WO2018218710A1 - 一种以robo1为靶点的嵌合抗原受体细胞及其制备和应用 - Google Patents

一种以robo1为靶点的嵌合抗原受体细胞及其制备和应用 Download PDF

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WO2018218710A1
WO2018218710A1 PCT/CN2017/089517 CN2017089517W WO2018218710A1 WO 2018218710 A1 WO2018218710 A1 WO 2018218710A1 CN 2017089517 W CN2017089517 W CN 2017089517W WO 2018218710 A1 WO2018218710 A1 WO 2018218710A1
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car
cell
nucleotide
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王保垒
韩昆昆
李华顺
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阿思科力(苏州)生物科技有限公司
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Priority to US16/700,401 priority Critical patent/US11697685B2/en
Priority to US18/319,638 priority patent/US20230287116A1/en

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Definitions

  • the invention relates to the field of biotechnology, in particular to a chimeric antigen receptor cell targeting ROBO1, in particular to an enhanced CAR-T cell and a CAR-NK cell targeting ROBO1, and preparation thereof and application.
  • Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) immunotherapy is a adoptive immunotherapy method that has been rapidly developed in recent years, in which CAR usually consists of antigen-binding domain and transmembrane structure. A domain, a costimulatory signaling region, and the like, which express and express an antibody capable of recognizing a tumor-associated antigen on the surface of an autologous T cell by a genetic modification technique, and the modified T cell thus has a targeted killing effect on the tumor cell.
  • CAR-T therapy has a significant therapeutic effect in the treatment of acute leukemia and non-Hodgkin's lymphoma and is considered to be one of the most promising treatments for cancer.
  • PD-L1 full-length programmed death receptor-ligand 1 the English name programmed cell death-Ligand 1 is a first type transmembrane protein with a size of 40 kDa.
  • PD-1 the full name of programmed death receptor 1, the English name for programmed death 1, is an important immunosuppressive molecule and is a member of the CD28 superfamily.
  • PD-1 programmeed death-1
  • PD-L1 and PD-L2 are widely expressed in antigen-presenting cells (APC).
  • APC antigen-presenting cells
  • the interaction of PD-1 with its ligand plays an important role in the negative regulation of immune response.
  • the expression of PD-L1 protein can be detected in many human tumor tissues.
  • the microenvironment of the tumor site can induce the expression of PD-L1 on tumor cells.
  • the expression of PD-L1 is beneficial to the occurrence and growth of tumors, and induces anti-tumor.
  • the apoptosis of T cells evades the attack of the immune system. Inhibition of the binding of PD-1 to its ligand can expose tumor cells to the killing field of the immune system, thereby achieving the effect of killing tumor tissue and treating cancer.
  • Patent application CN105505869A discloses a chimeric receptor T cell directed against a tumor stem cell which is fused with 2-3 independent antigen receptors, each chimeric antigen receptor being separately differentiated by a different tumor stem cell
  • the antigen binding portion of the antibody of the marker is composed of a combination of different functional proteins.
  • This chimeric receptor T cell activates T cell anti-tumor effects only after 2-3 chimeric antigen receptors recognize the antigen, and this method improves the specificity but the tumor cells that hide the antigen to some extent. Lower specificity.
  • CAR-T genetically modified chimeric antigen receptor T cell
  • the present invention provides a chimeric antigen receptor cell targeting ROBO1, particularly a boosted CAR-T cell and CAR-NK cell targeting ROBO1, and Its preparation and application.
  • a first aspect of the invention provides a coding nucleotide comprising a nucleotide encoding a chimeric antigen receptor and a nucleotide encoding an extracellular secretory protein, wherein the chimeric antigen receptor comprises an antigen binding domain, Transmembrane domain and costimulatory signaling region, said antigen binding junction
  • the domain is capable of specifically binding to the tumor-specific antigen ROBO1 and activating immune cells (eg, T cells, B cells, or natural killer cells) through a transmembrane domain and a costimulatory signaling region that is capable of blocking or inhibiting wild Binding of the PD-1 protein to the PD-L1 ligand.
  • the nucleotide encoding the chimeric antigen receptor and the nucleotide encoding the extracellular secreted protein are not in the same reading frame.
  • the extracellular secreted protein is a PD-1 protein fragment that binds to PD-L1 and/or PD-L2.
  • the extracellular secretory protein is an extracellular domain of a PD-1 protein or a mutant sequence thereof, wherein the mutant of the extracellular domain is capable of binding PD-L1 with high affinity;
  • the extracellular domain of the PD-1 protein or a mutant thereof has the amino acid sequence set forth in SEQ ID NO: 8.
  • the nucleotide encoding the extracellular domain of the PD-1 protein or a mutant thereof has the sequence set forth in SEQ ID NO: 1.
  • the extracellular domain of Robo1 includes five immunoglobulin conserved regions (named Ig1-5, respectively) and three Fibronectin type III repeats (named FNIII1-3, referred to as FN1-3, respectively), and the intracellular region includes four conserved regions. Areas (named: CC0, CC1, CC2, and CC3, respectively).
  • the antigen binding domain is capable of specifically binding to one or more of the Ig1, Ig2, Ig3, Ig4, Ig5, FN1, FN2 and FN3 domains of the tumor-specific antigen ROBO1.
  • the antigen-binding domain in the coding nucleotide, is capable of specifically binding to the FN3 domain of the tumor-specific antigen ROBO1 (hereinafter referred to as ROBO1-FN3).
  • the antigen-binding domain in the coding nucleotide, is an antibody or an antigen-binding fragment thereof; preferably, the antigen-binding domain is an FN3 structure that specifically binds to ROBO1. An antibody or antigen-binding fragment thereof.
  • the antigen-binding fragment in the coding nucleotide, is a Fab or scFv.
  • the antigen-binding domain in the coding nucleotide, is an anti-ROBO1-FN3scFv; preferably, the anti-ROBO1-FN3scFv has an amino acid sequence as shown in SEQ ID NO: .
  • the antigen-binding domain in the coding nucleotide, is an anti-ROBO1-FN3 scFv, and the nucleotide encoding the antigen-binding domain has the sequence shown in SEQ ID NO: the sequence of.
  • the encoding nucleotide further comprises an internal ribosome entry site IRES encoding nucleotide present in the nucleotide encoding the chimeric antigen receptor and encoding an extracellular secreted protein.
  • IRES encoding nucleotide has the sequence set forth in SEQ ID NO:3.
  • the encoding nucleotide comprises a coding nucleotide of an extracellular domain of an IRES-PD-1 protein or a mutant thereof.
  • the transmembrane domain is selected from the group consisting of: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134 a transmembrane domain of one or more of CD137, ICOS, and CD154.
  • the nucleotides encoding the transmembrane domain is the transmembrane CD8 (CD8 (TM)) domain.
  • the CD8 TM domain has as SEQ ID NO: 10 in the sequence shown in FIG.
  • the costimulatory signaling region comprises an intracellular domain of a costimulatory molecule, preferably, the costimulatory molecule is selected from the group consisting of: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22 One or more of CD79a, CD79b, CD66d, CD2, CD4, CD5, CD28, CD134, CD137, ICOS, CD154, 4-1BB and OX40.
  • the costimulatory signaling region comprises a 4-1BB and a CD3 ⁇ intracellular domain.
  • the 4-1BB intracellular domain has the sequence set forth in SEQ ID NO:11.
  • the CD3 ⁇ intracellular domain has the sequence set forth in SEQ ID NO:12.
  • the coding nucleotide of the 4-1BB intracellular domain has the sequence set forth in SEQ ID NO:5.
  • the coding nucleotide of the CD3 ⁇ intracellular domain has the sequence set forth in SEQ ID NO: 6.
  • the encoding nucleotide comprises a nucleotide encoding a CD8 transmembrane domain, a 4-1BB and a CD3 sputum intracellular domain.
  • said encoding comprises encoding nucleotide CD8 TM -4-1BB-CD3 ⁇ nucleotides.
  • the encoding nucleotide comprises an anti-ROBO1-FN3scFv-CD8 TM -4-1BB- CD3 ⁇ encoding nucleotides, preferably, the anti-ROBO1-FN3scFv-CD8 TM
  • the coding nucleotide of -4-1BB-CD3 ⁇ has the sequence shown in SEQ ID NO:13.
  • the encoding nucleotide sequence is isolated and/or synthesized.
  • the present invention comprises the coding nucleotide (the extracellular domain of PD-1 protein or a mutant thereof) the anti-ROBO1-FN3scFv-CD8 TM -4-1BB- CD3 ⁇ -IRES- encoding nucleotides, preferably, the ROBO1-FN3scFv-CD8 TM -4-1BB- CD3 ⁇ -IRES- antibody (the extracellular domain of PD-1 protein or a mutant thereof) encoding nucleotide having as SEQ ID NO The sequence shown in :7.
  • a second aspect of the invention provides a vector comprising the above-described encoding nucleotide of the invention.
  • the vector is selected from one or more of a plasmid, a bacterium, a virus, and the like.
  • the vector may use a viral vector such as a retroviral vector (including an oncogenic retroviral vector, a lentiviral vector, and a pseudotype vector) that lacks replication ability and is unable to self-replicate in transfected cells.
  • a viral vector such as a retroviral vector (including an oncogenic retroviral vector, a lentiviral vector, and a pseudotype vector) that lacks replication ability and is unable to self-replicate in transfected cells.
  • adenoviral vector including an oncogenic retroviral vector, a lentiviral vector, and a pseudotype vector
  • vaccinia virus vector or HSV Carrier and the like.
  • the vector is a lentiviral vector.
  • the lentiviral vector is a PRRSLIN vector.
  • the vector further comprises a promoter.
  • the promoter is an EF-1 ⁇ promoter.
  • a third aspect of the invention provides a chimeric antigen receptor cell (CAR-cell) encoding an extracellular secretory protein, wherein the extracellular secretory protein is capable of inhibiting or inhibiting binding of a wild-type PD-1 protein to a PD-L1 ligand .
  • CAR-cell chimeric antigen receptor cell
  • the extracellular secretory protein in the CAR-cell, is a PD-1 protein fragment that binds to PD-L1 and/or PD-L2.
  • the extracellular secretory protein in the CAR-cell, is an extracellular domain of a PD-1 protein or a mutant sequence thereof; preferably, the extracellular form of the PD-1 protein
  • the domain or mutant thereof has the amino acid sequence set forth in SEQ ID NO: 8.
  • the nucleotide encoding the extracellular domain of the PD-1 protein or a mutant thereof has the sequence shown in SEQ ID NO: 1.
  • the CAR-cell further encodes a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, and a costimulatory signaling domain, wherein the antigen binding domain is capable of The tumor-specific antigen ROBO1 is specifically bound and activated by the transmembrane domain and the costimulatory signaling domain (eg, CAR-T cells, B cells, or natural killer cells).
  • a chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, and a costimulatory signaling domain, wherein the antigen binding domain is capable of The tumor-specific antigen ROBO1 is specifically bound and activated by the transmembrane domain and the costimulatory signaling domain (eg, CAR-T cells, B cells, or natural killer cells).
  • the antigen-binding domain of the CAR-cell is capable of specifically binding to the Ig1, Ig2, Ig3, Ig4, Ig5, FN1, FN2 and FN3 domains of the tumor-specific antigen ROBO1 One or more of them.
  • the antigen binding domain in the CAR-cell, is capable of specifically binding to the tumor-specific antigen ROBO1-FN3.
  • the antigen-binding domain in the CAR-cell, is an antibody or an antigen-binding fragment thereof; preferably, the antigen-binding domain is a FN3 domain that specifically binds to ROBO1 Antibody or antigen-binding fragment thereof.
  • the antigen-binding fragment in the CAR-cell, is a Fab or scFv.
  • the antigen-binding domain in the CAR-cell, is an anti-ROBO1-FN3 scFv; preferably, the anti-ROBO1-FN3 scFv has an amino acid sequence as shown in SEQ ID NO: More preferably, the nucleotide encoding the antigen binding domain has the sequence set forth in SEQ ID NO: 2.
  • an internal ribosome entry site exists between the nucleotide encoding the chimeric antigen receptor and the nucleotide encoding the extracellular secretory protein in the CAR-cell in the CAR-cell.
  • IRES encodes a nucleotide having the sequence set forth in SEQ ID NO:3.
  • the transmembrane domain is selected from the group consisting of: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, Transmembrane domain of one or more of CD137, ICOS, and CD154.
  • the nucleotides encoding the transmembrane domain is the transmembrane CD8 (CD8 (TM)) domain.
  • the CD8 TM domain has as SEQ ID NO: 10 in the sequence shown in FIG.
  • the costimulatory signaling region comprises an intracellular domain of a costimulatory molecule, preferably, the costimulatory molecule is selected from the group consisting of: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22 One or more of CD79a, CD79b, CD66d, CD2, CD4, CD5, CD28, CD134, CD137, ICOS, CD154, 4-1BB and OX40.
  • the costimulatory signaling region comprises a 4-1BB and a CD3 ⁇ intracellular domain.
  • the 4-1BB intracellular domain has the sequence set forth in SEQ ID NO:11.
  • the CD3 ⁇ intracellular domain has the sequence set forth in SEQ ID NO:12.
  • the coding nucleotide of the 4-1BB intracellular domain has the sequence set forth in SEQ ID NO:5.
  • the coding nucleotide of the CD3 ⁇ intracellular domain has the sequence set forth in SEQ ID NO: 6.
  • the encoding nucleotide comprises a nucleotide encoding a CD8 transmembrane domain, a 4-1BB and a CD3 sputum intracellular domain.
  • the cell comprises encoding CAR- CD8 TM -4-1BB-CD3 ⁇ nucleotides.
  • the cells comprise CAR- ROBO1 FN3scFv--CD8 -4-1BB CD3 ⁇ -TM encoding the anti-nucleotides, preferably, the anti-ROBO1-FN3scFv-CD8 TM -
  • the coding nucleotide of 4-1BB-CD3 ⁇ has the sequence shown as SEQ ID NO: 13.
  • the CAR- (extracellular domain of PD-1 protein or a mutant) cell comprising an anti-ROBO1-FN3scFv-CD8 TM -4-1BB- CD3 ⁇ -IRES- of nucleotide, preferably, the anti-ROBO1-FN3scFv-CD8 TM -4-1BB- CD3 ⁇ -IRES- ( extracellular domain of PD-1 protein or a mutant thereof) encoding nucleotide having as SEQ ID NO: The sequence shown in 7.
  • the CAR-cell comprises the coding nucleotide of the first aspect of the invention.
  • the CAR-cell comprises the vector of the second aspect of the invention.
  • the CAR-cell is an immune cell selected from the group consisting of: T cells, natural killer (NK) cells, cytotoxic T One or more of lymphocyte (CTL) cells and regulatory T cells.
  • the T cell is a human T cell, preferably the human is a human having cancer.
  • the NK cells are human NK cells, preferably the human is a human having cancer, and more preferably, the NK cells are NK92 cells.
  • the CAR-cell is a human T cell (hereinafter abbreviated as CAR-T cell).
  • the CAR-cell is an NK cell (hereinafter abbreviated as CAR-NK cell).
  • the CAR-NK cell is a CAR-NK92 cell.
  • the CAR-cells of the invention may be administered alone or as a pharmaceutical composition in combination with other components such as IL-2 or other cytokines or cell populations.
  • a fourth aspect of the present invention provides a method for preparing the above CAR-cell, the method comprising the steps of:
  • step (3) Using the step (2) to obtain a virus infecting immune cells, the immune cells expressing the chimeric antigen receptor and the extracellular domain of the extracellular secreted PD-1 protein or a mutant protein thereof, thereby obtaining CAR-cells.
  • a fifth aspect of the invention provides a pharmaceutical composition comprising one or more selected from the group consisting of the above-described nucleotides, vectors and CAR-cells of the invention.
  • the pharmaceutical composition comprises an effective amount of CAR-cells, preferably CAR-T cells and/or CAR-NK92 cells.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.
  • the adjuvant is selected from one or more of the group consisting of a buffer, a carbohydrate, an antioxidant, a chelating agent, and a preservative.
  • the pharmaceutical compositions of the invention are preferably formulated for intravenous administration.
  • the administration of the pharmaceutical composition of the present invention can be carried out in any convenient manner, including by spraying, injection, oral, infusion, implantation or transplantation.
  • the pharmaceutical composition of the present invention can be subcutaneously or intradermally Intratumor, intraductal, intraspinal, intramuscular, intravenous or intraperitoneal administration to patients.
  • the pharmaceutical composition is administered to a patient by intravenous injection (i.v.).
  • a sixth aspect of the invention provides the use of one or more of the above-described encoding nucleotides, vectors, CAR-cells and pharmaceutical compositions of the invention for the manufacture of a medicament for the treatment and/or prevention of cancer.
  • the cancer is a tumor and a related disease that highly express Robo1, and the high expression described herein means that the expression level of Robo1 in the tumor cell is higher than that in the normal cell.
  • the expression level is higher than that in the normal cell.
  • the cancer is liver cancer, breast cancer, colon cancer, pancreatic cancer, prostate cancer, glioma or lung cancer.
  • the cancer is lung cancer.
  • the inventors of the present application have prepared a chimeric antigen receptor cell targeting ROBO1 based on its previous research work (such as Chinese patent application CN201610237593.2) and related to its preparation and application, especially one ROBO1 is a target of enhanced CAR-T cells and CAR-NK cells. These cells secrete the expression of the extracellular domain of PD-1 protein or its mutant molecules while stably expressing the CAR element, which can block PD. -1/PD-L1 molecular interaction, which was found by animal experiments to have a very good anti-tumor effect, and the above cells can significantly reduce the tumor recurrence rate compared with the conventional ROBO1-targeted CAR modified cells. To improve survival rate.
  • Example 1 is a schematic diagram showing the construction of a lentiviral expression vector provided in Example 1 of the present invention.
  • FIG. 2-A is a T cell not subjected to virus infection, and is used as a control
  • FIG. 2-B is a T cell after virus infection.
  • FIG. 3 is a flow chart of CAR-NK-92 flow provided by Example 4 of the present invention, and FIG. 3A is a NK-92 cell not infected with virus as a control; FIG. 3B is a virus infection and NK-92 cells after 1 month of culture were cultured by flow sorting.
  • Fig. 4 is a graph showing the results of analysis of the killing activity of CAR cells provided in Example 5 of the present invention.
  • Fig. 5 is a graph showing the results of evaluation of the efficacy of the drug according to Example 6 of the present invention in a mouse tumor model.
  • Figure 6 is a graph showing the survival rate of the experimental endpoint ROBO1-(extracellular domain of PD-1 protein or its mutant) in the CAR-T group and the ROBO1 CAR-T group, respectively, provided in Example 6 of the present invention.
  • Figure 7 is a graph showing the tumor recurrence rate of the experimental endpoint ROBO1-(extracellular domain of PD-1 protein or its mutant) in the CAR-T group and the ROBO1 CAR-T group, respectively, provided in Example 6 of the present invention.
  • the term "antibody” refers to an immunoglobulin molecule that specifically binds to an antigen.
  • the antibody may be a complete immunoglobulin derived from a natural source or derived from a recombinant source, and may be an immunoreactive portion of a complete immunoglobulin.
  • the antibody is typically a tetramer of immunoglobulin molecules.
  • the antibodies of the present invention may exist in various forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab) 2 , as well as single-chain antibodies and humanized antibodies (Harlow et al., 1999, In :Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al, 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al, 1988, Proc. Natl. Acad. Sci. 85: 5879-5883; Bird et al, 1988, Science 242: 423-426).
  • antibody fragment refers to a portion of an intact antibody and refers to the antigenic determining variable region of an intact antibody.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 and Fv fragments, linear antibodies formed from antibody fragments, scFv antibodies, and multispecific antibodies.
  • antigen-binding fragment consists of a complete light chain as well as a heavy chain VH, CH1 domain, which specifically binds to an antigen.
  • the term "encoding" refers to the intrinsic properties of polynucleotides such as genes, cDNAs or specific sequences of nucleotides in mRNA used as templates for the synthesis of other multimers and macromolecules in biological processes, said multimers and A macromolecule has any one of a defined sequence of nucleotides (ie, rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties produced thereby.
  • a defined sequence of nucleotides ie, rRNA, tRNA, and mRNA
  • a nucleotide sequence that is equivalent to an mRNA sequence and usually provides a coding strand in the sequence listing, and a non-coding strand that serves as a template for the transcriptional gene or cDNA, may be referred to as a protein or other product encoding that gene or cDNA.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA can include introns.
  • costimulatory molecule refers to an associated binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of a T cell, such as, but not limited to, proliferation, including but not limited to MHCI.
  • costimulatory ligand such as, but not limited to, proliferation, including but not limited to MHCI.
  • Class-like molecules, BTLA and Toll ligand receptors are examples of T cell that are used to mediating a costimulatory response of a T cell, such as, but not limited to, proliferation, including but not limited to MHCI.
  • lentivirus refers to the genus of the family Retroviridae, which is capable of efficiently infecting non-cyclical and post-mitotic cells; they can deliver significant amounts of genetic information into the host cell's DNA so that they are the most gene delivery vectors. One of the effective methods.
  • promoter is defined as required to initiate the specific transcription of a polynucleotide sequence, which is recognized by the cell's synthetic machinery or directs the DNA sequence of the synthesizing machine.
  • the term “specifically binds” refers to the recognition of a specific antigen but does not substantially recognize or bind other molecules in the sample.
  • vector is a composition of matter that includes an isolated nucleic acid and which can be used to deliver an isolated nucleic acid to the interior of a cell.
  • vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses.
  • vector includes autonomously replicating plasmids or viruses.
  • the term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.
  • isolated refers to changing or removing from a natural state.
  • a nucleic acid or peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide that is partially or completely separated from its coexisting material in its natural state is “isolated.”
  • the isolated nucleic acid or protein may be present in substantially purified form or, for example, may be present in a non-native environment, such as a host cell.
  • cancer is defined as a disease characterized by rapid and uncontrolled growth of distorted cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like.
  • anti-tumor effect refers to a biological effect which is clear by a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancerous condition. Said.
  • patient refers to any animal or cell thereof, whether in vitro or in situ, subject to the methods described herein.
  • the patient, subject or individual is a human.
  • the "-" linkage of the amino acid sequence of the present invention is such that the N-terminus of one fragment is directly linked to the C-terminus of the other fragment without any linker peptide therebetween.
  • the above gene sequences are sequentially sequenced according to the scFv gene, the CD8 gene, the human 4-1BB intracellular domain gene and the CD3 sputum intracellular domain gene, the IRES internal ribosome entry site, and the extracellular domain of the PD-1 protein or a mutant gene thereof. sequences joined, introducing different restriction sites at each junction sequence, to form a complete scFv-CD8 TM -4-1BB-CD3 ⁇ - IRES- ( extracellular domain of PD-1 protein or a mutant) gene sequences , as shown in SEQ ID NO: 7.
  • the scFv-CD8 TM -4-1BB-CD3 ⁇ - IRES- extracellular domain of PD-1 protein or a mutant gene sequences transformed into pRRSLIN connection by DNA vector, upstream of the gene for the EF-1 ⁇ promoter .
  • the vector was transformed into Stbl3 Escherichia coli strain, then transferred to a solid medium containing ampicillin for propagation, screened, positive clones were obtained, plasmids were extracted, clones were identified by restriction enzyme digestion, and the vector was successfully constructed by sequencing to obtain pRRSLIN-scFv- CD8 TM -4-1BB-CD3 ⁇ -IRES- (extracellular domain of PD-1 protein or a mutant) lentiviral vector, a lentiviral expression vector is shown in Fig.1.
  • Solution A 6.25 mL 2 x HEPES buffer buffer (the amount of packaging with 5 large dishes is the best).
  • Solution B A mixture of the following plasmids was separately added: 112.5 ⁇ g scFv-CD8TM-4-1BB-CD3 ⁇ -IRES- (extracellular domain of PD-1 protein or a mutant thereof) (target plasmid); 39.5 ⁇ g pMD2.G ( VSV-Genvelop); 73 ⁇ g pCMVR8.74 (gag, pol, tat, rev); 625 ⁇ L 2M calcium ion solution. Total volume of solution A: 6.25 mL.
  • CAR-T cells were prepared according to the procedure of Example 3 of CN201610226230.9.
  • Fig. 2 Flow cytometry to detect the positive expression rate of CAR-T
  • Fig. 2-A is the T cell without virus infection, as a control
  • Figure 2-B is the T cell after virus infection.
  • the antibody is FITC fluorescence Marking, indicated on the abscissa, if the T cell successfully expresses the CAR molecule, the signal value will increase significantly.
  • V/V volume ratio
  • polyglycolamine 8 ⁇ g/ml was added.
  • an equal amount of fresh complete medium (see the ATCC instructions for complete medium formulation) was added to adjust the cell density to 1 ⁇ 10 5 /ml to continue the culture.
  • the next day all the cells were centrifuged, fresh medium was added, and the culture was continued. Rehydration was performed every 1-2 days to maintain a cell density of 2-3 x 10 5 /ml.
  • CAR antibody staining was performed, and CAR-NK-92 positive cells were sorted by flow and expanded. The color change, cell density, and cell morphology of the medium were observed daily and recorded
  • the killing effect of CAR-T and CAR-NK-92 cells on tumor cells was detected by LDH release method, and LDH release was detected by ELISA.
  • target cells to a 96-well cell culture plate and add 100 ⁇ L per well. Three wells were used as effector cells (CART cells) to naturally release control wells, and no target cells were added, and only 100 ⁇ L of culture medium was added.
  • CART cells effector cells
  • Killing rate experimental group LDH (OD) / maximum LDH release group (OD).
  • H1299 lung cancer cell line (hereinafter referred to as H1299), stored in liquid nitrogen.
  • NOG mouse SPF grade, 4-6 weeks old when purchased, weight 15-25g, Beijing Weitong Lihua Experimental Animal Co., Ltd.
  • Quarantine domestication All animals are purchased and quarantined by a veterinarian. The quarantine and domestication period is 2-7 days. Animals that have passed the veterinary quarantine can enter this experiment.
  • Standard SPF grade murine feed Standard SPF grade murine feed.
  • the feed is provided by the production unit to provide a test report on the nutritional content of the feed.
  • the outer packaging is sterilized by ultraviolet irradiation, and then placed in a barrier environment and stored in a cold storage. Take 1 sachet (2.5kg) each time, weigh the required amount of feed and put it back and use it within 2 weeks.
  • the litter is replaced twice a week, and the feeding box is replaced at the same time.
  • the feeding box is replaced in time.
  • the animal room floor shall be disinfected at least once a day, and the wall of the cage and animal room shall be disinfected at least once a week. Wipe and disinfect with 0.1% benzalkonium or 0.5% of 84 disinfectant. The two disinfectants are used interchangeably, usually once a week. .
  • H1299 cells were cultured in DMEM medium containing 10% fetal bovine serum. H1299 cells in the exponential growth phase were collected, and the cells were resuspended in 1:1 PBS and Matrigel (0.1 mL/mouse), 5 ⁇ 10 6 cells/head, subcutaneously inoculated.
  • Observation time Observe 1 time each day in the morning and afternoon.
  • mice The tumor growth and systemic status of the mice were observed every two days after inoculation. The formation time and growth of the tumors were mastered, and the general activities, deaths, and feces of the rats were observed.
  • Weighing time The animal is weighed once during the reception and weighed once a week after the start of the experiment. The animal is also weighed and recorded when it is dead or dying.
  • Test animals all animals.
  • volume (mm 3 ) long diameter (mm) ⁇ short diameter 2 (mm 2 )/2. Take the average value and obtain the growth curve, as shown in Figure 5.
  • the program requires that the results of the measurements, observations, and data be recorded on an appropriate form or directly collected by a computer.
  • the variance homogeneity was checked by Levene's. If the variances were homogeneous (P>0.05), statistical analysis was performed by one-way analysis of variance (ANOVA). If ANOVA is statistically significant (P ⁇ 0.05), comparative analysis is performed using the LSD test.
  • ROBO1-(extracellular domain of PD-1 protein or its mutant) CAR-T group and ROBO1 CAR-T group had significant killing tumor activity compared with the control group.
  • the tumor recurrence rate and animal survival rate of ROBO1-(extracellular domain of PD-1 protein or its mutant) in the CAR-T group and ROBO1 CAR-T group were compared at the end of the experiment.
  • the results showed that ROBO1-(PD-1) The extracellular domain of the protein or its mutant) had a recurrence rate of 12.5% in the CAR-T group (recurrence of 1 mouse in 8 mice) and a survival rate of 100%.
  • the survival rate of the ROBO1 CAR-T group was 75% (six mice out of 8 mice survived), and the recurrence rate was 66.7% (4 of the 6 surviving mice had tumor recurrence).
  • the safety has a significant meaning.

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Abstract

一种以ROBO1为靶点的嵌合抗原受体细胞及其制备和应用，特别是一种以ROBO1为靶点的加强型CAR-T细胞和CAR-NK细胞，所述细胞在稳定表达CAR元件的同时，还可分泌表达PD-1蛋白的胞外结构域分子或其突变体，可阻断PD-1/PD-L1分子相互作用，通过动物实验发现，所述细胞具有非常好的抗肿瘤效果，并且与普通的以ROBO1为靶点的CAR修饰细胞相比，上述细胞可显著降低肿瘤复发率，提高存活率。

Description

一种以ROBO1为靶点的嵌合抗原受体细胞及其制备和应用 技术领域
本发明涉及生物技术领域，具体涉及一种以ROBO1为靶点的嵌合抗原受体细胞，特别是一种以ROBO1为靶点的加强型CAR-T细胞和CAR-NK细胞，及其制备和应用。
背景技术
嵌合抗原受体修饰T细胞(Chimeric Antigen Receptor T-Cell Immunotherapy，CAR-T)免疫疗法是近年来迅速发展起来的一种过继性免疫治疗方法，其中CAR通常由抗原结合结构域、跨膜结构域、共刺激信号传导区等组成，其通过基因修饰技术，将能识别肿瘤相关抗原的抗体融合表达于自体T细胞的表面，被修饰过的T细胞因此具有对肿瘤细胞的靶向杀伤力。CAR-T疗法在急性白血病和非霍奇金淋巴瘤的治疗上有着显著的疗效，被认为是最有前景的肿瘤治疗方式之一。除了血液***肿瘤外，研究者们一直在努力将CAR-T治疗扩展到实体瘤，但是利用CAR-T治疗实体瘤(是由异质的细胞群体组成的，这些异质的细胞具有不同的表面分子)并未取得理想的治疗效果。
PD-L1全称程序性死亡受体-配体1，英文名字programmed cell death-Ligand 1，是大小为40kDa的第一型跨膜蛋白。PD-1全称程序性死亡受体1，英文名字为programmed death 1，是一种重要的免疫抑制分子，为CD28超家族成员。PD-1(programmed death-1)主要表达于T细胞及初级B细胞表面，PD-1的两个配体(PD-L1和PD-L2)，广泛表达于抗原提呈细胞(APC)等。PD-1与其配体的相互作用，在免疫应答的负性调控方面发挥着重要作用。在许多人类肿瘤组织中均可检测到PD-L1蛋白的表达，肿瘤部位的微环境可诱导肿瘤细胞上的PD-L1的表达，表达的PD-L1有利于肿瘤的发生和生长，诱导抗肿瘤T细胞的凋亡，进而逃避免疫***的攻击。抑制PD-1与其配体的结合，可以使肿瘤细胞暴露于免疫***的杀伤视野，进而达到杀伤肿瘤组织及治疗癌症的作用。
在2016AACR年会上，华盛顿大学医学院皮肤病学科教授Paul T.Nghiem，MD，PhD做出报道，pembrolizumab这一具有抗PD-1作用的单克隆抗体可诱导高级别默克尔细胞癌的高反应性，中位无进展生存期PFS是9个月，传统化疗的患者PFS是3个月。近日百时美PD-1抑制剂Opdivo在临床试验方面取得新进展，发布了两组数据，其中一组显示，对当前任何药物均无治疗反应的晚期黑色素瘤的患者在使用Opdivo治疗中取得了34％五年生存率的结果。值得注意的是，IV期黑色素瘤患者的五年生存率通常只有15％到20％。另一组数据表明Opdivo和Yervoy联用治疗晚期黑色素瘤患者中取得22％的总缓解率，并且达到了69％两年总体生存率。另一项研究表明难治复发性或转移性头颈部鳞状细胞癌(SCCHN)患者在使用Opdivo治疗后生存期和存活率大大提高。数据表明，与对照组相比Opdivo治疗组死亡风险显著降低30％，中位总生存期显著延长。Opdivo治疗组一年存活率为36％，对照组为16.6％。此外，该研究还通过口咽部肿瘤HPV状态及PD-L1表达状态评估了Opdivo相对于对照方案的疗效。近期，Keytruda获FDA治疗复发性或难治性(R/R)经典型霍奇金淋巴瘤(cHL)的突破性药物资格。
专利申请CN105505869A公开了一种针对肿瘤干细胞的嵌合受体T细胞，此T细胞嵌合了2-3个独立的抗原受体，每个嵌合的抗原受体分别由不同的肿瘤干细胞特异性标记物的抗体的抗原结合部与不同的功能蛋白结合组成。此嵌合受体T细胞仅能在2-3个嵌合抗原受体均识别抗原后，才激活T细胞抗肿瘤作用，此方法在一定程度上提高了特异性但对于隐藏抗原的肿瘤细胞的特异性较低。
Liza B.John报道的文献(Blockade of PD-1 immunosuppression boosts CAR T-cell therapy)中通过加入抗PD-1抗体阻碍了PD-1免疫抑制途径即PD-1与配体PD-L的结合，从而在一定程度上提高基因修饰的表达嵌合抗原受体T细胞(CAR-T)的抗肿瘤功效。
发明内容
为克服现有技术的不足，本发明提供一种以ROBO1为靶点的的嵌合抗原受体细胞，特别是一种以ROBO1为靶点的加强型CAR-T细胞和CAR-NK细胞，及其制备和应用。
研究发现Robo1在多种癌症中过表达，如肝细胞癌、乳腺癌、结肠癌、胰腺癌、***癌、神经胶质瘤等，而在正常组织中只有少量表达，其可作为一种新的肿瘤相关抗原，是一种潜在的治疗和诊断靶标。
本发明第一方面提供一种编码核苷酸，其包含编码嵌合抗原受体的核苷酸和编码胞外分泌蛋白的核苷酸，其中，所述嵌合抗原受体包含抗原结合结构域、跨膜结构域和共刺激信号传导区，所述的抗原结合结 构域能够特异性结合肿瘤特异性抗原ROBO1，并通过跨膜结构域和共刺激信号传导区激活免疫细胞(例如T细胞、B细胞或自然杀伤细胞)，所述胞外分泌蛋白能够阻碍或抑制野生型PD-1蛋白与PD-L1配体的结合。
优选地，所述编码嵌合抗原受体的核苷酸和编码胞外分泌蛋白的核苷酸不在同一个阅读框。
在本发明的一个实施例中，所述胞外分泌蛋白是结合PD-L1和/或PD-L2的PD-1蛋白片段。
在本发明的一个优选实施例中，所述胞外分泌蛋白是PD-1蛋白的胞外结构域或其突变体序列，其中所述胞外结构域的突变体能够高亲和力结合PD-L1；优选地，所述PD-1蛋白的胞外结构域或其突变体具有如SEQ ID NO：8所示的氨基酸序列。
在本发明的一个优选实施例中，所述编码PD-1蛋白的胞外结构域或其突变体的核苷酸具有如SEQ ID NO：1所示的序列。Robo1的胞外区包括5个免疫球蛋白保守区(分别命名为Ig1-5)和3个FibronectinIII型重复序列(分别命名为FNIII1-3，简称为FN1-3)，胞内区包括4个保守区域(分别命名为：CC0、CC1、CC2和CC3)。所述的抗原结合结构域能够特异性结合肿瘤特异性抗原ROBO1的Ig1、Ig2、Ig3、Ig4、Ig5、FN1、FN2和FN3结构域中的一种或多种。
在本发明的一个实施例中，所述编码核苷酸中，所述的抗原结合结构域能够特异性结合肿瘤特异性抗原ROBO1的FN3结构域(以下简称为ROBO1-FN3)。
在本发明的一个实施例中，所述编码核苷酸中，所述的抗原结合结构域为抗体或其抗原结合片段；优选的，所述的抗原结合结构域为特异性结合ROBO1的FN3结构域的抗体或其抗原结合片段。
在本发明的一个实施例中，所述编码核苷酸中，所述抗原结合片段为Fab或scFv。
在本发明的一个实施例中，所述编码核苷酸中，所述抗原结合结构域为抗ROBO1-FN3scFv；优选地，所述抗ROBO1-FN3scFv具有如SEQ ID NO：9所示的氨基酸序列。
在本发明的一个优选实施例中，所述编码核苷酸中，所述抗原结合结构域为抗ROBO1-FN3scFv，所述编码抗原结合结构域的核苷酸具有如SEQ ID NO：2所示的序列。
在本发明的一个实施例中，所述编码核苷酸还包括内部核糖体进入位点IRES编码核苷酸，其存在于所述编码嵌合抗原受体的核苷酸和编码胞外分泌蛋白的核苷酸之间；优选地，所述IRES编码核苷酸具有如SEQ ID NO：3所示的序列。
在本发明的一个实施例中，所述编码核苷酸包括IRES-PD-1蛋白的胞外结构域或其突变体的编码核苷酸。
在本发明的一个实施例中，所述编码核苷酸中，所述的跨膜结构域选自：CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD134、CD137、ICOS和CD154中的一种或多种的跨膜结构域。
在本发明的一个优选实施例中，所述编码核苷酸中，所述的跨膜结构域为CD8跨膜(CD8TM)结构域。
在本发明的一个实施例中，所述CD8TM结构域具有如SEQ ID NO：10所示的序列。
在本发明的一个优选实施例中，所述CD8TM结构域的编码核苷酸具有如SEQ ID NO：4所示的序列。
在本发明的一个实施例中，所述的共刺激信号传导区包含共刺激分子的细胞内结构域，优选地，所述的共刺激分子选自：CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d、CD2、CD4、CD5、CD28、CD134、CD137、ICOS、CD154、4-1BB和OX40中的一种或多种。
在本发明的一个优选实施例中，所述编码核苷酸中，所述的共刺激信号传导区包含4-1BB和CD3ζ胞内结构域。
在本发明的一个实施例中，所述4-1BB胞内结构域具有如SEQ ID NO：11所示的序列。
在本发明的另一个实施例中，所述CD3ζ胞内结构域具有如SEQ ID NO：12所示的序列。
在本发明的一个实施例中，所述4-1BB胞内结构域的编码核苷酸具有如SEQ ID NO：5所示的序列。
在本发明的另一个实施例中，所述CD3ζ胞内结构域的编码核苷酸具有如SEQ ID NO：6所示的序列。
在本发明的一个优选实施例中，所述编码核苷酸包含编码CD8跨膜结构域、4-1BB和CD3ζ胞内结构域的核苷酸。
在本发明的一个更优选的实施例中，所述编码核苷酸包含CD8TM-4-1BB-CD3ζ的编码核苷酸。
在本发明的一个进一步优选的实施例中，所述编码核苷酸包含抗ROBO1-FN3scFv-CD8TM-4-1BB-CD3ζ的编码核苷酸，优选地，所述抗ROBO1-FN3scFv-CD8TM-4-1BB-CD3ζ的编码核苷酸具有SEQ ID NO：13所示的序列。
在本发明的一个实施例中，所述的编码核苷酸序列是分离和/或合成的。
在本发明的一个优选实施例中，所述编码核苷酸包含抗ROBO1-FN3scFv-CD8TM-4-1BB-CD3ζ-IRES-(PD-1蛋白的胞外结构域或其突变体)的编码核苷酸，优选地，所述抗ROBO1-FN3scFv-CD8TM-4-1BB-CD3ζ-IRES-(PD-1蛋白的胞外结构域或其突变体)的编码核苷酸具有如SEQ ID NO：7所示的序列。
本发明第二方面提供一种包含本发明上述编码核苷酸的载体。
在本发明的一个实施例中，所述载体选自质粒、细菌和病毒等中的一种或多种。
在本发明的一个实施例中，所述载体可使用缺乏复制能力、无法在转染细胞中自我复制的病毒载体例如逆转录病毒载体(包括致癌逆转录病毒载体、慢病毒载体和假型载体)、腺病毒载体、痘苗病毒载体或HSV 载体等。
在本发明的一个优选实施例中，所述载体为慢病毒载体。
在本发明的一个更优选实施例中，所述慢病毒载体为PRRSLIN载体。
在本发明的一个实施例中，所述载体还包括启动子。
在本发明的一个优选实施例中，所述启动子为EF-1α启动子。
本发明第三方面提供一种嵌合抗原受体细胞(CAR-细胞)，其编码胞外分泌蛋白，其中所述胞外分泌蛋白能够阻碍或抑制野生型PD-1蛋白与PD-L1配体的结合。
在本发明的一个实施例中，所述CAR-细胞中，所述胞外分泌蛋白是结合PD-L1和/或PD-L2的PD-1蛋白片段。
在本发明的一个优选实施例中，所述CAR-细胞中，所述胞外分泌蛋白是PD-1蛋白的胞外结构域或其突变体序列；优选的，所述PD-1蛋白的胞外结构域或其突变体具有如SEQ ID NO：8所示的氨基酸序列。
在本发明的一个优选实施例中，所述CAR-细胞中，所述编码PD-1蛋白的胞外结构域或其突变体的核苷酸具有如SEQ ID NO：1所示的序列。
优选地，所述CAR-细胞还编码嵌合抗原受体，其中，所述嵌合抗原受体包含抗原结合结构域、跨膜结构域和共刺激信号传导区，所述的抗原结合结构域能够特异性结合肿瘤特异性抗原ROBO1，并通过跨膜结构域和共刺激信号传导区激活所述CAR-细胞(例如CAR-T细胞、B细胞或自然杀伤细胞)。
在本发明的一个实施例中，所述CAR-细胞中，所述的抗原结合结构域能够特异性结合肿瘤特异性抗原ROBO1的Ig1、Ig2、Ig3、Ig4、Ig5、FN1、FN2和FN3结构域中的一种或多种。
在本发明的一个实施例中，所述CAR-细胞中，所述的抗原结合结构域能够特异性结合肿瘤特异性抗原ROBO1-FN3。
在本发明的一个实施例中，所述CAR-细胞中，所述的抗原结合结构域为抗体或其抗原结合片段；优选的，所述的抗原结合结构域为特异性结合ROBO1的FN3结构域的抗体或其抗原结合片段。
在本发明的一个实施例中，所述CAR-细胞中，所述抗原结合片段为Fab或scFv。
在本发明的一个实施例中，所述CAR-细胞中，所述抗原结合结构域为抗ROBO1-FN3scFv；优选地，所述抗ROBO1-FN3scFv具有如SEQ ID NO：9所示的氨基酸序列；更优选的，所述编码抗原结合结构域的核苷酸具有如SEQ ID NO：2所示的序列。
在本发明的一个实施例中，所述CAR-细胞中，所述CAR-细胞中编码嵌合抗原受体的核苷酸和编码胞外分泌蛋白的核苷酸之间存在内部核糖体进入位点IRES编码核苷酸，其具有如SEQ ID NO：3所示的序列。
在本发明的一个实施例中，所述CAR-细胞中，所述的跨膜结构域选自：CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD134、CD137、ICOS和CD154中的一种或多种的跨膜结构域。
在本发明的一个优选实施例中，所述编码核苷酸中，所述的跨膜结构域为CD8跨膜(CD8TM)结构域。
在本发明的一个实施例中，所述CD8TM结构域具有如SEQ ID NO：10所示的序列。
在本发明的一个优选实施例中，所述CD8TM结构域的编码核苷酸具有如SEQ ID NO：4所示的序列。
在本发明的一个实施例中，所述的共刺激信号传导区包含共刺激分子的细胞内结构域，优选地，所述的共刺激分子选自：CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d、CD2、CD4、CD5、CD28、CD134、CD137、ICOS、CD154、4-1BB和OX40中的一种或多种。
在本发明的一个优选实施例中，所述编码核苷酸中，所述的共刺激信号传导区包含4-1BB和CD3ζ胞内结构域。
在本发明的一个实施例中，所述4-1BB胞内结构域具有如SEQ ID NO：11所示的序列。
在本发明的另一个实施例中，所述CD3ζ胞内结构域具有如SEQ ID NO：12所示的序列。
在本发明的一个实施例中，所述4-1BB胞内结构域的编码核苷酸具有如SEQ ID NO：5所示的序列。
在本发明的另一个实施例中，所述CD3ζ胞内结构域的编码核苷酸具有如SEQ ID NO：6所示的序列。
在本发明的一个优选实施例中，所述编码核苷酸包含编码CD8跨膜结构域、4-1BB和CD3ζ胞内结构域的核苷酸。
在本发明的一个更优选的实施例中，所述CAR-细胞包含CD8TM-4-1BB-CD3ζ的编码核苷酸。
在本发明的一个进一步优选的实施例中，所述CAR-细胞包含抗ROBO1-FN3scFv-CD8TM-4-1BB-CD3ζ的编码核苷酸，优选地，所述抗ROBO1-FN3scFv-CD8TM-4-1BB-CD3ζ的编码核苷酸具有如SEQ ID NO：13所示的序列。
在本发明的一个优选实施例中，所述CAR-细胞包含抗ROBO1-FN3scFv-CD8TM-4-1BB-CD3ζ-IRES-(PD-1蛋白的胞外结构域或其突变体)的编码核苷酸，优选地，所述抗ROBO1-FN3scFv-CD8TM-4-1BB-CD3ζ-IRES-(PD-1蛋白的胞外结构域或其突变体)的编码核苷酸具有如SEQ ID NO：7所示的序列。
在本发明的一个实施例中，所述CAR-细胞包含本发明第一方面所述的编码核苷酸。
在本发明的一个实施例中，所述CAR-细胞包含本发明第二方面所述的载体。
本发明的一个实施例中，所述CAR-细胞为免疫细胞，选自：T细胞、自然杀伤(NK)细胞、细胞毒性T 淋巴(CTL)细胞和调节性T细胞中的一种或多种。
在本发明的一个实施例中，所述T细胞为人T细胞，优选地，所述的人为患有癌症的人。
在本发明的一个实施例中，所述NK细胞为人NK细胞，优选地，所述的人为患有癌症的人，更优选地，所述NK细胞为NK92细胞。
在本发明的一个优选实施例中，所述CAR-细胞为人T细胞(以下简称为CAR-T细胞)。
在本发明的一个优选实施例中，所述CAR-细胞为NK细胞(以下简称为CAR-NK细胞)。
在本发明的一个更优选的实施例中，所述CAR-NK细胞为CAR-NK92细胞。
本发明所述的CAR-细胞可单独施用或作为药物组合物与其他组分如IL-2或其他细胞因子或细胞群结合施用。
本发明第四方面提供一种上述CAR-细胞的制备方法，所述方法包括如下步骤：
(1)将上述编码核苷酸序列克隆至载体中；
(2)将步骤(1)得到的载体转染293T细胞，制备病毒；
(3)利用步骤(2)得到病毒侵染免疫细胞，免疫细胞表达嵌合抗原受体和胞外分泌的PD-1蛋白的胞外结构域或其突变体蛋白，得到CAR-细胞。
本发明第五方面提供一种药物组合物，其包含选自：本发明上述的编码核苷酸、载体和CAR-细胞的一项或多项。
在本发明的一个实施例中，所述药物组合物包含有效量的CAR-细胞，优选为CAR-T细胞和/或CAR-NK92细胞。
在本发明的一个实施例中，所述药物组合物还包括药学上可接受的辅料。
在本发明的一个实施例中，所述药物组合物中，所述辅料选自：缓冲液、碳水化合物、抗氧化剂、螯合剂和防腐剂中的一种或多种。本发明的药物组合物优选配制用于静脉内施用。
本发明所述的药物组合物的施用可以任何方便的方式进行，包括通过喷雾法、注射、口服、输注、植入或移植，优选的，本发明所述药物组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、静脉注射或腹膜内施用给患者。在本发明的一个实施例中，所述药物组合物通过静脉注射(i.v.)施用给患者。
本发明第六方面提供一种本发明上述的编码核苷酸、载体、CAR-细胞和药物组合物中的一项或多项在制备治疗和/或预防癌症的药物中的应用。
在本发明的一个实施例中，所述的应用中，所述的癌症为高表达Robo1的肿瘤及相关疾病，此处所述的高表达指肿瘤细胞中Robo1的表达水平高于正常细胞中其表达水平。
在本发明的一个实施例中，所述癌症为肝癌、乳腺癌、结肠癌、胰腺癌、***癌、神经胶质瘤或肺癌。
在本发明的一个优选实施例中，所述癌症为肺癌。
本申请的发明人在其之前研究工作(如中国专利申请CN201610237593.2)的基础上，以ROBO1为靶点制备了一种嵌合抗原受体细胞并涉及其制备和应用，特别是一种以ROBO1为靶点的加强型CAR-T细胞和CAR-NK细胞，上述细胞在稳定表达CAR元件的同时，还可分泌表达PD-1蛋白的胞外结构域或其突变体分子，可阻断PD-1/PD-L1分子相互作用，通过动物实验发现，所述细胞具有非常好的抗肿瘤效果，并且与普通的以ROBO1为靶点的CAR修饰细胞相比，上述细胞可显著降低肿瘤复发率，提高存活率。
附图说明
图1所示为本发明实施例1提供的慢病毒表达载体构建示意图。
图2所示为本发明实施例3提供的CAR-T流式检测图，图2-A为未进行病毒侵染的T细胞，作为对照；图2-B为病毒侵染后的T细胞。
图3所示为本发明实施例4提供的CAR-NK-92流式检测图，图3-A为未进行病毒侵染的NK-92细胞，作为对照；图3-B为病毒侵染且流式分选培养1月后的NK-92细胞。
图4所示为本发明实施例5提供的CAR细胞杀伤活性分析结果图。
图5所示为本发明实施例6提供的药物在小鼠肿瘤模型中的药效评估结果图。
图6所示为本发明实施例6提供的实验终点ROBO1-(PD-1蛋白的胞外结构域或其突变体)CAR-T组和ROBO1 CAR-T组各组动物存活率。
图7所示为本发明实施例6提供的实验终点ROBO1-(PD-1蛋白的胞外结构域或其突变体)CAR-T组和ROBO1 CAR-T组各组动物肿瘤复发率。
具体实施方式
除非另有定义，本发明中使用的所有技术和科学术语具有与本发明所述技术领域的普通技术人员通常理解的相同含义。
本发明中，术语“抗体”指的是与抗原特异性结合的免疫球蛋白分子。抗体可为源于自然源或源于重组源的完整的免疫球蛋白，并可为完整免疫球蛋白的免疫反应部分。抗体通常为免疫球蛋白分子的四聚物。本发明中的抗体可以以多种形式存在，包括例如，多克隆抗体、单克隆抗体、Fv、Fab和F(ab)2，以及单链抗体和人源化抗体等(Harlow等，1999,In:Using Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory Press,NY；Harlow等，1989,In:Antibodies:A Laboratory Manual,Cold Spring Harbor,New York；Houston等，1988,Proc.Natl.Acad.Sci.USA 85:5879-5883；Bird等，1988,Science 242:423-426)。
术语“抗体片段”指的是完整抗体的一部分，并指的是完整抗体的抗原决定可变区。抗体片段的例子包括但不限于Fab、Fab'、F(ab')2和Fv片段，由抗体片段形成的线性抗体、scFv抗体和多特异性抗体。
术语“抗原结合片段”由一条完整的轻链以及重链VH、CH1结构域组成，其可与抗原特异性结合。
术语“编码”指的是多核苷酸诸如基因、cDNA或mRNA中核苷酸的特异性序列用作模板合成在生物学过程中的其他多聚体和大分子的固有性质，所述多聚体和大分子具有核苷酸(即，rRNA、tRNA和mRNA)的限定序列或氨基酸的限定序列中的任一个和由其产生的生物学性质。因此，如果相应于那个基因的mRNA的转录和翻译在细胞或其他生物学***中产生蛋白质，则基因编码蛋白质。核苷酸序列等同mRNA序列并通常提供在序列表中的编码链，和用作转录基因或cDNA的模板的非编码链两者，都可被称为编码那个基因或cDNA的蛋白质或其他产物。
除非另有规定，“编码氨基酸序列的核苷酸序列”包括为彼此简并版本并编码相同的氨基酸序列的所有核苷酸序列。编码蛋白质和RNA的核苷酸序列可包括内含子。
术语“共刺激分子”指的是与共刺激配体特异性结合的T细胞上的关联结合伴侣，由此介导T细胞的共刺激应答，诸如但不限于增殖，共刺激分子包括但不限于MHCI类分子、BTLA和Toll配体受体。
术语“慢病毒”指的是逆转录病毒科的属，其能够有效感染非周期性和有丝***后的细胞；它们可传递显著量的遗传信息进入宿主细胞的DNA，以便它们是基因传递载体的最有效的方法之一。
术语“启动子”被定义为开始多核苷酸序列的特异性转录需要的，由细胞的合成机器识别或引导合成机器的DNA序列。
术语“特异性结合”指识别特异性抗原但基本上不识别或结合样本中的其他分子。
术语“载体”为物质组合物，其包括分离的核酸，并且其可用于传递分离的核酸至细胞内部。很多载体在本领域中是已知的，包括但不限于线性多核苷酸、与离子或两性分子化合物相关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制的质粒或病毒。该术语也应被解释为包括便于将核酸转移入细胞的非质粒和非病毒化合物，诸如例如聚赖氨酸化合物、脂质体等等。病毒载体的例子包括但不限于，腺病毒载体、腺伴随病毒载体、逆转录病毒载体等等。
术语“分离的”指从自然状态改变或移出。例如，天然存在于活动物中的核酸或肽不是“分离的”，但部分或完全与它的自然状态的共存物质分离的同一核酸或肽是“分离的”。分离的核酸或蛋白可以以基本上纯化的形式存在，或例如，可存在于非自然环境，诸如宿主细胞。
术语“癌症”被定义为以畸变细胞的快速和失控生长为特征的疾病。癌症细胞可局部蔓延或通过血流和淋巴***蔓延至身体的其他部分。各种癌症的例子包括但不限于乳腺癌、***癌、卵巢癌、子***、皮肤癌、胰腺癌、结肠直肠癌、肾癌、肝癌、脑癌、淋巴瘤、白血病、肺癌等等。
术语“抗肿瘤效应”，指的是生物学效应，其可由肿瘤体积的减少、肿瘤细胞数的减少、转移数的减少、预期寿命的增加或与癌性病症相关的各种生理症状的改善清楚表示。
术语“患者”、“对象”、“个体”等等在本文中可交换使用，并指的是服从本文描述方法的任何动物或其细胞，不论是体外或原位。在一些非限制性实施方式中，患者、对象或个体为人。
本发明所述的氨基酸序列的连接“-”为一片段的N末端与另一片段的C末端直接连接，中间没有任何连接肽。
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1慢病毒表达载体的制备
根据“Engineering high–affinity PD-1 variants for optimized immunotherapy and immune–PET imaging”(Maute R L,Gordon S R,Mayer A T,等.Proceedings of the National Academy of Sciences，2015，112(47)：E6506-E6514.)报道的PD-1突变分子HAC序列信息，合成PD-1蛋白的胞外结构域或其突变体基因序列， 如SEQ ID NO：1所示；从GenBank数据库中搜寻并合成已知的人抗ROBO1-FN3scFv(以下简称为scFv)基因序列、IRES内部核糖体进入位点、人CD8跨膜区基因序列、人4-1BB胞内区基因序列、CD3ζ胞内区基因序列，分别如SEQ ID NO：2-6所示。
将上述基因序列依次按scFv基因、CD8基因、人4-1BB胞内区基因和CD3ζ胞内区基因、IRES内部核糖体进入位点和PD-1蛋白的胞外结构域或其突变体基因的顺序进行连接，在各序列连接处引入不同的酶切位点，形成完整的scFv-CD8TM-4-1BB-CD3ζ-IRES-(PD-1蛋白的胞外结构域或其突变体)基因序列，如SEQ ID NO：7所示。
将scFv-CD8TM-4-1BB-CD3ζ-IRES-(PD-1蛋白的胞外结构域或其突变体)基因序列通过双酶切连接转化到pRRSLIN载体中，基因上游为EF-1α启动子。将载体转化到Stbl3大肠杆菌菌株后，转种到含有氨苄青霉素的固体培养基中进行繁殖，筛选，获得阳性克隆，提取质粒，酶切鉴定克隆，通过测序确认载体构建成功，获得pRRSLIN-scFv-CD8TM-4-1BB-CD3ζ-IRES-(PD-1蛋白的胞外结构域或其突变体)慢病毒表达载体，慢病毒表达载体构建示意图如图1所示。
实施例2慢病毒制备
1.转染前24小时，以每皿约8×106将293T细胞接种至15cm培养皿中。确保转染时细胞在80％左右的汇合度且均匀分布于培养皿中。
2.准备溶液A和溶液B
溶液A：6.25mL 2×HEPES buffer缓冲液(采用5个大皿一起包装的量，效果最好)。
溶液B：分别加入以下质粒的混合物：112.5μg scFv-CD8TM-4-1BB-CD3ζ-IRES-(PD-1蛋白的胞外结构域或其突变体)(目标质粒)；39.5μg pMD2.G(VSV-Genvelop)；73μg pCMVR8.74(gag，pol，tat，rev)；625μL 2M钙离子溶液。溶液A总体积：6.25mL。
3.充分混匀溶液B，轻轻涡旋溶液A的同时，逐滴加入溶液A，静置5-15分钟。轻轻涡旋上述A和B的混合溶液，逐滴加入含293T细胞的培养皿中，轻轻前后晃动培养皿使DNA与钙离子的混合物均匀分布。(不要旋转培养皿)放置于培养箱中培养16-18小时。
更换新鲜培养基，继续培养。
在转速500g，温度25℃下离心10min，使用PES膜(0.45μm)过滤；以70％乙醇消毒离心管(贝克曼库尔特ultra-clear SW28 centrifuge tubes)，并置于紫外灯下消毒30min；将已过滤的含慢病毒的上清液转移至离心管中，在离心管底部小心铺上一层20％蔗糖(每8mL上清液加1mL蔗糖)，以PBS平衡离心管，在转速25,000rpm(82,700g)，温度4℃下离心2h；小心取出离心管，倒掉上清液，倒置离心管去掉残余液体；加入100μL PBS，密封离心管，在4℃放置2h，每20min轻轻涡旋一次，500g离心1min(25℃)，收集病毒上清；冰上冷却后，置于-80℃保存。
实施例3 CAR-T细胞制备
参照CN201610226230.9实施例3的步骤制备CAR-T细胞。
病毒侵染后，用流式细胞仪进行CAR-T阳性率检测，流式检测图如图2所示，结果显示，阳性率为51.38％，成功制备CAR-T细胞。(图2：流式细胞仪检测CAR-T阳性表达率，图2-A为未进行病毒侵染的T细胞，作为对照；图2-B为病毒侵染后的T细胞。抗体为FITC荧光标记，在横坐标上进行表示，T细胞若成功表达CAR分子，该信号值将显著升高。)
实施例4 CAR-NK92稳定细胞株的制备
将NK-92细胞密度调整至2-3×105/ml，按体积比(V/V)病毒载体：细胞培养基＝1:5-10比例添加病毒载体，同时添加聚凝胺8μg/ml。4h之后，补加等量的新鲜的完全培养基(完全培养基配方参见ATCC说明)将细胞密度调整至1×105/ml继续培养。次日，将所有的细胞离心，加入新鲜的培养基，继续培养。每隔1-2天进行补液，使维持细胞密度在2-3×105/ml。72h后进行CAR抗体染色，同时流式分选CAR-NK-92阳性细胞并扩大培养。每天观察培养基的颜色变化、细胞密度、细胞形态并作相应记录。
流式分选后，阳性CAR-NK-92细胞进行持续培养，扩增1个月后，利用流式检测CAR NK-92细胞阳性率，流式检测图如图3所示，结果显示，CAR-NK92阳性率为96.57％，表明成功制备了稳定表达CAR元件测NK-92细胞(图3：流式细胞仪检测CAR-NK-92阳性表达率，图3-A为未进行病毒侵染的NK-92细胞，作为对照；图3-B为病毒侵染且流式分选培养1月后的NK-92细胞。抗体为APC荧光标记，在横坐标上进行表示，NK-92细胞若成功表达CAR分子，该信号值将显著升高。)
实施例5 CAR细胞体外活性检测
采用LDH释放法检测CAR-T和CAR-NK-92细胞对肿瘤细胞的杀伤效应，通过ELISA方法检测LDH释放。
1.用含5％小牛血清的RPMI-1640培养液将靶细胞调整到5×104/mL。
2.在96孔细胞培养板中加入靶细胞，每孔加100μL。取3个孔作为效应细胞(CART细胞)自然释放对照孔，不加靶细胞，仅加100μL培养液。
3.向各孔加100μL效应细胞，效应细胞与靶细胞的比例10:1；5:1；1:1。自然释放孔不加效应细胞只加100μL培养液，效应细胞与靶细胞共孵育6小时，每个实验置三个复孔。
4.最大释放孔中(阳性对照)加10μL Lysis Solution(10×)，孵育45-60min，每个实验置三个复孔。
5.取上述3和4中待测样品和对照样品各50μL，加入新鲜的96孔酶标板中，再加入反应液和底物，避光30min。
6.加入50μL终止液。
7.在酶联检测仪上测定各孔的光密度(OD值)，检测波长490nm或492nm，在1小时内测完。
8.特异性杀伤效率计算
杀伤率＝实验组LDH(OD)/最大LDH释放组(OD)。
计算公式：杀伤效率＝(实验组-效应自然释放-靶自然释放)/(靶最大释放-靶自然释放)×100％。
9.通过CBA试剂盒测定细胞因子分泌情况
CAR细胞杀伤活性分析结果如图4所示，结果显示：构建的CAR-T细胞和CAR-NK-92细胞均可显著杀伤肿瘤细胞，杀伤效率分别为：95％和83％，结果如图4。
实施例6 CAR细胞动物体内药效测试
1.所用细胞株：H1299肺癌细胞株(以下简称为H1299)，液氮保存。
2.实验动物：
2.1动物种系、等级、周龄、体重及来源：
NOG鼠，SPF级，购入时4-6周龄，体重15-25g，北京维通利华实验动物有限公司。
2.2检疫驯养：所有动物购入后，由兽医进行检疫检查。检疫驯养期2-7天。经兽医检疫合格的动物方能进入本实验。
2.3动物房：SPF级屏障动物房，苏州吉玛基因股份有限公司。
2.4动物饲料：标准SPF级鼠科饲料。饲料由生产单位提供饲料营养成分检测报告。饲料购入后用紫外照射消毒外包装后放入屏障环境，冷藏保存。每次取1小袋(2.5kg)，称好所需量饲料后夹好放回，2周内用完。
2.5动物饮用水：SPF纯化水，用饮水瓶供应，动物自由饮水。
2.6动物垫料：普通杨柳木刨花，高压灭菌后使用。
2.7饲养条件：
动物饲养于装有垫料的聚碳酸酯笼盒中，单笼饲养。除特殊说明的情况外，其余时间均自由摄食；动物自由饮水。
动物房室温20-26℃，湿度40％-70％，光照12小时明暗交替。垫料每周更换2次，同时更换饲养盒，出现饮水、尿液、稀便等严重污染垫料舒适性等情况时，及时更换饲养盒。每天更换高压消毒过的饮水瓶和瓶塞。所有换洗的笼具清洗过后均采用高压灭菌。
动物房地面至少每天消毒1次，笼架、动物房墙面至少每周消毒1次，用0.1％新洁尔灭或0.5％的84消毒液擦拭消毒，两种消毒液交叉使用，一般每周交叉1次。
3.实验方法
3.1肿瘤细胞的培养与浓度配置
肿瘤细胞的培养：H1299细胞培养在含10％胎牛血清的DMEM培养液中。收集指数生长期的H1299细胞，细胞重悬在1：1的PBS与基质胶中(0.1mL/只)，5×106cell/只，皮下接种。
3.2动物分组及给药剂量
各组动物分组后的情况及处理见下表：
表1动物分组及给药
Figure PCTCN2017089517-appb-000001
4.检测指标
4.1一般临床观察
观察时间：每天上、下午各观察1次。
观察内容：接种后每两天观察鼠肿瘤生长情况及其全身状况，掌握肿瘤的形成时间及生长情况，观察鼠一般活动、死亡情况、粪便情况等。
4.2体重
称量时间：动物接收时称重1次、实验开始后每周称量1次，动物在死亡或濒死时也需称重并记录。
检测动物：所有动物。
4.3肿瘤测量
每两天用游标卡尺测量肿瘤长径和短径，并记录下计算其体积：体积(mm3)＝长径(mm)×短径2(mm2)/2。取其平均值，得其生长曲线，如图5所示。
5.数据采集和统计分析
5.1数据采集
将方案要求测定、观察的结果和数据手记到适当的表格上或计算机直接采集数据。
5.2数据分析
本实验使用统计学软件SPSS20.0对数据进行处理，计量资料以平均值±标准差来表示。具体分析过程如下：
用Levene’s检验方差齐性，如果方差齐(P>0.05)，用单因素方差分析(ANOVA)进行统计分析。如果ANOVA有统计学意义(P≤0.05)，用LSD test(参数法)进行比较分析。
如果方差不齐(P≤0.05)，则用Kruskal-Wallis检验。如果Kruskal-Wallis检验有统计学意义(P≤0.05)，则用Mann-Whitney法进行均数间的两两比较。
结果显示，ROBO1-(PD-1蛋白的胞外结构域或其突变体)CAR-T组和ROBO1 CAR-T组与对照组比具有明显的杀伤肿瘤活性。但比较实验终点时ROBO1-(PD-1蛋白的胞外结构域或其突变体)CAR-T组和ROBO1 CAR-T组动物肿瘤复发率和动物存活率，结果显示，ROBO1-(PD-1蛋白的胞外结构域或其突变体)CAR-T组复发率为12.5％(8只小鼠1只出现复发)，存活率为100％。而ROBO1 CAR-T组存活率为75％(8只小鼠中6只小鼠存活)，复发率为66.7％(存活的6只小鼠中有4只发生肿瘤复发)。该结果说明：将封闭PD-1和PD-L1的PD-1蛋白的胞外结构域或其突变体分子引入CAR结构后，能显著降低CAR***后的肿瘤复发情况，同时对提高CAR药物的安全性有显著的意义。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。
Figure PCTCN2017089517-appb-000002
Figure PCTCN2017089517-appb-000003
Figure PCTCN2017089517-appb-000004
Figure PCTCN2017089517-appb-000005
Figure PCTCN2017089517-appb-000006
Figure PCTCN2017089517-appb-000007
Figure PCTCN2017089517-appb-000008
Figure PCTCN2017089517-appb-000009

Claims (27)

  1. 一种编码核苷酸，其包含编码嵌合抗原受体的核苷酸和编码胞外分泌蛋白的核苷酸，其中，所述嵌合抗原受体包含抗原结合结构域、跨膜结构域和共刺激信号传导区，所述的抗原结合结构域能够特异性结合肿瘤特异性抗原ROBO1，并通过跨膜结构域和共刺激信号传导区激活免疫细胞(例如T细胞、B细胞或自然杀伤细胞)，所述胞外分泌蛋白能够阻碍或抑制野生型PD-1蛋白与PD-L1配体的结合。
  2. 如权利要求1所述的编码核苷酸，其特征在于，所述编码嵌合抗原受体的核苷酸和编码胞外分泌蛋白的核苷酸不在同一个阅读框。
  3. 如权利要求1所述的编码核苷酸，其特征在于，所述的抗原结合结构域能够特异性结合肿瘤特异性抗原ROBO1的Ig1、Ig2、Ig3、Ig4、Ig5、FN1、FN2和FN3结构域中的一种或多种；和/或，所述胞外分泌蛋白是结合PD-L1和/或PD-L2的PD-1蛋白片段。
  4. 如权利要求3所述的编码核苷酸，其特征在于，所述的抗原结合结构域能够特异性结合肿瘤特异性抗原ROBO1的FN3结构域；和/或，所述胞外分泌蛋白是PD-1蛋白的胞外结构域或该胞外结构域的突变体，其中所述的胞外结构域的突变体能够高亲和力结合PD-L1。
  5. 如权利要求4所述的编码核苷酸，其特征在于，所述的抗原结合结构域为特异性结合ROBO1的FN3结构域的抗体或其抗原结合片段，所述抗原结合片段为Fab或scFv(例如，所述抗ROBO1-FN3 scFv具有如SEQ ID NO：9所示的氨基酸序列)；优选地，所述编码抗原结合结构域的核苷酸具有如SEQ ID NO：2所示的序列；和/或，所述编码所述胞外结构域或其突变体的核苷酸编码SEQ ID NO：8所示的氨基酸序列，优选地，所述编码所述胞外结构域或其突变体的核苷酸具有如SEQ ID NO：1所示的序列。
  6. 如权利要求5所述的编码核苷酸，其特征在于，所述编码嵌合抗原受体的核苷酸和编码胞外分泌蛋白的核苷酸之间存在内部核糖体进入位点IRES编码核苷酸，所述IRES编码核苷酸具有如SEQ ID NO：3所示的序列。
  7. 如权利要求1-6任一项所述的编码核苷酸，其特征在于，所述的跨膜结构域选自：CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD134、CD137、ICOS和CD154中的一种或多种的跨膜结构域；优选地，所述的跨膜结构域为CD8跨膜结构域(例如，所述CD8跨膜结构域具有如SEQ ID NO：10所示的序列)，更优选地，所述CD8跨膜结构域的编码核苷酸具有如SEQ ID NO：4所示的序列；和/或，所述的共刺激信号传导区包含共刺激分子的细胞内结构域，所述的共刺激分子选自：CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d、CD2、CD4、CD5、CD28、CD134、CD137、ICOS、CD154、4-1BB和OX40中的一种或多种；优选地，所述的共刺激信号传导区包含4-1BB(例如，所述4-1BB胞内结构域具有如SEQ ID NO：11所示的序列)和CD3ζ胞内结构域(例如，所述CD3ζ胞内结构域具有如SEQ ID NO：12所示的序列)，更优选地，所述4-1BB胞内结构域的编码核苷酸具有如SEQ ID NO：5所示的序列，和/或，所述CD3ζ胞内结构域的编码核苷酸具有如SEQ ID NO：6所示的序列。
  8. 如权利要求7所述的编码核苷酸，其特征在于，所述编码核苷酸包含抗ROBO1-FN3 scFv-CD8TM-4-1BB-CD3ζ的编码核苷酸；优选地，所述编码核苷酸包含抗ROBO1-FN3 scFv-CD8TM-4-1BB-CD3ζ-IRES-(PD-1蛋白的胞外结构域或其突变体)的编码核苷酸。
  9. 如权利要求8所述的编码核苷酸，其特征在于，所述编码核苷酸具有如SEQ ID NO：7所示的序列。
  10. 一种包含权利要求1-9任一项所述的编码核苷酸的载体。
  11. 如权利要求10所述的载体，其特征在于，所述载体选自质粒、细菌和病毒中的一种或多种。
  12. 一种CAR-细胞，其编码胞外分泌蛋白，其特征在于，所述胞外分泌蛋白能够阻碍或抑制野生型PD-1蛋白与PD-L1配体的结合。
  13. 如权利要求12所述的CAR-细胞，其特征在于，所述胞外分泌蛋白是结合PD-L1和/或PD-L2的PD-1蛋白片段。
  14. 如权利要求12所述的CAR-细胞，其特征在于，所述胞外分泌蛋白是PD-1蛋白的胞外结构域或其突变体序列，其中所述的胞外结构域的突变体能够高亲和力结合PD-L1，优选地，所述PD-1蛋白的胞外结构域或其突变体序列具有SEQ ID NO：8所示的氨基酸序列，更优选地，编码PD-1蛋白的胞外结构域或其突变体的核苷酸具有如SEQ ID NO：1所示的序列。
  15. 如权利要求12-14任一项所述的CAR-细胞，其特征在于，所述细胞还编码嵌合抗原受体，其中，所述嵌合抗原受体包含抗原结合结构域、跨膜结构域和共刺激信号传导区，所述的抗原结合结构域能够特异性结合肿瘤特异性抗原ROBO1，并通过跨膜结构域和共刺激信号传导区激活所述CAR-细胞(例如CAR-T细胞、B细胞或自然杀伤细胞)。
  16. 如权利要求15所述的CAR-细胞，其特征在于，所述的抗原结合结构域能够特异性结合肿瘤特异性抗原ROBO1的Ig1、Ig2、Ig3、Ig4、Ig5、FN1、FN2和FN3结构域中的一种或多种。
  17. 如权利要求16所述的CAR-细胞，其特征在于，所述的抗原结合结构域能够特异性结合肿瘤特异性抗原ROBO1的FN3结构域。
  18. 如权利要求17所述的CAR-细胞，其特征在于，所述的抗原结合结构域为特异性结合ROBO1的FN3结构域的抗体或其抗原结合片段，所述抗原结合片段为Fab或scFv(例如，所述抗ROBO1-FN3 scFv具有如SEQ ID NO：9所示的氨基酸序列)；优选地，所述CAR-细胞中编码抗原结合结构域的核苷酸具有如SEQ ID NO：2所示的序列。
  19. 如权利要求18所述的CAR-细胞，其特征在于，在所述CAR-细胞中编码嵌合抗原受体的核苷酸和编码胞外分泌蛋白的核苷酸之间存在内部核糖体进入位点IRES编码核苷酸，其具有如SEQ ID NO：3所示的序列。
  20. 如权利要求15所述的CAR-细胞，其特征在于，所述的跨膜结构域选自：CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD134、CD137、ICOS和CD154中的一种或多种的跨膜结构域；优选地，所述的跨膜结构域为CD8跨膜结构域(例如，所述CD8跨膜结构域具有如SEQ ID NO：10所示的序列)，更优选地，所述CD8跨膜结构域的编码核苷酸具有如SEQ ID NO：4所示的序列；和/或，
    所述的共刺激信号传导区包含共刺激分子的细胞内结构域，所述的共刺激分子选自：CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d、CD2、CD4、CD5、CD28、CD134、CD137、ICOS、CD154、4-1BB和OX40中的一种或多种；优选地，所述的共刺激信号传导区包含4-1BB(例如，所述4-1BB胞内结构域具有如SEQ ID NO：11所示的序列)和CD3ζ胞内结构域(例如，所述CD3ζ胞内结构域具有如SEQ ID NO：12所示的序列)，更优选地，所述4-1BB胞内结构域的编码核苷酸具有如SEQ ID NO：5所示的序列，和/或，所述CD3ζ胞内结构域的编码核苷酸具有如SEQ ID NO：6所示的序列。
  21. 如权利要求20所述的CAR-细胞，其特征在于，所述CAR-细胞包含抗ROBO1-FN3 scFv-CD8TM-4-1BB-CD3ζ(例如，具有如SEQ ID NO：13所示的序列的编码核苷酸；优选地，所述CAR-细胞包含抗ROBO1-FN3 scFv-CD8TM-4-1BB-CD3ζ-IRES-(PD-1蛋白的胞外结构域或其突变体))的编码核苷酸。
  22. 如权利要求21所述的CAR-细胞，其特征在于，所述CAR-细胞包含如SEQ ID NO：7所示的序列。
  23. 如权利要求12所述的CAR-细胞，其包含权利要求1-9任一项所述的编码核苷酸。
  24. 如权利要求12所述的CAR-细胞，其特征在于，所述CAR-细胞为免疫细胞，选自：T细胞、NK细胞、CTL细胞和调节性T细胞中的一种或多种。
  25. 如权利要求24所述的CAR-细胞，其特征在于，所述的T细胞为人T细胞；和/或，所述NK细胞为人NK细胞，优选的，所述NK细胞为NK92细胞。
  26. 一种药物组合物，其包含选自：权利要求1-9任一项所述的编码核苷酸、权利要求10或11所述的载体和权利要求12-25任一项所述的CAR-细胞中的一项或多项。
  27. 一种权利要求1-9任一项所述的编码核苷酸、权利要求10或11所述的载体或权利要求12-25任一项所述的CAR-细胞中的一项或多项在制备治疗和/或预防癌症的药物中的应用；优选的，所述的癌症为高表达Robo1的肿瘤及相关疾病。
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