or a human CD8a signal peptide.
  • the signal peptide amino acid sequence is as follows:
  • MALPVTALLLPLALLLHAARP human CD8a, SEQ ID NO. 22
  • the signal peptide amino acid sequence is as follows:
  • MAADSQTPWLLTFSLLCLLWPQEAGALP rat growth hormone signal peptide, SEQ ID NO. 24
  • the invention encompasses cells (e.g., T cells) that are transduced with a lentiviral vector (LV).
  • LV encodes a CAR that combines the antigen binding domain of a specific antibody with the intracellular domain of CD3- ⁇ , CD137.
  • transduced T cells can elicit a CAR-mediated T-cell response.
  • the invention also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue of a mammal comprising the step of administering to a mammal a T cell expressing CAR, wherein the CAR comprises specifically A binding moiety that interacts with a predetermined target, including, for example, the ⁇ chain portion of the intracellular domain of human CD3 ,, and a costimulatory signaling region.
  • the invention encompasses a type of cell therapy wherein T cells are genetically modified to express CAR, and CAR-T cells are injected into a recipient in need thereof.
  • the injected cells are capable of killing the recipient's tumor cells.
  • CAR-T cells replicate in vivo, producing long-lasting persistence that leads to sustained tumor control.
  • the CAR-T cells of the invention can undergo robust in vivo T cell expansion for an extended amount of time.
  • the CAR-mediated immune response can be part of a step of adoptive immunotherapy in which CAR-modified T cells induce an immune response specific for the antigen binding domain in the CAR.
  • anti CD19 CAR-T cells elicit a specific immune response against cells expressing CD19.
  • Treatable cancers include tumors that have not been vascularized or have not been vascularized, as well as vascularized tumors.
  • Cancer can include non-solid tumors (such as hematological tumors such as leukemias and lymphomas) or can include solid tumors.
  • Types of cancer treated with the CAR of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors such as sarcomas, carcinomas, and melanomas. Also included are adult tumors/cancers and childhood tumors/cancers.
  • Hematological cancer is a cancer of the blood or bone marrow.
  • hematological (or hematogenous) cancers include leukemia, including acute leukemia (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, granulocyte-monocyte type).
  • monocyte and erythroleukemia monocyte and erythroleukemia
  • chronic leukemia such as chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia
  • polycythemia vera lymphoma
  • Hodgkin's disease non Hodgkin's lymphoma (painless and high-grade forms)
  • multiple myeloma Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.
  • a solid tumor is an abnormal mass of tissue that usually does not contain a cyst or fluid area.
  • Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, mucinous sarcoma, liposarcoma mesothelioma, lymphoid malignancy, pancreatic cancer, ovarian cancer.
  • the antigen binding domain of a CAR of the invention is designed to treat a particular cancer.
  • CAR designed to target CD19 can be used to treat cancer and disorders including, but not limited to, pre-BALL (children's indications), adult ALL, mantle cell lymphoma, diffuse large B-cell lymphoma, allogeneic bone marrow transplantation After the remedy and so on.
  • the cancer and disorder include, but are not limited to, pre-BALL (children's indication), adult ALL, mantle cell lymphoma, diffuse large B-cell lymphoma, remedy after allogeneic bone marrow transplantation, etc. Treatment is performed using a combination of CARs that target CD19, CD20, CD22, and ROR1.
  • the CAR-modified T cells of the invention can also be used as vaccine types for ex vivo immunity and/or in vivo therapy in mammals.
  • the mammal is a human.
  • cells are isolated from a mammal, preferably a human, and genetically modified (i.e., transduced or transfected in vitro) with a vector that expresses the CAR disclosed herein.
  • CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit.
  • Mammalian recipients can be human, and CAR-modified cells can be autologous to the recipient.
  • the cells may be allogeneic, syngeneic or xenogeneic relative to the recipient.
  • the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against antigens in a patient.
  • cells activated and expanded as described herein can be used to treat and prevent diseases produced in individuals without an immune response.
  • the CAR-modified T cells of the invention are used to treat CCL.
  • the cells of the invention are used to treat a patient at risk of developing CCL. Therefore, the present invention provides treatment Or a method of preventing CCL comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the invention.
  • the CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition in combination with a diluent and/or with other components such as IL-2 or other cytokines or cell populations.
  • the pharmaceutical compositions of the present invention may comprise a population of target cells as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; Mixtures such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, sulfate buffered saline, and the like
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids
  • antioxidants such as EDTA or glutathione
  • adjuvants eg, aluminum hydroxide
  • preservatives eg, aluminum hydroxide
  • the pharmaceutical composition of the present invention can be administered in a form suitable for the disease to be treated (or prevented).
  • the amount and frequency of administration will be determined by factors such as the condition of the patient, and the type and severity of the patient's condition - although appropriate dosages may be determined by clinical trials.
  • a pharmaceutical composition comprising a T cell as described herein may be at a dose of from 10 4 to 10 9 cells/kg body weight, preferably from 10 5 to 10 6 cells/kg body weight (including all integers in those ranges) Value) application. T cell compositions can also be administered multiple times in these doses.
  • Cells can be administered by using injection 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 a person skilled in the medical arts by monitoring the patient's signs of disease and thus modulating the treatment.
  • compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection or intraperitoneally.
  • a T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection.
  • the T cell composition of the invention is preferably administered by i.v. injection.
  • Compositions of T cells can be injected directly into tumors, lymph nodes or infected sites.
  • cells activated and expanded using the methods described herein or other methods known in the art to extend T cells to therapeutic levels are combined with any number of related therapeutic modalities (eg, prior Administering to the patient, concurrently or afterwards, including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known For ARA-C) or natalizumab treatment for MS patients or for epilizumab treatment in patients with psoriasis or other treatment for patients with PML.
  • agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known For ARA-C) or natalizumab treatment for MS patients or for epilizumab treatment in patients with psoriasis or other treatment for patients with PML.
  • the T cells of the invention can be used in combination with chemotherapy, radiation, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies Or other immunotherapeutic agents.
  • the cell composition of the invention is administered to a bone marrow transplant, using a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (eg, before, simultaneously or after) patient.
  • a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (eg, before, simultaneously or after) patient.
  • XRT external beam radiation therapy
  • cyclophosphamide eg, before, simultaneously or after
  • the subject may undergo standard treatment of high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • the subject receives an injection of the expanded immune cells of the invention after transplantation.
  • the expanded cells are administered prior to or after surgery.
  • the dosage of the above treatment administered to the patient will vary with the precise nature of the condition being treated and the recipient of the treatment.
  • the dosage ratios administered by a human can be carried out according to practices accepted in the art.
  • 1 x 10 6 to 1 x 10 10 modified T cells of the invention e.g., CAR-T19 cells
  • the chimeric antigen receptor of the present invention wherein the N-terminus to C-terminus of the extracellular antigen-binding domain is an antibody light chain variable region and an antibody heavy chain variable region, which can significantly increase the activity of the antigen-binding receptor. It greatly enhances the killing ability of tumor cells, and the cell killing ability is improved compared with the control CAR (the N-terminal to C-terminal of the extracellular antigen-binding domain is the antibody heavy chain variable region and the antibody light chain variable region in turn). About doubled.
  • the extracellular amino acids of the optimized length of the extracellular domain (from CD8a) and the signal domain can significantly increase the killing activity of CAR-T cells against tumor target cells.
  • the coding plasmid was commissioned by Shanghai Boyi Biotechnology Co., Ltd. for full-length DNA synthesis and cloning construction.
  • the cloning vector was selected from the pWPT lentiviral vector (purchased from Addgene), and the cloning sites were BamH I and Sal I sites.
  • the cloned lentiviral expression vector plasmid was sequenced and the sequencing results were in agreement with expectations.
  • the constructed carrier structure is shown in FIG.
  • L rat growth hormone signal peptide SEQ ID No. 24
  • V H -V L -H longer CD8a hinge region SEQ ID No. 15
  • TM-4-1BB containing additional amino acids, SEQ ID No. 27 - CD3 ⁇ (SEQ ID NO. 20); its nucleotide sequence is as follows:
  • nucleotide sequence is as follows:
  • V H amino acid sequence shown in SEQ ID NO.8; V L amino acid sequence shown in SEQ ID NO.12; linker peptide sequence between the V H and V L are GGGGSGGGGSGGGGS (SEQ ID NO. 28); The TM amino acid sequence is shown in SEQ ID NO.
  • PBMCs mononuclear cells
  • PBMCs used serum-containing cell culture medium GT-T551 to adjust the final cell concentration to 2 ⁇ 10 6 cells/mL.
  • the cells were seeded in a cell culture flask previously coated with Retronectin (available from TAKORA) at a final concentration of 5 ⁇ g/m LCD3 monoclonal antibody (OKT3) and a final concentration of 10 ⁇ g/mL.
  • Retronectin available from TAKORA
  • IL-2 human interleukin 2
  • Example 3 Functional detection and comparison of CAR-T19.1 and CAR-T19.2 cells.
  • CAR-T19.1 and CAR-T19.2 When a group of independent experiments were performed on CAR-T19.1 and CAR-T19.2 cells, CAR-T cells were co-cultured with three tumor target cells on day 10, and the supernatant was taken for IFN after 18 hours. - gamma ELISA assay. Ramos is a CD19 positive cell and Molt4 and SEM are CD19 negative cells. The results are shown in Figure 2. Co-culture of the two CAR-T cells with Ramos produced positive results, but CAR-T19.1 and CAR-T19.2 did not show significant differences in the release of IFN- ⁇ . This result demonstrates that merely replacing the signal peptide sequence of CAR with the human CD8a signal peptide sequence is not significant for the improvement of CAR-T cell function. CAR-T19.2 will not be further studied.
  • CAR-T19 cells were co-cultured with four tumor cells, and ELISA for IFN- ⁇ release was performed. 10 5 CAR-T19 cells and 10 5 tumor cells were co-cultured, and after 18 hours, the culture supernatant was taken for ELISA detection. CAR-T19 cells were diluted 1:2 by uninfected donor cells (final CAR positive rate ⁇ 30%), Raji and Ramos were CD19 positive cells, and Molt4 and Karpas620 were CD19 negative cells. The results are shown in Figure 4. Co-culture with Raji and Ramos produced positive results, and CAR-T19.3 and CAR-T19.4 showed higher release of IFN- ⁇ than CAR-T19.1.
  • CD137 is a cell surface marker specifically activated by T cell antigens.
  • the results are shown in Figure 5.
  • CAR-T19.3 and CAR-T19.4 showed higher upregulation of CD137 activation than CAR-T19.1.
  • CD107a degranulation experiment was carried out using the CAR-T19 cells prepared in Example 1.
  • CD107a is a marker of T cell degranulation that directly leads to the release of perforin/granzyme and the killing of target cells.
  • 10 5 CAR-T19 cells and 10 5 tumor cells were co-cultured and stained with CD107a, and CD107a degranulation was detected by flow cytometry for 6 hours. The results are shown in Figure 6.
  • CAR-T19 cells were co-cultured with Raji and Ramos target cells, CAR-T19.3 and CAR-T19.4 showed higher degranulation than CAR-T19.1, and CAR-T19. 3 express the highest level of CD107a.
  • Peripheral blood cells of B-ALL patients were used as donors, PBMCs were isolated, OKT3 activated cells and CAR-19.1, CAR-19.3, and CAR-19.4 lentivirus infections were performed.
  • CD137 is a cell surface marker specifically activated by T cell antigens.
  • the results are shown in Figure 10.
  • CAR-T19.3 and CAR-T19.4 showed higher up-regulation of CD137 activation than CAR-T19.1.

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Abstract

本发明公开了一种优化的嵌合抗原受体及其基因和重组表达载体、工程化CD19靶向性的T细胞及其应用，所述嵌合抗原受体由CD19ScFv、CD8的铰链区和跨膜区、CD137的胞内信号结构域和CD3ζ的胞内信号结构域串联构成。采用本发明的嵌合抗原受体CD19ScFv-CD8-CD137-CD3ζ修饰的T细胞进行体外实验时，对CD19阳性靶细胞具有很好的特异杀伤活性，且比对照的已知的CD19靶向性的T细胞有更强的功能和更高的活性。

Description

CD19靶向性的嵌合抗原受体及其制法和应用 技术领域
本发明属于生物医药领域，具体地，本发明涉及CD19靶向性的嵌合抗原受体及其制法和应用。
背景技术
CD19分子是B淋巴细胞表面发挥特异性信号转导的受体，存在于B细胞成熟的各个阶段，在B祖细胞阶段出现并稳定持续地表达。CD19作为B细胞系特异的细胞表面分化抗原，仅表达于前B细胞和成熟B细胞表面，而在造血干细胞、浆细胞和其他正常组织细胞中不表达。而且，CD19分子在膜上比较暴露，容易接近，与单克隆抗体结合后无显著内化及脱落，也不会因为与抗体的结合而发生抗原调变,故CD19是最可靠的B细胞表面的生物标志物一。CD19通过B细胞受体调节信号传导，其在B细胞的发育、增殖和分化以及恶性转化中发挥重要作用。此外，近期关于癌基因MYC基因如何推动B淋巴细胞恶性肿瘤的发生与发展的研究，发现CD19对稳定Myc蛋白来说是绝对必要的，当Myc基因是稳定的，并处于较高水平时，Myc基因会促使癌症发展，并且，Myc蛋白水平高的患者更可能死于淋巴瘤。CD19可能是控制Myc基因通路上的开关，而控制通断开关可能代表了治疗淋巴细胞肿瘤的有力工具。基于以上现象，故CD19成为治疗CD19阳性B细胞恶性肿瘤的发生与发展的细胞的一个重要靶点。CD19在大多数B细胞淋巴瘤(如DLBCL、FL和套细胞淋巴瘤)、急性淋巴细胞白血病、慢性淋巴细胞白血病、多毛细胞白血病和一部分急性骨髓性白血病中都有高水平的表达。而CD19在造血干细胞、浆细胞及其他人体正常组织均不表达，因此CD19的靶向治疗不会与其他正常组织组织发生交叉反应，主要副作用是B细胞缺乏。目前抗CD19特异抗体Blinatumomab已获得美国FDA批准用于治疗复发或难治性费城染色体阴性B细胞急性淋巴细胞白血病。针对CD19的CAR T细胞治疗也在B细胞恶性肿瘤的临床研究获了良好的疗效。因此，CD19在临床实践中已被证明是治疗B细胞性血液肿安全、有效的靶点。
细胞免疫治疗是一种新兴的、具有显著疗效的肿瘤治疗模式，是一种自身免疫抗癌的新型治疗方法。它是运用生物技术和生物制剂对从病人体内采集的免疫细胞进行体外培养和扩增后回输到病人体内的方法，来激发、增强机体自身免疫功能，从而达到治疗肿瘤的目的。本领域技术人员一直致力于开发新的细胞免疫疗法，以提高细胞免疫疗法的效果，并降低其副作用。
发明内容
本发明的目的是优化CD19靶向性的嵌合抗原受体及其制法和应用。
本发明的第一方面，提供了一种嵌合抗原受体(CAR)(序列)，所述嵌合抗原受体的抗原结合结构域(即，scFv)如下式所示：
VL－VH
其中，VH为抗体重链可变区；VL为抗体轻链可变区；“－”为连接肽或肽键。
在另一优选例中，所述抗体重链可变区包括以下三个互补决定区CDR：
SEQ ID NO.5所示的CDR1，
SEQ ID NO.6所示的CDR2，和
SEQ ID NO.7所示的CDR3。
在另一优选例中，所述抗体重链可变区具有SEQ ID NO.8所示的氨基酸序列。
在另一优选例中，所述抗体轻链可变区包括一下三个互补决定区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，所述抗体轻链可变区具有SEQ ID NO.12所示的氨基酸序列。
在另一优选例中，所述嵌合抗原受体的结构如下式所示：
L－VL－VH－H－TM－4-1BB－CD3ζ
其中，
L为任选的引导序列(Leader sequence，即信号肽序列)；
H为绞链区；
TM为跨膜结构域；
4-1BB为为源于4-1BB的共刺激分子；
CD3ζ为源于CD3ζ的胞浆信号传导序列；
VH和VL分别如上所述。
在另一优选例中，L的序列如SEQ ID NO.22或SEQ ID NO.24所示。
在另一优选例中，H的序列包括SEQ ID NO.14所示的氨基酸序列；优选地，H的序列如SEQ ID NO.15所示。
在另一优选例中，TM的序列如SEQ ID NO.17所示。
在另一优选例中，4-1BB的序列如SEQ ID NO.18或27所示。
在另一优选例中，CD3ζ的序列如SEQ ID NO.20或SEQ ID NO.33所示。
在另一优选例中，所述嵌合抗原受体的抗原结合结构域如SEQ ID NO.13所示。
在另一优选例中，所述嵌合抗原受体的序列如SEQ ID NO.3或SEQ ID NO.1所示。
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，所述核酸分子包含选自下组的编码所述CD8a的铰链区核酸序列：
(a)编码如SEQ ID NO.14或15所示多肽的多核苷酸；
(b)序列如SEQ ID NO.16所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.16所示序列的同源性≥90％(较佳地≥95％)，并且编码SEQ ID NO.15所示氨基酸序列的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述核酸分子包含选自下组的编码所述CD8a的跨膜区的核酸序列：
(a)编码如SEQ ID NO.17所示多肽的多核苷酸；
(b)序列如SEQ ID NO.26所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.26所示序列的同源性≥90％(较佳地≥95％)，并且编码SEQ ID NO.17所示氨基酸序列的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述核酸分子包含选自下组的编码所述4-1BB(CD137)的胞内信号结构域的核酸序列：
(a)编码如SEQ ID NO.18或27所示多肽的多核苷酸；
(b)序列如SEQ ID NO.19所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.19所示序列的同源性≥90％(较佳地≥95％)，并且编码SEQ ID NO.18所示氨基酸序列的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述核酸分子包含选自下组的编码所述CD3ζ的胞内信号结构域的核酸序列：
(a)编码如SEQ ID NO.20或32所示多肽的多核苷酸；
(b)序列如SEQ ID NO.21或33所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.21或33所示序列的同源性≥90％(较佳地≥95％)，并且编码SEQ ID NO.20所示氨基酸序列的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述核酸分子包含选自下组的核酸序列：
(a)编码如SEQ ID NO.3或SEQ ID NO.1所示多肽的多核苷酸；
(b)序列如SEQ ID NO.4或SEQ ID NO.2所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.4或SEQ ID NO.2所示序列的同源性≥95％(较佳地≥98％)，并且编码SEQ ID NO.3或SEQ ID NO.1所示氨基酸序列的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述核酸分子为分离的。
在另一优选例中，所述核酸分子还包括编码前导序列(引导序列，信号肽)的多核苷酸，所述前导序列的氨基酸序列如SEQ ID NO.22或SEQ ID NO.24所示；优选地所述编码前导序列(信号肽)的多核苷酸如SEQ ID NO.23或25所示。
在另一优选例中，所述核酸分子的序列如SEQ ID NO.4或SEQ ID NO.2所示。
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一方面所述的核酸分子。
在另一优选例中，所述载体为慢病毒载体。
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。
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，所述细胞为哺乳动物细胞。
在另一优选例中，所述细胞为T细胞。
本发明的第五方面，提供了一种药物组合物，所述组合物含有药学上可接受的载体以及本发明第一方面所述的嵌合抗原受体、本发明第二方面所述的核酸分子、本发明第三方面所述的载体、或本发明第四方面所述的细胞。
本发明的第六方面，提供了本发明第一方面所述的嵌合抗原受体、本发明第二方面所述的核酸分子、本发明第三方面所述的载体、或本发明第四方面所述的细胞的用途，用于制备治疗肿瘤的药物或制剂。
在另一优选例中，所述的肿瘤包括CD19阳性B细胞恶性肿。
本发明的第七方面，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一方面所述的嵌合抗原受体、本发明第二方面所述的核酸分子、本发明第三方面所述的载体、或本发明第四方面所述的细胞、或本发明第五方面所述的药物组合物。
在另一优选例中，所述疾病为肿瘤。
本发明的第八方面，提供了一种制备CAR-T细胞(CAR-修饰的T细胞)的方法，所述CAR-T细胞表达本发明第一方面所述的嵌合抗原受体，
所述方法包括步骤：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T细胞内，从而获得所述CAR-T细胞。
本发明的其它特征和优点将在随后的具体实施方式部分予以详细说明。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为CAR-19.1,19.2CAR-19.3,和CAR-19.4的结构示意图。
图2为CAR-T19.1和19.2细胞与三种肿瘤细胞共培养的IFN-γ释放的ELISA检测。Ramos为CD19阳性细胞，Molt4和SEM为CD19阴性细胞。
图3为使用慢病毒转染制备的CAR-T19.1,CAR-T19.3,和CAR-T19.4的流式分 析，protein L染色被用于标记CAR阳性细胞，PBMC的供体为健康人。
图4为CAR-T19.1,19.3和19.4细胞与四种肿瘤细胞共培养的IFN-γ释放的ELISA检测。Raji和Ramos为CD19阳性细胞，Molt4和Karpas620为CD19阴性细胞。
图5为CAR-T19细胞与四种肿瘤细胞共培养的CD137表达上调的流式检测。
图6为使用CAR-T19细胞对四种肿瘤细胞的细胞杀伤实验，使用LDH试剂盒。
图7为CAR-T19细胞被四种肿瘤细胞刺激后细胞脱颗粒的流式检测，CD107a为脱颗粒的标记分子。
图8为使用慢病毒转染制备的CAR-T19.1,CAR-T19.3,和CAR-T19.4的流式分析，PBMC的供体为B-ALL病人。
图9为CAR-T19细胞与肿瘤细胞共培养的IFN-γ释放的ELISA检测。P，CD19阳性细胞，N，CD19阴性细胞，PBMC的供体为B-ALL病人。
图10为CAR-T19细胞与肿瘤细胞共培养的CD137表达上调的流式检测，PBMC的供体为B-ALL病人。
图11为根据本发明的嵌合抗原受体及载体结构示意图。
具体实施方式
本发明人通过广泛而深入的研究，获得一种优化的CD19靶向性的嵌合抗原受体及其制法和应用，所述嵌合抗原受体的细胞外抗原结合结构域N端到C端依次为抗体轻链可变区和抗体重链可变区。实验结果表明，本发明还提供的嵌合抗原受体，其特定的轻链可变区-重链可变区顺序的排布，膜外绞链区长度的调节，和信号传导结构域的调整能够显著提高抗原结合受体的活性，极大的提高了对肿瘤细胞的杀伤能力。
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。
嵌合抗原受体
本发明提供了包括细胞外结构域、跨膜结构域、和细胞内结构域的嵌合抗原受体(CAR)。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，本发明提供了经过基因工程改造以表达CAR的细胞(例如，T细胞)，其显示显著的抗肿瘤性质。本发明的CAR还可以包括胞外结构域，所述胞外结构域具有融合至T细胞抗原受体复合物ζ链(例如，CD3ζ)的细胞内信号传导结构域的抗原结合结构域。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改变抗原识别。示例性抗原为CD19，因为该抗原在恶性B细胞上表达。然而，本发明不限于靶向CD19。相反地，本发明包括任何抗原结合结构域，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CD137(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。
在一个实施方式中，本发明的CD19靶向性CAR包括包含本发明特定信号传导结构域(CD8的跨膜区、CD137和CD3ζ的胞内信号结构域串联而成)。与其他方式的CD19靶向性CAR相比，本发明的信号传导结构域显著增加了抗肿瘤活性和CAR-T细胞的体内持久性。
在本发明的一个较佳的实施方式中，本发明提供的嵌合抗原受体(CAR)的氨基酸序列如下：
CAR-19.3:
Figure PCTCN2017106135-appb-000001
其编码多核苷酸序列如下：
Figure PCTCN2017106135-appb-000002
在本发明的一个较佳的实施方式中，本发明提供的嵌合抗原受体(CAR)的氨基酸序列如下：
CAR-19.4:
Figure PCTCN2017106135-appb-000003
其编码多核苷酸序列如下：
Figure PCTCN2017106135-appb-000004
抗原结合结构域
在一个实施方式中，本发明的CAR包括被称为抗原结合结构域的靶-特异性结合元件。抗原结合结构域的选择取决于限定靶细胞表面的配体的类型和数目。例如，可选择抗原结合结构域，以识别用作与具体疾病状态相关的靶细胞上的细胞表面标记的配体。因此，细胞表面标记的例子包括与病毒、细菌和寄生虫感染，自身免疫疾病和癌细胞相关的那些标记。
在本发明优选的实施方式中，所述抗原为CD19。
在本发明优选的实施方式中，本发明的CAR的抗原结合结构域靶向CD19。优选地，本发明的CAR中的抗原结合结构域如下式(N端至C端)所示：
VL－VH
其中，VH为抗体重链可变区；VL为抗体轻链可变区；“－”为连接肽或肽键。
在本发明的一个优选地实施方式中，所述抗体重链可变区包括以下三个互补决定区CDR：
CDR1：GVSLPDYG，SEQ ID NO.5，
CDR2：IWGSETT，SEQ ID NO.6，和
CDR3：AKHYYYGGSYAMDY，SEQ ID NO.7。
在另一优选例中，所述抗体重链可变区的氨基酸序列如下：
Figure PCTCN2017106135-appb-000005
在本发明的一个优选地实施方式中，所述抗体轻链可变区包括以下三个互补决定区CDR：
CDR1':QDISKY，SEQ ID NO.9，
CDR2':HTS，SEQ ID NO.10，和
CDR3':QQGNTLPYT，SEQ ID NO.11。
在另一优选例中，所述抗体轻链可变区的氨基酸序列如下：
Figure PCTCN2017106135-appb-000006
在本发明的一个优选地实施方式中，本发明的CAR中的抗原结合结构域氨基酸序列如下：
Figure PCTCN2017106135-appb-000007
绞链区和跨膜区
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。
跨膜结构域可源于天然来源或合成来源。在天然来源中，该结构域可源于任何膜结合蛋白或跨膜蛋白。具体用于本发明的跨膜区可源于T-细胞受体的α、β或ζ链、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154(即至少包括上述中的跨膜区(一个或多个))。
优选地，本发明的CAR中的绞链区和跨膜区为CD8a的绞链区和跨膜区。
在本发明的一个优选的实施方式中，CD8a的绞链区包括以下氨基酸序列：
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO.14)；其编码多核苷酸序列如下：
Figure PCTCN2017106135-appb-000008
在本发明的一个优选的实施方式中，本发明的CAR中的绞链区氨基酸序列如下：
TRLSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO.15)；其编码多核苷酸序列如下：
Figure PCTCN2017106135-appb-000009
在本发明的一个优选的实施方式中，CD8a的跨膜区包括以下氨基酸序列
Figure PCTCN2017106135-appb-000010
其编码多核苷酸序列如下:
Figure PCTCN2017106135-appb-000011
胞内结构域
本发明的CAR的胞内结构域或另外的细胞内信号传导结构域是造成其中已放置CAR的免疫细胞的至少一种正常效应子功能的活化的原因。术语“效应子功能”指的是细胞的专有功能。例如，T细胞的效应子功能可为包括细胞因子分泌的细胞溶解活性或辅助活性。因此术语“细胞内信号传导结构域”指的是转导效应子功能信 号并指导细胞实施专有功能的蛋白部分。尽管通常可使用整个细胞内信号传导结构域，但在很多例子中，不必使用整个链。就使用细胞内信号传导结构域的截短部分而言，这种截短部分可用于代替完整的链，只要它转导效应子功能信号。术语细胞内信号传导结构域因此指包括足以转导效应子功能信号的细胞内信号传导结构域的任何截短部分。
用于本发明的CAR的细胞内信号传导结构域的优选例子包括T细胞受体(TCR)的胞浆序列和协同行动以在抗原受体结合后开始信号转导的共受体，以及这些序列的任何衍生物或变体和具有相同的功能能力的任何合成序列。
已知通过TCR单独产生的信号不足以完全活化T细胞，并且也需要次级或共刺激信号。因此，T细胞活化可被认为由两个不同类的胞浆信号传导序列介导：通过TCR(初级胞浆信号传导序列)开始抗原-依赖性初级活化的那些和以抗原-非依赖性方式起作用以提供次级或共刺激信号(次级胞浆信号传导序列)的那些。
初级胞浆信号传导序列以刺激方式或以抑制方式调节TCR复合物的初级活化。以刺激方式起作用的初级胞浆信号传导序列可包含信号传导基序，其已知为基于免疫受体酪氨酸的活化基序或ITAM。
包含在本发明中具有具体用途的初级胞浆信号传导序列的ITAM的例子包括源于TCRζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b和CD66d的那些。特别优选地，本发明的CAR中的胞浆信号传导分子包括源于CD3ζ的胞浆信号传导序列。
在优选的实施方式中，CAR的胞浆结构域可被设计以本身包括CD3-ζ信号传导结构域，或可与在本发明的CAR的内容中有用的任何其他期望的胞浆结构域(一个或多个)联合。例如，CAR的胞浆结构域可包括CD3ζ链部分和共刺激信号传导区。共刺激信号传导区指的是包括共刺激分子的细胞内结构域的一部分CAR。共刺激分子是淋巴细胞对抗原的有效应答所需的细胞表面分子，而不是抗原受体或它们的配体。这种分子的例子包括CD27、CD28、4-1BB(CD137)、OX40、CD30、CD40、PD-1、ICOS、淋巴细胞功能相关抗原-1(LFA-1)、CD2、CD7、LIGHT、
NKG2C、B7-H3和与CD83特异性结合的配体等等。因此，尽管本发明主要以4-1BB作为共刺激信号传导元件的例子，但其他共刺激元件也位于本发明的范围内。
本发明的CAR的胞浆信号传导部分内的胞浆信号传导序列可以随机或以规定的顺序相互连接。任选地，短的寡肽或多肽连接体，优选长度在2和10个氨基酸，可形成该连接。甘氨酸-丝氨酸双联体提供了特别合适的连接体。
在一个实施方式中，本发明的CAR中的胞浆结构域被设计以包括4-1BB的信号传导结构域和CD3ζ的信号传导结构域，其中4-1BB的信号传导结构域包括SEQ ID NO:4中提出的核酸序列和CD3-ζ的信号传导结构域包括SEQ ID NO:5中提出的核酸序列。
在一个实施方式中，本发明的CAR中的胞浆结构域被设计以包括4-1BB的信号传导结构域和CD3ζ的信号传导结构域，其中4-1BB的信号传导结构域包括编码SEQ ID NO:18的氨基酸序列的核酸序列，和CD3ζ的信号传导结构域包括编码SEQ ID NO:19的氨基酸序列的核酸序列。
在一个实施方式中，本发明的CAR中的胞浆结构域被设计以包括4-1BB的信号传导结构域和CD3ζ的信号传导结构域，其中4-1BB的信号传导结构域包括SEQ ID NO:18中提出的氨基酸序列，和CD3ζ的信号传导结构域包括SEQ ID NO:19中提出 的氨基酸序列。
优选地，4-1BB的胞内信号结构域包含如下氨基酸序列：
Figure PCTCN2017106135-appb-000012
其编码核苷酸序列如下：
Figure PCTCN2017106135-appb-000013
更优选地，4-1BB的胞内信号结构域包含如下氨基酸序列：
Figure PCTCN2017106135-appb-000014
其编码核苷酸序列如下
Figure PCTCN2017106135-appb-000015
优选地，CD3ζ的胞内信号结构域包含如下氨基酸序列：
Figure PCTCN2017106135-appb-000016
其编码核苷酸序列如下：
Figure PCTCN2017106135-appb-000017
在本发明的另一个优选地实施方式中，CD3ζ的胞内信号结构域包含如下氨基酸序列，其包含一个突变的氨基酸残基(下划线标示):
Figure PCTCN2017106135-appb-000018
其编码核苷酸序列如下
Figure PCTCN2017106135-appb-000019
载体
本发明包括包含CAR序列的DNA构建体，其中该序列包括可操作地连接至信号传导结构域的核酸序列的抗原结合结构域的核酸序列。可用于本发明的CAR的示例性的信号传导结构域包括抗-CD19scFv、CD8铰链和跨膜区、和CD137和CD3ζ胞内信号传导结构域。在一个实施方式中，本发明的CAR包括SEQ ID NO:10中提出的核酸序列。在另一个实施方式中，本发明的CAR包括编码SEQ ID NO:9的氨基酸序列的核酸序列。在另一个实施方式中，本发明的CAR包括SEQ ID NO:9中提出的氨基酸序列。
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DNA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，通常通过可操作地连接编码CAR多肽或其部分的核酸至启动子，并将构建体并入表达载体，实现编码CAR的天然或合成核酸的表达。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基因的基因(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，更优选地为pWPT-GFP慢病毒载体。本发明人研究证实，使用该慢病毒载体构建本发明CAR，对T细胞的转染效率较高，且具有高度的可重复性。
在本发明的一个优选地实施方式中，所述载体中还包括信号肽编码序列。优选地，所述信号肽序列连接在所述抗原结核结构域核酸序列的上游。优选地所述信号肽为大鼠生长激素信号肽或人源CD8a信号肽。
优选地，所述信号肽氨基酸序列如下：
MALPVTALLLPLALLLHAARP(人源CD8a，SEQ ID NO.22)；
其编码核酸序列如下：
Figure PCTCN2017106135-appb-000020
优选地，所述信号肽氨基酸序列如下：
MAADSQTPWLLTFSLLCLLWPQEAGALP(大鼠生长激素信号肽，SEQ ID NO.24)；
其编码核酸序列如下：
Figure PCTCN2017106135-appb-000021
治疗性应用
本发明包括用慢病毒载体(LV)转导的细胞(例如，T细胞)。例如，LV编码将特异性抗体的抗原结合结构域与CD3-ζ、CD137组合的细胞内结构域联合的CAR。因此，在一些例子中，转导的T细胞可引起CAR-介导的T-细胞应答。
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：施用给哺乳动物表达CAR的T细胞，其中CAR包括特异性地与预定标靶相互作用的结合部分，包括例如人CD3ζ的细胞内结构域的ζ链部分，和共刺激信号传导区。
在一个实施方式中，本发明包括一类细胞疗法，其中T细胞被基因修饰以表达CAR，和CAR-T细胞被注入需要其的接受者中。注入的细胞能够杀死接受者的肿瘤细胞。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 CD19 CAR-T细胞引起抗表达CD19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-CD19scFv、CD8铰链和跨膜区、和CD137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、。
在一个实施方式中，本发明的CAR的抗原结合结构域被设计以治疗具体的癌症。例如，被设计以靶向CD19的CAR可用于治疗癌症和紊乱，包括但不限于前-BALL(儿童指征)、成人ALL、套细胞淋巴瘤、扩散大B-细胞淋巴瘤，同种骨髓移植后的补救等等。
在一个实施方式中，癌症和紊乱包括但不限于前-BALL(儿童指征)，成人ALL、套细胞淋巴瘤、扩散大B-细胞淋巴瘤、同种骨髓移植后的补救等等，其可利用靶向CD19、CD20、CD22和ROR1的CAR的组合进行治疗。
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)扩展细胞，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。
通常地，如本文所述活化和扩展的细胞可用于治疗和预防无免疫应答的个体中产生的疾病。特别地，本发明的CAR-修饰的T细胞用于治疗CCL。在某些实施方式中，本发明的细胞用于治疗处于形成CCL风险中的患者。因此，本发明提供了治疗 或预防CCL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104至109个细胞/kg体重的剂量，优选105至106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1×106个至1×1010个本发明经修饰的T细胞(如，CAR-T19细胞)，通过例如静脉回输的方式，施用于患者。
本发明的优点包括：
1、本发明的嵌合抗原受体，其细胞外抗原结合结构域N端到C端依次为抗体轻链可变区和抗体重链可变区，能够显著提高抗原结合受体的活性，极大的提高了对肿瘤细胞的杀伤能力，与对照CAR(细胞外抗原结合结构域N端到C端依次为抗体重链可变区和抗体轻链可变区)相比，细胞杀伤能力提高了约一倍左右。
2、根据本发明的CAR，优化长度的胞外结构区(来自于CD8a)和信号结构域的额外氨基酸能够显著提高CAR-T细胞对肿瘤靶细胞的杀伤活力。
3、以上所述CAR-T活力的提高表现为显著增加的IFN-g的释放和CD137的上调，以及显著提高的直接细胞杀伤能力和CD107a脱颗粒能力。
实施例1 慢病毒表达载体的构建
编码质粒为委托上海博益生物科技有限公司做全长DNA合成和克隆构建。克隆载体选用的是pWPT慢病毒载体(购自Addgene公司)，克隆位点为BamH I和Sal I位点。将克隆慢病毒表达载体质粒进行测序,测序结果与预期相符。构建的载体结构如图11所示。
合成的CAR-19结构如下(图1)：
CAR-19.1
L(大鼠生长激素信号肽SEQ ID No.24)-VH－VL－H(较长CD8a铰链区SEQ ID No.15)－TM－4-1BB(包含额外氨基酸，SEQ ID No.27)－CD3ζ(SEQ ID NO.20)；其核苷酸序列如下：
Figure PCTCN2017106135-appb-000022
Figure PCTCN2017106135-appb-000023
CAR-19.2
L(人CD8a信号肽SEQ ID No.22)-VH－VL－H(较长CD8a铰链区SEQ ID No.15)－TM－4-1BB(包含额外氨基酸，SEQ ID No.27)－CD3ζ(SEQ ID NO.20)；
其核苷酸序列如下：
Figure PCTCN2017106135-appb-000024
CAR-19.3(SEQ ID NO.1)
L(人CD8a信号肽SEQ ID No.22)-VL－VH－H(较短CD8a铰链区SEQ ID No.14)－TM－4-1BB(无额外氨基酸，SEQ ID No.18)－含突变的CD3ζ(SEQ ID NO.33)；其核苷酸序列如SEQ ID NO.2所示。
CAR-19.4(SEQ ID NO.3)
L(人CD8a信号肽SEQ ID No.22)-VL－VH－H(较长CD8a铰链区SEQ ID No.15) －TM－4-1BB(包含额外氨基酸，SEQ ID No.27)－野生型的CD3ζ(SEQ ID NO.20)；其核苷酸序列如SEQ ID NO.4所示。
上述各CAR中，VH氨基酸序列如SEQ ID NO.8所示；VL氨基酸序列如SEQ ID NO.12所示；VL和VH之间的连接肽序列均为GGGGSGGGGSGGGGS(SEQ ID NO.28)；TM氨基酸序列如SEQ ID NO.17所示。
实施例2 CAR-T19.1,CAR-T19.2,CAR-T19.3,和CAR-T19.4细胞的制备
(1)取健康人静脉血，密度梯度离心方法分离获得单个核细胞(PBMCs)。
(2)第0天，PBMCs采用含有血清的细胞培养基GT-T551，调整细胞终浓度为2×106cell/mL。将细胞接种于预先经过终浓度为5μg/mLCD3单克隆抗体(OKT3)及终浓度为10μg/mL的Retronectin(购自TAKORA公司)包被的细胞培养瓶。培养基里加入终浓度为1000U/mL的重组人白介素2(IL-2)，在37℃，饱和湿度为5％CO2培养箱培养。
(3)第2天，加入新鲜GT-T551培养液，浓缩纯化的CAR-19.1,CAR-19.3,和CAR-19.4慢病毒液，1：1000的protamine sulfate，以及终浓度为1000U/mL IL-2。置于37℃，5％CO2培养箱中感染12小时后，弃培养液，加入新鲜的培养基，于37℃，5％CO2培养箱继续进行培养。
实施例3.CAR-T19.1和CAR-T19.2细胞的功能检测及对比。
一组独立实验对CAR-T19.1和CAR-T19.2细胞进行功能检测时，在第10天将CAR-T细胞与三种肿瘤靶细胞共培养，18小时后取培养液上清进行IFN-γ的ELISA检测。Ramos为CD19阳性细胞，Molt4和SEM为CD19阴性细胞。结果见图2，二种CAR-T细胞与Ramos的共培养产生阳性结果，但是CAR-T19.1和CAR-T19.2没有显示出明显的IFN-γ的释放的差异。这个结果证明仅仅将CAR的信号肽序列替换为人源CD8a信号肽序列对于CAR-T细胞功能改善并不明显。CAR-T19.2将不再被进一步研究。
实施例4 CAR-T19.1,CAR-T19.3,和CAR-T19.4细胞的功能检测及对比
(1)另一组独立的实验中，在第6天对CAR-T19细胞进行Protein L染色并用流式细胞仪检测CAR阳性表达的T细胞。结果见图3，慢病毒感染的CAR-T19.1,CAR-T19.3,和CAR-T19.4细胞的CAR阳性达到80～90％。
(2)第10天，分别将CAR-T19细胞与四种肿瘤细胞共培养，进行IFN-γ释放的ELISA检测。105CAR-T19细胞和105肿瘤细胞进行共培养，18小时后取培养液上清进行ELISA检测。CAR-T19细胞被未感染的同供体细胞1：2稀释(终CAR阳性率～30％)，Raji和Ramos为CD19阳性细胞，Molt4和Karpas620为CD19阴性细胞。结果见图4，与Raji和Ramos的共培养产生阳性结果，CAR-T19.3和CAR-T19.4显示出高于CAR-T19.1的IFN-γ的释放。
(3)第10天，使用(5)共培养的细胞进行CD137的抗体染色和流式检测。CD137是T细胞抗原特异性激活的细胞表面标记。结果见图5，CAR-T19.3和CAR-T19.4比CAR-T19.1表现出更高的CD137激活上调表达。
实施例5 CAR-T19.1,CAR-T19.3,和CAR-T19.4细胞的体外细胞杀伤性能的检测
(1)采用制备的CAR-T19细胞进行体外杀伤实验。试剂盒使用CytoTox 
Figure PCTCN2017106135-appb-000025
 Non-Radioactive Cytotoxicity Assay(Promega)并按照厂家提供的方法进行实验检测。采用4种肿瘤靶细胞和不同的CAR-T19细胞与靶细胞的比例，结果如图6所示。CAR-T19.3和CAR-T19.4比较CAR-T19.1表现出对Raji，Ramos更强的杀伤活性。同时CAR-T19.4具有最高的体外细胞杀伤力。
(2)采用实施例1中制备的CAR-T19细胞进行CD107a degranulation实验。CD107a是T细胞degranulation的标记,直接导致perforin/granzyme的释放和靶细胞的杀伤。105CAR-T19细胞和105肿瘤细胞进行共培养并进行CD107a的染色，6小时使用流式检测CD107a degranulation。结果见图6，在CAR-T19细胞与Raji和Ramos靶细胞进行共培养后，CAR-T19.3和CAR-T19.4表现出比CAR-T19.1更高的degranulation，同时CAR-T19.3表达最高水平的CD107a。
实施例6 采用B-ALL病人细胞作为供体制备CAR-T19.1,CAR-T19.3,和CAR-T19.4细胞，及细胞功能检测
(1)以B-ALL病人的外周血细胞为供体，分离PBMC，OKT3激活细胞并进行CAR-19.1,CAR-19.3,和CAR-19.4慢病毒感染。
(2)第12天，对CAR-T19细胞进行Protein L染色并用流式细胞仪检测CAR阳性表达的T细胞。结果见图8，慢病毒感染的CAR-T19.1,CAR-T19.3,和CAR-T19.4细胞的CAR阳性达到20～40％。
(3)第12天，分别将CAR-T19细胞与肿瘤靶细胞共培养，进行IFN-g释放的ELISA检测。105CAR-T19细胞和105肿瘤细胞进行共培养，18小时后取培养液上清进行ELISA检测。P为CD19阳性细胞，N为CD19阴性细胞。结果见图9，CAR-T19.3和CAR-T19.4显示出高于CAR-T19.1的IFN-γ的释放，同时CAR-T19.4具有最高的IFN-γ的水平。
(4)第12天，使用(3)共培养的细胞进行CD137的抗体染色和流式检测。CD137是T细胞抗原特异性激活的细胞表面标记。结果见图10，CAR-T19.3和CAR-T19.4比CAR-T19.1表现出更高的CD137激活上调表达。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。

Claims (11)

  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如下式所示：
    VL－VH
    其中，VH为抗体重链可变区；VL为抗体轻链可变区；“－”为连接肽或肽键；
    优选地，所述抗体重链可变区包括以下三个互补决定区CDR：
    SEQ ID NO.5所示的CDR1，
    SEQ ID NO.6所示的CDR2，和
    SEQ ID NO.7所示的CDR3；和/或
    所述抗体轻链可变区包括以下三个互补决定区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'。
  2. 如权利要求1所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体如下式所示：
    L－VL－VH－H－TM－4-1BB－CD3ζ
    其中，
    L为任选的引导序列(Leader sequence，即信号肽序列)；
    H为绞链区；
    TM为跨膜结构域；
    4-1BB为为源于4-1BB的共刺激分子；
    CD3ζ为源于CD3ζ的胞浆信号传导序列；
    VH和VL分别如上所述。
  3. 如权利要求2所述的嵌合抗原受体，其特征在于，L的序列如SEQ ID NO.22或24所示；和/或
    H的序列包括SEQ ID NO.14所示的氨基酸序列；优选地，H的序列如SEQ ID NO.15所示；和/或
    TM的序列如SEQ ID NO.17所示；和/或
    4-1BB的序列如SEQ ID NO.18所示；和/或
    CD3ζ的序列如SEQ ID NO.20或33所示。
  4. 如权利要求1所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体的序 列如SEQ ID NO.3或SEQ ID NO.1所示。
  5. 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)。
  6. 一种载体，其特征在于，所述的载体含有权利要求5中所述的核酸分子；优选地所述载体为慢病毒载体。
  7. 一种宿主细胞，其特征在于，所述的宿主细胞中含有权利要求6中所述的载体或染色体中整合有外源的权利要求5中所述的核酸分子；优选地，所述细胞为T细胞。
  8. 一种药物组合物，其特征在于，所述组合物含有药学上可接受的载体以及权利要求1中所述的嵌合抗原受体、权利要求5中所述的核酸分子、权利要求6中所述的载体、或权利要求7中所述的细胞。
  9. 权利要求1中所述的嵌合抗原受体、权利要求5中所述的核酸分子、权利要求6中所述的载体、或权利要求7中所述的细胞的用途，其特征在于，用于制备治疗肿瘤的药物或制剂；优选地，所述的肿瘤包括CD19阳性的B细胞肿瘤。
  10. 一种治疗疾病的方法，其特征在于，包括给需要治疗的对象施用适量的权利要求1中所述的嵌合抗原受体、权利要求5中所述的核酸分子、权利要求6中所述的载体、权利要求7中所述的细胞、或权利要求8中所述的药物组合物。
  11. 一种制备CAR-T细胞的方法，其特征在于，所述CAR-T细胞表达权利要求1所述的嵌合抗原受体，
    所述方法包括步骤：将权利要求5所述的核酸分子或权利要求6所述的载体转导入T细胞内，从而获得所述CAR-T细胞。
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