WO2017186121A1 - 一种改善免疫应答细胞功能的方法 - Google Patents

一种改善免疫应答细胞功能的方法 Download PDF

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WO2017186121A1
WO2017186121A1 PCT/CN2017/082024 CN2017082024W WO2017186121A1 WO 2017186121 A1 WO2017186121 A1 WO 2017186121A1 CN 2017082024 W CN2017082024 W CN 2017082024W WO 2017186121 A1 WO2017186121 A1 WO 2017186121A1
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cells
cell
immune response
antigen
seq
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PCT/CN2017/082024
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English (en)
French (fr)
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李宗海
狄升蒙
高慧萍
王华茂
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科济生物医药（上海）有限公司
上海市肿瘤研究所
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Application filed by 科济生物医药（上海）有限公司, 上海市肿瘤研究所 filed Critical 科济生物医药（上海）有限公司
Priority to CN202410382150.7A priority Critical patent/CN118165113A/zh
Priority to CN201780021791.8A priority patent/CN108884459B/zh
Priority to CN202410383209.4A priority patent/CN118146379A/zh
Priority to RU2019101430A priority patent/RU2793445C2/ru
Priority to US16/316,331 priority patent/US11111295B2/en
Priority to SG11201900171QA priority patent/SG11201900171QA/en
Priority to ES17823694T priority patent/ES2979068T3/es
Priority to CA3030257A priority patent/CA3030257A1/en
Priority to AU2017294276A priority patent/AU2017294276B2/en
Priority to EP17823694.9A priority patent/EP3483182B1/en
Priority to JP2019521178A priority patent/JP2019531084A/ja
Priority to KR1020197003874A priority patent/KR20190038564A/ko
Priority to CN201780042611.4A priority patent/CN109790222B/zh
Priority to IL264144A priority patent/IL264144B2/en
Priority to PCT/CN2017/092381 priority patent/WO2018006882A1/zh
Priority to BR112019000327A priority patent/BR112019000327A8/pt
Publication of WO2017186121A1 publication Critical patent/WO2017186121A1/zh
Priority to CL2019000061A priority patent/CL2019000061A1/es
Priority to US17/395,223 priority patent/US20220185880A1/en

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Definitions

  • the present invention is in the field of immunology, and more particularly, the present invention relates to a method of improving the function of immune response cells.
  • Chimeric antigen receptor is an artificially recombinant receptor that usually contains the antigen recognition domain of a monoclonal antibody located in the extracellular region, a transmembrane region, and an intracellular activation signal structure of an immune response cell. Domain composition.
  • CAR-T CAR-modified T-cell
  • Type I interferons were discovered more than half a century ago. Type I interferons contain IFN ⁇ protein (a class of identical proteins encoded by 13 human genes from IFNA1 to IFNA13), IFN ⁇ (encoded by a single individual and mouse gene IFNB1), and other less studied interferons such as IFN ⁇ , IFN ⁇ and IFN ⁇ (2. Trinchieri, G. Type Iinterferon: friend or foe? J. Exp. Med. 207, 2053-2063 (2010). 3. Kaur, S. & Platanias, LCIFN- ⁇ -specific signaling via a unique IFNAR1 interaction .Nat.Immunol. 14, 884-885 (2013)).
  • IFN ⁇ protein a class of identical proteins encoded by 13 human genes from IFNA1 to IFNA13
  • IFN ⁇ encoded by a single individual and mouse gene IFNB1
  • other less studied interferons such as IFN ⁇ , IFN ⁇ and IFN ⁇ (2. Trinchieri, G. Type I
  • Type I interferons are produced by the activation of pattern recognition receptors (PRRs) by various types of cells. PRRs respond to viral or bacterial components as well as ectopic endogenous molecules such as cytoplasmic DNA and extracellular DNA and RNA (Kawai, T. & Akira, S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 11 , 373–384 (2010)).
  • Type I interferon is transmitted by the same dimer IFN ⁇ / ⁇ receptor 1 (IFNAR1), which has a particularly high affinity for IFN ⁇ , or IFNAR1-IFNAR2 heterodimer (which binds to all type I interferons) signal.
  • IFNAR1 dimer IFN ⁇ / ⁇ receptor 1
  • IFN-stimulated genes ISGs
  • immunostimulatory effects Hervas-Stubbs, S. et al. Direct effects of type I interferons on cells of the immune system. Clin. Cancer Res .17,2619er Res.es.mmde Weerd,NAet al.Structural basis of a unique interferon- ⁇ signaling axis mediated via the receptor IFNAR1.Nat.Immunol.14,901ol.nol.is oMcNab,F., Mayer-Barber,K Sher, A., Wack, A. & O A. &, A.
  • Type I interferons in infectious disease Nat. Rev. Immunol. 15, 87 nol. Immun). Studies have shown that type I interferons have anticancer effects on some tumors, probably due to their immune stimulating function. However, systemic administration of type I interferons may have immunosuppressive effects (Lotrich, FEMajor depression during interferon- ⁇ treatment: vulnerability and prevention. Dialogues Clin. Neurosci. 11 , 417-425 (2009)) with major adverse events. The most common are fatigue, anorexia, hepatotoxicity, flu-like symptoms and severe depression (Kreutzer, K., Bonnekoh, B., Franke, I., Ulrich, J. & Gollnick, H.
  • the present invention overcomes the aforementioned problems and has additional advantages.
  • the present invention provides an immune response cell characterized in that the cell expresses an antigen-binding receptor; and an exogenous type I interferon.
  • the immune response cells of the invention comprise T cells, natural killer cells, cytotoxic T lymphocytes, natural killer T cells, DNT cells, and/or regulatory T cells.
  • the antigen binding receptor is endogenous. In some embodiments, the antigen binding receptor is recombinant. In some embodiments, the antigen binding receptor is a chimeric antigen receptor. In some embodiments, the antigen-binding receptor comprises a sequence-linked extracellular antigen binding region, a transmembrane region, and an intracellular signaling region. In some embodiments, the antigen binding unit is an antibody or fragment thereof that specifically binds to the antigen. In some embodiments, the intracellular signal region can contain a known signal motif for an immunoreceptor tyrosine activation motif (ITAM).
  • ITAM immunoreceptor tyrosine activation motif
  • examples of the ITAM comprising a cytoplasmic signaling sequence include those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, and CD66d.
  • the intracellular signaling region of the antigen binding receptor comprises one or more costimulatory domains.
  • the costimulatory domain is selected from one or more of those listed in Table 1.
  • the costimulatory domain is selected from the group consisting of CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BBL, MyD88, and 4-1BB Or A variety.
  • the costimulatory domain is selected from the group consisting of CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BBL, MyD88, and 4-1BB .
  • the amino acid sequence of the antigen-binding receptor is SEQ ID NO: 49; SEQ ID NO: 50; SEQ ID NO: 51; SEQ ID NO: 54; SEQ ID NO: 55; :56; SEQ ID NO: 61; SEQ ID NO: 62; SEQ ID NO: 63; SEQ ID NO: 64; SEQ ID NO: 65; SEQ ID NO: 66; SEQ ID NO: 67; SEQ ID NO: 68 SEQ ID NO: 69; SEQ ID NO: 70; SEQ ID NO: 71; SEQ ID NO: 72; SEQ ID NO: 73; SEQ ID NO: 74; SEQ ID NO: 75; and SEQ ID NO: 77
  • the antigen-binding receptor is encoded by a nucleotide sequence that is SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 Or SEQ ID NO: 76 has at least 90% identity.
  • the immune response cell does not comprise an exogenous co-stimulatory ligand.
  • the antigen capable of being bound by the antigen-binding receptor comprises a tumor antigen or a pathogen antigen.
  • the tumor antigen is selected from the group consisting of prostate specific membrane antigen (PSMA), carcinoembryonic antigen (CEA), IL13Ralpha, HER-2, CD19, NY-ESO-1, HIV-1 Gag, Lewis Y, MART -1, gp100, tyrosinase, WT-I, hTERT, mesothelin, EGFR, EGFRvIII, phosphatidylinositol 3, EphA2, HER3, EpCAM, MUC1, MUC16, CLDN18.2, folate receptor , CLDN6, CD30, CD138, ASGPR1, CDH16, GD2, 5T4, 8H9, ⁇ v ⁇ 6 integrin, B cell mature antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD20, CD22,
  • B cell mature antigen BC
  • the pathogen antigen comprises a viral antigen, a bacterial antigen, a fungal antigen, or a parasite antigen.
  • the pathogen is a virus.
  • the viruses include cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, and influenza virus.
  • the expression of the Type I interferon is constitutively expressed. In some embodiments, the expression of the type I interferon is inducible expression. In some embodiments, the type I interferon is expressed on the surface of the immune response cell. In some embodiments, the Type I interferon comprises: IFN ⁇ or IFN ⁇ .
  • the present invention provides an expression construct comprising: an expression cassette of the antigen-binding receptor of the present invention; and an expression cassette of type I interferon.
  • the expression of the Type I interferon is constitutively expressed.
  • the expression of the type I interferon is inducible expression.
  • the expression of the type I interferon is inducible expression and the expression for expressing the type I interferon is an inducible promoter.
  • the inducible promoter for expression of the type I interferon is the NFAT6 promoter.
  • the NFAT6 promoter comprises the nucleic acid sequence set forth in SEQ ID NO:78.
  • the invention provides a vector which expresses an antigen binding receptor of the invention and/or a type I interferon.
  • the viral vector is a lentiviral vector, a retroviral vector, or an adenoviral vector.
  • the viral vector is a retroviral vector.
  • the invention provides a method of increasing the viability of an immune response cell administered to an individual, wherein said immune response cell expresses an antigen binding receptor of the invention, and wherein said The method comprises administering to the individual the immune response cell and an effective amount of an exogenous type I interferon.
  • the exogenous type I interferon is administered sequentially or simultaneously with the immune response cell expressing the antigen binding receptor.
  • the exogenous type I interferon is administered to a patient simultaneously with the immune response cell by co-expression in an immune response cell.
  • the invention provides the use of an immune response cell of the invention in the manufacture of a pharmaceutical composition for treating a tumor, a pathogen infection, or enhancing an individual's immune tolerance in an individual in need thereof.
  • the invention also provides a method of treating a tumor or pathogen infection in an individual, or for enhancing an individual's immune tolerance, comprising administering to the individual in need thereof an immune response cell of the invention.
  • the methods of the invention result in cytotoxicity T in the peripheral blood of the individual after administration of the immune response cell to the individual compared to the absence of the exogenous type I interferon
  • the sum of the number of cells and helper T cells is increased by at least 50%.
  • the method comprises, after about 5 days of administering the immune response cell to the individual, the sum of the number of cytotoxic T cells and helper T cells in the peripheral blood of the individual is greater than 15,000/ ⁇ L; After about 5 days of the immune response cell, the sum of the number of cytotoxic T cells and helper T cells in the peripheral blood is greater than 500 / ⁇ L; or about 5 days after administration of the immune response cells, the peripheral blood in the body The sum of the number of cytotoxic T cells and helper T cells is greater than 50/ ⁇ L.
  • the tumor comprises: pancreatic cancer, liver cancer, lung cancer, gastric cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, renal cancer, leukemia, bone marrow Tumor, Ovarian cancer, cervical cancer, ovarian cancer, cervical cancer or glioma.
  • the pathogen comprises: a virus, a bacterium, a fungus, a protozoa or a parasite; preferably, the virus comprises: a cytomegalovirus, an Epstein-Barr virus, a human immunodeficiency virus or flu virus.
  • the tumor of the individual is reduced by at least 30% after treatment by the methods of the invention, according to computed tomography measurements. In some embodiments, the tumor of the individual completely disappears after treatment by the method of the invention, according to computed tomography measurements.
  • the invention provides a pharmaceutical composition comprising an immune response cell of the invention and a pharmaceutically acceptable carrier or excipient.
  • the invention provides a kit comprising an immune response cell of the invention and instructions for how to administer the immune response cell to an individual.
  • Figure 1 shows the structure of the recombinant lentiviral vector pRRL-EF-1 ⁇ -92-CAR (Fig. 1A) and the construction of the 92-28Z-NFAT6-IFN- ⁇ plasmid (Fig. 1B).
  • Figure 2 shows the detection of PBMC positive rate of lentivirus infection.
  • Figure 3 shows the detection of PBMC positive rate of lentivirus infection.
  • Figure 4 is a graph showing the release of cytokines from IFN-containing and IFN-free GPC3 CAR-T cells.
  • Figure 4A shows the induction of IFN- ⁇ expression by GPC3-28Z-IFN and GPC3-28Z CAR T cells. The results showed that only GPC3-28Z-IFN was incubated with Huh7 cells for IFN- ⁇ expression, indicating that GPC3-28Z-IFN was successfully induced to be expressed and secreted outside the cell after activation by the target antigen.
  • Figure 4B shows the comparison of INF- ⁇ -induced expression by GPC3-28Z-IFN and GPC3-28Z CAR.
  • Figure 4C shows a comparison of GPC3-28Z-IFN and GPC3-28Z CAR T cells leading to IL-2 release in vitro. The results showed that GPC3-28Z-IFN was more effective in causing cytokine release, indicating that CAR T cells containing IFN ⁇ can be activated more efficiently.
  • Figure 5 shows a comparison of the induction of cytokine release by 85-28Z T cells and 85-28Z-IFN T cells in vitro in different cell lines. The results show that CAR T cells containing IFN ⁇ can be activated more efficiently.
  • Figure 6 shows GPC3-28Z CAR T cells containing IFN ⁇ and GPC3 CAR T cells not containing IFN ⁇ in vitro against various cell lines (Fig. 6A: Huh7; Fig. 6B: Hep3B; Fig. 6C: PLC/PRR/5; Figure 6D: Hep G2; Figure 6E: SK-hep-1) comparison of killing efficiency.
  • Figure 7 is a graph showing the killing activity of CLD18A2 CAR-T cells containing IFN and no IFN.
  • FIG. 8 shows that CLD18A2 CAR-T cells containing IFN ⁇ and CLD18A2 CAR-T cells not containing IFN ⁇ were shown in the peripheral blood of mice for 5 days (Fig. 8A) for 7 days (Fig. 8B) and 10 days. (Fig. 8C) Comparison of the number of viable cells after (Fig. 8C). The results showed that the number of CLD18A2 CAR-T surviving cells containing IFN ⁇ was significantly higher than that of the CLD18A2 CAR-T cell group not containing IFN ⁇ at all time points.
  • Figure 9 is a comparison of the effect of GPC3-28Z CAR T cells containing IFN ⁇ and GPC3-28Z CAR T cells not containing IFN ⁇ on tumor volume over time in a mouse tumor model (Fig. 9A) and a comparison of tumor photographs (Fig. 9) Figure 9B).
  • the results showed that GPC3-28Z CAR T cells containing IFN ⁇ were able to more significantly reduce tumor volume than GPC3-28Z CAR T cells not containing IFN ⁇ and the control group.
  • Figure 10 is a graph showing the effect of CLD18A2 CAR-T cells containing IFN ⁇ and CLD18A2 CAR-T cells not containing IFN ⁇ on tumor volume in a mouse model of BGC-823-A2 subcutaneous transplantation in time (Fig. 10A). And a comparison of tumor photos (Fig. 10B). The results showed that CLD18A2 CAR-T cells containing IFN ⁇ were able to more significantly reduce tumor volume than CLD18A2 CAR-T cells not containing IFN ⁇ and the control group.
  • Figure 11 is a graph showing the antitumor activity of CLD18A2 CAR-T cells containing IFN and no IFN in a subcutaneous xenograft of gastric cancer PDX model. The results showed that one of the mice in the treatment group containing IFN completely resolved tumors.
  • Figure 12 is a graph showing tumor infiltration comparison of GPC3 CAR-T (92-28Z) cells containing IFN and no IFN in vivo.
  • 12A is a histochemical picture
  • FIG. 12B is a T cell number map. The results showed that there was no infiltrating CD3+ cells in the tumor tissues of the control group, and the number of CD3+ T cells in the INF ⁇ -CAR-T treatment group was higher than that in the 28Z CART group.
  • Figure 13 shows an immunohistochemical comparison of tumor infiltration of CLD18A2 CAR-T cells containing IFN and no IFN in vivo.
  • the results showed that Mock T cells showed almost no T cell infiltration around the tumor tissue, and 85-28Z and 85-2-28Z CAR T cells were visible at the edge of the tumor tissue, while 85-2-28Z-IFN T cells A certain infiltration can be observed inside the tumor tissue.
  • Figure 14 is a schematic representation of the structure of EGFR-CAR containing IFN and no IFN.
  • Figure 15 is a graph showing the positive rate of infection of T lymphocytes infected with retroviruses in mice.
  • Figure 16 is a graph showing the ability of EGFR CAR T cells containing IFN and IFN to secrete mIFN ⁇ in vitro. The results showed that mCAR-806-mIFN ⁇ was successfully activated and induced to express mIFN ⁇ after stimulation by target cells, and no expression of mIFN ⁇ was detected in the control group.
  • Figure 17 is a graph showing the induction of cytokine release in vitro (Figure 17A: mIL-2; Figure 17B: mIFN- ⁇ ; Figure 17C: mTNF- ⁇ ) of EGFR CAR-T cells containing IFN and no IFN.
  • Figure 18 is a comparison of in vitro toxicity tests of IFN-containing and IFN-free EGFR CAR-T cells.
  • the results showed that EGFR-CAR and EGFR-CAR-IFN had potent killing effect on target-positive CT26VIII cells compared with UT cells, the difference was significant (***P ⁇ 0.001), and the percentage of killing was dose-dependent.
  • untransfected UT cells had no killing effect on CT26 and CT26VIII
  • EGFR-CAR and EGFR-CAR-IFN two CAR-T cells had no killing effect on target-negative CT26 cells.
  • Figure 19 is a graph showing the in vivo toxicity test of EGFR CAR-T cells containing IFN and no IFN. The results showed that the tumor size of EGFR-CAR-T cells was basically the same as that of the control group, and no inhibition was observed. After EGFR-CAR-IFN cells were reinfused, tumor growth inhibition began on the 7th day. The rate was 5.9%, reaching the strongest on the 10th day, 18.5%. By the 17th day, the tumor inhibition rate could still reach 12.4%, which was significantly better than the EGFR-CAR-T cell group.
  • the present inventors conducted intensive studies and found that inducing expression of type I interferon by CAR-T cells can effectively increase the antitumor activity of CAR-T cells and reduce their toxic and side effects.
  • the present invention provides an immune response cell which expresses at least one receptor capable of binding an antigen (such as a tumor antigen or an antigen derived from a pathogen) and a type I interferon, and is applied to treat tumors and infections.
  • an antigen such as a tumor antigen or an antigen derived from a pathogen
  • activated and “activated” are used interchangeably herein and they, as well as other grammatical forms thereof, may refer to the process by which a cell transitions from a quiescent state to an active state.
  • the process can include a response to a phenotypic or genetic change in the antigen, migration, and/or functional activity state.
  • activation can refer to the process by which T cells are gradually activated.
  • T cells may require at least two signals to be fully activated. The first signal can occur after the TCR is bound by the antigen-MHC complex, while the second signal can occur by the conjugation of costimulatory molecules (see co-stimulatory molecules listed in Table 1).
  • anti-CD3 can simulate the first signal and anti-CD28 can simulate the second signal.
  • engineered T cells can be activated by the expressed CAR.
  • T cell activation or T cell triggering can refer to the state of a T cell that has been sufficiently stimulated to induce detectable cell proliferation, cytokine production, and/or detectable effector function.
  • co-stimulatory ligand includes a molecule on an antigen presenting cell (eg, aAPC, dendritic cell, B cell, etc.) that specifically binds to an identical costimulatory molecule on a T cell, thereby providing a signal,
  • the first signal provided by binding of, for example, a TCR/CD3 complex to a peptide-loaded MHC molecule, mediates a T cell response including, but not limited to, proliferation, activation, differentiation, and the like.
  • Costimulatory ligands can include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligands (ICOS-L) , intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, binding to Toll ligand receptor An agonist or antibody and a ligand that specifically binds to B7-H3.
  • Costimulatory ligands also specifically include antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function.
  • costimulatory molecule refers to an identical 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.
  • Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors.
  • co-stimulatory signal refers to a signal that, in combination with a first signal, such as TCR/CD3, results in T cell proliferation and/or up- or down-regulation of key molecules.
  • antigen binding unit refers to an immunoglobulin molecule and an immunologically active portion of an immune molecule, ie, a molecule containing an antigen binding site that specifically binds to an antigen ("immune response").
  • immunoglobulin molecules of various species including invertebrates and vertebrates. Structurally, the simplest naturally occurring antibody (eg, IgG) comprises four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Immunoglobulins represent a large family of molecules including several types of molecules, such as IgD, IgG, IgA, IgM, and IgE.
  • immunoglobulin molecule includes, for example, hybrid antibodies or altered antibodies and fragments thereof. It has been shown that the antigen binding function of antibodies can be carried out by fragments of naturally occurring antibodies. These fragments are collectively referred to as "antigen combining units.” Also included in the term “antigen binding unit” is any polypeptide chain-containing molecular structure having a specific shape that conforms to an epitope and recognizes an epitope, wherein one or more non-covalent binding interactions stabilize the structure between the molecule and the epitope Complex.
  • An antigen binding unit “specifically binds" to an antigen or is “immunoreactive” with an antigen if the antigen binding unit binds to the antigen with greater affinity or affinity than binding to other reference antigens, including polypeptides or other substances. ".
  • antigen refers to a substance that is recognized and specifically bound by an antigen binding unit.
  • Antigens can include peptides, proteins, glycoproteins, polysaccharides, and lipids, portions thereof, and combinations thereof.
  • Non-limiting exemplary antigens include tumor antigens or pathogen antigens.
  • Antigen can also refer to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immunologically-competent cells, or both. Those skilled in the art will appreciate that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.
  • immunoglobulin may refer to a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as chimeric antigen receptors or antigen receptors. The five members included in such proteins are IgA, IgG, IgM, IgD and IgE, with IgG being the most common circulating antibody. It is the most potent immunoglobulin in agglutination, complement fixation and other antibody reactions and is important in protecting against bacteria and viruses. For example, tumor cell antigens (or “tumor antigens”) or pathogen antigens can be identified by CAR.
  • tumor cell antigens or "tumor antigens”
  • pathogen antigens can be identified by CAR.
  • a sample eg, a cell
  • administered e.g, a patient
  • the autologous process differs from the allogeneic process in which the donor and recipient are different individuals.
  • xenograft and other grammatical forms thereof can include any procedure in which a recipient, a donor, and a donor are different species, transplanting, implanting, or infusing a cell, tissue, or organ into a recipient. Transplantation of cells, organs and/or tissues described herein can be used for xenografting into humans.
  • Xenografts include, but are not limited to, vascularized xenografts, partially vascularized xenografts, non-vascularized xenografts, xenogeneic dressings, xenogeneic Bandages and heterogeneous structures.
  • allogeneic transplantation may include transplantation of cells, tissues or organs in which the recipient and donor are the same species but different individuals, implanted. Or infusion to any of the recipients. Transplantation of cells, organs and/or tissues as described herein can be used for allogeneic transplantation into humans. Allografts include, but are not limited to, vascularized allografts, partially vascularized allografts, non-vascularized allografts, allogeneic dressings, allogeneic bandages, and allogeneic structures.
  • autologous transplantation and other forms of grammar thereof (eg, autologous transplantation) may include transplantation, implantation or infusion of a cell, tissue or organ into a recipient in which the recipient and donor are the same individual. Any program in . Transplantation of cells, organs and/or tissues described herein can be used for autologous transplantation into the human body. Autologous transplantation includes, but is not limited to, vascular autologous transplantation, partial vascular autologous transplantation, non-vascularized autografts, autologous dressings, autologous bandages, and autologous structures.
  • chimeric antigen receptor or "CAR” as used herein refers to an engineered molecule that can be expressed by immune cells including, but not limited to, T cells. CAR is expressed in T cells and T cells can be redirected to induce specific killing of target cells by a human receptor.
  • the extracellular binding domain of CAR can be derived from a murine, humanized or fully human monoclonal antibody.
  • epitope and its grammatical other forms as used herein may refer to a portion of an antigen that can be recognized by an antibody, B cell, T cell, or engineered cell.
  • an epitope can be a tumor epitope or a pathogen epitope recognized by a TCR. Multiple epitopes within the antigen can also be identified. Epitopes can also be mutated.
  • engineered and its grammatical other forms as used herein may refer to one or more changes in a nucleic acid, such as a nucleic acid within the genome of an organism.
  • engineered can refer to alterations, additions, and/or deletions of genes.
  • Engineered cells can also refer to cells having genes that are added, deleted, and/or altered.
  • cell or "engineered cell” and its grammatical other forms as used herein may refer to a cell of human or non-human animal origin. Engineered cells can also refer to cells that express CAR.
  • transfection refers to the introduction of an exogenous nucleic acid into a eukaryotic cell. Transfection can be achieved by a variety of means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polyamine transduce transfection, electroporation, microinjection, Liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.
  • stable transfection or “stable transfection” refers to the introduction and integration of exogenous nucleic acids, DNA or RNA into the genome of a transfected cell.
  • stable transfectant refers to a cell that stably integrates foreign DNA into genomic DNA.
  • nucleic acid molecule encoding refers to the order or sequence of deoxyribonucleotides along a deoxyribonucleic acid strand. The order of these deoxyribonucleotides determines the order of the amino acids along the polypeptide (protein) chain. Thus, the nucleic acid sequence encodes an amino acid sequence.
  • the term "individual” as used herein refers to any animal, such as a mammal or marsupial. Individuals of the invention include, but are not limited to, humans, non-human primates (e.g., rhesus or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and poultry of any kind.
  • non-human primates e.g., rhesus or other types of macaques
  • mice pigs, horses, donkeys, cows, sheep, rats, and poultry of any kind.
  • peripheral blood lymphocytes and other grammatical forms thereof as used herein may refer to lymphocytes circulating in blood (eg, peripheral blood).
  • Peripheral blood lymphocytes can refer to lymphocytes that are not limited to organs.
  • Peripheral blood lymphocytes can comprise T cells, NK cells, B cells, or any combination thereof.
  • immune response cell or “immunoreactive cell” as used herein may refer to a cell that can elicit an immune response, including but not limited to T cells, B cells, and NKT cells, their respective precursor cells, and their progeny.
  • An immune response cell can also refer to a cell of the lymphoid or myeloid lineage.
  • T cell and its grammatical other forms as used herein may refer to T cells of any origin.
  • the T cell can be a primary T cell such as an autologous T cell or the like.
  • T cells can also be human or non-human.
  • T cell activation or “T cell trigger” and other grammatical forms thereof as used herein may refer to a T cell that is sufficiently stimulated to induce detectable cell proliferation, cytokine production, and/or detectable effector function. status. In some embodiments, "complete T cell activation” can be similar to triggering cytotoxicity of T cells.
  • T cell activation can be measured using various assays known in the art.
  • the assay can be an ELISA for measuring cytokine secretion, ELISPOT, a flow cytometry assay for measuring intracellular cytokine expression (CD107), a flow cytometry assay for measuring proliferation, and for determining target cell elimination. Cytotoxicity assay (51Cr release assay).
  • the assay is typically compared to engineered cells (CAR T) using controls (non-engineered cells) to determine the relative activation of engineered cells compared to controls. Furthermore, the assay can be compared to engineered cells that are incubated or contacted with target cells that do not express the target antigen. For example, the comparison can be a comparison with CD19-CART cells incubated with target cells that do not express CD19.
  • sequence and its grammatical other forms as used herein, when used in reference to a nucleotide sequence, may include DNA or RNA, and may be single-stranded or double-stranded.
  • the nucleic acid sequence can be mutated.
  • the nucleic acid sequence can be of any length, for example, a nucleic acid having a length of from 2 to 1,000,000 or more nucleotides (or any integer value therebetween or above), for example, from about 100 to about 10,000 nucleotides in length or from about 200 to about 500. Nucleotides.
  • an effective amount refers to an amount that provides a therapeutic or prophylactic benefit.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed.
  • the expression vector contains sufficient cis-acting for expression Cis-acting elements; other elements for expression can be provided by host cells or in vitro expression systems.
  • Expression vectors include those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).
  • lentivirus refers to the genus of the family Retroviridae. Retroviruses are unique in retroviruses in their ability to infect non-dividing cells; they can deliver large amounts of genetic information into the DNA of host cells, making them one of the most efficient methods of gene delivery vectors. HIV, SIV and FIV are all examples of lentiviruses. Vectors derived from lentivirus provide a means to achieve significant levels of gene transfer in vivo.
  • operably linked refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the latter.
  • first nucleic acid sequence when the first nucleic acid sequence is functionally related to the second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects transcription or expression of the coding sequence.
  • the operably linked DNA sequences are contiguous and, where necessary, ligated two protein coding regions in the same reading frame.
  • promoter is defined as a DNA sequence that is recognized by the synthetic machinery or the introduced synthetic machinery required to initiate specific transcription of a polynucleotide sequence.
  • vector is a composition comprising 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 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.
  • sequence identity determines the percent identity by comparing two best matched sequences over a comparison window (eg, at least 20 positions), wherein portions of the polynucleotide or polypeptide sequence in the comparison window can comprise Addition or deletion (ie gap), for example 20% or less gap (eg 5 to 15%, or 10 to 12) compared to the reference sequence (which does not contain additions or deletions) for the best matched two sequences %).
  • a comparison window eg, at least 20 positions
  • portions of the polynucleotide or polypeptide sequence in the comparison window can comprise Addition or deletion (ie gap), for example 20% or less gap (eg 5 to 15%, or 10 to 12) compared to the reference sequence (which does not contain additions or deletions) for the best matched two sequences %).
  • the percentage is usually calculated by determining the number of positions in which the same nucleic acid base or amino acid residue occurs in both sequences to produce the number of correctly matched positions, dividing the number of correctly matched positions by the total number of positions in the reference sequence ( That is, the window size), and multiply the result by 100 to produce a percentage of sequence identity.
  • type I interferon as used herein includes IFN ⁇ , IFN ⁇ , and IFN- ⁇ , IFN- ⁇ , and IFN- ⁇ and the like. All type I interferons bind to specific cell surface receptors (so-called IFN-[alpha]/[beta] receptors) consisting of two strands of IFNARl and IFNAR2. In some embodiments, the term “type I stem” is used herein. "Interferon" is IFN ⁇ or IFN ⁇ . In some embodiments, the term “type I interferon” as used herein is IFN ⁇ . In some embodiments, a type I interferon as used herein includes human, mouse, or synthetic type I. Interferon.
  • the term "interferon alpha” as used herein may be a polypeptide having the sequence shown in NCBI aaa52724.1 or aaa52716.1 or aaa52725.1, or the sequence has at least 85% of the sequence The identity of the polypeptide.
  • the term "interferon beta” (INF- ⁇ ) as used herein may be at least 85% identical to NCBI aac41702.1 or np_002167.1 or aah96152.1p41273 or NP 001552. Protein, or a fragment thereof that has the function of a tumor necrosis factor (TNF) ligand.
  • TNF tumor necrosis factor
  • the element or the type I interferon applied to construct the antigen-binding receptor may be naturally occurring, such as may be isolated or purified from a mammal; or may be artificially prepared.
  • recombinant elements or type I interferons can be produced according to conventional genetic engineering recombination techniques.
  • the present invention may employ recombinant elements or type I interferons.
  • Amino acid sequences formed by substitution, deletion or addition of one or more amino acid residues are also included in the present invention on the basis of the respective elements or type I interferon polypeptide sequences.
  • Proper replacement of amino acids is a technique well known in the art that can be readily implemented and ensures that the biological activity of the resulting molecule is not altered. These techniques have taught one in the art that, in general, altering a single amino acid in a non-essential region of a polypeptide does not substantially alter biological activity.
  • bioactive fragments of the respective elements or polypeptides of type I interferon can be used in the present invention.
  • a biologically active fragment refers to a polypeptide which, as part of a full length polypeptide, still retains all or part of the function of the full length polypeptide.
  • the biologically active fragment retains at least 50% of the activity of the full length polypeptide.
  • the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full length polypeptide.
  • Modified or modified polypeptides can also be used in the present invention based on the various elements or type I interferon polypeptide sequences, for example, to promote their half-life, effectiveness, metabolism, and/or efficacy of the polypeptide.
  • disease refers to any alteration or disorder that impairs or interferes with the normal function of a cell, tissue or organ.
  • disease includes, but is not limited to, a tumor, a pathogen infection, an autoimmune disease, a T cell dysfunction disease, or a defect in immune tolerance (eg, transplant rejection).
  • tumor refers to a disease characterized by pathological hyperplasia of cells or tissues, and its subsequent migration or invasion of other tissues or organs. Tumor growth is usually uncontrolled and progressive, Does not induce or inhibit normal cell proliferation.
  • a tumor can affect a variety of cells, tissues or organs including, but not limited to, selected from the group consisting of bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, Nerve tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, urethra, ureter, urethra, uterus, vaginal organs, or tissue or corresponding cells.
  • Tumors include cancers such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells).
  • the tumor of the present invention may include, but is not limited to, leukemia (such as acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute granulocyte-monocytic leukemia, Acute monocytic leukemia, acute leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphoma (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia Disease, heavy chain disease, solid tumors such as sarcoma and cancer (such as fibrosarcoma, mucinous sarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endotheli
  • the "tumor” includes, but is not limited to, pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, Kidney cancer, leukemia, multiple myeloma, ovarian cancer, cervical cancer and glioma.
  • the type of tumor antigen referred to in the present invention may also be a tumor specific antigen (TSA) or a tumor associated antigen (TAA).
  • TSA is unique to tumor cells and does not occur on other cells in the body.
  • the TAA-associated antigen is not unique to tumor cells, but is expressed on normal cells under conditions in which the immune tolerance state to the antigen cannot be induced. Expression of the antigen on the tumor can occur under conditions that enable the immune system to respond to the antigen.
  • TAA may be antigens expressed on normal cells during fetal development, or they may be antigens that are normally present at very low levels on normal cells but are expressed at higher levels on tumor cells. .
  • TSA or TAA antigens include the following: differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific multicenter antigens Such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic resistance Originally as CEA; overexpressed oncogenes and mutant tumor suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens caused by chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens such as Epstein Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7.
  • differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel17), ty
  • the "tumor antigen” includes, but is not limited to, prostate specific membrane antigen (PSMA), carcinoembryonic antigen (CEA), IL13Ralpha, HER-2, CD19, NY-ESO-1, HIV- 1Gag, Lewis Y, MART-1, gp100, tyrosinase, WT-I, hTERT, mesothelin, EGFR, EGFRvIII, phosphatidylinositol 3, EphA2, HER3, EpCAM, MUC1, MUC16, CLDN18 .2, folate receptor, CLDN6, CD30, CD138, ASGPR1, CDH16, GD2, 5T4, 8H9, ⁇ v ⁇ 6 integrin, B cell mature antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD20, CD22 , kappa light chain, CD33, CD38, CD44, CD44v6, CD44v7/8, CD70,
  • PSMA
  • pathogen refers to protozoa that are capable of causing a disease, including: viruses, bacteria, fungi or parasites.
  • viral antigen refers to a polypeptide expressed by a virus capable of inducing an immune response.
  • Typical viruses include, but are not limited to, retroviridae (such as human immunodeficiency virus, such as HIV-1 (also known as HDTV-III, LAVE or HTLV-III/LAV, or HIV-III; and other strains, such as HIV-LP; picornavirus (such as poliovirus, hepatitis A virus; human enterovirus, coxsackie virus, rhinovirus, echovirus); calicivirus (such as gastroenteritis caused by strain); Covering viruses (such as equine encephalitis virus, rubella virus); flaviviruses (such as dengue virus, Japanese encephalitis virus, yellow fever virus); coronavirus (such as coronavirus); rhabdovirus (such as blisters) Stomatitis virus, rabies virus); filamentous virus family (such as Ebola Virus); paramyxoviridae (such as parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxovirus (
  • Typical bacteria include, but are not limited to, Pasteurella, Staphylococcus aureus, Streptococcus, Escherichia coli, Salmonella, and Pseudomonas aeruginosa.
  • infectious bacteria include, but are not limited to, Helicobacter pylori, spirochetes, Legionella pneumophila, Mycobacterium sp. (S. tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis, M. Kansaii, M.
  • an immune response cell of the invention is capable of recognizing and binding to a parasite antigen.
  • the parasites include endoparasites and ectoparasites.
  • the endoparasites include protozoa, helminths, mites, and flukes.
  • the parasitic antigen is, for example, an antigen derived from a species of the following family: Entamoeba histolytica; Babesia B.divergens, B.bigemina, B.equi, B.microfti, B.duncani; Balantidium coli;Blastocystis Spp.;Trypanosoma cruzi;Cryptosporidium spp.;Cyclospora cayetanensis;Dientamoeba fragilis;Giardia lamblia;Balamuthia mandrillaris;Acanthamoeba spp.;Isospora belli;Leishmania spp.;Plasmodium falciparum,Plasmodium vivax,Plasmodium ovale curtisi,Plasmodium ovale wallikeri,Plasmodium malariae, Plasmodium knowlesi; Naegleria
  • autoimmune disease as used herein is defined as a condition caused by an autoimmune response.
  • Autoimmune diseases are the result of inappropriate and overreaction to autoantigens.
  • autoimmune diseases include, but are not limited to, appendicitis, alopecia, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, diabetes (type I), malnourished bullous epidermis Disorder, epididymitis, glomerulonephritis, Graves' disease, Gilanbar syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris , psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, viti
  • Tolerance or "immune tolerance” is the failure of the immune system to produce a defensive immune response to a particular antigen. Tolerance can be natural or self-independent, in which the body does not attack its own proteins and antigens, or can be induced by manipulation of the immune system. Central tolerance occurs during lymphocyte development and plays a role in the thymus and bone marrow. During this process, T lymphocytes and B lymphocytes that recognize autoantigens are deleted before they develop into fully immunocompetent cells. This process is most active during fetal development, but lasts for the rest of the life as immature lymphocytes are produced.
  • Peripheral T cell tolerance refers to the functional anergy of autoantigens present in peripheral tissues and occurs after T and B cells mature and enter the periphery. These processes include the inhibition of autoreactive cells by "regulatory" T cells, and the generation of hyporeactivity (non-reactivity) in lymphocytes that encounter antigen without co-stimulatory signals accompanying inflammation. "Acquired” or “induced tolerance” refers to the adaptation of the immune system to external antigens, characterized by specific non-reactivity of lymphoid tissue with a given antigen, and in other cases may induce cell-mediated or humoral immunity. .
  • tolerance can be clinically induced by repeated administration of very large doses of antigen or a small dose below the threshold required to stimulate an immune response (eg, by intravenous or sublingual administration of soluble antigen).
  • the antigen that induces the formation of immune tolerance is called Tolerogen. Immunosuppression is also beneficial for inducing tolerance. Destruction of self-tolerance can lead to autoimmunity.
  • the second case is when the tolerant state has been induced, either prior to exposure to the donor's antigen in a manner that results in immune tolerance rather than sensitization to the recipient, or after chronic rejection.
  • Successful allogeneic transplantation requires a degree of immune tolerance to allogeneic antigens.
  • the achievement of immune tolerance prevents host-versus graft response leading to transplant rejection and failure, and prevents graft versus host response (GVHD).
  • enhancing immune response cell function includes, for example, enhancing T cell function.
  • enhancing T cell function involves inducing, causing or stimulating T cells to have sustained or enhanced biological function, or to renew or reactivate depleted or inactive T cells.
  • enhanced T cell function include increased levels of interferon secreted by CD8+ T cells, increased proliferation, and increased antigenic reactivity (eg, viral or pathogen clearance) relative to pre-intervention levels.
  • the level of enhancement is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The manner in which such enhancement is measured is known to those of ordinary skill in the art.
  • T cell dysfunctional disease includes a condition or disorder of a T cell characterized by a decrease in antigenic stimulatory reactivity.
  • the T cell dysfunctional disorder is a disorder associated with inappropriately increased signaling specificity by PD-1.
  • the T cell dysfunction disease is a disease in which T cells are incapable or secrete cytokines, proliferate, or have reduced ability to perform cytolytic activity. Examples of T cell dysfunction diseases characterized by T cell dysfunction include unabsorbed acute infection, chronic infection, and tumor immunity.
  • exogenous refers to a nucleic acid molecule or polypeptide that is not endogenously expressed in a cell, or that has a level of expression insufficient to achieve overexpression.
  • exogenous includes recombinant nucleic acid molecules or polypeptides expressed in a cell, such as exogenous, heterologous and overexpressed nucleic acid molecules and polypeptides.
  • receptor refers to a polypeptide, or a portion thereof, that selectively binds one or more ligands on a cell membrane.
  • an antigen binding receptor of the invention specifically binds to an antigen to which it binds.
  • the antigen binding receptor of the invention is a chimeric antigen receptor.
  • Chimeric Antigen Receptor refers to a tumor antigen binding domain fused to an intracellular signal transduction domain that activates T cells.
  • the extracellular binding domain of CAR is derived from a mouse or humanized or human monoclonal antibody.
  • a chimeric antigen receptor typically comprises an extracellular antigen binding region or antigen binding unit.
  • the extracellular antigen binding region can be fully human. In other cases, the extracellular antigen binding region can be humanized. In other instances, the extracellular antigen binding region can be murine or the chimera in the extracellular antigen binding region consists of amino acid sequences from at least two different animals. In some embodiments, the extracellular antigen binding region can be non-human.
  • antigen binding regions can be designed. Non-limiting examples include single-chain variable fragments (scFv) derived from antibodies, fragment antigen binding regions (Fabs) selected from libraries, single domain fragments, or natural ligands that bind to their cognate receptors.
  • the extracellular antigen binding region can comprise an scFv, Fab, or natural ligand, as well as any derivatives thereof.
  • An extracellular antigen binding region can refer to a molecule other than an intact antibody, which can comprise a portion of an intact antibody and can bind to an antigen to which the intact antibody binds.
  • antibody fragments can include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; bifunctional antibodies, linear antibodies; single-chain antibody molecules (eg, scFv); and formed from antibody fragments Multispecific antibodies.
  • An extracellular antigen binding region such as a scFv, Fab or natural ligand, can be part of a CAR that determines antigen specificity.
  • the extracellular antigen binding region can bind to any complementary target.
  • Extracellular antigen binding regions can be derived from known An antibody to the variable region sequence.
  • the extracellular antigen binding region can be obtained from antibody sequences obtained from available mouse hybridomas.
  • extracellular antigen binding regions can be obtained from whole-out cleavage of tumor cells or primary cells, such as tumor infiltrating lymphocytes (TIL).
  • TIL tumor infiltrating lymphocytes
  • the binding specificity of the extracellular antigen binding region can be determined by a complementarity determining region or CDR, such as a light chain CDR or a heavy chain CDR.
  • CDR complementarity determining region
  • binding specificity can be determined by light chain CDRs and heavy chain CDRs.
  • a given combination of heavy chain CDRs and light chain CDRs can provide a given binding pocket that can confer greater affinity and/or specificity to an antigen (eg, GPC3) than other reference antigens.
  • an antigen eg, GPC3
  • a CDR specific for Glypican-3 can be expressed in an extracellular binding region of a CAR such that a GPC3-targeting CAR can target an immune response cell to a GPC3-expressing tumor cell.
  • the extracellular antigen binding region can comprise a light chain CDR specific for the antigen.
  • the light chain CDR can be a complementarity determining region of an antigen binding unit, such as the scFv light chain of a CAR.
  • the light chain CDRs may comprise contiguous amino acid residue sequences, or two or more contiguous sequence of amino acid residues separated by non-complementarity determining regions (eg, framework regions).
  • a light chain CDR can comprise two or more light chain CDRs, which can be referred to as a light chain CDR-1, CDR-2, and the like.
  • the light chain CDRs can comprise three light chain CDRs, which can be referred to as light chain CDR-1, light chain CDR-2 and light chain CDR-3, respectively.
  • a set of CDRs present on a common light chain can be collectively referred to as a light chain CDR.
  • the extracellular antigen binding region can comprise a heavy chain CDR that is specific for the antigen.
  • the heavy chain CDRs can be heavy chain complementarity determining regions of antigen binding units such as scFv.
  • the heavy chain CDRs may comprise a contiguous sequence of amino acid residues, or a contiguous sequence of two or more amino acid residues separated by a non-complementarity determining region (eg, a framework region).
  • the heavy chain CDRs can comprise two or more heavy chain CDRs, which can be referred to as heavy chain CDR-1, CDR-2, and the like.
  • the heavy chain CDRs can comprise three heavy chain CDRs, which can be referred to as heavy chain CDR-1, heavy chain CDR-2 and heavy chain CDR-3, respectively.
  • a set of CDRs present on a common heavy chain can be collectively referred to as a heavy chain CDR.
  • the extracellular antigen binding region can be modified in various ways by using genetic engineering.
  • the extracellular antigen binding region can be mutated such that the extracellular antigen binding region can be selected to have a higher affinity for its target.
  • the affinity of the extracellular antigen binding region for its target can be optimized for targets that can be expressed at low levels on normal tissues. This optimization can be done to minimize potential toxicity.
  • a clone of an extracellular antigen binding region having a higher affinity for the membrane-bound form of the target may be A counterpart that is superior to its soluble form. This modification can be made because different levels of soluble forms of the target can also be detected and their targeting can cause undesirable toxicity.
  • the extracellular antigen binding region comprises a hinge or spacer.
  • the terms hinge and spacer are used interchangeably.
  • the hinge can be considered as part of a CAR for providing flexibility to the extracellular antigen binding region.
  • the hinge can be used to detect CAR on the cell surface of a cell, particularly when detecting antibodies to the extracellular antigen binding region are ineffective or available.
  • the length of the hinge derived from an immunoglobulin may need to be optimized, depending on the location of the extracellular antigen binding region that targets the epitope on the target.
  • the hinge may not belong to an immunoglobulin, but to another molecule, such as the native hinge of a CD8 alpha molecule.
  • the CD8 alpha hinge may contain cysteine and proline residues known to play a role in the interaction of the CD8 co-receptor and the MHC molecule. The cysteine and proline residues can affect the performance of the CAR.
  • the CAR hinge can be adjustable in size. This morphology of the immunological synapse between the immune response cell and the target cell also defines the distance that cannot be functionally bridged by the CAR due to the distal membrane epitope on the cell surface target molecule, ie, the use of a short hinge CAR does not The synaptic distance reaches an approximation of the signal's ability to conduct. Similarly, the membrane proximal CAR target epitope was only observed for signal output in the context of a long hinged CAR.
  • the hinge can be adjusted depending on the extracellular antigen binding region used. The hinge can be of any length.
  • the transmembrane domain can anchor the CAR to the plasma membrane of the cell.
  • the natural transmembrane portion of CD28 can be used for CAR.
  • the natural transmembrane portion of CD8 ⁇ can also be used in the CAR.
  • CD8 may be a protein having at least 85, 90, 95, 96, 97, 98, 99 or 100% identity to the NCBI reference number: NP_001759 or a fragment thereof having stimulatory activity.
  • a “CD8 nucleic acid molecule” may be a polynucleotide encoding a CD8 polypeptide, and in some cases, the transmembrane region may be a natural transmembrane portion of CD28, and “CD28” may refer to NCBI reference number: NP_006130 or its stimulating activity.
  • a fragment has a protein of at least 85, 90, 95, 96, 97, 98, 99 or 100% identity.
  • a "CD28 nucleic acid molecule” can be a polynucleotide encoding a CD28 polypeptide.
  • the transmembrane portion can comprise a CD8 alpha region.
  • the (fine) intracellular signaling region of CAR may be responsible for activating at least one of the effector functions of the immune response cells into which the CAR has been placed.
  • CAR can induce effector functions of T cells, for example, the effector function is cytolytic activity or helper activity, including secretion of cytokines.
  • intracellular signaling region refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a specific function. Although the entire intracellular signaling region can generally be used, in many cases it is not necessary to use the entire chain of the signal domain. In some embodiments, a truncated portion of an intracellular signaling region is used. In some embodiments, the term intracellular signaling region is therefore intended to include a cell sufficient to transduce an effector function signal. Any truncated portion of the inner signal conducting region.
  • Preferred examples of signal domains for use in CAR may include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act synergistically to initiate signal transduction after target-receptor binding, as well as any derivatives thereof or Variant sequences and any synthetic sequences of these sequences that have the same functionality.
  • TCRs T cell receptors
  • co-receptors that act synergistically to initiate signal transduction after target-receptor binding
  • the intracellular signaling region can contain a known signal motif for an immunoreceptor tyrosine activation motif (ITAM).
  • ITAMs containing cytoplasmic signaling sequences include those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, and CD66d.
  • the intracellular signal domain is derived from a CD3 ⁇ chain.
  • T cell signaling domain containing one or more ITAM motifs is the CD3 ⁇ domain, also known as the T cell receptor T3 ⁇ chain or CD247.
  • This domain is part of the T cell receptor-CD3 complex and plays an important role in binding antigen recognition of several intracellular signal transduction pathways to the main effector activation of T cells.
  • CD3 ⁇ primarily refers to human CD3 ⁇ and its isoforms, as known from the Swissprot entry P20963, including proteins having substantially the same sequence.
  • the full T cell receptor T3 ⁇ chain is not required and that any derivative of the signal domain comprising the T cell receptor T3 ⁇ chain is suitable, including any functional equivalent thereof. .
  • the intracellular signaling domain can be selected from any one of the domains of Table 1.
  • the domain can be modified such that identity to the reference domain can range from about 50% to about 100%.
  • Any of the domains of Table 1 can be modified such that the modified form can comprise about 50, 60, 70, 80, 90, 95, 96, 97, 98, 99 or up to about 100% identity.
  • the intracellular signaling region of CAR may further comprise one or more costimulatory domains.
  • the intracellular signaling region may comprise a single costimulatory domain, such as an ⁇ chain (first generation CAR) or it is with CD28 or 4-1BB (second generation CAR).
  • the intracellular signaling region can comprise two costimulatory domains, such as CD28/OX40 or CD28/4-1BB (third generation).
  • CD28 phosphatidylinositol-4,5-diphosphate 3-kinase
  • 4-1BB/OX40 TNF-receptor-associated factor adapter protein
  • signals generated by the CAR may be combined with an auxiliary or costimulatory signal.
  • costimulatory signaling domains chimeric antigen receptor-like complexes can be designed to contain several possible costimulatory signal domains.
  • a second co-stimulatory signal is required for complete productive T cell activation.
  • receptors have been reported to provide co-stimulation for T cell activation including, but not limited to, CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BBL, MyD88, and 4- 1BB.
  • the signaling pathways used by these costimulatory molecules work synergistically with the primary T cell receptor activation signal.
  • the signals provided by these costimulatory signaling regions can act synergistically with primary effect activation signals derived from one or more ITAM motifs (eg, the CD3zeta signal transduction domain) and can fulfill the requirements for T cell activation.
  • the addition of a costimulatory domain to a chimeric antigen receptor-like complex can enhance the efficacy and durability of engineered cells.
  • the T cell signal domain and the costimulatory domain are fused to each other to form a signaling region.
  • the chimeric antigen receptor binds to the target antigen.
  • the target antigen can be obtained or isolated from various sources.
  • a target antigen as used herein is an antigenic epitope on an antigen or antigen that is critical in mammals for immune recognition and ultimately elimination or control of pathogenic factors or disease states.
  • the immune recognition can be a cell and/or a body fluid. In the case of intracellular pathogens and cancer, the immune recognition can be, for example, a T lymphocyte reaction.
  • the target antigen can be derived or isolated from an antigen such as a viral microorganism such as the virus described herein above.
  • the chimeric antigen receptor binding of the invention includes, for example, HIV (Korber et al, eds HIV Molecular Immunology Database, Los Alamos National Laboratory, Los Alamos, N. Mex. 1977), influenza, herpes, herpes simplex Papillomavirus (U.S. Patent No. 5,719,054), Hepatitis B (US Patent No. 5,780,036), Hepatitis C (US Patent No. 5,709,995), EBV, cytomegalovirus (CMV) virus, and the like.
  • HIV Korean et al, eds HIV Molecular Immunology Database, Los Alamos National Laboratory, Los Alamos, N. Mex. 1977
  • influenza herpes
  • herpes simplex Papillomavirus U.S. Patent No. 5,719,054
  • Hepatitis B
  • the target antigen can also be derived from or isolated from the pathogenic bacteria described herein.
  • the chimeric antigen receptor binding of the invention is, for example, from Chlamydia (U.S. Patent No. 5,869,608), Mycobacterium, Legionella, Meningitis, Group A Streptococcus, Salmonella, Listeria, Haemophilus influenzae (U.S. Patent No. 5,955,596) and the like.
  • the target antigen can be derived or isolated, for example, from Aspergillus, Invasive Candida (US Pat. No. 5,645,992), Nocardia, Histoplasmosis, Cryptosporidium And other pathogenic yeasts.
  • the target antigen can be derived or isolated from, for example, pathogenic protozoa and pathogenic parasites including, but not limited to, Pneumocystis carinii, trypanosomiasis, Leishmania (U.S. Patent No. 5,965,242), Plasmodium (U.S. Patent No. 5,589,343) and Toxoplasma gondii.
  • pathogenic protozoa and pathogenic parasites including, but not limited to, Pneumocystis carinii, trypanosomiasis, Leishmania (U.S. Patent No. 5,965,242), Plasmodium (U.S. Patent No. 5,589,343) and Toxoplasma gondii.
  • the target antigen comprises an antigen associated with a pre-cancerous or proliferative state.
  • Target antigens may also be associated with or caused by cancer.
  • a chimeric antigen receptor of the invention recognizes and binds to a tumor antigen comprising TSA and TAA as described herein before.
  • modulation refers to a positive or negative change. Modification examples include 1%, 2%, 10%, 25%, 50%, 75%, or 100% variation.
  • treatment refers to the process of attempting to alter a disease caused by an individual or a cell.
  • Clinical interventions can be either preventive or clinically pathologically.
  • Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing the direct or indirect pathological consequences of any disease, preventing metastasis, slowing the progression of the disease, improving or ameliorating the condition, alleviating or improving the prognosis.
  • immune dysfunction means that the subject has an immunodeficiency that is easily infected. Organisms that cause opportunistic infections usually do not cause illnesses that have a healthy immune system, but can infect people with weakened immune systems or suppressed immune systems.
  • induced expression refers to expression under conditions such as when T cells bind to an antigen.
  • T cells bind to an antigen.
  • One skilled in the art how to perform conventional "induced expression”.
  • the invention provides an immune response cell that expresses an antigen binding receptor and an exogenous type I interferon.
  • an immune response cell of the invention can target an antigen expressed on a cancer. Its antigen or epitope can be expressed on cancer or cancer related tissues. In some cases, the target antigen may be overexpressed on cancer and have reduced or no expression on normal tissues. In some cases, cancer-specific antigens and epitopes thereof can be targeted using the immune response cells of the invention. Antigens can be derived from a wide variety of tumor antigens, such as tumor antigens produced by mutations, shared tumor-specific antigens, differentiation antigens, and antigens that are overexpressed in tumors.
  • the antigens that can be targeted or bound by the immune response cells of the present invention may be or are derived from, by way of example only, including, but not limited to, folate receptor alpha, 707-AP, adipophilin, AFP, AIM-2, ALDH1A1.
  • Tumor-associated antigens may be antigens that are not normally expressed by the host, which may be mutated, truncated, misfolded or otherwise abnormally expressed by the host; they may be identical to the normally expressed molecules but expressed at abnormally high levels; or they may Expressed in an abnormal environment.
  • the tumor associated antigen can be, for example, a protein or protein fragment, a complex carbohydrate, a ganglioside, a hapten, a nucleic acid, other biomolecules, or any combination thereof.
  • the antigen can be a neo-antigen.
  • the new antigen can be derived from somatic mutations in cancer cells.
  • the new antigen can be a mutant form of triphosphate isomerase (TPI).
  • Mutated fibronectin (FN) is another example of a novel antigen that can be targeted by the immune response cells of the invention.
  • the new antigen can be identified by a screening platform, such as biochemistry, whole-out cleavage sequencing, genetically targeted expression (GTE), or a combination thereof.
  • a target that can be bound by an immune response cell of the invention may be associated with a cancer stroma.
  • the cancer stroma may be associated with the tumor microenvironment.
  • the antigen can be a matrix antigen.
  • matrix antigens and epitopes can be present on, but not limited to, tumor endothelial cells, tumor vasculature, tumor fibroblasts, pericancetes, tumor stroma, and/or tumor mesenchymal cells.
  • Those antigens may, for example, be selected from the group consisting of CD34, MCSP, FAP, CD31, PCNA, CD117, CD40, MMP4 and/or tenascin.
  • Tissue expression of the antigen can be measured by immunohistochemistry (IHC) analysis and/or flow cytometry. Tissue expression can also be measured by quantitative copy number obtained by PCT (qPCR).
  • the target antigen can be expressed on the surface of cancer cells.
  • antigens that can be targeted may not be expressed in the context of MHC or HLA.
  • the immune response cells of the invention can be targeted in a non-MHC restricted manner Cell surface antigen.
  • antigens that can be targeted with CAR-T may be overexpressed as compared to expression on normal tissues.
  • Overexpression can be about 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold expression on normal tissues as measured by IHC, qPCR or flow cytometry. 10, 20, 30, 40, 50, 60, 70, 80, 90 or up to 100 times.
  • the antigen binding receptor comprises an antigen binding domain (extracellular binding region) and an intracellular signal domain capable of activating immune response cells.
  • a transmembrane region is also included between the antigen binding domain and the intracellular signal domain (intracellular signal region).
  • the extracellular binding region comprises an antibody to an antigen that is a tumor antigen or a pathogen antigen. Expression of the antigen-binding receptor on the surface of the immune response cell allows the immune response cell to have a highly specific cytotoxic effect on the tumor cell or pathogen expressing the antigen.
  • the antigen-binding receptor of the present invention comprises an antibody that is a single-chain antibody that is linked to a transmembrane region, and a transmembrane region that is immediately followed by an intracellular signal region.
  • an antigen binding domain of the invention is a binding domain that binds to a tumor antigen.
  • the tumor antigen is a differentiation antigen selected from the group consisting of MART-1/MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-center antigen
  • MART-1/MelanA MART-I
  • gp100 Pmel17
  • TRP-1, TRP-2 tumor-specific multi-center antigen
  • MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15 overexpressed embryonic antigens such as CEA
  • overexpressed oncogenes and mutant tumor suppressor genes such as p53, Ras, HER-2/neu Unique tumor antigens caused by chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR
  • viral antigens such as
  • the tumor antigen is selected from the group consisting of prostate specific membrane antigen (PSMA), carcinoembryonic antigen (CEA), IL13Ralpha, HER-2, CD19, NY-ESO-1, HIV-1 Gag, Lewis Y, MART -1, gp100, tyrosinase, WT-I, hTERT, mesothelin, EGFR, EGFRvIII, phosphatidylinositol 3, EphA2, HER3, EpCAM, MUC1, MUC16, CLDN18.2, folate receptor , CLDN6, CD30, CD138, ASGPR1 CDH16, GD2, 5T4, 8H9, ⁇ v ⁇ 6 integrin, B cell mature antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD20, CD22, kappa light chain, CD33, CD38, CD44 , CD44v6, CD44v7/8, CD70, CD123, CD17
  • PSMA
  • the tumor antigen is selected from the group consisting of prostate specific membrane antigen, carcinoembryonic antigen, IL13Ralpha, HER-2, CD19, NY-ESO-1, HIV-1 Gag, Lewis Y, MART-1, gp100, cheese Lysinase, WT-I, hTERT, mesothelin, EGFR, EGFRvIII, phosphatidylinositol 3, EphA2, HER3, EpCAM, MUC1, MUC16, claudin 18.2, folate receptor, claudin 6, CD30, CD138 One or more of MAGE3, ASGPR1 and CDH16.
  • the transmembrane region of the antigen binding receptor can be selected from a transmembrane region of a protein such as CD8 or CD28.
  • the human CD8 protein is a heterodimer composed of two chains, ⁇ or ⁇ .
  • the transmembrane region is selected from the transmembrane region of CD8 alpha or CD28.
  • the CD8 ⁇ hinge region is a flexible region, and therefore, CD8 or CD28 and a transmembrane region plus a hinge region are used to link the target recognition domain scFv of the antigen-binding receptor CAR to the intracellular signal region. .
  • the intracellular signal domain of the invention may be selected from the group consisting of CD3 ⁇ , Fc ⁇ RI ⁇ , CD28 costimulatory signal domain, CD137 costimulatory signal domain, and combinations thereof.
  • the CD3 molecule consists of five subunits, of which the CD3 ⁇ subunit (also known as CD3zeta, abbreviated as Z) contains three ITAM motifs, which are important signal transduction regions in the TCR-CD3 complex.
  • CD28 and CD137 are costimulatory signaling molecules, and the costimulation of their intracellular signal segments after binding to their respective ligands causes sustained proliferation of immune response cells (mainly T lymphocytes), and It can increase the level of cytokines such as IL-2 and IFN- ⁇ secreted by immune response cells, and improve the survival cycle and anti-tumor effect of CAR immune response cells in vivo.
  • the intracellular signal transduction domain is a combination of a CD3 ⁇ signal domain or a CD3 ⁇ signal domain with other costimulatory signals such as CD28.
  • an expression construct can be included in an immune response cell of the invention, wherein the expression construct has elements that are sequentially linked as follows: antibody, CD28 costimulatory signal domain, CD3 ⁇ , and in opposition to the aforementioned elements Linked NFAT6, type I interferon expression unit.
  • the antibody and the CD28 costimulatory signal domain are joined by a CD8 alpha transmembrane region and a CD8 alpha hinge region.
  • the activated T cell nuclear factor of NFAT (Nuclear factor of activated T cells) Transcriptional expression of cytokines plays an important role in T cell activation.
  • the inventors placed the IFN-beta coding sequence under the regulation of the NFAT6 promoter, so that IFN-beta can be expressed at a high level only when the CAR-T cells contact the antigen to induce T cell activation.
  • the NFAT6 promoter is a promoter composed of a combination of six NFAT binding positions and a minimal promoter of IL2 (Hooijberg E, Bakker AQ, Ruizendaal JJ, Spits H. NFAT-controlled expression of GFP permits visualization and Isolation of antigen-stimulated primary human Tcells. Blood. 2000 Jul 15; 96(2): 459-66), which can be used to regulate the expression of cytokines such as IL12 in T lymphocytes such as TCR-T (Zhang L, Kerkar SP, Yu Z, Zheng Z, Yang S, Restifo NP, Rosenberg SA, Morgan RA. Immunity adoptive T cell therapy by targeting and controlling IL-12 expression to the tumor environment. Mol Ther. 2011 Apr; 19(4): 751-9).
  • the invention also encompasses a nucleic acid encoding the antigen-binding receptor.
  • the invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the invention.
  • the present invention also provides a vector comprising the above nucleic acid encoding a receptor protein that binds to an antigen expressed on the surface of an immune response cell.
  • the vector used in the present invention is a lentiviral plasmid vector pRRLSIN-cPPT.PGK-GFP.WPRE. It should be understood that other types of viral vectors as well as non-viral vectors are also applicable.
  • the invention also includes viruses comprising the vectors described above.
  • the virus of the present invention includes a packaged infectious virus, and also includes a virus to be packaged containing components necessary for packaging as an infectious virus.
  • Other viruses known in the art that can be used to transduce foreign genes into immune response cells and their corresponding plasmid vectors can also be used in the present invention.
  • the immune response cell of the present invention is transduced with a construct capable of expressing an antigen-binding receptor and an exogenous type I interferon, or an expression vector, or a virus comprising the plasmid.
  • a construct capable of expressing an antigen-binding receptor and an exogenous type I interferon or an expression vector, or a virus comprising the plasmid.
  • Conventional nucleic acid transduction methods including non-viral and viral transduction methods, can be used in the present invention.
  • the immune response cell of the present invention may further carry a coding sequence of a foreign cytokine; the cytokine includes, but not limited to, IL-12, IL-15 or IL-21 and the like.
  • cytokine includes, but not limited to, IL-12, IL-15 or IL-21 and the like.
  • These cytokines have further immunomodulatory or anti-tumor activity, enhance the function of effector T cells and activated NK cells, or directly exert anti-tumor effects.
  • cytokines will help the immune response cells to function better.
  • the immune response cell of the present invention can also express another antigen in addition to the antigen-binding receptor described above.
  • An antigen-binding receptor An antigen-binding receptor.
  • the immune response cells of the invention may also express a chemokine receptor; the chemokine receptors include, but are not limited to, CCR2. Those skilled in the art will appreciate that the CCR2 chemokine receptors may allow CCR2 binding in vivo to compete with it, which is advantageous for blocking tumor metastasis.
  • the immune response cells of the present invention can also express siRNA that reduces PD-1 expression or a protein that blocks PD-L1.
  • siRNA that reduces PD-1 expression
  • the immune response cells of the present invention may also express a safety switch; preferably, the safety switch comprises: iCaspase-9, Truncated EGFR or RQR8.
  • the immune response cells of the invention do not express a costimulatory ligand such as 4-1BBL.
  • a transgene encoding a receptor or a CAR that binds to an antigen can be incorporated into the cell.
  • a transgene can be incorporated into an immune response cell, such as a T cell.
  • the transgene can be a complementary DNA (cDNA) fragment that is a copy of messenger RNA (mRNA); or the gene itself (with or without introns) located in the original region of its genomic DNA.
  • cDNA complementary DNA
  • mRNA messenger RNA
  • a nucleic acid encoding a transgene sequence, such as DNA can be randomly inserted into the chromosome of the cell. Random integration can be produced by any method that introduces a nucleic acid, such as DNA, into a cell.
  • the method can include, but is not limited to, electroporation, ultrasound, use of a gene gun, lipofection, calcium phosphate transfection, use of dendrimers, microinjection, and use of viruses including adenovirus, AAV, and retroviral vectors.
  • Vector, and/or type II ribozyme can be produced by any method that introduces a nucleic acid, such as DNA, into a cell.
  • the method can include, but is not limited to, electroporation, ultrasound, use of a gene gun, lipofection, calcium phosphate transfection, use of dendrimers, microinjection, and use of viruses including adenovirus, AAV, and retroviral vectors.
  • Vector, and/or type II ribozyme
  • the DNA encoding the transgene can also be designed to include a reporter gene such that the presence of the transgene or its expression product can be detected by activation of the reporter gene. Any reporter gene can be used, such as those described above.
  • the cells containing the transgene can be selected by selecting cells in the cell culture in which the reporter gene has been activated.
  • Expression of CAR can be verified by expression assays such as qPCR or by measuring the level of RNA.
  • the level of expression can also indicate the number of copies. For example, if the level of expression is very high, this may indicate that more than one copy of the CAR is integrated into the genome. Alternatively, high expression may indicate that the transgene is integrated in a high transcribed region, such as near a highly expressed promoter. Expression can also be verified by measuring protein levels, for example by Western blotting.
  • an immune response cell of the invention may comprise one or more transgenes.
  • the one or more transgenes can express a CAR protein that recognizes and binds to at least one epitope on the antigen or binds to a mutant epitope on the antigen.
  • CAR can be a functional CAR.
  • the immune response cells of the invention may comprise one or more CARs, or they may comprise a single CAR and two Secondary engineered receptors.
  • the transgene can encode a suicide gene.
  • CAR immune response cells cause tumor regression but can be associated with toxicity.
  • the target antigen when the target antigen is shared in normal tissues and tumor cells, the CAR immune response cells may not be able to distinguish between tumors and normal tissues ("target/off-target toxicity").
  • a systemic disturbance of the immune system called cytokine release syndrome (CRS)
  • CRS may comprise a systemic inflammatory response syndrome or a cytokine storm, which may be a consequence of rapid expansion of the CAR immune response cells in vivo.
  • CRS is a condition characterized by fever and hypotension, which can lead to multiple organ failure.
  • the toxicity is associated with in vivo expansion of infused CAR immune response cells, which can cause an overall disturbance of the immune system, as well as release high levels of pro-inflammatory cytokines such as TNF[alpha] and IL-6.
  • Suicide genes can induce the elimination of CAR immunoreactive cells.
  • the suicide gene may be any gene that induces apoptosis in the CAR immunoreactive cells.
  • a suicide gene can be encoded in the viral vector together with the antigen-binding receptor. The coding of the suicide gene allows for the mitigation or complete abortion of the toxicity caused by in vivo expansion of the infused CAR immune response cells under specific conditions.
  • CAR immunoreactive cells that are present in antigens of normal tissues can be produced such that they transiently express CAR, eg, after electroporating the mRNA encoding the receptor.
  • a major effort to further strengthen CAR immunoreactive cells by including a safety switch can substantially eliminate CAR immunoreactive cells in the case of severe target toxicity.
  • the vector encoding CAR can be associated with, for example, an inducible caspase-9 gene (activated by a dimeric chemical inducer) or a truncated form of EGF receptor R (activated by the monoclonal antibody cetuximab) or RQR8. Safety switch combination.
  • transgenes used herein may be from different species.
  • one or more of the transgenes can comprise a human gene, a mouse gene, a rat gene, a porcine gene, a bovine gene, a dog gene, a cat gene, a monkey gene, a chimpanzee gene, or any combination thereof.
  • a transgene can be from a human having a human genetic sequence.
  • One or more transgenes may comprise a human gene. In some cases, one or more of the transgenes are not adenoviral genes.
  • the transgene can be inserted into the genome of the immunoreactive cell in a random or site-specific manner.
  • a transgene can be inserted into a random site in the genome of an immune cell.
  • These transgenes can be functional, for example, fully functional when inserted into any part of the genome.
  • a transgene can encode its own promoter or can be inserted into a position controlled by its internal promoter.
  • the transgene can be inserted into a gene, such as an intron of a gene or an exon, promoter or non-coding region of a gene.
  • a transgene can be inserted to insert a disruptive gene, such as an endogenous immune checkpoint.
  • more than one copy of the transgene can be inserted into multiple random sites within the genome. For example, multiple copies can be inserted into random sites in the genome. This may result in an increase in overall expression compared to random insertion of the transgene once.
  • a copy of the transgene can be inserted into the gene and another copy of the transgene can be inserted into a different gene.
  • the transgene can be targeted such that it can be inserted into a specific site in the genome of the immunoreactive cell.
  • a polynucleic acid comprising a receptor sequence encoding an antigen binding agent can take the form of a plasmid vector.
  • the plasmid vector may comprise a promoter. In some cases, the promoter can be constitutive. In some embodiments, the promoter is inducible. The promoter may be or may be derived from CMV, U6, MND or EF1a. In some embodiments, the promoter can be adjacent to the CAR sequence. In some embodiments, the plasmid vector further comprises a splice acceptor. In some embodiments, the splice acceptor can be adjacent to the CAR sequence.
  • the promoter sequence can be a PKG or MND promoter.
  • the MND promoter may be a synthetic promoter of the U3 region of the MoMuLV LTR modified with myeloproliferative sarcoma virus enhancer.
  • a polynucleic acid encoding a receptor of interest can be designed to be delivered to a cell by non-viral techniques.
  • the polynucleic acid can be a Good Manufacturing Practice (GMP) compatible reagent.
  • GMP Good Manufacturing Practice
  • Promoters can be ubiquitous, constitutive (unrestricted promoters, allowing for continuous transcription of related genes), tissue-specific promoters or inducible promoters. Expression of a transgene inserted adjacent to or proximate to the promoter can be modulated. For example, a transgene can be inserted near or beside a ubiquitous promoter.
  • Some ubiquitous promoters may be the CAGGS promoter, the hCMV promoter, the PGK promoter, the SV40 promoter or the ROSA26 promoter.
  • Promoters can be endogenous or exogenous.
  • one or more transgenes can be inserted adjacent to or proximate to the endogenous or exogenous ROSA26 promoter.
  • the promoter may be specific for immunoreactive cells.
  • one or more transgenes can be inserted adjacent to or proximate to the porcine ROSA26 promoter.
  • Tissue-specific promoters or cell-specific promoters can be used to control the location of expression.
  • one or more transgenes can be inserted into proximity or proximity of a tissue-specific promoter.
  • Tissue-specific promoters may be FABP promoter, Lck promoter, CamKII promoter, CD19 promoter, keratin promoter, albumin promoter, aP2 promoter, insulin promoter, MCK promoter, MyHC promoter, WAP Promoter, or Col2A promoter.
  • Inducible promoters can also be used. These inducible promoters can be turned on and off by adding or removing an inducer if necessary.
  • the inducible promoter is contemplated to be, but not limited to, Lac, tac, trc, trp, araBAD, phoA, recA, proU, cst-1, tetA, cadA, nar, PL, cspA, T7, VHB, Mx, and/or Trex.
  • inducible promoter is a controlled promoter which does not express or underexpress a gene operably linked thereto before the desired condition is reached, and is achieved under the expected conditions.
  • a gene that is operably linked to it is expressed or expressed at a high level.
  • an inducible promoter of the present application does not express or underexpress a gene operably linked thereto under normal or high oxygen content conditions in a cell, and in response to a reduced oxygen content in the cell, A gene that is operably linked thereto is expressed or overexpressed under hypoxic conditions.
  • an inducible promoter for use herein includes Hypoxia-Inducible Transcription factor-1 ⁇ (HIF-1 ⁇ ).
  • the term "inducible promoter” as used herein refers to an "immune cell-inducible promoter” that does not express or underexpresses an immune response cell prior to its exposure to the antigen or when the immune response cell is not activated.
  • the "immune cell-inducible promoter” comprises a NFAT (activated T cell nuclear factor) type promoter.
  • NFAT-type promoter refers to a class of promoters that regulate the expression of a gene to which they are operably linked based on NFAT binding activity.
  • NFAT is a general term for a family of transcription factors that play an important role in immune responses. One or more members of the NFAT family are expressed in most cells of the immune system. NFAT is also involved in the development of the heart, skeletal muscle and nervous system.
  • the NFAT transcription factor family consists of five members, NFAT1, NFAT2, NFAT3, NFAT4, and NFAT5.
  • NFAT1 to NFAT4 are regulated by calcium signals.
  • Calcium signaling is critical for NFAT activation because calmodulin (CaM) activates serine/threonine phosphatase calcineurin (CN).
  • CaM calmodulin
  • CN serine/threonine phosphatase calcineurin
  • Activated CN rapidly dephosphorylates the amino-terminal serine-rich region (SRR) and SP repeats of the NFAT protein, resulting in a conformational change that exposes nuclear localization signals, resulting in NFAT input into the nucleus.
  • NFAT in the transcriptional expression of cytokines during T cell activation, which can be used to modulate the immune cell-inducible promoters described herein, thereby expressing or expressing high levels of expression when the immune response cells are exposed to antigen activation.
  • a nucleic acid of the invention may comprise any suitable nucleotide sequence encoding a NFAT type promoter (or a functional part or a functional variant thereof).
  • NFAT-type promoter refers to one or more NFAT response elements linked to the minimal promoter of any gene expressed by a T cell.
  • the minimal promoter of the gene expressed by T cells is the smallest human IL-2 promoter.
  • the NFAT response element can include, for example, NFAT1, NFAT2, NFAT3, and/or NFAT4 response elements.
  • more than one NFAT binding motif can be included in a "NFAT-type promoter" as described herein.
  • the "NFAT-type promoter” can include 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NFAT binding motifs.
  • the "NFAT-type promoter” includes up to 12 NFAT binding motifs.
  • the "NFAT-type promoter” can be a promoter consisting of a plurality of the NFAT-binding motifs in series with a promoter, such as the IL2 minimal promoter.
  • the NFAT type promoters described herein comprise six NFAT binding motifs, designated (NFAT) 6 .
  • the (NFAT) 6 is also referred to as NFAT6.
  • the NFAT6 also represents a 6-repeat NFAT binding motif (SEQ ID NO: 78) in the NFAT-type promoter.
  • the transgenic sequences may also include transcriptional or translational regulatory sequences, such as promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals.
  • transcriptional or translational regulatory sequences such as promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals.
  • the transgene encodes a receptor or CAR that binds to the antigen, wherein the transgene is inserted into a safe harbor such that the antigen-binding receptor is expressed.
  • the transgene is inserted into the PD1 and/or CTLA-4 locus.
  • the transgene is delivered as a lentivirus to the cells for random insertion, while a PD1- or CTLA-4 specific nuclease can be provided as mRNA.
  • the transgene is delivered by a viral vector system such as retrovirus, AAV or adenovirus, and mRNA encoding a nuclease specific for safe harbor (eg, AAVS1, CCR5, albumin, or HPRT). Cells can also be treated with mRNA encoding PD1 and/or CTLA-4 specific nucleases.
  • the polynucleotide encoding the CAR is provided by a viral delivery system with an mRNA encoding a HPRT-specific nuclease and a PD1- or CTLA-4 specific nuclease.
  • CARs that can be used with the methods and compositions disclosed herein can include all types of these chimeric proteins, including the first, second, and third generation designs previously described herein.
  • a transgene can be introduced into an immunoreactive cell using a retroviral vector (gamma-retroviral or lentiviral vector).
  • a transgene encoding a CAR or any receptor that binds an antigen, or a variant or fragment thereof can be cloned into a retroviral vector and can be derived from an endogenous promoter, a retroviral long terminal repeat, or a target Cell type-specific promoter drive.
  • Non-viral vectors can also be used.
  • Non-viral vector delivery systems can include DNA plasmids, naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes or poloxamers.
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • a vector derived from a retrovirus such as a lentivirus is A suitable tool for achieving long-term gene transfer because they allow long-term stable integration of the transgene and its propagation in daughter cells.
  • Lentiviral vectors have the added advantage over vectors derived from retroviruses such as murine leukemia virus because they can transduce non-proliferating cells. They also have the added advantage of low immunogenicity.
  • An advantage of adenoviral vectors is that they do not fuse into the genome of the target cell, thereby bypassing negative integration-related events.
  • the cells can be transfected with a transgene encoding the antigen-binding receptor.
  • the transgenic concentration can range from about 100 picograms to about 50 micrograms.
  • the amount of nucleic acid (eg, ssDNA, dsDNA, or RNA) introduced into the cell can be altered to optimize transfection efficiency and/or cell viability. For example, 1 microgram of dsDNA can be added to each cell sample for electroporation.
  • the amount of nucleic acid (eg, double stranded DNA) required for optimal transfection efficiency and/or cell viability varies depending on the cell type.
  • the amount of nucleic acid (eg, dsDNA) used for each sample can directly correspond to transfection efficiency and/or cell viability. For example, a range of transfection concentrations.
  • the transgene encoded by the vector can be integrated into the genome of the cell. In some embodiments, the transgene encoded by the vector is forward integrated. In other cases, the reverse integration of the transgene encoded by the vector.
  • the immunoreactive cell can be a dry memory consisting of CD45RO(-), CCR7(+), CD45RA(+), CD62L+ (L-selectin), CD27+, CD28+, and/or IL-7R ⁇ + T SCM cells, which also express CD95, IL-2R ⁇ , CXCR3, and/or LFA-1, and exhibit many different functional properties from the stem memory cells.
  • the immunoreactive cells may also be central memory T CM cells comprising L-selectin and CCR7, wherein the central memory cells may secrete, for example, IL-2 but not IFNy or IL-4.
  • the immunoreactive cells may also be effector memory T EM cells comprising L-selectin or CCR7 and produce, for example, effector cytokines such as IFNy and IL-4.
  • the vector by administration to an individual patient is typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial infusion) or topical application, as described below.
  • the vector can be delivered ex vivo to the cells, such as cells removed from an individual patient (eg, lymphocytes, T cells, bone marrow aspirate, tissue biopsy), and then typically after re-selecting the cells into which the vector is incorporated Implanted in a patient. Cells can be expanded before or after selection.
  • Suitable immunoreactive cells for expression of a receptor that binds to an antigen may be cells that are autologous or non-autologous to the individual in need thereof.
  • a suitable source of immune response cells can be obtained from the individual.
  • T cells can be obtained.
  • the T cells can be obtained from a number of sources, including PBMC, bone marrow, lymph node tissue, cord blood, thymus tissue, and tissues from infected sites, ascites, pleural effusion, spleen tissue, and tumors.
  • any number of known to those skilled in the art, such as separation Ficoll TM, from one body from the collected blood T cells were obtained.
  • cells from circulating blood of an individual are obtained by apheresis.
  • Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • cells collected by apheresis collection can be washed to remove plasma fractions and placed in a suitable buffer or medium for subsequent processing steps.
  • cells can be derived from a healthy donor, from a patient diagnosed with cancer, or a patient diagnosed with an infection.
  • the cells can be part of a mixed cell population with different phenotypic characteristics.
  • Cell lines can also be obtained from transformed T cells according to the methods previously described.
  • Cells can also be obtained from a cell therapy library.
  • Modified cells that are resistant to immunosuppressive therapy can be obtained by any of the methods described herein. It is also possible to select a suitable cell population prior to modification.
  • the engineered cell population can also be selected after modification.
  • Engineered cells can be used for autologous transplantation.
  • the cells can be used for allogeneic transplantation.
  • the cells are administered to a sample for identification of the same patient of a cancer associated target sequence. In other instances, the cells are administered to a patient different from the patient whose sample is used to identify the cancer-related target sequence.
  • suitable primary cells include peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL), and other blood cell subpopulations such as, but not limited to, T cells, natural killer cells, monocytes, Natural killer T cells, monocyte precursor cells, hematopoietic stem cells or non-pluripotent stem cells.
  • the cell can be any immune cell, including any T cell such as a tumor infiltrating cell (TIL), such as a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, or any other type of T cell.
  • T cells can also include memory T cells, memory stem T cells, or effector T cells.
  • T cells can also be expanded from a large population.
  • T cells may also be inclined to specific populations and phenotypes.
  • a T cell can be tilted to a phenotype comprising CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+), and/or IL-7R ⁇ (+).
  • Suitable cells may be selected from one or more of the following list: CD45RO (-), CCR7 (+), CD45RA (+), CD62L (+), CD27 (+), CD28 (+) and/or IL-7R ⁇ (+).
  • Suitable cells also include stem cells such as, for example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, and mesenchymal stem cells.
  • stem cells such as, for example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, and mesenchymal stem cells.
  • Suitable cells can comprise any number of primary cells, such as human cells, non-human cells, and/or mouse cells.
  • Suitable cells can be progenitor cells.
  • Suitable cells can be derived from a subject (eg, a patient) to be treated.
  • the amount of therapeutically effective cells required in a patient can vary depending on the viability of the cells and the efficiency with which the cells are genetically modified (eg, the efficiency with which the transgene is integrated into one or more cells, or the level of expression of the protein encoded by the transgene) ).
  • the product (eg, doubling) of the cell viability after genetic modification and the efficiency of transgene integration can correspond to a therapeutic amount of cells available for administration to a subject.
  • an increase in cell viability after genetic modification may correspond to a reduction in the amount of essential cells effective to administer the treatment to the patient.
  • an increase in the efficiency of integration of the transgene into one or more cells can correspond to a reduction in the number of cells necessary to administer a therapeutically effective in a patient.
  • determining the amount of therapeutically effective cells required can include determining a function associated with changes in cells over time.
  • determining the amount of cells that are required to be therapeutically effective can include determining a function corresponding to a change in efficiency of integrating the transgene into one or more cells according to a time-dependent variable (eg, cell culture time, electroporation time, Cell stimulation time).
  • the therapeutically effective cell can be a population of cells comprising about 30% to about 100% of the expression of a receptor that binds to the antigen on the surface of the cell.
  • the therapeutically effective cells can express about 30%, 35%, 40%, 45%, 50%, 55%, 60 of the antigen-binding receptor on the cell surface as measured by flow cytometry. %, 65%, 70%, 75% 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9 % or more than about 99.9%.
  • the antigen-binding receptor when the antigen-binding receptor is present on the plasma membrane of a cell, and when activated by binding to a target, it can result in a receptor having a binding antigen for its cell surface capable of binding The toxicity of the target cells.
  • the cells when cells are present in the plasma membrane of a cell, the cells can be cytotoxic cells (eg, NK cells or cytotoxic T lymphocytes), antigen-binding receptors described herein, and when It can increase the cytotoxic activity of cytotoxic cells against target cells when activated by binding to their targets.
  • an antigen-binding receptor described herein when activated by binding of its target, can increase cytotoxicity by at least 10 compared to cytotoxicity in the absence of cells that bind to the target. %, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 75%, at least 2 times, at least 2.5 times, at least 5 times, at least 10 times or more 10 times .
  • the immune response cells of the invention can be used to prepare pharmaceutical compositions.
  • the pharmaceutical composition may comprise a pharmaceutically acceptable carrier in addition to an effective amount of an immune response cell.
  • pharmaceutically acceptable means that when the molecular body and composition are suitably administered to an animal or a human, they do not produce an adverse, allergic or other untoward reaction.
  • sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof such as carboxymethyl fibers Sodium, ethyl cellulose and methyl cellulose; western yellow gum powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, Sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Wetting agents, such as sodium lauryl sulfate; colorants; flavoring agents; compressed tablets, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffers
  • composition of the present invention can be formulated into various dosage forms as needed, and can be administered by a physician in accordance with factors such as patient type, age, body weight, and general disease condition, mode of administration, and the like.
  • the mode of administration can be, for example, parenteral administration (e.g., injection) or other treatment.
  • parenteral administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) or intrasternal injection or infusion techniques.
  • Formulations comprising an immunoreactive cell population administered to an individual comprise a plurality of immunoreactive cells effective to treat and/or prevent a particular indication or disease.
  • a therapeutically effective population of immunoreactive cells can be administered to an individual.
  • administration contain from about 1 ⁇ 10 4 to about 1 ⁇ 10 10 cells reactive immunization formulation.
  • the formulation will contain from about 1 ⁇ 10 5 to about 1 ⁇ 10 9 cells reactive immunization, about 5 ⁇ 10 5 to about 5 ⁇ 10 8 cells reactive immunization, or from about 1 ⁇ 10 6 to About 1 ⁇ 10 7 immunoreactive cells.
  • the number of CAR immunoreactive cells administered to the individual will vary from wide range. The doctor will finalize the appropriate dose to use.
  • a chimeric antigen receptor is used to stimulate an immune cell mediated immune response.
  • a T cell mediated immune response is an immune response involving T cell activation.
  • Activated antigen-specific cytotoxic T cells are capable of inducing apoptosis in target cells that exhibit a foreign antigenic epitope on the surface, such as cancer cells that display tumor antigens.
  • a chimeric antigen receptor is used to provide anti-tumor immunity in a mammal. Subjects will develop anti-tumor immunity due to T cell-mediated immune responses.
  • a method of treating a subject having cancer can involve administering one or more immune response cells of the invention to a subject in need of treatment.
  • the immune response cell binds to a tumor target molecule and induces cancer cell death.
  • the invention also provides a method of treating a pathogen infection in an individual comprising administering to the individual a therapeutically effective amount of an immune response cell of the invention.
  • the frequency of administration of the immunoreactive cells of the present invention will depend on factors including the disease being treated, the elements of the particular immunoreactive cells, and the mode of administration. For example, it can be administered 4 times, 3 times, 2 times a day, once a day, every other day, every three days, every four days, every five days, every six days, once a week, once every eight days, every time. Dosing once every nine days, every ten days, once a week, or twice a month.
  • the immune response cells of the present application have improved viability, they can be administered not only in a therapeutically effective amount that is lower than an immune response cell that is similar but does not express exogenous type I interferon, and can Administration at a lower frequency to achieve at least a similar, and preferably more pronounced, effect.
  • an immune response cell of the invention can be administered in combination with another therapeutic agent.
  • the additional therapeutic agent is a chemotherapeutic agent.
  • Chemotherapeutic agents that can be used in conjunction with the immune response cells of the invention include, but are not limited to, mitotic inhibitors (vinca alkaloids), including vincristine, vinblastine, vindesine, and novibin (TM) (vinorelbine) , 5'-dehydro sulfide); topoisomerase I inhibitors such as camptothecin compound, including Camptosar TM (irinotecan HCL), Hycamtin TM (topotecan HCL) and derived from camptothecin Other compounds of its analogs; podophyllotoxin derivatives such as etoposide, teniposide and midozozoz; alkylating agents cisplatin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide , carmustine, busulfan,
  • chemotherapeutic agents that can be used in conjunction with the immune response cells of the invention include, but are not limited to, anti-angiogenic agents, including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligos) Nucleotide) and other angiogenesis inhibitors such as angiostatin, endostatin, interferon, interleukin-1 (including alpha and beta) interleukin 12, retinoic acid and metalloproteinase-1 and -2 tissue inhibition Agent.
  • anti-angiogenic agents including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligos) Nucleotide) and other angiogenesis inhibitors such as angiostatin, endostatin, interferon, interleukin-1 (including alpha and beta) interleukin 12, retinoic acid and metalloproteinase-1 and -2 tissue inhibition Agent.
  • the compositions may be isotonic, ie they may have the same osmotic pressure as blood and tears.
  • the desired isotonicity of the compositions of the present invention can be achieved using sodium chloride or other pharmaceutically acceptable agents such as glucose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes.
  • the viscosity of the composition can be maintained at a selected level using a pharmaceutically acceptable thickening agent.
  • Suitable thickeners include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like. The preferred concentration of thickener will depend on the reagent selected. It will be apparent that the choice of suitable carrier and other additives will depend on the exact route of administration and the nature of the particular formulation, such as a liquid dosage form.
  • kits comprising the immune response cells of the invention.
  • the kit can be used to treat or prevent cancer, pathogen infection, immune disorders or allogeneic transplantation.
  • a kit can include a therapeutic or prophylactic composition comprising an effective amount of an immune response cell comprising one or more unit dosage forms.
  • the kit comprises a sterile container that can contain a therapeutic or prophylactic composition; such a container can be a cartridge, ampule, bottle, vial, tube, bag, blister pack, or other suitable as is known in the art.
  • Container form Such containers may be made of plastic, glass, laminated paper, metal foil or other materials suitable for holding the drug.
  • immunoreactive cells such as CAR T cells
  • instructions for administering CAR immunoreactive cells to a subject at risk of developing a cancer, a pathogen infection, an immune disorder, or an allogeneic transplant will generally include information regarding the composition used to treat or prevent cancer, pathogen infection, immune disease, or allogeneic transplantation.
  • the kit can include from about 1 x 10 4 cells to about 1 x 10 6 cells.
  • the kit can include at least about 1 x 10 5 cells, at least about 1 x 10 6 cells, at least about 1 x 10 7 cells, at least about 4 x 10 7 cells, at least about 5 x 10 7 cells, at least about 6 ⁇ 10 7 cells, at least about 6 ⁇ 10 7 cells, 8 ⁇ 10 7 cells, at least about 9 ⁇ 10 7 cells, at least about 1 ⁇ 10 8 cells, at least about 2 x 108 cells, at least about 3 x 10 8 cells, at least about 4 x 10 8 cells, at least about 5 x 10 8 cells, at least about 6 x 10 8 cells, at least about 6 x 10 8 cells, At least about 8 x 10 8 cells, at least about 9 x 10 8 cells, at least about 1 x 10 9 cells, at least about 2 x 10 9 cells, at least about 3 x 10 9 cells, at least about 4 x 10 9 cells, at least about 5 ⁇ 10 9 cells, at least about 6 ⁇ 10 9 cells, at least about 8 ⁇ 10 9 cells, at least about 9
  • the kit can include allogeneic cells.
  • a kit can include cells that can include genomic modifications.
  • the kit can comprise "off the shelf" cells.
  • the kit can include cells that can be expanded for clinical use. In some cases, the kit may contain content for research purposes.
  • the instructions include at least one of: a description of a therapeutic agent; a dosage regimen and administration for treating or preventing a tumor, a pathogen infection, an immune disease, or an allograft or a symptom thereof; a preventive measure, a warning , contraindications, excessive information, adverse reactions, animal pharmacology, clinical studies, and/or citations.
  • Instructions can be printed directly on the container (if any), or as a label on the container, or as a separate paper, booklet, card or folder in the container or in the container.
  • the instructions provide methods of administering an immune response cell of the invention for treating or preventing a tumor, a pathogen infection, an immune disease, or an allograft or a symptom thereof.
  • the instructions provide methods of administering an immunoreactive cell of the invention before, after or simultaneously with the administration of a chemotherapeutic agent.
  • the invention also provides a method of treating a tumor or pathogen infection in an individual, Or a method for enhancing an individual's immune tolerance.
  • the methods comprise administering to an individual in need thereof an immune response cell of the invention, the immune cell expressing the antigen binding receptor and an exogenous type I interferon.
  • the methods comprise administering to the individual in need thereof an antigen binding receptor of the invention and an exogenous type I interferon.
  • the exogenous type I interferon is administered sequentially or simultaneously with the immune response cell expressing the antigen binding receptor.
  • the exogenous type I interferon is administered to a patient simultaneously with the immune response cell by co-expression in an immune response cell.
  • the present invention provides a method of increasing the viability of an immune response cell administered to an individual, wherein the immune response cell expresses an antigen binding receptor of the present invention, and wherein the method comprises administering to the individual
  • the immune response cell is also an effective amount of exogenous type I interferon.
  • the exogenous type I interferon is administered sequentially or simultaneously with the immune response cell expressing the antigen binding receptor.
  • the exogenous type I interferon is administered to a patient simultaneously with the immune response cell by co-expression in an immune response cell.
  • the exogenous type I interferon is not co-expressed with the exogenous type I interferon or the immune response cell.
  • the immune response cells of the invention can be administered at lower doses and/or lower frequencies.
  • the invention is administered to an individual in need thereof as compared to the case where the exogenous type I interferon is not administered or the exogenous type I interferon is not co-expressed
  • the amount of immune response cells is reduced by at least 10%, 20%, 30, 40, 50%, 60, 70%, 80% or 90%.
  • the invention is administered to an individual in need thereof as compared to the case where the exogenous type I interferon is not administered or the exogenous type I interferon is not co-expressed
  • the frequency of immune response cells is reduced by at least 10%, 20%, 30, 40, 50%, 60, 70%, 80%, or 90%.
  • the present invention is required to be administered to an individual in need thereof multiple times as compared with the case where the exogenous type I interferon is not administered or the exogenous type I interferon is not co-expressed.
  • the interval between each administration is extended by at least 10%, 20%, 30, 40, 50%, 60, 70%, 80%, 90%, 100%, 120%, 140%, 160 %, 180%, 200%, 500%, 750%, 1000%.
  • the methods of the invention result in cytotoxicity T in the peripheral blood of the individual after administration of the immune response cell to the individual compared to the absence of the exogenous type I interferon
  • the sum of the number of cells and helper T cells is increased by at least 10%, 20%, 30, 40, 50%, 60, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, 200%, 500%, 750%, 1000%.
  • the method results in the in vitro administration of the immune response cell to the individual about 5 days later
  • the sum of the number of cytotoxic T cells and helper T cells in the peripheral blood is greater than 5,000/ ⁇ L, 10,000/ ⁇ L, 15,000/ ⁇ L, 20,000/ ⁇ L, 25,000/ ⁇ L; about 7 days after administration of the immune response cells
  • the sum of the number of cytotoxic T cells and helper T cells in the peripheral blood is greater than 100/ ⁇ L, 200/ ⁇ L, 300/ ⁇ L, 400/ ⁇ L, 500/ ⁇ L, 600/ ⁇ L, 700 / ⁇ L, 800 / ⁇ L, 900 / ⁇ L, 1,000 / ⁇ L, 1,500 / ⁇ L, 2,000 / ⁇ L, 2,500 / ⁇ L, 3,000 / ⁇ L, 3,500 / ⁇ L, 4,000 / ⁇ L, 4,500 / ⁇ L, or 5,000 / ⁇ L; or about 10 days after administration of the immune response cells, the sum of the number of cyto
  • the invention also provides a method of modulating an immune response in an individual, the method comprising administering to the individual an effective amount of an immune response cell of any of the invention.
  • the invention also provides a method of enhancing immune tolerance in a subject, the method comprising administering to the individual an effective amount of an immune response cell of the invention comprising a receptor that binds to a tumor antigen and a vector encoding a type I interferon.
  • the method prevents or reduces autoimmune diseases or diseases associated with allografts.
  • the invention also provides a method of treating or preventing infection by a pathogen in a subject, the method comprising administering an effective amount of an immune response cell comprising a receptor that binds to a viral antigen and a vector encoding a type I interferon.
  • Autologous lymphocyte infusion can be used for treatment.
  • Autologous peripheral blood mononuclear cells PBMC
  • T cells can be activated and expanded using methods described herein and known in the art and then injected into a patient.
  • allogeneic cells can be used to treat a patient.
  • Transplantation can refer to adoptive transplantation of cellular products.
  • the transplant can be autograft, allogeneic, xenograft or any other transplant.
  • the transplant can be a xenograft.
  • Transplantation can also be allogeneic transplantation.
  • the subject can administer immunoreactive cells, wherein the immunoreactive cells that can be administered can be from about 1 to about 35 days of age.
  • the cells administered may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or up to about 40 days.
  • the age of CAR immunoreactive cells can be calculated from the time of stimulation.
  • the age of the immunoreactive cells can be calculated from the time of blood collection.
  • the age of the immunoreactive cells can be calculated from the time of transduction.
  • the immunoreactive cells that can be administered to the subject are from about 10 to about 14 or about 20 days of age.
  • the "age" of an immunoreactive cell can be determined by the telomere length.
  • a "young" immune response cell can have a longer telomere length than "depleted” or "old” immunoreactive cells.
  • immunoreactive cells lose an estimated telomere length of about 0.8 kb per week in culture, and young immunoreactive cell cultures can have telomeres that are about 1.4 kb longer than about 44 days of immunoreactive cells.
  • a longer telomere length can be associated with a positive objective clinical response in a patient and persistence of cells in vivo.
  • Cells can be functional before, after, and/or during transplantation.
  • the transplanted cells may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 after transplantation. 20, 21, 22, 23, 24, 25, 6, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90 or 100 days.
  • the transplanted cells can function at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months after transplantation.
  • the transplanted cells can function at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 years after transplantation.
  • the transplanted cells can function during the life of the recipient.
  • transplanted cells can function at 100% of their normal expected function.
  • the transplanted cells can also perform their normal expected functions of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, , 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 , 96, 97, 98, or up to about 100% of the functionality.
  • Transplanted cells can also perform more than 100% of their normal intended function.
  • the transplanted cells can function as approximately 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000 as normal expected functions. Or up to about 5,000% of the functionality.
  • Porting can be done by any type of transplant.
  • Topography may include, but is not limited to, subhepatic sac space, subsplenic sac space, subcapsular space, omentum, gastric or intestinal submucosa, small intestinal vascular segment, venous sac, testis, brain, spleen, or cornea.
  • the transplant can be a subcapsular transplant.
  • Transplantation can also be intramuscular transplantation.
  • the transplant can be a portal vein transplant.
  • transplant rejection can be improved after treatment with the immune response cells of the present invention as compared to when one or more wild type cells are transplanted to the recipient.
  • transplant rejection can be a hyperacute rejection.
  • Transplant rejection can also be an acute rejection.
  • Other types of rejection may include chronic rejection.
  • Transplant rejection can also be cell-mediated rejection or T cell-mediated rejection.
  • Transplant rejection can also be a natural killer cell mediated rejection.
  • Improving transplantation may mean alleviating hyperacute rejection, which may include reducing, reducing or reducing malnutrition Role or symptom.
  • Transplantation can refer to adoptive transplantation of cellular products.
  • Another indication of successful transplantation may be the number of days the recipient does not need immunosuppressive therapy.
  • the recipient may not require at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days of immunosuppressive therapy. This can indicate that the transplant was successful. This can also indicate that the transplanted cells, tissues and/or organs are not repelled.
  • the recipient does not require immunosuppressive therapy for at least 1 day.
  • the recipient may also not require immunosuppressive therapy for at least 7 days.
  • the recipient does not require immunosuppressive therapy for at least 14 days.
  • the recipient does not require immunosuppressive therapy for at least 21 days.
  • the recipient does not require immunosuppressive therapy for at least 28 days.
  • the recipient does not require immunosuppressive therapy for at least 60 days.
  • the recipient may not require at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years of immunosuppressive therapy.
  • Another sign of successful transplants may be the number of days that recipients need to reduce their immunosuppressive therapy. For example, after the treatment provided herein, the recipient may require at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days of reduced immunosuppressive therapy. This can indicate that the transplant was successful. This may also indicate that there is no or only minimal rejection of the transplanted cells, tissues and/or organs.
  • a recipient may require at least 1 day of reduced immunosuppressive therapy.
  • Recipients may also require at least 7 days of reduced immunosuppressive therapy.
  • the recipient may require at least 14 days of reduced immunosuppressive therapy.
  • Recipients require at least 21 days of reduced immunosuppressive therapy.
  • Recipients require at least 28 days of reduced immunosuppressive therapy.
  • Recipients require at least 60 days of reduced immunosuppressive therapy.
  • the recipient may require at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years of reduced immunosuppressive therapy.
  • Reduced immunosuppressive therapy can refer to less immunosuppressive therapy as compared to the immunosuppressive therapy required when transplanting one or more wild-type cells into a recipient.
  • Immunosuppressive therapy can include any treatment that inhibits the immune system. Immunosuppressive therapy can help alleviate, reduce or eliminate transplant rejection in patients.
  • immunosuppressants can be used before, during, and/or after transplantation, including MMF (Cellcept), ATG (anti-thymocyte globulin), anti-CD154 (CD4OL), anti-CD40 (2C10) , immunosuppressive drugs, anti-IL-6R antibodies (tocilizumab, Actemra), anti-IL-6 antibodies (sarilumab, olokizumab), CTLA4-Ig (Abatacept/Orencia), anti-IL-6 antibodies (ASKP1240, CCFZ533X2201) ), amphetamine (Campath), anti-CD20 (rituximab), bevacizumab (LEA29Y), sirolimus (Rapimune), everolimus, tacrolimus (Prograf), Zele-napax, Silimict, Remicade,
  • one or more immunosuppressive agents/drugs may be used together or sequentially.
  • One or more immunosuppressive agents/drugs can be used to induce therapy or to maintain treatment.
  • the same or different drugs can be used in the induction and maintenance phases.
  • daclizumab Zenapax
  • tacrolimus Prograf
  • sirolimus Rostune
  • Non-pharmacological regimens can also be used to achieve immunosuppression, including but not limited to whole body irradiation, thymic irradiation, and total and/or partial splenectomy. These techniques can also be used in combination with one or more immunosuppressive drugs.
  • the CD28 costimulatory signal domain is abbreviated as 28; CD3 ⁇ is abbreviated as Z; 4-1BB or CD137 is abbreviated as BB.
  • a chimeric antigen receptor constructed as an intracellular signal domain with an scFv code of 85-2 and a CD3 ⁇ and a CD28 costimulatory signal domain can be designated as 85-2-28Z. This is true for the construction of CARs for different antigens.
  • liver cancer cell lines SK-HEP-1 and PLC/PRF/5 were purchased from the ATCC cell bank, and Huh-7 was purchased from the Japanese RIKEN cell bank.
  • PBMC is from the Shanghai Blood Center.
  • AIM V medium CTS, Cat #1665773.
  • FCS Fetal bovine serum
  • IL-2 Shanghai Huaxin, recombinant human interleukin-2 for injection.
  • PE-Streptavidin BD pharmingen, Cat #554061.
  • CytoTox Non-radioactive cytotoxicity assay Promega, Cat# G1780.
  • the following vector system used to construct the lentiviral plasmid vector of the present invention belongs to the third generation auto-inactivated lentiviral vector system, which has four plasmids: a packaging plasmid pMDLg RRE encoding the protein Gag/Pol (purchased from addgene).
  • the packaging plasmid pRSV-REV (purchased from addgene) encoding the Rev protein
  • the envelope plasmid pCMV-VSV-G (purchased from addgene) encoding the VSV-G protein
  • the empty vector pRRLSIN-cPPT.PGK-GFP.WPRE A recombinant expression vector encoding the gene of interest, purchased from addgene, which is effective in reducing the risk of forming replicable lentiviral particles.
  • the inventors first modified the empty vector pRRLSIN-cPPT.PGK-GFP.WPRE by a conventional molecular cloning technique, and started with elongation factor-1 ⁇ (elongation factor-1 ⁇ , EF-1 ⁇ ).
  • the promoter replaces the promoter of the original vector and adds a MluI cleavage site between the promoter and the CD8 ⁇ sp signal peptide.
  • a ClaI/SalI (purchased from NEB) double-digested vector pWPT-EGFP (purchased from addgene) was used to recover a 1.1 kb DNA fragment, and ligated to the ClaI/SalI double-digested vector pRRLSIN-cPPT with T4 DNA ligase. .PGK-GFP.WPRE, and transformed into the host strain TOP10, picked clones, identified positive clones by colony PCR and confirmed by sequencing to obtain recombinant plasmid pRRLSIN-cPPT.EF-1 ⁇ -EGFP.WPRE.
  • a downstream primer 5'-GCGGTGTCCTCGCTCCGCAGGCTGCTCAGCTCCATGTAGGCGGTG-3' (SEQ ID NO: 2) amplifying a heavy chain variable region fragment; a plasmid comprising a fragment of the 92 light chain variable region (SEQ ID NO: 79 in patent 201510481235.1)
  • a light chain variable region fragment was amplified using the upstream primer 5'-GCGGAGCGAGGACACCGCCGTGTACTACTGCGCCCGGTTCTACAGCTAC-3' (SEQ ID NO: 3) and the downstream primer 5'-CGGCGCTGGCGTCGTGGTACGTTTGATCTCCAGTTTGGTG-3' (SEQ ID NO: 4).
  • the above heavy and light chain variable region primers were further amplified by a duplex PCR with a 92 scFv fragment (SEQ ID NO: 5) containing a repeat sequence with the upstream CD8 ⁇ signal peptide and the downstream hinge region, designated as fragment 1, 765 bp in size.
  • the PCR amplification conditions were pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 40 s; annealing: 58 ° C, 40 s; extension: 68 ° C, 40 s; 25 cycles, and then extended at 68 ° C for 10 min.
  • the PCR amplified bands were confirmed by agarose gel electrophoresis to match the expected fragment size.
  • the vector plasmid pRRLSIN-cPPT.EF- constructed in this example was used.
  • 1 ⁇ -EGFP.WPRE was used as a template to amplify the EF-1 ⁇ promoter (SEQ ID NO: 8) containing the CD8 ⁇ signal peptide (containing the MluI restriction site), and was named as fragment 2, and the size was 442 bp.
  • the PCR amplification conditions were pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 30 s; annealing: 53 ° C, 30 s; extension: 68 ° C, 30 s; 25 cycles, then total extension 68 ° C, 10 min.
  • the PCR amplified bands were confirmed by agarose gel electrophoresis to match the expected fragment size.
  • the upstream primer 5'-accacgacgccagcgccg-3' (SEQ ID NO: 9) and the downstream primer 5'-aatccagaggttgattgtcgacctagcgagggggcagggcctgc-3' (SEQ ID NO: 10) were used as pWPT-eGFP-F2A-GPC3-BBZ, pWPT-eGFP, respectively.
  • the PCR amplification conditions were pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 30 s; annealing: 60 ° C, 30 s; extension: 68 ° C, 30 s; 25 cycles, then total extension 68 ° C, 10 min.
  • the PCR amplified bands were confirmed by agarose gel electrophoresis to match the expected fragment size.
  • the equimolar amount of about 50 ng fragment 2, fragment 1 and fragment 3 were respectively subjected to splicing PCR.
  • the splicing conditions were: pre-denaturation 94 ° C, 4 min; denaturation: 94 ° C, 40 s; annealing: 60 ° C, 40 s; extension: 68 ° C, 140 s, 5 cycles, then total extension 68 ° C, 10 min, supplement DNA polymerase and upstream primer 5'-gcaggggaaagaatagtagaca-3' (SEQ ID NO: 6) and downstream primer 5'-aatccagaggttgattgtcgacctagcgagggggggggcctgc-3' (SEQ ID NO: 10 25 cycles of PCR amplification, pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 40 s; annealing: 60 ° C, 40 s; extension: 68 ° C, 140 s, total
  • the above vector plasmids pRRLSIN-cPPT.EF-1 ⁇ -EGFP.WPRE and fragments 92-BBZ, 92-28Z and 92-28BBZ were digested with restriction endonucleases Mlu I and SalI (purchased from NEB), respectively.
  • the T4 ligase purchased from NEB was ligated, transformed into TOP10, and cloned for PCR to identify positive bacteria, which were sent to Invitrogen for sequencing to confirm the correct sequence, thereby obtaining pRRL-EF-1 ⁇ -92-BBZ, pRRL-EF- 1 ⁇ -92-28Z and pRRL-EF-1 ⁇ -92-28BBZ.
  • the upstream primer 5'-gcaggggaaagaatagtagaca-3' (SEQ ID NO: 6) was used first. Fragment 6 was amplified by PCR by the downstream primer 5'-TCAGAAGGTCAAAATTCAAAGTCTGTTTCACGCGAGGGGGCAGGGCCTGCATGTGAA-3' (SEQ ID NO: 17).
  • plasmid HG15693-G (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., the 562th base of the 41BBL gene contains a mutation from G to A) as a template, respectively, using the upstream primer 5' -gagacgttgagtccaaccctgggcccatggaatacgcctctgacgc-3' (SEQ ID NO: 18) and the downstream primer 5'-TCGGAGGAGGCGGGTGGCAGGTCCACGGTC-3' (SEQ ID NO: 19), fragment 7 was amplified by PCR; using the upstream primer 5'-ctgccacccgcctcctcccgaggctcggaa-3' (SEQ ID NO: 20) and the downstream primer 5'-TGATTGTCGACTTATTCCGACCTCGGTGAAGGGA-3' (SEQ ID NO: 21), fragment 8 was amplified by PCR, and
  • equimolar fragments 6 and 9 were spliced and amplified with primer pairs (SEQ ID NO: 6 and SEQ ID NO: 21) to give 92-28Z-F2A-41BBL (SEQ ID NO: 22).
  • This fragment was digested with Mlu I and SalI, and inserted into the same digested vector pRRLSIN-cPPT.EF-1 ⁇ -EGFP.WPRE by the same method as above, and confirmed by sequencing to obtain plasmid pRRL-EF-1 ⁇ -92. -28Z-F2A-41BBL.
  • first primers SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35
  • Synthetic fragment 10 followed by pWPT-EGFP plasmid as template, upstream primer (SEQ ID NO: 36) and downstream primer (SEQ ID NO: 37) ), fragment 11 was amplified.
  • Fragments 10 and 11 were mixed equimolarly, by bridge PCR, and using primer pairs (SEQ ID NO: 35 and SEQ ID NO: 38) After amplification, the fragment was digested with ClaI and SalI, and inserted into the same digested vector pRRLSIN-cPPT-PGK-EGFP.WPRE by the same method as above, and confirmed by sequencing to obtain three NFATs.
  • fragment 14 was amplified by primer pair (SEQ ID NO: 43 and SEQ ID NO: 44) using the vector pGMT-IFN- ⁇ (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) as a template.
  • PCR amplification was performed using primer pairs (SEQ ID NO: 43 and SEQ ID NO: 38), and the amplified product was digested with Mlu I and Cla I and ligated to the same vector pRRLSIN-NFAT6- EGFP-PA2 was confirmed by sequencing to obtain the pRRLSIN-NFAT6-huIFN ⁇ -PA2 plasmid.
  • the EGFP fragment 16 carrying the NdeI restriction site was amplified by primer pair (SEQ ID NO: 45 and SEQ ID NO: 46);
  • a good plasmid pRRLSIN-NFAT6-EGFP-PA2 plasmid was used as a template and amplified by primer pair (SEQ ID NO: 47 and SEQ ID NO: 48) to obtain NFAT6 fragment 17 with NdeI restriction site (requires amplification of 6 Fragments of the unit, while eliminating the restriction site of SalI).
  • Fragments 16 and 17 were mixed equimolarly, amplified using primer pairs (SEQ ID NO: 45 and SEQ ID NO: 48), digested with EcoRI and KpnI, ligated into the same digested vector pRRLSIN-cPPT.EF In the -1 ⁇ -EGFP.WPRE, it was verified by sequencing that the plasmid pRRLSIN-EF1 ⁇ -EGFP-NFAT6-huIFN ⁇ -PA2 was obtained.
  • the plasmid pRRL-EF-1 ⁇ -92-28Z was digested with MluI and SalI to obtain a 92-28Z fragment, which was ligated into the same double-digested pRRLSIN-EF1 ⁇ -EGFP-NFAT6-huIFN ⁇ -PA2 vector.
  • the correct plasmid pRRLSIN-EF1 ⁇ -92-28Z-NFAT6-hu IFN ⁇ -PA2 was sequenced.
  • pRRL-EF-1 ⁇ -92-BBZ The above five plasmids pRRL-EF-1 ⁇ -92-BBZ, pRRL-EF-1 ⁇ -92-28Z, pRRL-EF-1 ⁇ -92-28BBZ, pRRL-EF-1 ⁇ -92-28Z-F2A-41BBL and pRRL- EF-1 ⁇ -92-28Z-NFAT6-huIFN ⁇ -PA2 is commonly referred to as pRRL-EF-1 ⁇ -92-CAR (Fig. 1).
  • amino acid sequences corresponding to 92-BBZ, 92-28Z, 92-28BBZ and 92-28Z-F2A-41BBL are SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, respectively.
  • the amino acid sequence of -28Z-NFAT6-IFN- ⁇ expression comprises two segments, respectively, a CAR constructed as 92-28Z as shown in SEQ ID NO: 50 and IFN as shown in SEQ ID NO: 53, the vector construction of which is Figure 1B shows.
  • 293T cells were seeded at a density of 4.5 ⁇ 10 6 in a 10 cm culture dish, and cultured at 37 ° C, 5% CO 2 overnight to prepare a packaged virus, the medium was containing DMEM, and 10% fetal calf serum was added;
  • the plasmid mixture is added to the PEI mixture, and immediately after the addition, vortex or gently mix, incubate for 20 min at room temperature;
  • the concentrated lentiviral suspension is divided into 50 ⁇ l portions, stored in the finished tube and stored at -80 °C; the monotropic retrovirus is unstable, and needs to be used as soon as possible after packaging. It is not recommended to freeze at -80 °C. .
  • 293T cells were seeded in a 12-well culture plate at a number of 1 ⁇ 10 5 cells;
  • the concentrated lentivirus was added to the cell suspension at 1 uL, 0.2 uL and 0.04 uL, respectively, and polybrene was added to a final concentration of 6 ug/mL;
  • the 293T cells were trypsinized, and after adding the same amount of medium, the cells were evenly blown, and the cell suspension was transferred into a 1.5 mL centrifuge tube;
  • T-lymphocyte activation from human PBMC Blood Center of Shanghai, medium was AIM V + 2% AB serum + IL-2 (500U / mL ) to adjust the density of PBMC 1 ⁇ 10 6 / mL, 1: 1 The ratio of anti-human CD3 and CD28 antibody-coated magnetic beads was activated for 48 h;
  • Retronectin coated 48-well plates 160 ⁇ l of retronectn solution (5 ⁇ g/mL) was added to each well and incubated overnight at 4 °C;
  • the target cells corresponding to 92-CAR are SK-HEP-1 (GPC3-) and Huh-7 (GPC3+);
  • Effector cells CAR-T cells and control T cells were added to 96-well plates at a target-to-target ratio of 0.3:1, 1:1, and 3:1;
  • Each experimental group each target cell + CTL expressing a different chimeric antigen receptor
  • Control group 1 maximum release of LDH from target cells
  • Control group 2 spontaneous release of LDH from target cells
  • Control group 3 effector cells spontaneously release LDH
  • CytoTox 96 non-radioactive cytotoxicity detection kit (Promega) was used. This method is based on the colorimetric detection method and can replace the 51 Cr release method. CytoTox The degree of cell lysis is reflected by detecting the amount of lactate dehydrogenase (LDH). LDH is a stable cytoplasmic enzyme that is released when cells are lysed and released in much the same way as 51 Cr is released in radioactive analysis. The released LDH medium supernatant can be detected by a 30 minute coupled enzyme reaction in which LDH converts a tetrazolium salt (INT) to red formazan. The amount of red product produced is directly proportional to the number of cells lysed. Refer specifically to the instructions for the CytoTox 96 non-radioactive cytotoxicity test kit.
  • LDH lactate dehydrogenase
  • the cytotoxicity calculation formula is:
  • the T cell (GPC3-CD28Z in Figure 2) expressing 92-28Z (SEQ ID NO: 15, encoding the nucleotide sequence as shown in SEQ ID NO: 57) had an infection efficiency of 35.1% and expressed 92-28Z-NFAT6-
  • the infection efficiency of IFN- ⁇ T cells (GPC3-CD28Z-IFN in Figure 2) was 19.2%, and the control vector MOCK infection efficiency was 49%, as shown in Fig. 2.
  • 92-CAR T after detection of infection positive rate was detected as T lymphocytes expressing 92-28Z-NFAT6-IFN- ⁇ , 92-28Z and empty vector MOCK according to the effective target ratio of 0.3:1, 1:1, 3:1.
  • the in vitro killing effect of liver cancer cell lines SK-HEP-1 (GPC3 - ) and Huh-7 (GPC3 + ) and PLC/PRF/5 (GPC3 + ) was detected after 18 hours of co-culture, and the content of LDH in the supernatant was detected.
  • the present inventors further integrated 92-28Z-NFAT6-IFN- ⁇ with other GPC3-BBZ, GPC3-28BBZ and GPC3-41BBL constructed by the same extracellular antigen binding unit 92 (SEQ ID NO: 5) (on a CD28Z basis). Expression of 4-1BBL) compared to CAR-T cells.
  • FACS detects the expression of various CARs (see Figure 3). The expression ratio of various CARs is about 40%.
  • Example 3 In vitro induction of cytokine release assay by GPC3 CAR-T cells containing IFN and no IFN
  • the cytokines released by untransfected T cells, 92-28Z T cells, and 92-28Z-IFN T cells were detected, respectively. Collect the above three T cells with good growth within 1-2 weeks after lentivirus infection, inoculate 5 ⁇ 10 4 /200 ⁇ L (positive cell number) in 24-well plates, and inoculate 5 ⁇ 10 4 /200 ⁇ L/24 wells in the same manner.
  • the huh7 cells were incubated with CAR T cells for 24 hours, and the supernatant was collected to measure the concentrations of IFN- ⁇ , IFN- ⁇ , and IL-2, and the results are shown in Figures 4A-4C.
  • FIG. 4A only GPC3-28Z-IFN T was incubated with Huh7 cells for IFN ⁇ expression, indicating that GPC3-28Z-IFN T cells were successfully induced to express and secreted outside the cell after being activated by the target antigen.
  • the results indicate that GPC3-28Z-IFN T cells can be more effective in a variety of GPC3-positive cells, such as Huh7, PLC ⁇ PRF ⁇ 5, Hep-3B and other cells. Is activated.
  • Example 4 In vitro induction of cytokine release assay by CLD18A2 CAR-T cells containing IFN and no IFN
  • the second generation chimeric antigen receptors 85-28Z (SEQ ID NO: 55) and 85-2-28Z (SEQ ID NO:) expressing antibodies 85 and 85-2 were constructed using PRRLSIN-cPPT.EF-1 ⁇ as a vector. 54) Lentiviral plasmid.
  • the 85-28Z sequence consists of the CD8 ⁇ signal peptide, 85scFV, CD8hinge, CD28 transmembrane and intracellular signaling domain regions, and the intracellular CD3 ⁇ of CD3; the sequence of 85-2-28Z is composed of CD8 ⁇ signal peptide, hu8E5-2IscFV, The CD8 hinge region, the CD28 transmembrane region and the intracellular signaling domain region, and the intracellular CD3 ⁇ of CD3 are composed.
  • 85-28Z-IFNb CAR (encoding nucleotide sequence as shown in SEQ ID NO: 58) expressing IFNb cytokine was constructed on the basis of 85-28Z and 85-2-28Z, at 85-2-28Z CAR Based on the construction of 85-2-28Z-IFNb CAR (encoding nucleotide sequence as shown in SEQ ID NO: 59) which can express IFNb cytokines.
  • Cytokines released from transfected T cells (Mock), 85-28Z T cells, and 85-28Z-IFN T cells were detected, respectively. Collect the above three T cells with good growth within 1-2 weeks after lentivirus infection, inoculate 5 ⁇ 104/200 ⁇ L (positive cell number) in 24-well plates, and inoculate 5 ⁇ 104/200 ⁇ L according to the effective target ratio of 1:1. /24 pore target cells.
  • Target cells include 293T-A1, 293T-A2, AGS, AGS-A2, BGC-823, and BGC-823-A2 cells.
  • the supernatant was collected after 24 hours of co-cultivation.
  • the IFN- ⁇ cytokine released during the co-culture of CAR T lymphocytes with target cells in the supernatant was detected by sandwich ELISA.
  • SK-HEP-1 is a GPC3-negative human hepatocellular carcinoma cell line
  • PLC/PRF/5 is a GPC3-positive human hepatocellular carcinoma cell line
  • HepG2 is a GPC3-positive human hepatocellular carcinoma cell line
  • Hep3B is a GPC3-positive person.
  • Hepatocellular carcinoma cell lines were purchased from the American Type Culture Collection (ATCC); Huh-7 (also known as Huh7) was a GPC3-positive human hepatocellular carcinoma cell line purchased from the Japanese RIKEN cell bank.
  • CytoTox 96 non-radioactive cytotoxicity test kit (Promega) was used for detection (specific method can refer to CytoTox 96 non-radioactive cytotoxicity test kit instructions), and detected CAR T lymphocytes to Huh 7, Hep 3B, PLC /PRF/5, Hep G2 and SK-HEP-1 liver cancer cells in vitro toxicity killing effect.
  • the untransfected T cells, 92-28Z T cells and 92-28Z-IFN T cells were co-cultured with tumor cells at a ratio of 1:3, 1:1 and 3:1 for 18 h.
  • the settings of each experimental group and each control group are as follows:
  • the experimental group was set up: each target cell + T lymphocytes expressing different chimeric antigen receptors;
  • Control group 1 spontaneous release of LDH from effector cells
  • Control group 2 spontaneous release of LDH from target cells
  • Control group 3 maximum LDH release from target cells
  • Control group 4 volume corrected control
  • Control group 5 medium background control.
  • 293T-A1 and 293T-A2 cells are human kidney epithelial cell lineages stably expressing CLD18A1 and CLD18A2 in vitro.
  • AGS and BGC-823 are human gastric cancer cell lines, and on this basis, AGS-A2 and BGC-823-A2 cell lines stably expressing CLD18A2 were constructed.
  • CytoTox 96 non-radioactive cytotoxicity test kit (Promega) was used for detection (refer to the CytoTox 96 non-radioactive cytotoxicity test kit for specific methods), and CAR T lymphocytes were detected for 293T-A1, 293T-A2. , AGS, AGS-A2, BGC-823, BGC-823-A2 cells in vitro toxicity killing effect.
  • effector cells spontaneous LDH release correcting the spontaneous release of LDH by effector cells
  • target cell spontaneous LDH release correcting the spontaneous release of LDH from target cells
  • volume correction control correct volume change due to the addition of lysate (10 ⁇ );
  • % cytotoxicity [(experimental group - effector cell control - target cell control) / (maximum amount of target cell lysis - target cell control)] x 100.
  • the effector cell control, the target cell control, and the experimental group lost the medium control; the target cell maximum lysis amount was subtracted from the volume control.
  • CAR-T GPC3-28ZT cells or GPC3-28Z-IFN T cells
  • CAR-T cells GPC3-28Z cells
  • T cells or GPC3-28Z-IFN T cells survive in vivo.
  • Table 4 The number of T cells (CD3+) and CAR-T cells per ⁇ l of peripheral blood in the GPC3-28Z-IFN T cell group were higher than those in the GPC3-28ZT cell group and the Mock group.
  • CD3+ (pieces / ⁇ L)
  • CAR-T (pieces / ⁇ L) Mock 193.1453 0
  • GPC3-28Z 375.802 232.2
  • GPC3-28Z-IFN 1034.315 439.5
  • Example 8 Determination of in vivo survival time of CLD18A2 CAR-T cells
  • the gastric cancer PDX tumor of about 2 ⁇ 2 ⁇ 2 mm was inoculated subcutaneously into the right axillary part of NOD/SCID mice, and the day of tumor cell inoculation was recorded as D0 days.
  • CAR-T cells 85-2-28Z T cells or 85-2-28Z-
  • IFN T cells IFN T cells
  • Peripheral blood was collected from mouse saphenous veins on D5, D7 and D10 after CAR-T infusion, respectively, and CAR-T cells (empty T cells (Mock), 85-2-28Z T cells or 85- were detected. Survival of 2-28Z-IFN T cells in vivo.
  • Example 9 In vivo killing activity of GPC3CAR-T (92-28Z) cells containing IFN and no IFN
  • Anti-tumor treatment experiments of Huh7 subcutaneous xenografts were performed on untransfected T cells (Mock), GPC3-28Z T cells and GPC3-28Z-IFN T cells.
  • Huh7 cells in a logarithmic growth phase and in good growth state were collected and adjusted to a density of 1 ⁇ 10 7 /mL using physiological saline, and a mouse model was made by inoculating NOD-SCID mice, and the injection volume was about 200 ⁇ L. (2 ⁇ 10 6 /only), the day of tumor cell inoculation is the 0th day.
  • Example 10 In vivo killing activity of CLD18A2 CAR-T cells containing IFN and no IFN
  • BGC-823-A2 cells collected in logarithmic growth phase and grown well were adjusted to a density of 2.5 ⁇ 10 7 /mL using physiological saline, and the volume of the injected cell suspension was 200 ⁇ L ( 5 ⁇ 10 6 / only) subcutaneously in the right axilla of mice. The day of tumor cell inoculation is recorded as day 0.
  • Adoptive transfer of T cells 100 mg/kg of cyclophosphamide was intraperitoneally injected at a tumor volume of 100-150 mm 3 (Day 11), and 1 ⁇ 10 7 CAR T cells were infused through the tail vein 24 hours after injection (Mock) Cells, 85-28Z T, 85-2-28Z T cells or 85-2-28Z-IFN cells), while using the untransfected T cell group (Mock group) as a control, were observed to measure the growth of subcutaneous xenografts.
  • Example 11 Anti-tumor test of CLD18A2 CAR-T cells containing IFN and no IFN in subcutaneous xenografts of gastric cancer PDX model
  • T cells 100 mg/kg of cyclophosphamide was intraperitoneally injected at a tumor volume of 30 mm3, and 1.0 ⁇ 107 CAR-T cells (85-2-28Z T cells or 85-) were infused through the tail vein 24 hours after the injection. 2-28Z-IFN T cells), while the untransfected T cell group was used as a control. Observe and measure the growth of subcutaneous xenografts of gastric cancer PDX.
  • Example 12 Effect of GPC3CAR-T (92-28Z) cells containing IFN and no IFN on tumor invasion in vivo
  • Example 13 Effect of CLD18A2 CAR-T cells containing IFN and no IFN on tumor invasion in vivo
  • Mock T cells showed almost no T cell infiltration around the tumor tissue.
  • 85-28Z and 85-2-28Z CAR T cells were visible at the edge of the tumor tissue, while 85-2-28Z - IFN T cells can observe some infiltration within the tumor tissue.
  • mice infected with retrovirus were retroviral packaging system, which were EGFR-CAR and EGFR-CAR-IFN, respectively.
  • the positive infection rates were 65.1% and 35.2%, respectively (Fig. 15).
  • Example 15 Determination of in vitro secretion of mIFN ⁇ by EGFR CAR (806-28Z) T cells containing IFN and no IFN
  • Example 17 in vitro toxicity test of EGFR CAR (806-28Z)-T cells containing IFN and no IFN
  • Example 18 in vivo toxicity test of EGFR CAR (806-28Z)-T cells containing IFN and no IFN

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Abstract

提供了一种改善免疫应答细胞功能的方法以及一种免疫应答细胞，该细胞表达至少一种能结合抗原的受体以及I型干扰素，具有显著的杀伤肿瘤或病原体的能力，可被用于治疗肿瘤和传染病。

Description

一种改善免疫应答细胞功能的方法
交叉引用
本申请要求2016年4月26日提交的中国专利申请201610265614.1、2016年7月8日提交的中国专利申请201610536449.9以及2016年12月13日提交的中国专利申请201611148447.9的权益，该申请通过引用将其全部内容并入本文。
技术领域
本发明属于免疫学领域，更具体地，本发明涉及一种改善免疫应答细胞功能的方法。
背景技术
嵌合抗原受体(Chimeric antigen receptor，CAR)是一种人工重组受体，通常含有位于胞外区的单克隆抗体的抗原识别结构域、跨膜区、及免疫应答细胞的胞内激活信号结构域组成。近年来，在临床试验中使用靶向CD19的CAR修饰的T细胞(CAR-T)细胞治疗B细胞白血病取得了巨大的成功。然而，有不少白血病患者会有复发。而且并不是所有的血液肿瘤都非常有效。此外，CAR-T细胞治疗实体性肿瘤也疗效不太显著。因此，如何改善现有CAR-T细胞技术，增加其抗肿瘤活性仍然是非常重要。
I型干扰素(Type I interferons)大概于半个多世纪前发现。I型干扰素包含IFNα蛋白(一类从IFNA1到IFNA13共13个人基因编码的同一性蛋白)，IFNβ(由单一个人和小鼠基因IFNB1编码)以及其他研究较少的干扰素如IFNε，IFNκ and IFNω(2.Trinchieri，G.Type Iinterferon:friend or foe？J.Exp.Med.207，2053-2063(2010).3.Kaur，S.& Platanias，L.C.IFN-β-specific signaling via a unique IFNAR1 interaction.Nat.Immunol.14，884-885(2013))。I型干扰素由多种类型的细胞在模式识别受体(pattern recognition receptors(PRRs))活化后产生。PRRs对病毒或者细菌成分以及异位的内源分子(如细胞浆DNA与细胞外DNA和RNA)产生应答(Kawai，T.& Akira，S.The role of pattern-recognition receptors in innate immunity:update on Toll-like receptors.Nat.Immunol.11，373–384(2010))。I型干扰素通过同一性二聚体IFNα/β受体1(IFNAR1)(该受体对IFNβ的亲和力特别高)或IFNAR1-IFNAR2异源二聚体(能与所有I型干扰素结合)传递信号。这些受体的活化，导致IFN-刺激基因(ISGs)的转录上调，引发很多免疫刺激效应(Hervas-Stubbs，S.et al.Direct  effects of type I interferons on cells of the immune system.Clin.Cancer Res.17，2619er Res.es.mmde Weerd，N.A.et al.Structural basis of a unique interferon-βsignaling axis mediated via the receptor IFNAR1.Nat.Immunol.14，901ol.nol.is oMcNab，F.，Mayer-Barber，K.，Sher，A.，Wack，A.& O A.&，A.Type I interferons in infectious disease.Nat.Rev.Immunol.15，87nol.Immun)。已有研究表明，I型干扰素对一些肿瘤有抗癌作用，可能归因于它们的免疫刺激功能。但是，I型干扰素的全身用药可能发生免疫抑制作用(Lotrich，F.E.Major depression during interferon-αtreatment:vulnerability and prevention.Dialogues Clin.Neurosci.11，417-425(2009))，并伴有重大的不良事件，最常见的有乏力、厌食、肝毒性、流感样症状和严重的抑郁症(Kreutzer，K.，Bonnekoh，B.，Franke，I.，Ulrich，J.& Gollnick，H.Sarcoidosis，myasthenia gravis and anterior ischaemic optic neuropathy:severe side effects of adjuvant interferon-αtherapy in malignant melanoma？.J.Dtsch.Dermatol.Ges.2，689-694(in German)(2004))，这类严重的毒副作用严重限制了其应用。
发明内容
本发明克服了前述问题，并且具有额外的优势。
根据本发明的一个方面，本发明提供了一种免疫应答细胞，其特征在于，该细胞表达结合抗原的受体；和外源性I型干扰素。
在一些实施方案中，本发明的免疫应答细胞包括T细胞、自然杀伤细胞、细胞毒性T淋巴细胞、自然杀伤T细胞、DNT细胞、和/或调节性T细胞。
在一些实施方案中，所述结合抗原的受体是内源性的。在一些实施方案中，所述结合抗原的受体是重组的。在一些实施方案中，所述结合抗原的受体是嵌合抗原受体。在一些实施方案中，所述结合抗原的受体包括顺序连接的胞外抗原结合区、跨膜区和胞内信号区。在一些实施方案中，所述抗原结合单元是特异性结合所述抗原的抗体或其片段。在一些实施方案中，所述胞内信号区可以含有已知的免疫受体酪氨酸激活基序(ITAM)的信号基序。在一些实施方案中，所述含有细胞质信号传导序列的ITAM的实例包括衍生自TCRζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b和CD66d的那些。
在一些实施方案中，所述结合抗原的受体的胞内信号区包括一个或多个共刺激结构域。在一些实施方案中，所述共刺激结构域选自表1中所列举的之中的一种或多种。在一些实施方案中，所述共刺激结构域选自CD28、OX40、CD27、CD2、CD5、ICAM-1、LFA-1(CD11a/CD18)、4-1BBL、MyD88和4-1BB中的一种或 多种。在一些实施方案中，所述共刺激结构域选自CD28、OX40、CD27、CD2、CD5、ICAM-1、LFA-1(CD11a/CD18)、4-1BBL、MyD88和4-1BB中的两种。
在一些实施方案中，所述结合抗原的受体的氨基酸序列与SEQ ID NO:49；SEQ ID NO:50；SEQ ID NO:51；SEQ ID NO:54；SEQ ID NO:55；SEQ ID NO:56；SEQ ID NO:61；SEQ ID NO:62；SEQ ID NO:63；SEQ ID NO:64；SEQ ID NO:65；SEQ ID NO:66；SEQ ID NO:67；SEQ ID NO:68；SEQ ID NO:69；SEQ ID NO:70；SEQ ID NO:71；SEQ ID NO:72；SEQ ID NO:73；SEQ ID NO:74；SEQ ID NO:75；以及SEQ ID NO:77之一具有至少90％的同一性。
在一些实施方案中，所述结合抗原的受体由核苷酸序列编码，所述核苷酸序列与SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60或SEQ ID NO:76具有至少90％的同一性。
在一些实施方案中，所述免疫应答细胞中不包含外源的共刺激配体。
在一些实施方案中，能够被所述的结合抗原的受体结合的抗原包括肿瘤抗原或病原体抗原。在一些实施方中，所述肿瘤抗原选自前列腺特异性膜抗原(PSMA)、癌胚抗原(CEA)、IL13Ralpha、HER-2、CD19、NY-ESO-1、HIV-1Gag、Lewis Y、MART-1、gp100、酪氨酸酶、WT-I、hTERT、间皮素、EGFR、EGFRvIII、磷脂酰肌醇蛋白聚糖3、EphA2、HER3、EpCAM、MUC1、MUC16、CLDN18.2、叶酸受体、CLDN6、CD30、CD138、ASGPR1、CDH16、GD2、5T4、8H9、αvβ6整合素、B细胞成熟抗原(BCMA)、B7-H3、B7-H6、CAIX、CA9、CD20、CD22、κ轻链(kappa light chain)、CD33、CD38、CD44、CD44v6、CD44v7/8、CD70、CD123、CD171、CSPG4、EGP2、EGP40、ERBB3、ERBB4、ErbB3/4、FAP、FAR、FBP、胚胎型AchR、GD2、GD3、HLA-AI MAGE A1、MAGE3、HLA-A2、IL11Ra、KDR、Lambda、MCSP、NCAM、NKG2D配体、PRAME、PSCA、PSC1、ROR1、Sp17、SURVIVIN、TAG72、TEM1、TEM8、VEGRR2、HMW-MAA、VEGF受体、和/或纤连蛋白、腱生蛋白或肿瘤坏死区的癌胚变体。
在一些实施方案中所述病原体抗原包括病毒抗原、细菌抗原、真菌抗原或寄生虫抗原。在一些实施方案中，所述的病原体是病毒。所述病毒包括巨细胞病毒、爱泼斯坦-巴尔病毒、人类免疫缺陷病毒及流感病毒。
在一些实施方案中，所述I型干扰素的表达是组成型表达。在一些实施方案中，所述I型干扰素的表达是诱导型表达。在一些实施方案中，所述I型干扰素表达在所述免疫应答细胞的表面。在一些实施方案中，所述I型干扰素包括：IFNα或IFN β。
根据本发明的一个方面，本发明提供了一种表达构建物，其包括顺序连接的：本发明的结合抗原的受体的表达盒；和I型干扰素的表达盒。在一些实施方案中，所述I型干扰素的表达是组成型表达。在一些实施方案中，所述I型干扰素的表达是诱导型表达。在一些实施方案中，所述I型干扰素的表达是诱导型表达，并且用于表达所述I型干扰素的表达是诱导型启动子。在一些实施方案中，用于表达所述I型干扰素的诱导型启动子是NFAT6启动子。在一些实施方案中，所述NFAT6启动子包括如SEQ ID NO:78所述的核酸序列。
根据本发明的一个方面，本发明提供了一种载体，其表达本发明的结合抗原的受体和/或I型干扰素。在一些实施方案中，所述的病毒载体是慢病毒载体、逆转录病毒载体或腺病毒载体。在一些实施方案中，所述的病毒载体是逆转录病毒载体。
根据本发明的一个方面，本发明提供了一种提高向个体给予的免疫应答细胞活力的方法，其特征在于，所述免疫应答细胞表达本发明所述的结合抗原的受体，并且其中所述方法包括向所述个体给予所述免疫应答细胞以及有效量的外源性I型干扰素。在一些实施方案中，所述外源性I型干扰素与所述表达结合抗原的受体的免疫应答细胞顺序给予或者同时给予。在一些实施方案中，所述外源性I型干扰素通过在免疫应答细胞中共表达，与所述免疫应答细胞同时向患者给予。
根据本发明的一个方面，本发明提供了本发明的免疫应答细胞在制备用于治疗有此需要的个体的肿瘤、病原体感染、或增强个体免疫耐受能力的药物组合物中的用途。本发明还提供了一种治疗个体的肿瘤或病原体感染，或用于增强个体免疫耐受能力方法，包括向有此需要的个体给予本发明的免疫应答细胞。
在一些实施方案中，本发明的方法使得在向所述个体给予所述免疫应答细胞后，与不存在所述外源性I型干扰素的情况相比，所述个体外周血中细胞毒性T细胞和辅助T细胞的数量之和提高至少50％。在一些实施方案中，所述方法使得在向所述个体给予所述免疫应答细胞约5天后，所述个体外周血中细胞毒性T细胞和辅助T细胞的数量之和大于15,000个/μL；给予所述免疫应答细胞约5天后，所述个体外周血中细胞毒性T细胞和辅助T细胞的数量之和大于500个/μL；或者给予所述免疫应答细胞约5天后，所述个体外周血中细胞毒性T细胞和辅助T细胞的数量之和大于50个/μL。
在一些实施方案中，所述的肿瘤包括：胰腺癌、肝癌、肺癌、胃癌、头颈部鳞状细胞癌、前列腺癌、结肠癌、乳腺癌、淋巴瘤、胆囊癌、肾癌、白血病、骨髓瘤、 卵巢癌、宫颈癌、卵巢癌、宫颈癌或胶质瘤。在一些实施方案中，所述的病原体包括：病毒、细菌、真菌、原生动物或寄生虫；较佳地，所述的病毒包括：巨细胞病毒、爱泼斯坦-巴尔病毒、人类免疫缺陷病毒或流感病毒。
在一些实施方案中，根据计算机断层扫描测量，在通过本发明方法治疗后，所述个体的肿瘤减小至少30％。在一些实施方案中，根据计算机断层扫描测量，在通过本发明方法治疗后，所述个体的肿瘤完全消失。
根据本发明的一个方面，本发明提供了一种药物组合物，其包括本发明的免疫应答细胞以及药学上可接受的载体或赋形剂。
根据本发明的一个方面，本发明提供了一种试剂盒，其包括本发明的免疫应答细胞以及指导如何向个体给予所述免疫应答细胞的说明书。
以引用的方式并入
本说明书中提及的所有公布、专利和专利申请都以引用的方式并入本文，所述引用的程度就如同已特定地和个别地指示将各个别公布、专利或专利申请以引用的方式并入一般。
附图说明
附图更进一步说明了本说明书所公开的新特性。参照这些附图将能更好地理解本说明书中所公开的特性和优点，但应当理解，这些附图仅用于说明应用本文所公开原理的具体的实施方案，而非意欲对所附权利要求的范围加以限制。
图1所示为重组慢病毒载体pRRL-EF-1α-92-CAR结构示意图(图1A)，以及92-28Z-NFAT6-IFN-β质粒的构建(图1B)。
图2所示为慢病毒感染PBMC阳性率检测图。
图3所示为慢病毒感染PBMC阳性率检测图。
图4所示为含有IFN和不含IFN的GPC3 CAR-T细胞的细胞因子的释放对比图。图4A所示为GPC3-28Z-IFN及GPC3-28Z CAR T细胞对IFN-β诱导表达情况。结果显示，只有GPC3-28Z-IFN与Huh7细胞共孵育时有IFN-β的表达，说明GPC3-28Z-IFN在被靶抗原激活后，IFNβ能够被成功诱导表达，并分泌至细胞外。图4B所示为GPC3-28Z-IFN及GPC3-28Z CAR对INF-γ诱导表达对比。图4C所示为GPC3-28Z-IFN及GPC3-28Z CAR T细胞在体外导致IL-2释放的对比图。结果显示，GPC3-28Z-IFN能更有效地导致细胞因子释放，说明包含IFNβ的CAR T细胞能够被更有效地被激活。
图5所示为85-28Z T细胞和85-28Z-IFN T细胞在不同细胞系中体外诱导细胞因子释放的对比图。结果显示，包含IFNβ的CAR T细胞能够被更有效地被激活。
图6所示为包含IFNβ的GPC3-28Z CAR T细胞与不包含IFNβ的GPC3 CAR T细胞在体外对各种细胞系(图6A：Huh7；图6B：Hep3B；图6C：PLC/PRR/5；图6D：Hep G2；图6E：SK-hep-1)的杀伤效率对比图。
图7所示为含有IFN和不含IFN的CLD18A2 CAR-T细胞的杀伤活性图。
图8所示为包含IFNβ的CLD18A2 CAR-T细胞与不包含IFNβ的CLD18A2 CAR-T细胞在小鼠外周血中在回输5天(图8A)所示为7天(图8B)和10天(图8C)后的存活细胞数对比图。结果显示，在所有时间点，包含IFNβ的CLD18A2 CAR-T存活细胞数均显著高于不包含IFNβ的CLD18A2 CAR-T细胞组。
图9所示为包含IFNβ的GPC3-28Z CAR T细胞与不包含IFNβ的GPC3-28Z CAR T细胞在小鼠肿瘤模型中随时间对肿瘤体积影响的对比图(图9A)以及肿瘤照片对比图(图9B)。结果显示，包含IFNβ的GPC3-28Z CAR T细胞与不包含IFNβ的GPC3-28Z CAR T细胞和对照组相比，能够更显著地减小肿瘤体积。
图10所示为包含IFNβ的CLD18A2 CAR-T细胞与不包含IFNβ的CLD18A2 CAR-T细胞在小鼠BGC-823-A2细胞皮下移植肿瘤模型中随时间对肿瘤体积影响的对比图(图10A)以及肿瘤照片对比图(图10B)。结果显示，包含IFNβ的CLD18A2 CAR-T细胞与不包含IFNβ的CLD18A2 CAR-T细胞和对照组相比，能够更显著地减小肿瘤体积。
图11所示为含有IFN和不含IFN的CLD18A2 CAR-T细胞在胃癌PDX模型皮下移植瘤中的抗肿瘤活性对比图。结果显示，含有IFN的治疗组中有1只小鼠肿瘤完全消退。
图12所示为含有IFN和不含IFN的GPC3 CAR-T(92-28Z)细胞在体内对肿瘤浸润对比图。其中图12A为组织化学图片，图12B为T细胞数量图。结果表明，对照组肿瘤组织中无明显浸润的CD3+细胞，INFβ-CAR-T治疗组中CD3+T细胞数量高于28Z CART组。
图13所示为含有IFN和不含IFN的CLD18A2 CAR-T细胞在体内对肿瘤浸润的免疫组化图对比。结果显示，Mock T细胞在肿瘤组织周围几乎观察不到T细胞的浸润，85-28Z和85-2-28Z CAR T细胞在肿瘤组织的边缘可以看到，而85-2-28Z-IFN T细胞在肿瘤组织内部可以观察到一定的浸润。
图14所示为含有IFN和不含IFN的EGFR-CAR结构示意图。
图15所示为逆转录病毒感染小鼠T淋巴细胞的感染阳性率图。
图16所示为含有IFN和不含IFN的EGFR CAR T细胞体外分泌mIFNβ能力对比图。结果显示，经过靶细胞刺激后，mCAR-806-mIFNβ能够被成功激活，并诱导表达mIFNβ，对照组则未检出有mIFNβ的表达
图17所示为含有IFN和不含IFN的EGFR CAR-T细胞在体外诱导细胞因子释放(图17A：mIL-2；图17B：mIFN-γ；图17C：mTNF-α)的对比图。
图18所示含有IFN和不含IFN的EGFR CAR-T细胞的体外毒性试验对比图。结果显示，EGFR-CAR和EGFR-CAR-IFN与UT细胞相比，对靶点阳性的CT26VIII细胞具有强效的杀伤作用，差异有显著意义(***P＜0.001)，杀伤百分比呈剂量依赖性，而未经转染的UT细胞对CT26及CT26VIII均没有杀伤作用，EGFR-CAR和EGFR-CAR-IFN两种CAR-T细胞对靶点阴性的CT26细胞没有杀伤作用。
图19所示为含有IFN和不含IFN的EGFR CAR-T细胞的体内毒性试验对比图。结果显示，EGFR-CAR-T细胞对对肿瘤大小与对照组基本一致，未观察到抑制作用，而EGFR-CAR-IFN细胞回输后，在第7天开始出现抑制肿瘤生长的现象，抑瘤率为5.9％，第10天时达到最强，为18.5％，到第17天时，抑瘤率仍可达到12.4％，明显优于EGFR-CAR-T细胞组。
具体实施方式
以下具体说明详尽地展示了本文所公开的实施方案。应当理解，本说明书并非意欲仅限于此处所公开的具体的实施方案，而是可以发生改变。本领域技术人员将理解，本说明书中所公开的内容可以有多种改变或变化，而均涵盖于所公开的范围和原则之内。除非另有说明，每个实施方案均可与任何其他实施方案任意组合。
本文所公开的某些实施方案包含了数值范围，并且本发明的某些方面可采用范围的方式描述。除非另有说明，应当理解数值范围或者以范围描述的方式仅是出于简洁、便利的目的，并不应当认为是对本发明的范围的严格限定。因此，采用范围方式的描述应当被认为具体地公开了所有可能的子范围以及在该范围内的所有可能的具体数值点，正如这些子范围和数值点在本文中已经明确写出。例如，从1至6的范围的描述应当被认为具体公开了从1至3、1至4、1至5、2至4、2至6、3至6等的子范围，以及在这些范围内的具体的数值点，例如1、2、3、4、5、6。不论所述数值的宽窄，上述原则均同等适用。当采用范围描述时，该范围包括范围的端点。
为了克服现有技术中的缺陷，本发明进行了深入的研究，发现使CAR-T细胞诱导型表达I型干扰素，可有效地增加CAR-T细胞的抗肿瘤活性，而且降低其毒副作用。在此基础上，本发明提供了一种免疫应答细胞，其表达至少一种能结合抗原(如肿瘤抗原或来自病原体的抗原)的受体和I型干扰素，将其应用于治疗肿瘤、传染病和其他疾病，具有显著优异的杀伤肿瘤或病原体的能力。
本文所用的术语“激活”和“活化”可互换使用，它们以及其语法上的其他形式可以指细胞从静止状态转变为活性状态的过程。该过程可以包括对抗原、迁移和/或功能活性状态的表型或遗传变化的响应。例如，术语“激活”可以指T细胞逐步活化的过程。例如，T细胞可能需要至少两个信号才能完全激活。第一信号可以在由抗原-MHC复合物接合TCR之后发生，而第二信号可以通过共刺激分子(参见表1中所列举的共刺激分子)的接合发生。在体外，抗CD3可以模拟所述第一信号，抗CD28可以模拟所述第二信号。例如，工程化T细胞可以被表达的CAR激活。本文所用的T细胞激活或T细胞触发可以指已经被充分刺激以诱导可检测的细胞增殖、细胞因子产生和/或可检测的效应物功能的T细胞的状态。
本文所用的术语“共刺激配体”包括特异性结合T细胞上的同一性共刺激分子的抗原呈递细胞(例如，aAPC、树突状细胞、B细胞等)上的分子，由此提供信号，与由例如TCR/CD3复合物与加载有肽的MHC分子的结合提供的第一信号共同介导T细胞应答，包括但不限于增殖、激活、分化等。共刺激配体可以包括但不限于CD7、B7-1(CD80)、B7-2(CD86)、PD-L、PD-L2、4-1BBL、OX40L、诱导型共刺激配体(ICOS-L)、细胞间粘附分子(ICAM)、CD30L、CD40、CD70、CD83、HLA-G、MICA、MICB、HVEM、淋巴毒素β受体、3/TR6、ILT3、ILT4、HVEM、结合Toll配体受体的激动剂或抗体以及与B7-H3特异性结合的配体。共刺激配体还特别包括与T细胞上存在的共刺激分子特异性结合的抗体，例如但不限于CD27、CD28、4-1BB、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。
本文所用的术语“共刺激分子”是指与共刺激配体特异性结合的T细胞上的同一性结合配偶体，从而介导T细胞的共刺激应答，例如但不限于增殖。共刺激分子包括但不限于MHC I类分子、BTLA和Toll配体受体。
如本文所用的“共刺激信号”是指与第一信号，例如TCR/CD3结合，组合导致T细胞增殖和/或关键分子的上调或下调的信号。
本文所用的术语“抗原结合单元”是指免疫球蛋白分子和免疫分子的免疫活性部分，即含有与抗原特异性结合(“免疫反应”)的抗原结合位点的分子。术语“抗原结合单元”中还包括各种物种来源的免疫球蛋白分子，包括无脊椎动物和脊椎动物。结构上，最简单的天然存在的抗体(例如IgG)包含四条多肽链，通过二硫键相互连接的两条重(H)链和两条轻链(L)链。免疫球蛋白代表包括几种类型的分子的一大家族的分子，如IgD、IgG、IgA、IgM和IgE。术语“免疫球蛋白分子”包括例如杂交抗体或改变的抗体及其片段。已经显示抗体的抗原结合功能可以通过天然存在的抗体的片段进行。这些片段统称为“抗原结合单元”。术语“抗原结合单元”中还包括具有与表位吻合并识别表位的特定形状的任何含多肽链的分子结构，其中一个或多个非共价结合相互作用稳定分子结构和表位之间的复合物。
如果抗原结合单元以与其它参考抗原(包括多肽或其他物质)结合相比更大亲和力或亲合力结合抗原，则所述抗原结合单元与抗原“特异性结合”或与抗原是“免疫反应性的”。
本文所用的“抗原”是指被抗原结合单元识别并特异性结合的物质。抗原可以包括肽、蛋白质、糖蛋白、多糖和脂质，其部分及其组合。非限制性示例性抗原包括肿瘤抗原或病原体抗原。“抗原”也可以指引发免疫反应的分子。这种免疫反应可能涉及抗体产生或特定免疫活性细胞(immunologically-competent cells)的活化，或两者兼有。本领域技术人员将理解，任何大分子，包括实际上所有的蛋白质或肽，都可以作为抗原。
本文所用的术语“免疫球蛋白”或“Ig”可以指作为抗体起作用的一类蛋白质。由B细胞表达的抗体有时称为嵌合抗原受体或抗原受体。包括在这类蛋白质中的五个成员是IgA、IgG、IgM、IgD和IgE，其中IgG是最常见的循环抗体。它是凝集、补体固定和其他抗体反应中最有效的免疫球蛋白，在防御细菌和病毒方面是重要的。例如，可以通过CAR识别肿瘤细胞抗原(或“肿瘤抗原”)或病原体抗原。
本文使用的术语“自体”及其语法上的其他形式可以指来自相同的本源。例如，样品(例如，细胞)可以被去除、处理并在稍后的时间给予相同的个体(例如，患者)。自体过程与其中供体和受体是不同个体的同种异体过程不同。
本文使用的“异种移植”及其语法上的其他形式可以包括其中接受者和供体是不同的物种的将细胞、组织或器官移植、植入或输注到受体中的任何程序。本文所述的细胞、器官和/或组织的移植可用于异种移植入人中。异种移植包括但不限于血管化异种移植物、部分血管化异种移植物、非血管化异种移植、异种敷料、异种 绷带和异种结构等。
本文使用的“同种异体移植”及其语法上的其他形式(例如，同种异体的移植)可以包括其中受体和供体是同一物种但不同个体的将细胞、组织或器官移植，植入或输注到接受者中的任何程序。本文所述的细胞、器官和/或组织的移植可以用于同种异体移植入人体。同种异体移植包括但不限于血管化同种异体移植、部分血管化的同种异体移植、无血管化的同种异体移植、同种异体敷料、同种异体绷带、和同种异体结构。
本文所用的“自体移植”及其语法上的其他形式(例如，自体的移植)可以包括其中接受者和供体是相同的个体的将细胞、组织或器官移植、植入或输注到接受者中的任何程序。本文所述的细胞、器官和/或组织的移植可用于自体移植入人体。自体移植包括但不限于血管化自体移植、部分血管化自体移植、非血管化自体移植、自体敷料、自体绷带和自体结构。
本文所用的术语“嵌合抗原受体”或“CAR”是指可以由包括但不限于T细胞的免疫细胞表达的工程化分子。CAR在T细胞中表达并且可以重定向T细胞以诱导以由人造受体决定的特异性杀死靶细胞。CAR的细胞外结合结构域可以衍生自鼠、人源化或完全人单克隆抗体。
本文所用的术语“表位”及其语法上的其他形式可以指可被抗体、B细胞、T细胞或工程细胞识别的部分抗原。例如，表位可以是被TCR识别的肿瘤表位或病原体表位。也可以识别抗原内的多个表位。表位也可以突变。
本文所用的术语“工程化”及其语法上的其他形式可以指核酸的一个或多个改变，例如生物体基因组内的核酸。术语“工程化”可以指基因的改变、添加和/或缺失。工程细胞还可以指具有加入、缺失和/或改变的基因的细胞。
本文所用的术语“细胞”或“工程细胞”及其语法上的其他形式可以指人或非人动物来源的细胞。工程细胞也可以指表达CAR的细胞。
本文所用的术语“转染”是指将外源核酸引入真核细胞。转染可以通过本领域已知的各种手段来实现，包括磷酸钙-DNA共沉淀、DEAE-葡聚糖介导的转染、聚凝胺介导的转染、电穿孔、显微注射、脂质体融合、脂质转染、原生质体融合、逆转录病毒感染和生物弹道技术(biolistics)。
术语“稳定转染”或“稳定地转染”是指将外源核酸、DNA或RNA引入和整合到转染细胞的基因组中。术语“稳定转染体”(stable transfectant)是指将外来DNA稳定地整合到基因组DNA中的细胞。
如本文所用，术语“核酸分子编码”、“编码DNA序列”和“编码DNA”是指沿着脱氧核糖核酸链的脱氧核糖核苷酸的顺序或顺序。这些脱氧核糖核苷酸的顺序决定了沿着多肽(蛋白质)链的氨基酸的顺序。因此，核酸序列编码氨基酸序列。
本文所用的术语“个体”是指任何动物，例如哺乳动物或有袋动物。本发明的个体包括但不限于人类、非人类灵长类动物(例如恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠和任何种类的家禽。
本文所用的术语“外周血淋巴细胞”(PBL)及其语法上的其他形式可以指在血液(例如外周血)中循环的淋巴细胞。外周血淋巴细胞可以指不局限于器官的淋巴细胞。外周血淋巴细胞可以包含T细胞、NK细胞、B细胞或其任何组合。
本文所用的术语“免疫应答细胞”或“免疫反应性细胞”可以指可以引发免疫应答的细胞，包括但不限于T细胞、B细胞和NKT细胞、它们各自的前体细胞及其后代。免疫应答细胞还可以指淋巴或骨髓谱系的细胞。
本文所用的术语“T细胞”及其语法上的其他形式可以指任何来源的T细胞。例如，T细胞可以是原代T细胞例如自体T细胞等。T细胞也可以是人或非人的。
本文所用的术语“T细胞活化”或“T细胞触发”及其语法上的其他形式可以指被充分刺激以诱导可检测的细胞增殖、细胞因子产生和/或可检测的效应物功能的T细胞的状态。在一些实施方案中，“完全T细胞活化”可以类似于触发T细胞的细胞毒性。可以使用本领域已知的各种测定来测量T细胞活化。所述测定可以是测量细胞因子分泌的ELISA、ELISPOT、用于测量细胞内细胞因子表达的流式细胞术测定(CD107)、用于测量增殖的流式细胞术测定、和用于确定靶细胞消除的细胞毒性测定(51Cr释放测定)。所述测定通常使用对照(非工程细胞)与工程细胞(CAR T)进行比较，以确定与对照相比，工程细胞的相对激活。此外，所述测定可以与未表达靶抗原的靶细胞孵育或接触的工程细胞进行比较。例如，所述比较可以是与不表达CD19的靶细胞孵育的CD19-CART细胞进行的比较。
当用于指核苷酸序列时，本文所用的术语“序列”及其语法上的其他形式可以包括DNA或RNA，并且可以是单链或双链。核酸序列可以突变。核酸序列可以具有任何长度，例如长度为2至1,000,000或更个核苷酸(或其间或之上的任何整数值)核酸，例如长度为约100至约10,000个核苷酸或约200至约500个核苷酸。
本文所用的术语“有效量”是指提供治疗或预防益处的量。
本文所用的术语“表达载体”是指包含重组多核苷酸的载体，其包含与待表达的核苷酸序列有效连接的表达调控序列。表达载体包含用于表达的足够的顺式作用 元件(cis-acting elements)；用于表达的其它元件可以由宿主细胞或体外表达系统提供。表达载体包括本领域所有已知的那些，例如粘粒、质粒(例如裸露或包含在脂质体中的)和病毒(例如，慢病毒、逆转录病毒、腺病毒和腺相关病毒)。
本文所用的术语“慢病毒”是指逆转录病毒科的属。逆转录病毒在能够感染非分裂细胞方面是逆转录病毒中独特的；它们可以将大量的遗传信息递送到宿主细胞的DNA中，因此它们是基因递送载体最有效的方法之一。HIV、SIV和FIV都是慢病毒的实例。源自慢病毒的载体提供了在体内实现显著水平的基因转移的手段。
本文所用的术语“可操作地连接”是指在调控序列和异源核酸序列之间的功能性连接，该连接导致后者的表达。例如，当第一核酸序列与第二核酸序列成功能关系时，第一核酸序列与第二核酸序列可操作地连接。例如，如果启动子影响编码序列的转录或表达，则启动子可操作地连接到编码序列。通常，可操作地连接的DNA序列是连续的，并且在必要时在相同的阅读框中连接两个蛋白质编码区。
本文使用的术语“启动子”定义为由启动多核苷酸序列的特异性转录所需的细胞的合成机制或引入的合成机制识别的DNA序列。
本文使用的术语“载体”是包含分离的核酸并可用于将分离的核酸递送至细胞内部的组合物。在本领域中已知许多载体，包括但不限于线性多核苷酸、与离子或两亲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。该术语还应被解释为包括促进核酸转移到细胞中的非质粒和非病毒化合物，例如聚赖氨酸化合物、脂质体等。病毒载体的实例包括但不限于腺病毒载体、腺相关病毒载体、逆转录病毒载体等。
本文使用的术语序列“同一性”通过在比较窗口(例如至少20个位置)上比较两个经最佳匹配的序列来确定同一性百分比，其中比较窗口中多核苷酸或多肽序列的部分可以包含添加或缺失(即间隙)，例如对于最佳匹配的两个序列而言与参考序列(其不包含添加或缺失)相比20％或更少的间隙(例如5至15％、或10至12％)。通常通过确定在两个序列中发生相同的核酸碱基或氨基酸残基的位置的数目来计算百分比，以产生正确匹配的位置的数目，将正确匹配位置的数目除以参考序列中的位置总数(即窗口大小)，并将结果乘以100，以产生序列同一性的百分比。
本文所用的术语“I型干扰素”包括IFNα、IFNβ、以及IFN-ε、IFN-κ以及IFN-ω等。所有的I型干扰素均与由IFNAR1和IFNAR2两条链组成的特定的细胞表面受体(即所谓的IFN-α/β受体)结合。在一些实施方案中，本文所用的术语“I型干 扰素”为IFNα或IFNβ。在一些实施方案中，本文所用的术语“I型干扰素”为IFNβ。在一些实施方案中，本文所用I型干扰素包括人类、小鼠、或者合成的I型干扰素。在一些实施方案中，本文所用的术语“干扰素α”可以是具有NCBI aaa52724.1或aaa52716.1或aaa52725.1所示序列的多肽，或者是序列与这些序列具有至少有85％的同一性的多肽。在一些实施方案中，本文所用的术语“干扰素β”(INF-β)可以是具有NCBI aac41702.1或np_002167.1或aah96152.1p41273或NP 001552至少有85％同一性的蛋白，或者是其具有肿瘤坏死因子(TNF)配体功能的片段。
在一些实施方案中，应用于构建所述的结合抗原的受体的元件或所述的I型干扰素可以是天然存在的，比如其可被分离或纯化自哺乳动物；也可以是人工制备的，比如可以根据常规的基因工程重组技术来生产重组各元件或I型干扰素。优选的，本发明可采用重组的各元件或I型干扰素。
在所述各元件或I型干扰素多肽序列的基础上，经过一个或多个氨基酸残基的取代、缺失或添加而形成的氨基酸序列也包括在本发明中。适当替换氨基酸是本领域公知的技术，所述技术可以很容易地被实施并且确保不改变所得分子的生物活性。这些技术使本领域人员认识到，一般来说，在一种多肽的非必要区域改变单个氨基酸基本上不会改变生物活性。
所述各元件或I型干扰素的多肽的生物活性片段都可以应用到本发明中。在这里，所述的生物活性片段的含义是指作为一种多肽，其作为全长多肽的一部分，仍然能保持全长的多肽的全部或部分功能。通常情况下，所述的生物活性片段至少保持50％的全长多肽的活性。在更优选的条件下，所述活性片段能够保持全长多肽的60％、70％、80％、90％、95％、99％、或100％的活性。
在所述各元件或I型干扰素多肽序列的基础上，经修饰或改良的多肽也可以应用到本发明中，比如，可采用为了促进其半衰期、有效性、代谢、和/或多肽的效力而加以修饰或改良的多肽。也就是说，任何不影响多肽的生物活性的变化形式都可用于本发明中。
本文所用的术语“疾病”或“病症”或“紊乱”等是指任何损害或干扰细胞、组织或器官的正常功能的改变或失调。例如，所述的“疾病”包括但不限于：肿瘤、病原体感染、自身免疫性疾病、T细胞功能障碍性疾病、或免疫耐受能力缺陷(如移植排斥)。
本文所用的术语“肿瘤”指的是一种以细胞或组织的病理性增生为特征的疾病，及其随后的迁移或侵袭其他组织或器官。肿瘤生长通常是不受控制的和进行性的， 不诱导或抑制正常细胞增殖。肿瘤可影响多种细胞、组织或器官，包括但不限于选自膀胱、骨、脑、乳腺、软骨、神经胶质细胞、食管、输卵管、胆囊、心脏、肠、肾、肝、肺、淋巴结、神经组织、卵巢、胰腺、前列腺、骨骼肌、皮肤、脊髓、脾、胃、睾丸、胸腺、甲状腺、气管、尿道、输尿管、尿道、子宫、阴道器官，或组织或相应的细胞。肿瘤包括癌症，如肉瘤，癌，或浆细胞瘤(浆细胞的恶性肿瘤)。本发明所述的肿瘤，可包括，但不限于白血病(如急性白血病、急性淋巴细胞白血病、急性髓细胞性白血病，急性粒细胞白血病，急性早幼粒细胞白血病、急性粒-单核细胞白血病、急性单核细胞白血病、急性白血病、慢性白血病、慢性粒细胞白血病、慢性淋巴细胞白血病、真性红细胞增多症)，淋巴瘤(霍奇金病、非霍奇金病)、原发性巨球蛋白血症，重链病，实体瘤如肉瘤和癌症(如纤维肉瘤、粘液肉瘤、脂肪肉瘤、软骨肉瘤、骨肉瘤、脊索瘤、内皮肉瘤、淋巴管肉瘤、血管肉瘤、淋巴管内皮肉瘤，滑膜vioma，间皮瘤，尤文氏瘤、平滑肌肉瘤、横纹肌肉瘤、结肠癌、胰腺癌、乳腺癌、卵巢癌、前列腺癌、鳞状细胞癌、基底细胞癌、腺癌、汗腺癌、皮脂腺癌、乳头状癌、乳头状腺癌、癌、支气管癌、髓样癌、肾细胞癌、肝癌，尼罗河管癌，绒癌、精原细胞瘤、胚胎癌、肾母细胞瘤、宫颈癌、子宫癌、睾丸癌、肺癌、小细胞肺癌、膀胱癌、上皮癌、胶质瘤、星形细胞瘤、髓母细胞瘤，颅咽管瘤、室管膜瘤、松果体瘤、血管母细胞瘤，听神经瘤，少突胶质瘤、神经鞘瘤、脑膜瘤、黑色素瘤、神经母细胞瘤、视网膜母细胞瘤)、食管癌、胆囊癌、肾癌、多发性骨髓瘤。较佳地，所述的“肿瘤”包括但不限于：胰腺癌、肝癌、肺癌、胃癌、食管癌、头颈部鳞状细胞癌、前列腺癌、结肠癌、乳腺癌、淋巴瘤、胆囊癌、肾癌、白血病、多发性骨髓瘤、卵巢癌、宫颈癌和胶质瘤。
本发明中提及的肿瘤抗原的类型也可以是肿瘤特异性抗原(TSA)或肿瘤相关抗原(TAA)。TSA是肿瘤细胞独特的，不发生在体内的其他细胞上。TAA相关抗原不是肿瘤细胞独有的，而是在不能诱导对抗原的免疫耐受状态的条件下在正常细胞上表达。抗原在肿瘤上的表达可以在使得免疫系统能够对抗原作出反应的条件下发生。当免疫系统不成熟并且不能应答时，TAA可以是在胎儿发育期间在正常细胞上表达的抗原，或者它们可以是通常以正常细胞上极低水平存在但在肿瘤细胞上以更高水平表达的抗原。
TSA或TAA抗原的非限制性实例包括以下：分化抗原如MART-1/MelanA(MART-I)、gp100(Pmel17)、酪氨酸酶、TRP-1、TRP-2和肿瘤特异性多中心抗原如MAGE-1、MAGE-3、BAGE、GAGE-1、GAGE-2、p15；过表达的胚胎抗 原如CEA；过表达的癌基因和突变的肿瘤抑制基因如p53、Ras、HER-2/neu；由染色体易位引起的独特的肿瘤抗原，例如BCR-ABL、E2A-PRL、H4-RET、IGH-IGK、和MYL-RAR；以及病毒抗原，如爱泼斯坦巴尔病毒抗原EBVA和人乳头瘤病毒(HPV)抗原E6和E7等。其他大的、基于蛋白质的抗原包括TSP-180、MAGE-4、MAGE-5、MAGE-6、RAGE、NY-ESO、p185erbB2、p180erbB-3、c-met、nm-23H1、PSA、TAG-72、CA 19-9、CA 72-4、CAM 17.1、NuMa、K-ras、β-联蛋白、CDK4、Mum-1、p 15、p 16、43-9F、5T4、791Tgp72、甲胎蛋白、beta-HCG、BCA225、BTAA、CA 125、CA 15-3\CA 27.29\BCAA、CA 195、CA 242、CA-50、CAM43、CD68\P1、CO-029、FGF-5、G250、Ga733\EpCAM、HTgp-175、M344、MA-50、MG7-Ag、MOV18、NB/70K、NY-CO-1、RCAS1、SDCCAG16、TA-90\Mac-2结合蛋白\亲环蛋白C相关蛋白、TAAL6、TAG72、TLP、以及TPS。
在一些实施方案中，所述的“肿瘤抗原”包括但不限于：前列腺特异性膜抗原(PSMA)、癌胚抗原(CEA)、IL13Ralpha、HER-2、CD19、NY-ESO-1、HIV-1Gag、Lewis Y、MART-1、gp100、酪氨酸酶、WT-I、hTERT、间皮素、EGFR、EGFRvIII、磷脂酰肌醇蛋白聚糖3、EphA2、HER3、EpCAM、MUC1、MUC16、CLDN18.2、叶酸受体、CLDN6、CD30、CD138、ASGPR1、CDH16、GD2、5T4、8H9、αvβ6整合素、B细胞成熟抗原(BCMA)、B7-H3、B7-H6、CAIX、CA9、CD20、CD22、κ轻链(kappa light chain)、CD33、CD38、CD44、CD44v6、CD44v7/8、CD70、CD123、CD171、CSPG4、EGP2、EGP40、ERBB3、ERBB4、ErbB3/4、FAP、FAR、FBP、胚胎型AchR、GD2、GD3、HLA-AI MAGE A1、MAGE3、HLA-A2、IL11Ra、KDR、Lambda、MCSP、NCAM、NKG2D配体、PRAME、PSCA、PSC1、ROR1、Sp17、SURVIVIN、TAG72、TEM1、TEM8、VEGRR2、HMW-MAA、VEGF受体、和/或纤连蛋白、腱生蛋白或肿瘤坏死区的癌胚变体。
本文所用的术语“病原体”是指能够引起疾病的原生动物，包括：病毒、细菌、真菌或寄生虫。所述的“病毒抗原”是指病毒表达的，能够诱导免疫反应的多肽。
典型的病毒包括但不限于，逆转录病毒科(如人类免疫缺陷病毒，比如HIV-1(也被称为HDTV-III，LAVE或HTLV-Ⅲ/LAV，或HIV-III；和其他菌株，如HIV-LP；小核糖核酸病毒(如脊髓灰质炎病毒、甲型肝炎病毒；人类肠道病毒，柯萨奇病毒、鼻病毒、埃可病毒)；杯状病毒(例如菌株引起的肠胃炎)；披盖病毒(如马脑炎病毒、风疹病毒)；黄病毒科(如登革热病毒、乙型脑炎病毒、黄热病病毒)；冠状病毒科(例如冠状病毒)；弹状病毒科(如水泡性口炎病毒、狂犬病毒)；丝状病毒科(如埃博拉 病毒)；副粘病毒科(如副流感病毒、腮腺炎病毒、麻疹病毒、呼吸道合胞病毒)；正粘病毒(如流感病毒)；亚病毒科(例如汉坦病毒，部分病毒，白蛉和内罗病毒)；舞台病毒科(出血热病毒)；呼肠病毒科(如呼肠孤病毒，环状病毒和轮状病毒)；双核糖核酸病毒；肝病毒(乙型肝炎病毒)；微小病毒科(细小病毒)；乳多空病毒科(乳头状瘤病毒，多瘤病毒)；腺病毒科(大多数腺病毒)；疱疹病毒(单纯疱疹病毒(HSV)的1和2，水痘-带状疱疹病毒、巨细胞病毒(CMV)、单纯疱疹病毒；痘病毒(天花病毒、牛痘病毒，痘病毒)；虹彩病毒科(如非洲猪瘟病毒)；和未分类的病毒(如三角洲肝炎剂(认为是一个有缺陷的卫星TE乙型病毒性肝炎)、非甲非乙型肝炎的药物，(1级＝内部传递；232类非肠道传播(即丙型肝炎)；诺瓦克和相关病毒和星状病毒)。
典型的细菌，包括但不限于，巴氏杆菌、金黄色葡萄球菌、链球菌、大肠杆菌、沙门氏菌和铜绿假单胞菌。传染性细菌具体的实例包括，但不限于，幽门螺杆菌，螺旋体，嗜肺军团菌，分枝杆菌SPS(如结核杆菌，鸟型结核杆菌，内分枝杆菌，M.Kansaii，M.gordonae)，金黄色葡萄球菌、淋病奈瑟菌、脑膜炎奈瑟菌、单增李斯特菌、化脓性链球菌(A组链球菌)，无乳链球菌(B组链球菌链球菌(草绿色链球菌组)、粪链球菌、牛链球菌、链球菌(厌氧SPS)，肺炎链球菌，弯曲杆菌属、肠球菌属、流感嗜血杆菌、炭疽杆菌、白喉杆菌、棒状杆菌属、梭菌属丹毒丝菌，产气荚膜梭菌、破伤风杆菌、阴沟肠杆菌、肺炎克雷伯菌、多杀性巴氏杆菌、类杆菌属、具核梭杆菌、念珠状链，梅毒密螺旋体，雅司螺旋体、钩端螺旋体、立克次体、以色列放线菌。
在一些实施方案中，本发明的免疫应答细胞能够识别并结合寄生虫抗原。所述寄生虫包括内寄生虫和外寄生虫。所述内寄生虫包括原生动物、蠕虫、蛔虫、和吸虫。在一些实施方案中，所述寄生虫抗原例如是源自以下科的物种的抗原：Entamoeba histolytica；Babesia B.divergens,B.bigemina,B.equi,B.microfti,B.duncani；Balantidium coli；Blastocystis spp.；Trypanosoma cruzi；Cryptosporidium spp.；Cyclospora cayetanensis；Dientamoeba fragilis；Giardia lamblia；Balamuthia mandrillaris；Acanthamoeba spp.；Isospora belli；Leishmania spp.；Plasmodium falciparum,Plasmodium vivax,Plasmodium ovale curtisi,Plasmodium ovale wallikeri,Plasmodium malariae,Plasmodium knowlesi；Naegleria fowleri；Rhinosporidium seeberi；Sarcocystis bovihominis,Sarcocystis suihominis；Trypanosoma brucei；Toxoplasma gondii；Trichomonas vaginalis；Taenia saginata；Bertiella mucronata, Bertiella studeri；Taenia solium；Diphyllobothrium latum；Echinococcus granulosus,Echinococcus multilocularis,E.vogeli,E.oligarthrus；Hymenolepis nana,Hymenolepis diminuta；Spirometra erinaceieuropaei；Cestoda,Taenia multiceps；Clonorchis sinensis；Clonorchis viverrini；Dicrocoelium dendriticum；Fasciola hepatica,Fasciola gigantica；Fasciolopsis buski；Gnathostoma spinigerum,Gnathostoma hispidum；Metagonimus yokogawai；Metorchis conjunctus；Opisthorchis viverrini,Opisthorchis felineus,Clonorchis sinensis；Paragonimus westermani；Paragonimus africanus；Paragonimus caliensis；Paragonimus kellicotti；Paragonimus skrjabini；Paragonimus uterobilateralis；Schistosoma sp.；Schistosoma mansoni and Schistosoma intercalatum；Schistosoma haematobium；Schistosoma japonicum；Schistosoma mekongi；Echinostoma echinatum；Trichobilharzia regenti,Schistosomatidae；Ancylostoma duodenale,Necator americanus；Angiostrongylus costaricensis；Anisakis；Ascaris sp.Ascaris lumbricoides；Baylisascaris procyonis；Brugia malayi,Brugia timori；Dioctophyme renale；Dracunculus medinensis；Enterobius vermicularis,Enterobius gregorii；Halicephalobus gingivalis；Loa loa filaria；Mansonella streptocerca；Onchocerca volvulus；Strongyloides stercoralis；Thelazia californiensis,Thelazia callipaeda；Toxocara canis,Toxocara cati；Trichinella spiralis,Trichinella britovi,Trichinella nelsoni,Trichinella nativa；Trichuris trichiura,Trichuris vulpis；Wuchereria bancrofti；Archiacanthocephala,Moniliformis moniliformis；Linguatula serrata；Oestroidea；Calliphoridae,Sarcophagidae；Cochliomyia hominivorax；Tunga penetrans；Cimex lectularius；Dermatobia hominis；Pediculus humanus；Pediculus humanus corporis；Pthirus pubis；Demodex folliculorum/brevis/canis；Sarcoptes scabiei；Trombiculidae；Pulex irritans；Ixodidae和Argasidae。
本文所用的术语“自身免疫性疾病”定义为由自身免疫应答引起的病症。自身免疫性疾病是对自身抗原的不适当和过度反应的结果。自身免疫性疾病的实例包括但不限于阑尾炎、秃头症、强直性脊柱炎、自身免疫性肝炎、自身免疫性腮腺炎、克罗恩病、糖尿病(I型)、营养不良的大疱性表皮松解症、附睾炎、肾小球性肾炎、格雷夫斯病、吉兰巴尔综合征、桥本病、溶血性贫血、系统性红斑狼疮、多发性硬化症、重症肌无力、寻常型天疱疮、牛皮癣、风湿热、类风湿关节炎、结节病、硬皮病、干燥综合征、脊柱关节病、甲状腺炎、血管炎、白癜风、粘液性水肿、恶性贫血、溃疡性结肠炎等。
“耐受”或“免疫耐受”是免疫系统对特定抗原产生防御性免疫应答的失败。耐受性可以是天然的或自身的，其中身体不会攻击其自身的蛋白质和抗原，或者可以通过免疫系统的操作而诱导。中枢耐受发生在淋巴细胞发育过程中，并在胸腺和骨髓中起作用。在此过程中，识别自身抗原的T淋巴细胞和B淋巴细胞在发育成完全免疫活性细胞之前被缺失。这个过程在胎儿发育过程中是最活跃的，但是随着未成熟的淋巴细胞的生成而持续终生。外周T细胞耐受性是指存在于外周组织中的自身抗原的功能性无反应性，并且在T和B细胞成熟并进入周边后发生。这些过程包括通过“调节性”T细胞抑制自身反应性细胞，以及在没有伴随炎症的共刺激信号的情况下遇到抗原的淋巴细胞中产生低反应性(无反应性)。“获得性”或“诱导耐受性”是指免疫系统对外部抗原的适应，其特征在于淋巴组织与给定抗原的特异性非反应性，在其他情况下可能诱导细胞介导的或体液免疫。在成年人中，可以通过重复施用非常大剂量的抗原或低于刺激免疫应答所需阈值的小剂量(例如通过静脉内或舌下施用可溶性抗原)来临床诱导耐受性。诱导免疫耐受性形成的抗原称为耐受原(Tolerogen)。免疫抑制也有利于诱导耐受。自我耐受的破坏可导致自身免疫。
非自身抗原的免疫识别通常使来自相同物种(同种异体移植物)的生物的外来组织的移植和移植复杂化，导致移植排斥。淋巴细胞，特别是T淋巴细胞，在同种异体移植排斥、移植失败和GVHD中起关键作用。通常有两种情况可以接受同种异体移植。一个是当细胞或组织移植到从免疫监视系统分离的免疫豁免位点(如在眼睛或睾丸中)时，或具有强的分子信号以防止危险的炎症(如在脑中)。第二种情况是当已经诱导出耐受状态时，无论是先前以致使免疫耐受而不是接受者致敏的方式暴露于供体的抗原，或者在慢性排斥之后。成功的同种异体移植需要针对同种异体抗原生出一定程度的免疫耐受性。免疫耐受性的实现可以防止导致移植排斥和失败的宿主抗移植物反应，并防止移植物抗宿主反应(GVHD)。
本文所用的术语“增强免疫应答细胞功能”包括例如增强T细胞功能。以T细胞为例，增强T细胞功能包括诱导、引起或刺激T细胞使其具有持续的或增强的生物功能，或更新或重新激活耗竭的(exhausted)或非活性的T细胞。增强T细胞功能的实例包括：相对于干预前水平，CD8+T细胞分泌的干扰素增加、增殖增加、抗原反应性(例如病毒或病原体清除率)增加。在一个实施方案中，增强水平至少为50％，或者60％，70％，80％，90％，100％，120％，150％，200％。测量这种增强的方式是本领域普通技术人员已知的。
本文所用的术语“T细胞功能障碍性疾病”包括以抗原刺激反应性降低为特征的T细胞的病症或病症。在一些实施方案中，所述T细胞功能障碍性病症是与通过PD-1的不适当增加的信号传导特异性相关的病症。在一些实施方案中，T细胞功能障碍性疾病是其中T细胞是无能的或分泌细胞因子、增殖或执行溶细胞活性的能力降低的疾病。以T细胞功能障碍为特征的T细胞功能障碍性疾病的实例包括未吸收的急性感染、慢性感染和肿瘤免疫。
本文所用的术语“外源性”指的是一个核酸分子或多肽，其在细胞内没有内源性表达，或表达水平不足以实现过表达时具有的功能。因而，“外源性”包括在细胞内表达的重组核酸分子或多肽，如外源性、异源性和过表达的核酸分子和多肽。
本文所用的术语“受体”是指一种多肽，或其部分，其在细胞膜上选择性地结合一个或多个配体。
在一些实施方案中，本发明的结合抗原的受体与能够与之结合的抗原特异性结合。
在一些实施方案中，本发明的结合抗原的受体是嵌合抗原受体。本文所用的术语“嵌合抗原受体(Chimeric Antigen Receptor，CAR)”指一种融合到细胞内信号转导域的肿瘤抗原结合结构域，能激活T细胞。常见地，CAR的胞外结合结构域来源于小鼠或人源化或人的单克隆抗体。
嵌合抗原受体通常包含胞外抗原结合区或者抗原结合单元。在一些实施方案中，胞外抗原结合区可以是完全人的。在其他情况下，胞外抗原结合区域可以被人源化。在其他情况下，胞外抗原结合区可以是鼠源的，或者所述胞外抗原结合区中的嵌合体由来自至少两种不同动物的氨基酸序列组成。在一些实施方案中，所述胞外抗原结合区可以是非人的。
可以设计多种抗原结合区域。非限制性实例包括衍生自抗体的单链可变片段(scFv)、选自文库的片段抗原结合区(Fab)、单结构域片段或与接合其同源受体的自然配体。在一些实施方案中，胞外抗原结合区域可以包含scFv、Fab或天然配体，以及它们的任何衍生物。胞外抗原结合区可以指除完整抗体之外的分子，其可以包含完整抗体的一部分并且可以与完整抗体所结合的抗原结合。抗体片段的实例可以包括但不限于Fv、Fab、Fab'、Fab'-SH、F(ab')2；双功能抗体、线性抗体；单链抗体分子(例如scFv)；和由抗体片段形成的多特异性抗体。
胞外抗原结合区，例如scFv、Fab或天然配体，可以是确定抗原特异性的CAR的一部分。胞外抗原结合区可以结合任何互补靶。胞外抗原结合区可以衍生自已知 可变区序列的抗体。胞外抗原结合区可以从获自可获得的小鼠杂交瘤的抗体序列中得到。或者，可以从肿瘤细胞或原代细胞例如肿瘤浸润淋巴细胞(TIL)的全外切割测序获得胞外抗原结合区。
在一些实施方案中，胞外抗原结合区的结合特异性可以通过互补决定区或CDR，如轻链CDR或重链CDR来确定。在许多情况下，结合特异性可以通过轻链CDR和重链CDR来确定。与其他参考抗原相比，给定的重链CDR和轻链CDR的组合可以提供给定的结合袋，其可以赋予抗原(例如GPC3)更大的亲和力和/或特异性。例如，特异于磷脂酰肌醇蛋白聚糖-3的CDR可以在CAR的细胞外结合区域中表达，使得靶向GPC3的CAR可以将免疫应答细胞靶向表达GPC3的肿瘤细胞。
在本文公开的任何实施方案的某些方面，胞外抗原结合区，例如scFv可以包含对抗原特异性的轻链CDR。轻链CDR可以是抗原结合单元例如CAR的scFv轻链的互补决定区。轻链CDR可以包含连续的氨基酸残基序列，或由非互补决定区(例如框架区)隔开的两个或更多个连续的氨基酸残基序列，。在一些实施方案中，轻链CDR可以包含两个或更多个轻链CDR，其可以被称为轻链CDR-1，CDR-2等。在一些实施方案中，轻链CDR可以包含三个轻链CDR，其可分别称为轻链CDR-1，轻链CDR-2和轻链CDR-3。在一些实例中，存在于普通轻链上的一组CDR可统称为轻链CDR。
在本文公开的任何实施方案的某些方面，胞外抗原结合区，例如scFv可以包含对抗原特异的重链CDR。重链CDR可以是抗原结合单元例如scFv的重链互补决定区。重链CDR可以包含氨基酸残基的连续序列，或由非互补决定区(例如框架区)隔开的两个或更多个氨基酸残基的连续序列。在一些实施方案中，重链CDR可以包含两个或更多个重链CDR，其可以称为重链CDR-1，CDR-2等。在一些实施方案中，重链CDR可以包含三个重链CDR，其可分别称为重链CDR-1，重链CDR-2和重链CDR-3。在一些实施方案中，存在于共同重链上的一组CDR可统称为重链CDR。
通过使用基因工程，可以以各种方式修饰胞外抗原结合区。在一些实施方案中，可以突变胞外抗原结合区域，从而可以选择胞外抗原结合区域以对其靶标具有更高的亲和力。在一些实施方案中，胞外抗原结合区域对其靶标的亲和力可针对可在正常组织上以低水平表达的靶标进行优化。可以进行此优化，以尽量减少潜在的毒性。在其他情况下，对靶标的膜结合形式具有更高亲和力的胞外抗原结合区域的克隆可 以优于其可溶形式的对应物。可以进行这种修饰，因为也可以检测到不同水平的可溶形式的靶标，并且它们的靶向可引起不期望的毒性。
在一些实施方案中，胞外抗原结合区域包括铰链或间隔区。术语铰链和间隔区可以互换使用。铰链可以被认为是用于向胞外抗原结合区提供柔性的CAR的一部分。在一些实施方案中，铰链可用于检测细胞的细胞表面上的CAR，特别是当检测胞外抗原结合区的抗体不起作用或可用时。例如，衍生自免疫球蛋白的铰链的长度可能需要优化，这取决于胞外抗原结合区域靶向靶上的表位的位置。
在一些实施方案中，铰链可能不属于免疫球蛋白，而是属于另一种分子，如CD8α分子的天然铰链。CD8α铰链可以含有已知在CD8辅助受体和MHC分子的相互作用中起作用的半胱氨酸和脯氨酸残基。所述半胱氨酸和脯氨酸残基可影响所述CAR的性能。
CAR铰链可以是尺寸可调的。免疫应答细胞和靶细胞之间的免疫突触的这种形貌还限定了由于细胞表面靶分子上的膜远端表位而不能由CAR进行功能桥接的距离，即使用短铰链CAR也不能使突触距离达到信号能够传导的近似值。同样，膜近端CAR靶抗原表位仅在长铰链CAR的背景下观察到信号输出。可以根据所使用的胞外抗原结合区域来调节铰链。铰链可以是任何长度的。
跨膜结构域可以将CAR锚定在细胞的质膜上。CD28的天然跨膜部分可用于CAR。在其他情况下，也可以在CAR中使用CD8α的天然跨膜部分。“CD8”可以是与NCBI参考号：NP_001759或其具有刺激活性的片段具有至少85、90、95、96、97、98、99或100％同一性的蛋白质。“CD8核酸分子”可以是编码CD8多肽的多核苷酸，在某些情况下，跨膜区可以是CD28的天然跨膜部分，“CD28”可以指与NCBI参考号：NP_006130或其具有刺激活性的片段具有至少85、90、95、96、97、98、99或100％同一性的蛋白质。“CD28核酸分子”可以是编码CD28多肽的多核苷酸。在一些实施方案中，跨膜部分可以包含CD8α区域。
CAR的(细)胞内信号传导区域可以负责活化CAR已经置于其中的免疫应答细胞的效应子功能中的至少一种。CAR可以诱导T细胞的效应子功能，例如，所述效应子功能是细胞溶解活性或辅助活性，包括细胞因子的分泌。因此，术语细胞内信号传导区域是指转导效应子功能信号并引导细胞进行特异功能的蛋白质部分。虽然通常可以使用整个细胞内信号传导区域，但是在许多情况下，不必使用信号结构域的整个链。在一些实施方案中，使用细胞内信号传导区的截短部分。在一些实施方案中，术语细胞内信号传导区域因此意在包括足以转导效应子功能信号的细胞 内信号传导区的任何截短部分。
在CAR中使用的信号结构域的优选实例可以包括T细胞受体(TCR)的细胞质序列和协同作用以在靶-受体结合之后启动信号转导的共同受体，以及它们的任何衍生物或变体序列和这些序列的具有相同功能性的任何合成序列。
在一些实施方案中，所述细胞内信号传导区域可以含有已知的免疫受体酪氨酸激活基序(ITAM)的信号基序。含有细胞质信号传导序列的ITAM的实例包括衍生自TCRζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b和CD66d的那些。然而，在优选的实施方案中，细胞内信号结构域衍生自CD3ζ链。
含有一个或多个ITAM基序的T细胞信号结构域的实例是CD3ζ结构域，也称为T细胞受体T3ζ链或CD247。该结构域是T细胞受体-CD3复合物的一部分，并且在将几种细胞内信号转导途径的抗原识别与T细胞的主效应激活相结合方面起重要作用。如本文所用，CD3ζ主要是指人类CD3ζ及其同种型，如从Swissprot条目P20963所知的，包括具有基本相同序列的蛋白质。作为嵌合抗原受体的一部分，再次重申，不需要全T细胞受体T3ζ链，并且其包含T细胞受体T3ζ链的信号结构域的任何衍生物都是合适的，包括其任何功能等同物。
细胞内信号传导结构域可以选自表1的任何一个结构域。在一些实施方案中，可以修饰结构域，使得与参考结构域的同一性可以为约50％至约100％。可以修饰表1的任何一个结构域，使得修饰形式可以包含约50、60、70、80、90、95、96、97、98、99或至多约100％的同一性。
CAR的细胞内信号传导区可以进一步包含一个或多个共刺激结构域。细胞内信号传导区可以包含单个共刺激结构域，例如ζ链(第一代CAR)或其与CD28或4-1BB(第二代CAR)。在其他实例中，细胞内信号传导区可以包含两个共刺激结构域，例如CD28/OX40或CD28/4-1BB(第三代)。
与细胞内信号结构域如CD8一起，这些共刺激结构域可以产生激酶途径的下游激活，从而支持基因转录和功能性细胞反应。CAR的共刺激结构域可以激活与CD28(磷脂酰肌醇-4,5-二磷酸3-激酶)或4-1BB/OX40(TNF-受体相关因子衔接蛋白)途径以及MAPK和Akt激活相关的近端信号蛋白。
在某些情况下，通过CAR产生的信号可能与辅助或共刺激信号相结合。对于共刺激信号结构域，嵌合抗原受体样复合物可被设计成包含若干可能的共刺激信号结构域。如本领域众所周知的，在幼稚T细胞中，T细胞受体的单独接合不足以诱 导T细胞的完全活化为细胞毒性T细胞。完整的生产性T细胞激活需要第二共刺激信号。已经报道对T细胞活化提供共刺激的几种受体，包括但不限于CD28、OX40、CD27、CD2、CD5、ICAM-1、LFA-1(CD11a/CD18)、4-1BBL、MyD88和4-1BB。这些共刺激分子使用的信号传导途径均能与主T细胞受体激活信号协同作用。这些共刺激信号传导区域提供的信号可以与源自一个或多个ITAM基序(例如CD3zeta信号转导域)的主效应激活信号协同作用，并且可以完成T细胞激活的要求。
在一些实施方案中，向嵌合抗原受体样复合物添加共刺激结构域可以增强工程细胞的功效和耐久性。在另一个实施方案中，T细胞信号结构域和共刺激结构域彼此融合从而构成信号传导区。
表1.共刺激结构域
Figure PCTCN2017082024-appb-000001
Figure PCTCN2017082024-appb-000002
嵌合抗原受体结合靶抗原。当在体外或离体测定T细胞活化时，可以从各种来源获得或分离靶抗原。本文使用的靶抗原是抗原或抗原上的免疫表位，其在哺乳动物中对于免疫识别和最终消除或控制致病因素或疾病状态中至关重要。免疫识别可以是细胞和/或体液。在细胞内病原体和癌症的情况下，免疫识别可以是例如T淋巴细胞反应。
靶抗原可以衍生或分离自例如病毒性微生物例如本文前述病毒的抗原。在一些实施方案中，本发明的嵌合抗原受体结合例如包括HIV(Korber等，eds HIV Molecular Immunology Database，Los Alamos National Laboratory，Los Alamos，N.Mex.1977)、流感，疱疹、单纯疱疹人乳头状瘤病毒(美国专利号5,719,054)、乙型肝炎(美国专利号5,780,036)、丙型肝炎(美国专利号5,709,995)、EBV、巨细胞病毒(CMV)的病毒等。
靶抗原还可以衍生自或从本文前述的病原细菌分离。在一些实施方案中，本发明的嵌合抗原受体结合例如来自衣原体(美国专利号5,869,608)、分枝杆菌、军团菌、脑膜炎、A组链球菌、沙门氏菌、李斯特菌、流感嗜血杆菌(美国专利号5,955,596)等的抗原。
在一些实施方案中，靶抗原可以衍生或分离自例如包括曲霉属(Aspergillus)、侵入性假丝酵母属(美国专利号5,645,992)、诺卡氏菌属、组织胞浆菌病、隐孢子虫病等的病原性酵母。
在一些实施方案中，靶抗原可以衍生或分离自例如致病性原生动物和病原性寄生虫，包括但不限于卡氏肺囊虫、锥虫病、利什曼原虫(美国专利号5,965,242)、疟原虫(美国专利号5,589,343)和弓形虫(Asxoplasma gondii)等。
在一些实施方案中，靶抗原包括与癌前或增生状态相关的抗原。靶抗原也可能与癌症相关或起因于癌症。例如，在一些实施方案中，本发明的嵌合抗原受体识别并结合包括本文前述的TSA和TAA的肿瘤抗原。
本文所用的术语“调节”是指正向或负向改变。调节范例包括1％、2％、10％、25％、50％、75％、或100％变化。
本文所用的术语“治疗”是指在试图改变个人或处理细胞引起的的疾病过程中 的临床干预，既可以进行预防也可以在临床病理过程干预。治疗效果包括但不限于，防止疾病的发生或复发、减轻症状、减少任何疾病直接或间接的病理后果、防止转移、减慢疾病的进展速度、改善或缓解病情、缓解或改善预后等。
本文所用的术语“免疫功能低下”是指受试者具有免疫缺陷，其很容易被感染。引起机会感染的生物体通常情况下不会导致具有健康免疫系统生病，但能感染免疫系统功能低下或免疫系统受抑制的人。
本文所用的术语“组成性表达”是指在所有的生理条件下的表达。
本文所用的术语“诱导表达”是指在一定条件下的表达，所述的条件例如T细胞与抗原发生结合的时候。本领域技术人员如何进行常规的“诱导表达”。
在一些实施方案中，本发明提供了一种免疫应答细胞，该细胞表达结合抗原的受体和外源性I型干扰素。
在一些实施方案中，本发明所述的免疫应答细胞可以靶向在癌症上表达的抗原。其抗原或表位可以在癌症或癌症相关组织上表达。在一些情况下，靶抗原可能在癌症上过表达，并且在正常组织上具有减少或无表达。在某些情况下，癌症的特异性抗原及其表位可以用本发明的免疫应答细胞靶向。抗原可以衍生自各种各样的肿瘤抗原，例如由突变产生的肿瘤抗原、共享的肿瘤特异性抗原、分化抗原和在肿瘤中过表达的抗原。仅仅举若干实例，可以被本发明所述的免疫应答细胞靶向或者结合的抗原可是是或者来源于，包括但不限于：叶酸受体α,707-AP,adipophilin,AFP,AIM-2,ALDH1A1,Annexin II,ART-4,ARTC1,BAGE,BAGE-1,BCLX(L),BCMA,BING-4,BRACHYURY(IVS7T/C多态性),BRACHYURY(TIVS7-2,多态性),BRACHYURY,B-RAF,CAMEL,CAR-ABL融合蛋白(b3a2),CASP-5,CASP-8,Cdc27,CDC27/m,CDK4,CDK-4/m,CDKN2A,CEA,COA-1,CPSF,Cyp-B,DAM-6,-10,dek-can融合蛋白,DKK1,EFTUD2,EGFR,ELF2M,ENAH(hMena),EP-CAM,EphA3,ESO-1/LAGE-2,ETV6-AML1融合蛋白,ETV6/AML,EZH2,FGF5,FLT3-ITD,FN1,G250,G250/MN/CAIX,Gage 3,4,5,6,7,GAGE-1,2,8,GAGE-3,-4,-5,-6,-7B,GnT-V,GnTVf,Gp100,gp100/Pmel17,GPC3,GPNMB,Her2/neu,Her3,HERV-K-MEL,HLA-A1ld,HLA-A2d,HPV E6,HPV E7,hsp70-2,HSP70-2M,HST-2,hTERT,hTRT,iCE,IL13Rα2,KIAA0205,KK-LC-1,KM-HN-1,K-ras,LDLR/FUT,LDLR-岩藻糖基转移酶融合蛋白,MAGE-A1,MAGE-A10,MAGE-A12,MAGE-A2,MAGE-A3,MAGE-A4,MAGE-A6,MAGE-A9,MAGE-C2,MART-1,MART2,MC1R,M-CSFT,MCSP,mdm-2,ME1,Melan-A/MART-1,MMP-2,MUC1(VNTR多 态性)-c,MUC1,MUC1-n,MUC2,MUM-1,MUM-1f,MUM-2,MUM-3,NA-88,NA88-A,NFYC,N-ras,NY-BR-1,NY-ESO-1,OA1,OGT,OS-9,P15,p53,PAP,PBF,Pml/RARα和TEL/AML1pml-RARα融合蛋白,PRAME,PRDX5,PSA,PSCA,PSMA,PTPRK,RAB38/NY-MEL-1,RAGE,RAGE-1,RBAF600,RGS5,RNF43,RU1,RU2,RU2AS,SAGE,SART-1,SART-2,SART-3,secernin 1,SIRT2,SNRPD1,SOX10,Sp17,SSX-2,SSX-4,STEAP,STEAP1,SYT-SSX1-或-SSX2融合蛋白质,TBRACHYURY,T,TAG-1,TAG-2,TGF-βRII,TPI/mbcr-abl,TRAG-3,TRP-2,TRP-1,TRP-1/gp75,TRP-2,TRP-2/INT2,TRP2-INT2g,VEGF和/或WT1,XAGE-1b,α-辅肌动蛋白-4,β-连环蛋白,β-连环蛋白/m,三磷酸异构酶,乳腺球蛋白-A,人乳头瘤病毒(HPV),人表皮生长因子受体2(HER2/neu),人表皮生长因子受体3(HER3),伸长因子2,前列腺特异性抗原(PSA),存活蛋白,新PAP,激肽释放酶4,甲胎蛋白,端粒酶,粘蛋白,细胞周期蛋白D1,肌球蛋白/m,肌球蛋白I,肠羧酸酯酶,胱天蛋白酶-8/m,酪氨酸酶。
此外，本发明的免疫应答细胞可以靶向肿瘤相关抗原。肿瘤相关抗原可以是宿主不正常表达的抗原，它们可能被宿主表达的分子突变，截短，错误折叠或其他异常表现；它们可以与通常表达的分子相同但却以异常高水平表达；或者它们可以在异常的环境中表达。肿瘤相关抗原可以是例如蛋白质或蛋白质片段，复合碳水化合物，神经节苷脂，半抗原，核酸，其他生物分子或其任何组合。在一些情况下，抗原可以是新抗原(neo-antigen)。新抗原可以衍生自癌细胞的体细胞突变。例如，新抗原可以是三磷酸异构酶(TPI)的突变形式。突变的纤连蛋白(FN)是可以用本发明的免疫应答细胞靶向的新抗原的另一个实例。新抗原可以通过筛选平台进行鉴定，例如生物化学，全外切割测序，遗传靶向表达(GTE)或其组合。
在某些情况下，可以被本发明的免疫应答细胞结合的靶标可能与癌症基质相关联。癌基质可能与肿瘤微环境相关。抗原可以是基质抗原。举例而言，基质抗原和表位可以存在于包括但不限于肿瘤内皮细胞、肿瘤脉管系统、肿瘤成纤维细胞、肿瘤周细胞、肿瘤基质和/或肿瘤间充质细胞上。那些抗原例如可以选自CD34、MCSP、FAP、CD31、PCNA、CD117、CD40、MMP4和/或腱生蛋白。
可以通过免疫组织化学(IHC)分析和/或流式细胞术来测量抗原的组织表达。组织表达也可以通过定量PCT(qPCR)获得的拷贝数来测量。在一些情况下，靶抗原可以在癌细胞的表面表达。在某些情况下，可被靶向的抗原可能不会在MHC或HLA的环境中表达。本发明的免疫应答细胞可以以非MHC限制的方式靶向细 胞表面抗原。在一些情况下，与其在正常组织上的表达相比，可以用CAR-T靶向的抗原可能过表达。通过IHC、qPCR或流式细胞术来测量，过表达可以是在正常组织上的表达的约1倍、2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍、20倍、30倍、40倍、50倍、60倍、70倍、80倍、90倍或至多100倍。
在一些实施方案中，所述的结合抗原的受体包含抗原结合结构域(胞外结合区)与能活化免疫应答细胞的胞内信号结构域。较佳地，在抗原结合结构域与胞内信号结构域(胞内信号区)之间，还包括跨膜区。作为一种实施方式，所述胞外结合区包含针对抗原的抗体，所述抗原是肿瘤抗原或病原体抗原。将该结合抗原的受体表达于免疫应答细胞的表面，可使得免疫应答细胞对表达该抗原的肿瘤细胞或病原体具有高度特异性的细胞毒性作用。
在一些实施方案中，本发明所述的结合抗原的受体中，包含的抗体为单链抗体，其与跨膜区相连接，跨膜区后紧接胞内信号区。
在一些实施方案中，本发明所述的抗原结合结构域是结合肿瘤抗原的结合域。在一些实施方案中，所述肿瘤抗原是分化抗原选自MART-1/MelanA(MART-I)、gp100(Pmel17)、酪氨酸酶、TRP-1、TRP-2和肿瘤特异性多中心抗原如MAGE-1、MAGE-3、BAGE、GAGE-1、GAGE-2、p15；过表达的胚胎抗原如CEA；过表达的癌基因和突变的肿瘤抑制基因如p53、Ras、HER-2/neu；由染色体易位引起的独特的肿瘤抗原，例如BCR-ABL、E2A-PRL、H4-RET、IGH-IGK、和MYL-RAR；以及病毒抗原，如爱泼斯坦巴尔病毒抗原EBVA和人乳头瘤病毒(HPV)抗原E6和E7等。其他大的、基于蛋白质的抗原包括TSP-180、MAGE-4、MAGE-5、MAGE-6、RAGE、NY-ESO、p185erbB2、p180erbB-3、c-met、nm-23H1、PSA、TAG-72、CA 19-9、CA 72-4、CAM 17.1、NuMa、K-ras、β-联蛋白、CDK4、Mum-1、p 15、p 16、43-9F、5T4、791Tgp72、甲胎蛋白、beta-HCG、BCA225、BTAA、CA 125、CA 15-3\CA 27.29\BCAA、CA 195、CA 242、CA-50、CAM43、CD68\P1、CO-029、FGF-5、G250、Ga733\EpCAM、HTgp-175、M344、MA-50、MG7-Ag、MOV18、NB/70K、NY-CO-1、RCAS1、SDCCAG16、TA-90\Mac-2结合蛋白\亲环蛋白C相关蛋白、TAAL6、TAG72、TLP、以及TPS中的一种或多种。在一些实施方案中，所述肿瘤抗原选自前列腺特异性膜抗原(PSMA)、癌胚抗原(CEA)、IL13Ralpha、HER-2、CD19、NY-ESO-1、HIV-1Gag、Lewis Y、MART-1、gp100、酪氨酸酶、WT-I、hTERT、间皮素、EGFR、EGFRvIII、磷脂酰肌醇蛋白聚糖3、EphA2、HER3、EpCAM、MUC1、MUC16、CLDN18.2、叶酸受体、CLDN6、CD30、CD138、ASGPR1、 CDH16、GD2、5T4、8H9、αvβ6整合素、B细胞成熟抗原(BCMA)、B7-H3、B7-H6、CAIX、CA9、CD20、CD22、κ轻链(kappa light chain)、CD33、CD38、CD44、CD44v6、CD44v7/8、CD70、CD123、CD171、CSPG4、EGP2、EGP40、ERBB3、ERBB4、ErbB3/4、FAP、FAR、FBP、胚胎型AchR、GD2、GD3、HLA-AIMAGE A1、MAGE3、HLA-A2、IL11Ra、KDR、Lambda、MCSP、NCAM、NKG2D配体、PRAME、PSCA、PSC1、ROR1、Sp17、SURVIVIN、TAG72、TEM1、TEM8、VEGRR2、HMW-MAA、VEGF受体、和/或纤连蛋白、腱生蛋白或肿瘤坏死区的癌胚变体中的一种或多种。在一些实施方案中，所述肿瘤抗原选自前列腺特异性膜抗原、癌胚抗原、IL13Ralpha，HER-2，CD19，NY-ESO-1，HIV-1Gag，Lewis Y，MART-1，gp100，酪氨酸酶，WT-I，hTERT，间皮素，EGFR、EGFRvIII，磷脂酰肌醇蛋白聚糖3、EphA2，HER3，EpCAM，MUC1，MUC16，claudin 18.2，叶酸受体，claudin 6、CD30、CD138、MAGE3、ASGPR1和CDH16中的一种或多种。
在一些实施方案中，所述结合抗原的受体的跨膜区可以选自CD8或CD28等蛋白的跨膜区。人CD8蛋白是个异二聚体，由αβ或者γδ两条链组成。在一些实施方案中，跨膜区选自CD8α或者CD28的跨膜区。此外，CD8α铰链区(hinge)是一个柔性区域，因此，CD8或CD28和跨膜区加上铰链区被用于将结合抗原的受体CAR的靶点识别结构域scFv和胞内信号区连接起来。
本发明的胞内信号结构域可以选自CD3ζ、FcεRIγ、CD28共刺激信号结构域、CD137共刺激信号结构域、及其组合。CD3分子由五个亚单位组成，其中CD3ζ亚单位(又称CD3zeta，简称Z)含有3个ITAM基序，该基序是TCR-CD3复合体中重要的信号转导区。此外，如前所述，CD28和CD137是共刺激信号分子，在与各自配体结合后其胞内信号区段产生的共刺激作用引起免疫应答细胞(主要是T淋巴细胞)的持续增殖，并能够提高免疫应答细胞分泌IL-2和IFN-γ等细胞因子的水平，提高CAR免疫应答细胞在体内的存活周期和抗肿瘤效果。在一些实施方案中，所述的细胞内的信号转导域是CD3ζ信号结构域或CD3ζ信号结构域与其它共刺激信号如CD28的组合。
在一些实施方案中，本发明的免疫应答细胞中可以包括表达构建物，该表达构建物中存在按如下方式顺序连接的元件：抗体、CD28共刺激信号结构域、CD3ζ，以及与前述元件反向连接的NFAT6、I型干扰素表达单元。较佳地，所述的抗体与CD28共刺激信号结构域之间通过CD8α跨膜区和CD8α铰链区相连。
在一些实施方案中，活化T细胞核因子NFAT(Nuclear factor of activated T cells) 在T细胞活化过程中细胞因子的转录表达起着重要的作用。基于这样考虑，本发明人将IFN-beta编码序列置于NFAT6启动子的调控之下，这样只有当CAR-T细胞接触抗原引发T细胞活化，IFN-beta才能高水平表达。
NFAT6启动子是利用6个NFAT的结合位子与IL2的最小启动子(minimal promoter)串联在一起组成的启动子(Hooijberg E,Bakker AQ,Ruizendaal JJ,Spits H.NFAT-controlled expression of GFP permits visualization and isolation of antigen-stimulated primary human Tcells.Blood.2000Jul 15；96(2):459-66)，可用于调节细胞因子如IL12等在T淋巴细胞如TCR-T中的表达(Zhang L,Kerkar SP,Yu Z,Zheng Z,Yang S,RestifoNP,Rosenberg SA，Morgan RA.Improving adoptive T cell therapy by targeting and controlling IL-12expression to thetumor environment.Mol Ther.2011Apr；19(4):751-9)。
根据本发明的一个方面，本发明也包括编码所述结合抗原的受体的核酸。本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或多肽的片段、类似物和衍生物。
本发明还提供了包含上述编码表达于免疫应答细胞表面的结合抗原的受体蛋白的核酸的载体。在一个具体实施方案中，本发明使用的载体是一种慢病毒质粒载体pRRLSIN-cPPT.PGK-GFP.WPRE。应理解，其它类型的病毒载体以及非病毒载体，也是可以应用的。
本发明还包括包含上述载体的病毒。本发明的病毒包括包装后的具有感染力的病毒，也包括包含包装为具有感染力的病毒所必需成分的待包装的病毒。本领域内已知的其它可用于将外源基因转导入免疫应答细胞的病毒及其对应的质粒载体也可用于本发明。
本发明的免疫应答细胞，其被转导有能表达结合抗原的受体和外源性I型干扰素的构建物，或表达载体，或包含该质粒的病毒。本领域常规的核酸转导方法，包括非病毒和病毒的转导方法都可以用于本发明。
本发明所述的免疫应答细胞还可以进一步携带外源的细胞因子的编码序列；所述的细胞因子包括但不限于：IL-12，IL-15或IL-21等。这些细胞因子具有进一步的免疫调节或抗肿瘤的活性，能增强效应T细胞及活化的NK细胞的功能，或直接发挥抗肿瘤作用。因此，本领域技术人员可以理解，这些细胞因子的运用有助于所述的免疫应答细胞更好地发挥作用。
本发明所述的免疫应答细胞还可以表达除了上述结合抗原的受体以外的另一 种结合抗原的受体。
本发明所述的免疫应答细胞还可以表达趋化因子受体；所述的趋化因子受体包括但不限于CCR2。本领域技术人员可以理解，所述的CCR2趋化因子受体可以使得体内的CCR2与之竞争性结合，对于阻断肿瘤的转移是有利的。
本发明所述的免疫应答细胞还可以表达能降低PD-1表达的siRNA或者阻断PD-L1的蛋白。本领域技术人员可以理解，竞争性阻断PD-L1与其受体PD-1的相互作用，有利于恢复抗肿瘤T细胞反应，从而抑制肿瘤生长。
本发明所述的免疫应答细胞还可以表达安全开关；较佳地，所述的安全开关包括：iCaspase-9，Truancated EGFR或RQR8。
在一些实施方案中，本发明的免疫应答细胞不表达诸如4-1BBL这类的共刺激配体。
可以将编码目标结合抗原的受体或CAR的转基因并入细胞中。例如，可将转基因并入免疫应答细胞，例如T细胞。当插入细胞时，转基因可以是互补DNA(cDNA)片段，其是信使RNA(mRNA)的拷贝；或者位于其基因组DNA原始区域的基因本身(含或不含内含子)。
编码转基因序列的核酸如DNA可以随机插入细胞的染色体。随机整合可以由将核酸(例如DNA)引入细胞的任何方法产生。例如，该方法可以包括但不限于电穿孔、超声、使用基因枪、脂转染、磷酸钙转染、使用树枝状大分子、显微注射和使用包括腺病毒、AAV和逆转录病毒载体的病毒载体、和/或II型核酶。
编码转基因的DNA也可以设计成包括报告基因，从而可以通过报告基因的活化检测转基因或其表达产物的存在。可以使用任何报道基因，例如上述那些。通过在细胞培养物中选择其中报道基因已经被活化的细胞，可以选择含有转基因的细胞。
CAR的表达可以通过表达测定法，例如qPCR或通过测量RNA的水平来验证。表达水平也可以指示拷贝数。例如，如果表达水平非常高，这可以表明CAR的多于一个拷贝被整合到基因组中。或者，高表达可以指示转基因整合在高转录区域中，例如高度表达的启动子附近。也可以通过测量蛋白质水平来验证表达，例如通过Western印迹。
在一些实施方案中，本发明的免疫应答细胞可以包含一种或多种转基因。所述一种或多种转基因可以表达CAR蛋白，而该CAR蛋白识别并结合抗原上的至少一个表位或结合抗原上的突变表位。CAR可以是功能性CAR。在一些实施方案中本发明的免疫应答细胞可以包含一种或多种CAR，或者其可以包含单一CAR和二 次工程化受体。
在一些实施方案中，转基因可编码自杀基因。如癌症患者的许多有效治疗所证明的，CAR免疫应答细胞使得肿瘤消退但可伴随毒性。在一些实施方案中，当靶标抗原在正常组织和肿瘤细胞中共享时，CAR免疫应答细胞可能不能区分肿瘤和正常组织(“在靶/脱靶毒性”)。在另一些情况下，可以发生免疫系统的全身扰动，称为细胞因子释放综合征(CRS)。所述CRS可以包含全身炎症反应综合征或细胞因子风暴，这可能是CAR免疫应答细胞体内快速膨胀的后果。CRS是以发热和低血压为特征的病症，严重者可导致多器官功能衰竭。在大多数情况下，所述毒性与输注的CAR免疫应答细胞的体内扩增相关，其可引起免疫系统的整体扰动，以及释放高水平的促炎细胞因子，例如TNFα和IL-6。自杀基因可以诱导消除CAR免疫反应性细胞。自杀基因可以是在所述CAR免疫反应性细胞中诱导细胞凋亡的任何基因。自杀基因可以与所述结合抗原的受体一起编码在病毒载体内。编码自杀基因使得在特定的情况下可以缓解或者彻底中止由输注的CAR免疫应答细胞在体内扩增引起的毒性。
在一些实施方案中，可以产生存在于正常组织的抗原的CAR免疫反应性细胞，使得它们瞬时表达CAR，例如在电穿孔编码受体的mRNA之后。此外，通过包括安全开关来进一步加强CAR免疫反应性细胞的重大努力，在严重的在靶毒性的情况下，可以大大消除CAR免疫反应性细胞。编码CAR的载体可以与诸如可诱导的半胱天冬酶-9基因(由二聚化学诱导剂激活)或截短形式的EGF受体R(由单克隆抗体西妥昔单抗激活)或RQR8的安全开关组合。
本文所用的一个或多个转基因可以来自不同的物种。例如，一个或多个转基因可以包含人基因、小鼠基因、大鼠基因、猪基因、牛基因、狗基因、猫基因、猴基因、黑猩猩基因或其任何组合。例如，转基因可以来自具有人类遗传序列的人。一个或多个转基因可以包含人基因。在某些情况下，一个或多个转基因不是腺病毒基因。
如上所述，转基因可以以随机或位点特异性方式插入免疫反应性细胞的基因组中。例如，可以将转基因插入到免疫细胞的基因组中的随机位点。这些转基因可以是功能性的，例如，在插入到基因组中的任何地方中时是完全功能性的。例如，转基因可以编码其自身的启动子，或者可以插入其内部启动子控制的位置。或者，可以将转基因插入基因，例如基因的内含子或基因的外显子、启动子或非编码区。可以插入转基因使得插入破坏基因，例如内源性免疫检查点。
在一些实施方案中，一个以上拷贝的转基因可以插入到基因组内的多个随机位点。例如，可将多个拷贝插入基因组中的随机位点。与转基因随机插入一次相比，这可能导致整体表达增加。或者，转基因的拷贝可以插入到基因中，转基因的另一拷贝可以插入到不同的基因中。可以靶向转基因，使其可以插入到免疫反应性细胞的基因组中的特定位点。
在一些实施方案中，包含编码结合抗原的受体序列的多核酸可以采取质粒载体的形式。质粒载体可以包含启动子。在某些情况下，启动子可以是组成型的。在一些实施方案中，启动子是可诱导的。启动子可以是或可以衍生自CMV、U6、MND或EF1a。在一些实施方案中，启动子可以与CAR序列相邻。在一些实施方案中，质粒载体还包含剪接受体。在一些实施方案中，剪接受体可以与CAR序列相邻。启动子序列可以是PKG或MND启动子。MND启动子可以是含有骨髓增生性肉瘤病毒增强子修饰的MoMuLV LTR的U3区域的合成启动子。
在一些实施方案中，可以设计编码目标受体的多核酸，以通过非病毒技术递送至细胞。在某些情况下，多核酸可以是良好的制造规范(GMP)兼容试剂。
编码目标结合抗原的受体或CAR的多核酸的表达可以由一种或多种启动子控制。启动子可以是普遍存在的，组成型(不受限制的启动子，允许相关基因的连续转录)，组织特异性启动子或诱导型启动子。可以调节插入邻近或接近启动子的转基因的表达。例如，转基因可插入到普遍存在的启动子附近或旁边。一些普遍存在的启动子可以是CAGGS启动子、hCMV启动子、PGK启动子、SV40启动子或ROSA26启动子。
启动子可以是内源的或外源的。例如，可以将一个或多个转基因插入到内源或外源ROSA26启动子的邻近或接近处。此外，启动子可以是免疫反应性细胞的特异性。例如，一个或多个转基因可以插入猪ROSA26启动子的邻近或接近。
组织特异性启动子或细胞特异性启动子可用于控制表达的位置。例如，可以将一个或多个转基因插入组织特异性启动子的邻近或接近。组织特异性启动子可以是FABP启动子、Lck启动子、CamKII启动子、CD19启动子、角蛋白启动子、白蛋白启动子、aP2启动子、胰岛素启动子、MCK启动子、MyHC启动子、WAP启动子、或Col2A启动子。
也可以使用诱导型启动子。如果需要，可以通过添加或除去诱导剂来开启和关闭这些诱导型启动子。预期诱导型启动子可以是但不限于Lac、tac、trc、trp、araBAD、phoA、recA、proU、cst-1、tetA、cadA、nar、PL、cspA、T7、VHB、Mx和/或Trex。
本文所用的术语“诱导型启动子”是一种受控的启动子，其在所期待的条件未达成前不表达或者低表达与其可操作地连接的基因，而在所期待的条件达成的情况下表达或者高水平表达与其可操作地连接的基因。例如，在一些实施方中，本申请的诱导型启动子在细胞中的正常或高氧含量的条件下不表达或者低表达与其可操作地连接的基因，而响应于细胞中降低的氧含量，在缺氧条件下表达或者高表达与其可操作地连接的基因。在一些实施方案中，本文所用的诱导型启动子包括低氧可诱导的转录因子-1α(Hypoxia-Inducible Transcription factor-1α,HIF-1α)。在在一些实施方案中，本文所用的术语“诱导型启动子”是指“免疫细胞诱导型启动子”，其在免疫应答细胞接触抗原之前或免疫应答细胞未被激活之时不表达或者低表达与其可操作地连接的基因，而仅在免疫应答细胞接触抗原或免疫应答细胞被激活时，才会驱动与其操作性连接的基因发生高水平表达或者在缺氧等条件下表达。在一些实施方案中，所述的“免疫细胞诱导型启动子”包括NFAT(活化T细胞核因子)型启动子。
本文所用的“NFAT型启动子”是指基于NFAT结合活性进行调控与其可操作地连接的基因的表达的一类启动子。
NFAT是一个在免疫反应中具有重要作用的转录因子家族的统称。NFAT家族的一个或多个成员在免疫系统的大多数细胞中表达。NFAT也参与心脏、骨骼肌和神经系统的发育。
NFAT转录因子家族由五个成员NFAT1、NFAT2、NFAT3、NFAT4和NFAT5组成。NFAT1至NFAT4受钙信号调节。钙信号对NFAT激活至关重要，因为钙调蛋白(CaM)激活丝氨酸/苏氨酸磷酸酶钙调神经磷酸酶(CN)。活化的CN使NFAT蛋白氨基末端的丝氨酸丰富区域(SRR)和SP重复序列快速脱磷酸化，导致构象变化，暴露核定位信号，导致NFAT输入核中。
基于NFAT在T细胞活化过程中细胞因子的转录表达所起的作用，其可用于调控本文所述的免疫细胞诱导型启动子，从而在免疫应答细胞接触抗原活化时，表达或者高水平表达与其可操作地连接的基因。
本发明的核酸可以包含编码NFAT型启动子(或其功能部分或功能变体)的任何合适的核苷酸序列。本文所用的“NFAT型启动子”是指与T细胞表达的任何基因的最小启动子连接的一个或多个NFAT应答元件。优选地，由T细胞表达的基因的最小启动子是最小的人IL-2启动子。NFAT应答元件可以包括例如NFAT1、NFAT2、NFAT3和/或NFAT4应答元件。在一些实施方案中，本文所述的“NFAT 型启动子”中可包括多于1个NFAT结合基序。例如，所述“NFAT型启动子”可包括2、3、4、5、6、7、8、9、10或更多个NFAT结合基序。在一些实施方案中，所述的“NFAT型启动子”中包括多达12个NFAT结合基序。在一些实施方案中，所述“NFAT型启动子”可以是多个所述NFAT结合基序与启动子如IL2最小启动子串联所组成的启动子。在一些实施方案中，本文所述的NFAT型启动子包括6个NFAT结合基序，记作(NFAT)6。出于便利的目的，所述(NFAT)6也记作NFAT6。在一些实施方案中，所述NFAT6也表示在所述NFAT型启动子中的6个重复的NFAT结合基序(SEQ ID NO:78)。
此外，尽管不是表达必需的，但转基因序列还可以包括转录或翻译调控序列，例如启动子、增强子、绝缘体、内部核糖体进入位点、编码2A肽和/或多腺苷酸化信号的序列。
在一些实施方案中，转基因编码目标结合抗原的受体或CAR，其中将转基因插入安全港，使得表达所述结合抗原的受体。在一些实施方案中，将转基因插入PD1和/或CTLA-4基因座。在其他情况下，将转基因以慢病毒递送至细胞随机插入，而PD1-或CTLA-4特异性核酸酶可作为mRNA提供。在一些实施方案中，转基因通过病毒载体系统如逆转录病毒、AAV或腺病毒以及编码对于安全港特异的核酸酶(例如AAVS1、CCR5、白蛋白或HPRT)的mRNA递送。也可以用编码PD1和/或CTLA-4特异性核酸酶的mRNA处理细胞。在一些实施方案中，编码CAR的多核苷酸通过病毒递送系统与编码HPRT特异性核酸酶和PD1-或CTLA-4特异性核酸酶的mRNA一起提供。可以与本文公开的方法和组合物一起使用的CAR可以包括所有类型的这些嵌合蛋白，包括本文前述的第一、第二和第三代设计。
在一些实施方案中，可以使用逆转录病毒载体(γ-逆转录病毒或慢病毒载体)将转基因导入免疫反应性细胞。例如，编码CAR的转基因或结合抗原的任何受体或其变体或片段可被克隆到逆转录病毒载体中，并且可以由其内源性启动子、逆转录病毒长末端重复序列、或对靶细胞类型特异性的启动子驱动。也可以使用非病毒载体。非病毒载体递送系统可以包括DNA质粒、裸核酸和与递送载体如脂质体或泊洛沙姆复合的核酸。
已经开发了许多基于病毒的系统用于将基因转移到哺乳动物细胞中。例如，逆转录病毒为基因递送系统提供了便利的平台。可以使用本领域已知的技术将所选择的基因插入载体并包装在逆转录病毒颗粒中。衍生自逆转录病毒如慢病毒的载体是 实现长期基因转移的合适工具，因为它们允许转基因的长期稳定整合及其在子细胞中的繁殖。慢病毒载体与衍生自逆转录病毒如鼠类白血病病毒的载体相比具有附加优点，因为它们可以转导非增殖细胞。它们还具有低免疫原性的附加优点。腺病毒载体的优点是它们不融合到靶细胞的基因组中，从而绕过负面的整合相关事件。
可以用编码所述结合抗原的受体的转基因转染细胞。转基因浓度可以为约100皮克至约50微克。在一些实施方案中，可以改变引入细胞的核酸(例如，ssDNA、dsDNA或RNA)的量以优化转染效率和/或细胞活力。例如，可以向每个细胞样品中加入1微克dsDNA用于电穿孔。在一些实施方案中，最佳转染效率和/或细胞活力所需的核酸(例如，双链DNA)的量根据细胞类型而不同。在一些实施方案中，用于每个样品的核酸(例如，dsDNA)的量可以直接对应于转染效率和/或细胞活力。例如，一系列转染浓度。由载体编码的转基因可以整合到细胞基因组中。在一些实施方案中，由载体编码的转基因前向整合。在其他情况下，由载体编码的转基因的反向整合。
在一些实施方案中，免疫反应性细胞可以是由CD45RO(-)、CCR7(+)、CD45RA(+)、CD62L+(L-选择素)、CD27+、CD28+和/或IL-7Rα+组成的干记忆TSCM细胞，所述干记忆细胞还可以表达CD95、IL-2Rβ、CXCR3和/或LFA-1，并且显示出与所述干记忆细胞不同的许多功能属性。或者，免疫反应性细胞还可以是包含L-选择素和CCR7的中枢记忆体TCM细胞，其中中枢记忆细胞可以分泌例如IL-2，但不分泌IFNγ或IL-4。免疫反应性细胞还可以是包含L-选择蛋白或CCR7的效应记忆TEM细胞，并产生例如效应细胞因子如IFNγ和IL-4。
通常通过全身给药(例如静脉内、腹膜内、肌内、皮下或颅内输注)或局部应用，通过给予个体患者体内递送载体，如下所述。或者，载体可以离体递送到细胞，例如从个体患者(例如，淋巴细胞、T细胞、骨髓抽吸物、组织活检)移出的细胞，然后通常在选择并入了该载体的细胞后将细胞再植入患者体内。在选择之前或之后，可以扩增细胞。
用于表达结合抗原的受体的合适的免疫反应性细胞可以是对于有需要的个体是自体的或非自体的细胞。
可以从个体获得合适的免疫应答细胞的来源。在某些情况下，可以获得T细胞。所述T细胞可以从许多来源获得，包括PBMC、骨髓、淋巴结组织、脐带血、胸腺组织和来自感染部位、腹水、胸腔积液、脾组织和肿瘤的组织。在某些情况下，可以使用任何数量的本领域技术人员已知的技术，例如FicollTM分离，从自所述个 体收集的血液获得T细胞。在一个实施方案中，通过单采血获得来自个体的循环血液的细胞。单采制品通常含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核白细胞、红细胞和血小板。在一个实施方案中，可以洗涤通过单采采集收集的细胞以除去血浆级分并将细胞置于合适的缓冲液或培养基中用于随后的加工步骤。
或者，可以从健康供体，来自诊断患有癌症的患者或诊断为感染的患者衍生细胞。在一些实施方案中，细胞可以是具有不同表型特征的混合细胞群体的一部分。还可以根据前述方法从转化的T细胞获得细胞系。还可以从细胞治疗库获得细胞。可以通过本文所述的任何方法获得对免疫抑制治疗有抗性的修饰细胞。还可以在修饰前选择合适的细胞群。修饰后也可以选择工程细胞群。工程细胞可用于自体移植。或者，细胞可用于同种异体移植。在一些实施方案中，将细胞施用于样品用于鉴定癌症相关靶序列的同一患者。在其他情况下，将细胞施用于不同于其样本用于鉴定癌症相关靶序列的患者的患者。
在一些实施方案中，合适的原代细胞包括外周血单核细胞(PBMC)、外周血淋巴细胞(PBL)和其它血液细胞亚群，例如但不限于T细胞、天然杀伤细胞、单核细胞、天然杀伤剂T细胞、单核细胞前体细胞、造血干细胞或非多能干细胞。在一些实施方案中，细胞可以是任何免疫细胞，包括任何T细胞如肿瘤浸润细胞(TIL)，如CD3+T细胞、CD4+T细胞、CD8+T细胞或任何其他类型的T细胞。T细胞还可以包括记忆T细胞、记忆干T细胞或效应T细胞。也可以从大量群体中选择T细胞，例如从全血中选择T细胞。T细胞也可以从大量群体中扩增。T细胞也可能倾向于特定种群和表型。例如，T细胞可以倾斜于表型包含CD45RO(-)、CCR7(+)、CD45RA(+)、CD62L(+)、CD27(+)、CD28(+)和/或IL-7Rα(+)。合适的细胞可以选自以下列表中的一种或多种标志物：CD45RO(-)、CCR7(+)、CD45RA(+)、CD62L(+)、CD27(+)、CD28(+)和/或IL-7Rα(+)。合适的细胞还包括干细胞，例如，例如胚胎干细胞、诱导的多能干细胞、造血干细胞、神经元干细胞和间充质干细胞。合适的细胞可以包含任何数量的原代细胞，例如人细胞、非人细胞和/或小鼠细胞。合适的细胞可以是祖细胞。合适的细胞可以衍生自要治疗的受试者(例如，患者)。
患者中需要的治疗有效的细胞的量可以根据细胞的存活力和细胞被遗传修饰的效率而变化(例如，转基因被整合到一个或多个细胞中的效率，或者由转基因编码的蛋白质的表达水平)。在一些实施方案中，遗传修饰后细胞存活力的产物(例如，倍增)和转基因整合的效率可以对应于可用于给予受试者的细胞的治疗量。在 一些实施方案中，遗传修饰后细胞存活力的增加可能对应于给予治疗对患者有效的必需细胞量的减少。在一些实施方案中，转基因整合到一个或多个细胞中的效率的增加可以对应于给予在患者中治疗有效的必需的细胞数量的减少。在一些实施方案中，确定所需的治疗有效的细胞的量可以包括确定与细胞随时间变化相关的功能。在一些实施方案中，确定需要治疗有效的细胞的量可以包括确定与根据时间相关变量将转基因整合到一个或多个细胞中的效率变化相对应的功能(例如，细胞培养时间、电穿孔时间、细胞刺激时间)。在一些实施方案中，治疗有效的细胞可以是细胞群，其包含在细胞表面上约30％至约100％的结合抗原的受体的表达。在一些实施方案中，通过流式细胞术测量，治疗有效的细胞可以在细胞表面上表达所述结合抗原的受体约30％、35％、40％、45％、50％、55％、60％、65％、70％、75％80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、99.9％或超过约99.9％。
在一些实施方案中，当所述结合抗原的受体存在于细胞的质膜上时，并且当通过结合靶标而被活化时，可以导致具有对于其细胞表面表达所述结合抗原的受体能够结合的靶标的细胞的毒性。例如，在某些情况下，当细胞存在于细胞的质膜中时，细胞可以是细胞毒性细胞(例如，NK细胞或细胞毒性T淋巴细胞)，本文所述的结合抗原的受体，并且当其通过结合其靶标而被激活时可以增加细胞毒性细胞对靶细胞的细胞毒性活性。例如，在一些实施方案中，本文所述的结合抗原的受体，当通过其靶标的结合而被激活时，与对不存在结合靶的细胞的细胞毒性相比，可以使细胞毒性增加至少10％、至少15％、至少20％、至少25％、至少30％、至少40％、至少50％、至少75％、至少2倍、至少2.5倍、至少5倍、至少10倍或更多10倍。
本发明的免疫应答细胞可以应用于制备药物组合物。所述的药物组合物除了包括有效量的免疫应答细胞，还可包含药学上可接受的载体。术语“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。
可作为药学上可接受的载体或其组分的一些物质的具体例子是糖类，如乳糖、葡萄糖和蔗糖；淀粉，如玉米淀粉和土豆淀粉；纤维素及其衍生物，如羧甲基纤维素钠、乙基纤维素和甲基纤维素；西黄蓍胶粉末；麦芽；明胶；滑石；固体润滑剂，如硬脂酸和硬脂酸镁；硫酸钙；植物油，如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油；多元醇，如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇；海藻 酸；乳化剂，如
Figure PCTCN2017082024-appb-000003
润湿剂，如月桂基硫酸钠；着色剂；调味剂；压片剂、稳定剂；抗氧化剂；防腐剂；无热原水；等渗盐溶液；和磷酸盐缓冲液等。
本发明的组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。给药方式例如可以采用肠胃外给药(如注射)或其它治疗方式。
免疫原性组合物的“肠胃外”施用包括例如皮下(s.c.)、静脉内(i.v.)、肌内(i.m.)或胸骨内注射或输注技术。
给予个体的包含免疫反应性细胞群体的制剂包含有效治疗和/或预防特定适应症或疾病的多个免疫反应性细胞。因此，可以向个体施用免疫反应性细胞的治疗有效群体。通常，施用包含约1×104至约1×1010个免疫反应性细胞的制剂。在大多数情况下，制剂将包含约1×105至约1×109个免疫反应性细胞、约5×105至约5×108个免疫反应性细胞、或约1×106至约1×107个免疫反应性细胞。然而，根据癌症的位置、来源、身份、程度和严重程度、待治疗的个体的年龄和身体状况等，对个体施用的CAR免疫反应性细胞的数量将在宽的范围之间变化。医生将最终确定要使用的适当剂量。
在一些实施方案中，使用嵌合抗原受体来刺激免疫细胞介导的免疫应答。例如，T细胞介导的免疫应答是涉及T细胞活化的免疫应答。活化的抗原特异性细胞毒性T细胞能够在表面上显示外源抗原表位的靶细胞中诱导细胞凋亡，例如显示肿瘤抗原的癌细胞。在另一个实施方案中，使用嵌合抗原受体在哺乳动物中提供抗肿瘤免疫。由于T细胞介导的免疫应答，受试者将产生抗肿瘤免疫。
在某些情况下，治疗患有癌症的受试者的方法可以涉及向需要治疗的受试者施用一种或多种本发明所述的免疫应答细胞。所述免疫应答细胞可结合肿瘤靶分子并诱导癌细胞死亡。如前文所述，本发明还提供治疗个体中的病原体感染的方法，包括向所述个体施用治疗有效量的本发明的免疫应答细胞。
本发明的免疫反应性细胞的给药频率将根据包括所治疗疾病的因素、特定免疫反应性细胞的元件和给药方式。例如可以每日给药4次、3次、2次或每日一次、每隔一天、每三天、每四天、每五天、每六天一次、每周一次、每八天一次、每九天一次、每十天、每周一次、或者每月两次给药。如本文所述，由于本申请的免疫应答细胞具有改善的活力，从而可以不仅以与类似的但不表达外源性I型干扰素的免疫应答细胞更低的治疗有效的量给药，并且可以以更低的频率给药，以获得至少类似、并且优选更加显著的疗效。
在一些实施方案中，本发明的免疫应答细胞可以与另一治疗剂联合给药。在一些实施方案中，所述另一治疗剂是化疗药。可以与本发明的免疫应答细胞联合应用的化疗药物包括但不限于有丝分裂抑制剂(长春花生物碱)，包括长春新碱、长春花碱、长春地辛和诺维宾(TM)(长春瑞滨，5'-去氢硫化氢)；拓扑异构酶I抑制剂，例如喜树碱化合物，包括CamptosarTM(伊立替康HCL)、HycamtinTM(托泊替康HCL)和衍生自喜树碱及其类似物的其它化合物；鬼臼毒素衍生物，例如依托泊苷、替尼泊苷和米多昔佐兹；烷基化剂顺铂、环磷酰胺、氮芥、三亚甲基硫代磷酰胺、卡莫司汀、白消安、苯丁酸氮芥、布列喹嗪、尿嘧啶芥末、氯洛芬和达卡巴嗪；抗代谢物，包括阿糖胞苷、氟尿嘧啶、甲氨蝶呤、巯嘌呤、硫唑嘌呤和丙卡巴肼；抗生素，包括但不限于多柔比星、博来霉素、更生霉素、柔红霉素、霉素霉素、丝裂霉素、肉瘤霉素C和道诺霉素；以及其它化疗药物，包括但不限于抗肿瘤抗体、达卡巴嗪、氮胞苷、阿姆沙康、美法仑、异环磷酰胺和米托蒽醌。
在一些实施方案中，可以与本发明的免疫应答细胞联合应用的化疗药物包括但不限于抗血管生成剂，包括抗VEGF抗体(包括人源化和嵌合抗体、抗VEGF适体和反义寡核苷酸)以及其他血管发生抑制剂，例如血管抑素、内皮抑制素、干扰素、白细胞介素1(包括α和β)白介素12、视黄酸和金属蛋白酶-1和-2的组织抑制剂。
在一些实施方案中，组合物可以是等渗的，即它们可以具有与血液和泪液相同的渗透压。本发明组合物的期望等渗性可以使用氯化钠或其它药学上可接受的试剂如葡萄糖、硼酸、酒石酸钠、丙二醇或其它无机或有机溶质来实现。如果需要，组合物的粘度可以使用药学上可接受的增稠剂维持在选定的水平。合适的增稠剂包括，例如，甲基纤维素、黄原胶、羧甲基纤维素、羟丙基纤维素、卡波姆等。增稠剂的优选浓度将取决于所选择的试剂。显然，合适的载体和其它添加剂的选择将取决于确切的给药途径和特定剂型的性质，例如液体剂型。
本发明还提供了包含本发明免疫应答细胞的试剂盒。所述试剂盒可用于治疗或预防癌症、病原体感染、免疫病症或同种异体移植。在一个实施方案中，试剂盒可以包括含有有效量的包含一种或多种单位剂型的免疫应答细胞的治疗或预防组合物。在一些实施方案中，试剂盒包含可含有治疗或预防性组合物的无菌容器；这样的容器可以是盒、安瓿、瓶、小瓶、管、袋、泡罩包装或本领域已知的其它合适的容器形式。这种容器可以由塑料、玻璃、层压纸、金属箔或其他适合于保持药物的材料制成。在一些实施方案中，可以提供免疫反应性细胞，例如CAR T细胞，以 及将CAR免疫反应性细胞给予具有发生癌症、病原体感染、免疫病症或同种异体移植的风险的受试者的说明书。说明书通常将包括关于组合物用于治疗或预防癌症、病原体感染、免疫疾病或同种异体移植的信息。在一些实施方案中，试剂盒可以包括约1×104个细胞至约1×106个细胞。在一些实施方案中，试剂盒可以包括至少约1×105个细胞，至少约1×106个细胞，至少约1×107个细胞，至少约4×107个细胞，至少约5×107个细胞，至少约6×107个细胞，至少约6×107个细胞，8×107个细胞，至少约9×107个细胞，至少约1×108个细胞，至少约2×108个细胞，至少约3×108个细胞，至少约4×108个细胞，至少约5×108个细胞，至少约6×108个细胞，至少约6×108细胞，至少约8×108个细胞，至少约9×108细胞，至少约1×109个细胞，至少约2×109个细胞，至少约3×109个细胞，至少约4×109个细胞，至少约5×109个细胞，至少约6×109个细胞，至少约8×109个细胞，至少约9×109个细胞，至少约1×1010个细胞，至少约2×1010个细胞，至少约3×1010个细胞，至少约4×1010个细胞，至少约5×1010个细胞，至少约6×1010个细胞，至少为ab至少约9×1010个细胞，至少约9×1010个细胞，至少约1×1011个细胞，至少约2×1011个细胞，至少约3×1011个细胞，至少约4×1011个细胞，至少约5×1011个细胞，至少约8×1011个细胞，至少约9×1011个细胞，或至少约1×1012个细胞。例如，可以在试剂盒中包括大约5×1010个细胞。在另一个实例中，试剂盒可以包括3×106个细胞；细胞可以扩增至约5×1010个细胞并施用于受试者。
在一些实施方案中，试剂盒可以包括同种异体细胞。在一些实施方案中，试剂盒可以包括可以包含基因组修饰的细胞。在一些实施方案中，试剂盒可以包含“现成的”细胞。在一些实施方案中，试剂盒可以包括可以扩展用于临床使用的细胞。在某些情况下，试剂盒可能包含用于研究目的的内容物。
在一些实施方案中，说明书包括以下中的至少一个：治疗剂的描述；用于治疗或预防肿瘤、病原体感染、免疫疾病或同种异体移植或其症状的剂量方案和给药；预防措施、警示、禁忌症、过量信息、不良反应、动物药理学、临床研究、和/或引用文献。说明书可以直接打印在容器上(如果有的话)，或作为容器上的标签，或作为容器内或容器中提供的单独的纸张、小册子、卡片或文件夹打印。在一些实施方案中，说明书提供施用本发明所述的免疫应答细胞用于治疗或预防肿瘤、病原体感染、免疫疾病或同种异体移植或其症状的方法。在某些情况下，说明书提供了施用化学治疗剂之前、之后或同时给予本发明的免疫反应性细胞的方法。
根据本发明的一个方面，本发明还提供了一种治疗个体的肿瘤或病原体感染， 或用于增强个体免疫耐受能力方法。在一些实施方案中，所述方法包括向有此需要的个体给予本发明的免疫应答细胞，所述免疫细胞表达所述结合抗原的受体以及外源性I型干扰素。在一些实施方案中，所述方法包括向有此需要的个体给予本发明所述的结合抗原的受体以及外源性I型干扰素。在一些实施方案中，所述外源性I型干扰素与所述表达结合抗原的受体的免疫应答细胞顺序给予或者同时给予。在一些实施方案中，所述外源性I型干扰素通过在免疫应答细胞中共表达，与所述免疫应答细胞同时向患者给予。
本发明提供了一种提高向个体给予的免疫应答细胞活力的方法，其特征在于，所述免疫应答细胞表达本发明所述的结合抗原的受体，并且其中所述方法包括向所述个体给予所述免疫应答细胞以及有效量的外源性I型干扰素。在一些实施方案中，所述外源性I型干扰素与所述表达结合抗原的受体的免疫应答细胞顺序给予或者同时给予。在一些实施方案中，所述外源性I型干扰素通过在免疫应答细胞中共表达，与所述免疫应答细胞同时向患者给予。
在一些实施方案中，正是由于本发明的免疫应答细胞活力得以提高，从而与不给予外源性I型干扰素或者所述免疫应答细胞不共表达所述外源性I型干扰素的情况下相比，能够以更低的剂量和/或更低的频率向给予本发明的免疫应答细胞。
在一些实施方案中，与不给予外源性I型干扰素或者所述免疫应答细胞不共表达所述外源性I型干扰素的情况下相比，向有此需要的个体给予的本发明的免疫应答细胞的量降低至少10％、20％、30、40、50％、60、70％、80％或90％。在一些实施方案中，与不给予外源性I型干扰素或者所述免疫应答细胞不共表达所述外源性I型干扰素的情况下相比，向有此需要的个体给予的本发明的免疫应答细胞的频率降低至少10％、20％、30、40、50％、60、70％、80％、或90％。或者，与不给予外源性I型干扰素或者所述免疫应答细胞不共表达所述外源性I型干扰素的情况下相比，在需要多次向有此需要的个体给予的本发明的免疫应答细胞的情况下，每次给予的间隔时间延长至少10％、20％、30、40、50％、60、70％、80％、90％、100％、120％、140％、160％、180％、200％、500％、750％、1000％。
在一些实施方案中，本发明的方法使得在向所述个体给予所述免疫应答细胞后，与不存在所述外源性I型干扰素的情况相比，所述个体外周血中细胞毒性T细胞和辅助T细胞的数量之和提高至少10％、20％、30、40、50％、60、70％、80％、90％、100％、120％、140％、160％、180％、200％、500％、750％、1000％。在一些实施方案中，所述方法使得在向所述个体给予所述免疫应答细胞约5天后，所述个体外 周血中细胞毒性T细胞和辅助T细胞的数量之和大于5,000个/μL、10,000个/μL、15,000个/μL、20,000个/μL、25,000个/μL；给予所述免疫应答细胞约7天后，所述个体外周血中细胞毒性T细胞和辅助T细胞的数量之和大于100个/μL、200个/μL、300个/μL、400个/μL、500个/μL、600个/μL、700个/μL、800个/μL、900个/μL、1,000个/μL、1,500个/μL、2,000个/μL、2,500个/μL、3,000个/μL、3,500个/μL、4,000个/μL、4,500个/μL、或5,000个/μL；或者给予所述免疫应答细胞约10天后，所述个体外周血中细胞毒性T细胞和辅助T细胞的数量之和大于10个/μL、20个/μL、30个/μL、40个/μL、50个/μL、60个/μL、70个/μL、80个/μL、90个/μL、或100个/μL。
本发明还提供了一种在个体中调节免疫反应的方法，该方法包括给予个体本发明的任意一种有效量的免疫应答细胞。
本发明还提供了一种加强个体免疫耐受的方法，该方法包括给予个体施用有效量本发明的免疫应答细胞，该细胞包含结合肿瘤抗原的受体和编码I型干扰素的载体。较佳地，该方法能防止或减少自身免疫性疾病或与同种异体移植相关的疾病。
本发明还提供了一种治疗或预防个体病原体感染的方法，该方法包括施用有效量的免疫应答细胞，该细胞包含结合病毒抗原的受体和编码I型干扰素的载体。
自体淋巴细胞输注可用于治疗。可以从需要治疗的患者收集自体外周血单核细胞(PBMC)，并且可以使用本文所述和本领域已知的方法活化和扩增T细胞，然后注入患者体内。在其他情况下，同种异体细胞可用于治疗患者。
本文公开的方法可以包括移植。移植可以指细胞产品的过继性移植。移植可以是自体移植、同种异体移植、异种移植或任何其他移植。例如，移植可以是异种移植。移植也可以是同种异体移植。
在一些实施方案中，受试者可以给予免疫反应性细胞，其中可以施用的免疫反应性细胞可以是约1至约35天龄。例如，所施用的细胞可以是1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34或最多约40天。可以从刺激时起计算CAR免疫反应性细胞的年龄。可以从血液采集的时间开始计算免疫反应性细胞的年龄。可以从转导时起计算免疫反应性细胞的年龄。在一些实施方案中，可以给予受试者的免疫反应性细胞为约10至约14或约20天龄。在一些实施方案中，免疫反应性细胞的“年龄”可以通过端粒长度来确定。例如，“年轻”的免疫反应细胞可以具有比“耗尽”或“老”的免疫反应性细胞更长的端粒长度。不受特定理论的束缚， 可以认为免疫反应性细胞在培养物中每周丢失约0.8kb的估计端粒长度，并且年轻的免疫反应性细胞培养物可以具有比约44天的免疫反应性细胞长约1.4kb的端粒。不受特定理论的束缚，人们认为更长的端粒长度可以与患者中的阳性客观临床反应和体内细胞的持久性相关联。
在移植之前、之后和/或期间，细胞(例如，工程细胞或工程化的原代T细胞)可以是功能性的。例如，移植的细胞可以是在移植后至少约1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18，19、20、21、22、23、24、25、6、27、28、29、30、40、50、60、70、80、90或100天起作用。移植细胞可以在移植后至少约1、2、3、4、5、6、7、8、9、10、11或12个月起作用。移植细胞可以在移植后至少约1、2、3、4、5、6、7、8、9、10、15、20、25或30年起作用。在一些实施方案中，移植细胞可以在接受者的寿命期间起作用。
此外，移植细胞可以以其正常预期功能的100％起作用。移植细胞还可以发挥其正常预期功能约1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、或高达约100％的功能。
移植细胞也可以发挥其正常预期功能的超过100％的作用。例如，移植的细胞可以起到正常预期功能的约110、120、130、140、150、160、170、180、190、200、250、300、400、500、600、700、800、900、1000或至多约5000％的功能。
移植可以通过任何类型的移植。局部可以包括但不限于肝下囊空间、脾囊下空间、肾囊下空间、网膜、胃或肠粘膜下层、小肠血管节段、静脉囊、睾丸、脑、脾或角膜。例如，移植可以是囊下移植。移植也可以是肌内移植。移植可以是门静脉移植。
与当一个或多个野生型细胞被移植到接受者之时相比，采用本发明的免疫应答细胞治疗之后可以改善移植排斥反应。例如，移植排斥可以是超急性排斥反应。移植排斥也可以是急性排斥反应。其他类型的排斥可能包括慢性排斥反应。移植排斥也可以是细胞介导的排斥反应或T细胞介导的排斥反应。移植排斥也可以是自然杀伤细胞介导的排斥反应。
改善移植可能意味着减轻超急性排斥反应，其可以包括减少、减轻或降低不良 作用或症状。移植可以指细胞产品的过继性移植。
移植成功的另一个迹象可以是接受者不需要免疫抑制治疗的天数。例如，在提供本发明的免疫应答细胞之后，接受者可以不要求至少约1、2、3、4、5、6、7、8、9、10或更多天的免疫抑制治疗。这可以表明移植成功。这也可以表明移植的细胞，组织和/或器官没有排斥。
在某些情况下，接受者不需要免疫抑制治疗至少1天。接受者也可不需要免疫抑制治疗至少7天。接受者不需要免疫抑制治疗至少14天。接受者不需要免疫抑制治疗至少21天。接受者不需要免疫抑制治疗至少28天。接受者不需要免疫抑制治疗至少60天。此外，接受者可能不需要至少1、2、3、4、5、6、7、8、9、10或更多年的免疫抑制治疗。
移植成功的另一个迹象可能就是接受者需要减少的免疫抑制治疗的天数。例如，在本文提供的所述治疗之后，接受者可能需要至少1、2、3、4、5、6、7、8、9、10或更多天的减少的免疫抑制治疗。这可以表明移植成功。这也可以表明移植的细胞、组织和/或器官没有或仅有很小的排斥。
例如，接受者可能需要至少1天的减少的免疫抑制治疗。接受者也可能需要至少7天的减少的免疫抑制治疗。接受者可能需要至少14天的减少的免疫抑制治疗。接受者需要至少21天的减少的免疫抑制治疗。接受者需要至少28天的减少的免疫抑制治疗。接受者需要至少60天的减少的免疫抑制治疗。此外，接受者可能需要至少1、2、3、4、5、6、7、8、9、10或更多年的减少的免疫抑制治疗。
减少的免疫抑制治疗可以指与当将一种或多种野生型细胞移植到接受者中时所需的免疫抑制治疗相比而言较少的免疫抑制治疗。
免疫抑制治疗可以包括抑制免疫系统的任何治疗。免疫抑制治疗可以帮助缓解、减少或消除患者的移植排斥反应。例如，免疫抑制剂可以在移植之前、期间和/或之后使用，包括MMF(霉酚酸酯(Cellcept))、ATG(抗胸腺细胞球蛋白)、抗-CD154(CD4OL)、抗-CD40(2C10)、的免疫抑制药物、抗-IL-6R抗体(tocilizumab、Actemra)、抗-IL-6抗体(sarilumab、olokizumab)、CTLA4-Ig(Abatacept/Orencia)、抗-IL-6抗体(ASKP1240、CCFZ533X2201)、安非他明(Campath)、抗CD20(利妥昔单抗)、贝伐单抗(LEA29Y)、西罗莫司(Rapimune)、依维莫司、他克莫司(Prograf)、达替珠单抗(Ze-napax)、巴利昔单抗(Similect)、英夫利昔单抗(Remicade)、环孢菌素、脱氧精蛋白、可溶性补体受体1、眼镜蛇毒素、抗C5抗体(eculizumab/Soliris)、甲基泼尼松龙、FTY720、依维莫司、来氟米特、 抗IL-2R-Ab、雷帕霉素、抗CXCR3抗体、抗ICOS抗体、抗OX40抗体和抗CD122抗体。此外，一种或多种免疫抑制剂/药物可一起使用或依次使用。一种或多种免疫抑制剂/药物可用于诱导治疗或维持治疗。诱导和维持阶段可以使用相同或不同的药物。在某些情况下，daclizumab(Zenapax)可用于诱导治疗，他克莫司(Prograf)和西罗莫司(Rapimune)可用于维持治疗。还可以使用非药物方案来实现免疫抑制，包括但不限于全身照射，胸腺照射和全部和/或部分脾切除术。这些技术也可以与一种或多种免疫抑制药组合使用。
实施例
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
在本发明以下实施例中，当构建所述的结合抗原的受体或CAR时，CD28共刺激信号结构域简称为28；CD3ζ简称为Z；4-1BB或CD137简称为BB。例如，将代码为85-2的scFv与CD3ζ以及CD28共刺激信号结构域作为胞内信号结构域所构建的嵌合抗原受体可以记作85-2-28Z。针对不同抗原的CAR的构建均是如此。
1.实验材料
肝癌细胞系SK-HEP-1和PLC/PRF/5购自ATCC细胞库，Huh-7购自日本RIKEN细胞库。
PBMC来自上海市血液中心。
AIM V培养基：CTS，Cat#1665773。
Figure PCTCN2017082024-appb-000004
 Human T-Activator CD3/CD28：Life technologies，Cat#11161D。
胎牛血清(FCS)：Gibco，Cat#10099-141。
IL-2：上海华新，注射用重组人白介素-2。
羊抗人F(ab’)2抗体：Jackson ImmunoResearch,Cat#109-066-006。
PE-Streptavidin：BD pharmingen，Cat#554061。
CytoTox 
Figure PCTCN2017082024-appb-000005
非放射性细胞毒性检测：Promega，Cat#G1780。
2.实验方法
2.1慢病毒载体构建
2.1.1pRRL-EF1α-92-CAR慢病毒载体的构建
作为示例，以下构建本发明的慢病毒质粒载体使用的载体系统属于第三代自灭活慢病毒载体系统，该系统共有四个质粒：即编码蛋白Gag/Pol的包装质粒pMDLg RRE(购自addgene)，编码Rev蛋白的包装质粒pRSV-REV(购自addgene)，编码VSV-G蛋白的包膜质粒pCMV-VSV-G(购自addgene)及基于空载体pRRLSIN-cPPT.PGK-GFP.WPRE(购自addgene)的编码目的基因CAR的重组表达载体，该系统可以有效降低形成可复制性慢病毒颗粒的风险。
在本系统中，本发明人首先通过常规分子克隆的技术方式，对空载体pRRLSIN-cPPT.PGK-GFP.WPRE进行改造，用延长因子-1α(elongation factor-1α，简称EF-1α)的启动子替代了原载体的启动子，并在启动子和CD8αsp信号肽之间增加了MluI酶切位点。具体地，使用ClaI/SalI(购自NEB)双酶切载体pWPT-EGFP(购自addgene)，回收1.1Kb的DNA片段，用T4DNA连接酶连接于经ClaI/SalI双酶切的载体pRRLSIN-cPPT.PGK-GFP.WPRE，并转化于宿主菌TOP10中，挑取克隆通过菌落PCR鉴定阳性克隆并通过测序确认，获得重组质粒pRRLSIN-cPPT.EF-1α-EGFP.WPRE。
在中国专利201510481235.1中阐述了经过人源化改造、能特异性识别人源GPC3蛋白的抗体92。为了构建92-CAR慢病毒质粒，以包含92重链可变区(专利201510481235.1中的SEQ ID NO:80)片段的质粒为模板，采用上游引物5’-ctccacgccgccaggccggaggtgcagctggtgcag-3’(SEQ ID NO:1)和下游引物5’-GCGGTGTCCTCGCTCCGCAGGCTGCTCAGCTCCATGTAGGCGGTG-3’(SEQ ID NO:2)扩增出重链可变区片段；以包含92轻链可变区(专利201510481235.1中的SEQ ID NO:79)片段的质粒为模板，采用上游引物5’-GCGGAGCGAGGACACCGCCGTGTACTACTGCGCCCGGTTCTACAGCTAC-3’(SEQ ID NO:3)和下游引物5’-CGGCGCTGGCGTCGTGGTACGTTTGATCTCCAGCTTGGTG-3’(SEQ ID NO:4)扩增出轻链可变区片段。上述重链和轻链可变区引物通过搭桥PCR，进一步扩增出含有与上游CD8α信号肽和下游铰链区重复序列的92scFv片段(SEQ ID NO:5)，命名为片段1，大小765bp。PCR扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：58℃，40s；延伸：68℃，40s；进行25个循环，然后在68℃延伸10min。 PCR扩增条带通过琼脂糖凝胶电泳确认符合预计的片段大小。
采用上游引物5’-gcaggggaaagaatagtagaca-3’(SEQ ID NO:6)和下游引物5’-CGGCCTGGCGGCGTGGAG-3’(SEQ ID NO:7)，以本实施例中构建的载体质粒pRRLSIN-cPPT.EF-1α-EGFP.WPRE为模板，扩增出含有CD8α信号肽的EF-1α promoter(SEQ ID NO:8)(内含MluI酶切位点)，命名为片段2，大小442bp。PCR扩增条件为预变性：94℃，4min；变性：94℃，30s；退火：53℃，30s；延伸：68℃，30s；进行25个循环，然后总延伸68℃，10min。PCR扩增条带通过琼脂糖凝胶电泳确认符合预计的片段大小。
采用上游引物5’-accacgacgccagcgccg-3’(SEQ ID NO:9)和下游引物5’-aatccagaggttgattgtcgacctagcgagggggcagggcctgc-3’(SEQ ID NO:10)，分别以pWPT-eGFP-F2A-GPC3-BBZ、pWPT-eGFP-F2A-GPC3-28Z和pWPT-eGFP-F2A-GPC3-28BBZ为模板(具体参考中国专利CN 104140974A)，扩增出含有Hinge-BBZ(SEQ ID NO:11)的片段3、Hinge-28Z(SEQ ID NO:12)的片段4和Hinge-28BBZ(SEQ ID NO:13)的片段5(均内含Sal I酶切位点)，大小分别为694bp、703bp和829bp。PCR扩增条件为预变性：94℃，4min；变性：94℃，30s；退火：60℃，30s；延伸：68℃，30s；进行25个循环，然后总延伸68℃，10min。PCR扩增条带通过琼脂糖凝胶电泳确认符合预计的片段大小。
分别将等摩尔约50ng片段2、片段1和片段3进行拼接PCR，拼接条件为：预变性94℃，4min；变性：94℃，40s；退火：60℃，40s；延伸：68℃，140s，进行5个循环，然后总延伸68℃，10min，补充DNA聚合酶及上游引物5’-gcaggggaaagaatagtagaca-3’(SEQ ID NO:6)和下游引物5’-aatccagaggttgattgtcgacctagcgagggggcagggcctgc-3’(SEQ ID NO:10)，通过PCR扩增25个循环，扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：60℃，40s；延伸：68℃，140s，总延伸68℃，10min。扩增获得92-BBZ的DNA片段(SEQ ID NO:14)理论大小分别为1865bp。扩增产物经琼脂糖电泳确认与理论大小一致。
分别将等摩尔约50ng片段2、片段1和片段4进行拼接PCR，同上拼接反应条件，扩增获得92-28Z的DNA片段(SEQ ID NO:15)理论大小分别为1874bp。扩增产物经琼脂糖电泳确认与理论大小一致。
分别将等摩尔约50ng片段2、片段1和片段5进行拼接PCR，同上拼接反应条件，扩增获得92-28BBZ的DNA片段(SEQ ID NO:16)理论大小分别为2000bp，。扩增产物经琼脂糖电泳确认与理论大小一致。
采用限制性内切酶Mlu I和SalI(购自NEB)分别酶切上述载体质粒pRRLSIN-cPPT.EF-1α-EGFP.WPRE和片段92-BBZ、92-28Z以及92-28BBZ。通过T4连接酶(购自NEB)进行连接，转化TOP10，挑克隆进行进行PCR鉴定阳性菌，送到Invitrogen进行测序确认序列正确，由此获得pRRL-EF-1α-92-BBZ、pRRL-EF-1α-92-28Z和pRRL-EF-1α-92-28BBZ。
2.1.2共表达4-1BBL的92-CAR慢病毒载体的构建
此外，为构建共表达92-28Z和41BBL的质粒，以上述构建的pRRL-EF-1α-92-28Z质粒为模板，先使用上游引物5’-gcaggggaaagaatagtagaca-3’(SEQ ID NO:6)和下游引物5’-TCAGAAGGTCAAAATTCAAAGTCTGTTTCACGCGAGGGGGCAGGGCCTGCATGTGAA-3’(SEQ ID NO:17)，通过PCR扩增出片段6。为得到F2A-41BBL片段，以质粒HG15693-G(购自北京义翘神州生物技术有限公司，该41BBL基因的第562碱基包含一个由G到A的突变)为模板，分别使用上游引物5’-gagacgttgagtccaaccctgggcccatggaatacgcctctgacgc-3’(SEQ ID NO:18)和下游引物5’-TCGGAGGAGGCGGGTGGCAGGTCCACGGTC-3’(SEQ ID NO:19)，通过PCR扩增出片段7；使用上游引物5’-ctgccacccgcctcctccgaggctcggaa-3’(SEQ ID NO:20)和下游引物5’-TGATTGTCGACTTATTCCGACCTCGGTGAAGGGA-3’(SEQ ID NO:21)，通过PCR扩增出片段8，再将片段7和8等摩尔拼接后，用引物对(SEQ ID NO:18和SEQ ID NO:21)进行PCR扩增，得到片段9。最后将等摩尔的片段6和9进行拼接，用引物对(SEQ ID NO:6和SEQ ID NO:21)进行扩增，得到92-28Z-F2A-41BBL(SEQ ID NO:22)。采用Mlu I和SalI酶切此片段，使用上述同样的方法，插入到经同样酶切的载体pRRLSIN-cPPT.EF-1α-EGFP.WPRE，经测序确认正确，得到质粒pRRL-EF-1α-92-28Z-F2A-41BBL。
2.1.3可调控共表达IFN的92-CAR慢病毒载体的构建
为构建可以共表达92-28Z及IFN beta(同时通过在IFN beta前面插入NFAT元件以实现可控表达)的质粒，首先通过引物搭桥的方式，利用引物(SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35)合成片段10，然后以pWPT-EGFP质粒为模板，以上游引物(SEQ ID NO:36)和下游引物(SEQ ID NO:37)，扩增出片段11。将片段10和11等摩尔混合，通过搭桥PCR，并使用引物对(SEQ ID NO:35和SEQ ID NO: 38)进行扩增后，采用ClaI和SalI酶切此片段，使用上述同样的方法，插入到经同样酶切的载体pRRLSIN-cPPT-PGK-EGFP.WPRE，经测序确认正确，获得包含三个NFAT重复序列的载体pRRLSIN-NFAT3-EGFP-PA2。以此pRRLSIN-NFAT3-EGFP-PA2为模板，分别用引物对(SEQ ID NO:39和SEQ ID NO:40)及引物对(SEQ ID NO:41和SEQ ID NO:42)扩增出片段12和13，然后通过搭桥PCR，以引物对(SEQ ID NO:39和SEQ ID NO:42)进行扩增得到片段14，随后用Mlu I和SalI酶切此片段，连接于同样酶切的pRRLSIN-NFAT3-EGFP-PA2中，经测序确认，得到包含六个NFAT重复序列的载体pRRLSIN-NFAT6-EGFP-PA2。以载体pGMT-IFN-β(购自北京义翘神州生物技术有限公司)为模板，通过引物对(SEQ ID NO:43和SEQ ID NO:44)扩增得到片段15。以此片段为模板，使用引物对(SEQ ID NO:43和SEQ ID NO:38)进行PCR扩增，扩增产物经过Mlu I和Cla I酶切，连接于同样酶切的载体pRRLSIN-NFAT6-EGFP-PA2，经测序确认正确，获得pRRLSIN-NFAT6-huIFNβ-PA2质粒。以此构建好的pRRLSIN-NFAT6-huIFNβ-PA2质粒为模板，通过引物对(SEQ ID NO:45和SEQ ID NO:46)扩增得到带有NdeI酶切位点的EGFP片段16；以上述构建好的质粒pRRLSIN-NFAT6-EGFP-PA2质粒为模板，通过引物对(SEQ ID NO:47和SEQ IDNO:48)扩增，获得带有NdeI酶切位点的NFAT6片段17(需要扩增6个单位的片段，同时消除SalI的酶切位点)。将片段16和17等摩尔混合，使用引物对(SEQ ID NO:45和SEQ ID NO:48)扩增，使用EcoRI和KpnI双酶切此片段，连接于同样酶切的载体pRRLSIN-cPPT.EF-1α-EGFP.WPRE中，经测序验证正确，得到pRRLSIN-EF1α-EGFP-NFAT6-huIFNβ-PA2质粒。最后，使用MluI和SalI双酶切质粒pRRL-EF-1α-92-28Z，获得92-28Z片段，将此片段连接于同样双酶切的pRRLSIN-EF1α-EGFP-NFAT6-huIFNβ-PA2载体，得到测序正确的质粒pRRLSIN-EF1α-92-28Z-NFAT6-hu IFNβ-PA2。
以上五种质粒pRRL-EF-1α-92-BBZ、pRRL-EF-1α-92-28Z、pRRL-EF-1α-92-28BBZ、pRRL-EF-1α-92-28Z-F2A-41BBL和pRRL-EF-1α-92-28Z-NFAT6-huIFNβ-PA2通称为pRRL-EF-1α-92-CAR(图1)。92-BBZ、92-28Z、92-28BBZ和92-28Z-F2A-41BBL对应的氨基酸序列分别为SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52。92-28Z-NFAT6-IFN-β表达的氨基酸序列包含两段，分别为如SEQ ID NO:50所示的构建为92-28Z的CAR和如SEQ ID NO:53所示的IFN，其载体构建如图1B所示。
2.2病毒制备
1)以4.5×106的密度接种293T细胞于10cm培养皿中，37℃，5％CO2培养过夜准备包装病毒，培养基为含DMEM，添加10％胎牛血清；
2)将慢病毒穿梭载体pRRL-92-28Z-NFAT6-IFN-β5.2ug与包装质粒pRsv-REV 6.2μg、pRRE-PMDLg 6.2μg、VSVg 2.4μg溶入800μL无血清DMEM培养液，混匀；
3)将60μg PEI(1μg/μl)溶解于800μl的无血清DMEM培养液中，轻轻混匀(或1000rpm涡旋5秒钟)，室温孵育5min；
4)转染复合物的形成：将质粒混合液加入PEI混合液中，加入后立即涡旋混合或轻轻混匀，室温下孵育20min；
5)将转染复合物1.6ml滴加入含11ml DMEM培养基的10cm培养皿中，4-5h小时后，更换新鲜培养基；
6)72h后，收集病毒液上清。
2.3病毒浓缩
1)5X PEG8000NaCl配制：称取NaCl 8.766g、PEG8000 50g溶解在200ml Milli-Q纯水中，121℃30min湿热灭菌30min，降至室温后放4℃冰箱保存；
2)将收集的病毒上清液使用0.45μm滤头过滤，加入1/4的5X PEG-8000NaCl母液7.5ml，上下颠倒混匀；
3)每20～30min混合一次，共进行3-5次；
4)4℃放置过夜；
5)4℃，4000g，离心60min；
6)去上清后，加入适量的AIM V培养基(含2％AB血清)溶解重悬病毒沉淀；
7)浓缩后的慢病毒悬液分装成50μl每份，保存在成品管中，储存在-80℃；单嗜性逆转录病毒不稳定，包装后需尽快使用，不推荐-80℃冻存。
2.4慢病毒滴度测定
以1×105细胞数目接种293T细胞于12孔培养板；
浓缩后的慢病毒分别以1uL、0.2uL和0.04uL添加到细胞悬液中，并添加polybrene至终浓度6ug/mL；
37℃，5％CO2培养过夜后，更换新鲜培养基；
感染72h后，胰酶消化293T细胞，加等量培养基终止后，吹打均匀，将细胞悬液转移入1.5mL离心管中；
400g离心5min，弃上清，PBS+2％FBS溶液洗一次；
6)取适量细胞，按1:50稀释比例加入Biotin标记的羊抗人Fab抗体，冰上孵育30min；
7)加入1mL PBS+2％FBS溶液清洗一次后，按1:50稀释比例加入PE标记的streptavidin，冰上孵育30min；
8)PBS+2％FBS溶液清洗两次后，加入适量体积的PBS+2％FCS溶液重悬细胞，转移至流式管中；
9)流式细胞仪检测后，取阳性率为5～20％的细胞样品为宜，计算滴度(TU/mL)＝细胞数量(105)×阳性率/病毒体积(mL)。
2.5慢病毒转导T淋巴细胞-CAR-T淋巴细胞的制备
1)T淋巴细胞活化：人PBMC来源于上海市血液中心，培养基为AIM V+2％AB血清+IL-2(500U/mL)调整PBMC密度为1×106/mL，按1:1的比例加入抗人CD3和CD28抗体包被的磁珠活化48h；
2)Retronectin包被48孔板：每孔加入160μl retronectn溶液(5μg/mL)，4℃孵育过夜；
3)弃去48孔板中的Retronectin溶液，1ml PBS洗两次；
4)将细胞接种于Retronectin包被的48孔板中，每孔细胞数目3×105，按照MOI＝10加入慢病毒，补充培养基至300μL；
5)32℃，1800rpm，离心40min后，转移至细胞培养箱继续培养24h；
6)更换新鲜培养基，调整细胞密度为5×105/mL，每2-3天进行传代。
2.6T淋巴细胞嵌合抗原受体表达
1)感染7天后，取4×105的T细胞，4℃，400g，离心5min，弃上清，PBS+2％FCS清洗一次；
2)加50μL PBS+2％FCS重悬细胞，加入1μL Biotin标记的羊抗人Fab抗体，冰上孵育30min；
3)PBS+2％FCS清洗两次后，加入50μL PBS+2％FCS重悬细胞，加入1μL PE标记的Streptavidin，冰上孵育30min；
4)PBS+2％FCS清洗两次后，加入400μL PBS+2％FCS重悬细胞，转移至流式管中，采用流式细胞仪检测感染效率。
2.7体外毒性实验
靶细胞
92-CAR对应的靶细胞为SK-HEP-1(GPC3-)和Huh-7(GPC3+)；
调整靶细胞浓度为1×106/mL，取100μL接种到96well板；
效应细胞：按效靶比0.3:1、1:1和3:1向96孔板中加入CAR-T细胞及对照T细胞；
各组均设5个复孔，取5个复孔的平均值。
其中各实验组和各对照组如下：
各实验组：各靶细胞+表达不同嵌合抗原受体的CTL；
对照组1：靶细胞最大释放LDH；
对照组2：靶细胞自发释放LDH；
对照组3：效应细胞自发释放LDH；
检测方法：效应细胞与靶细胞共培养18h后，采用CytoTox 96非放射性细胞毒性检测试剂盒(Promega公司)进行。该方法是基于比色法的检测方法，可替代51Cr释放法。CytoTox 
Figure PCTCN2017082024-appb-000006
通过检测乳酸脱氢酶(LDH)的含量反映细胞的裂解程度。LDH是一种稳定的胞质酶，在细胞裂解时会释放出来，其释放方式与51Cr在放射性分析中的释放方式基本相同。释放出的LDH培养基上清中，可通过30分钟偶联的酶反应来检测，在酶反应中LDH可使一种四唑盐(INT)转化为红色的甲臜(formazan)。生成的红色产物的量与裂解的细胞数成正比。具体参照CytoTox 96非放射性细胞毒性检测试剂盒说明书。
细胞毒性计算公式为：
Figure PCTCN2017082024-appb-000007
3、结果
实施例1、92-CAR在人T淋巴细胞上的表达
人T淋巴细胞采用抗CD3和CD28抗体包被的磁珠刺激48h后采用高滴度的慢病毒以MOI＝10离心感染。感染后第7天通过流式细胞术检测慢病毒感染T淋巴细胞阳性率。表达92-28Z(SEQ ID NO:15，编码核苷酸序列如SEQ ID NO:57所示)的T细胞(图2中的GPC3-CD28Z)感染效率为35.1％，表达92-28Z-NFAT6-IFN-β的T细胞(图2中的GPC3-CD28Z-IFN)感染效率为19.2％，对照载体MOCK感染效率为49％，如图2。
实施例2、92-CAR T细胞的体外抗肿瘤活性
将感染阳性率检测后的92-CAR T分别按效靶比0.3:1、1:1、3:1检测表达92-28Z-NFAT6-IFN-β、92-28Z及空载体MOCK的T淋巴细胞对肝癌细胞系SK-HEP-1(GPC3-)和Huh-7(GPC3+)及PLC/PRF/5(GPC3+)的体外杀伤效果，共培养18h后，检测上清中LDH的含量。结果中可以看出表达92-28Z CAR-T细胞特异性杀伤GPC3阳性的Huh-7及PLC/PRF/5细胞，而不杀伤GPC3阴性的SK-HEP-1细胞；共表达huIFN-β的CAR-T细胞的杀伤能力高于同等效靶比的92-28Z CAR-T细胞，见表2。
表2、92-CAR T细胞对靶细胞的毒性杀伤检测
Figure PCTCN2017082024-appb-000008
本发明人进一步将92-28Z-NFAT6-IFN-β与其它通过同一保外抗原结合单元92(SEQ ID NO:5)构建的GPC3-BBZ、GPC3-28BBZ和GPC3-41BBL(在CD28Z基础上共表达了4-1BBL)CAR-T细胞相比。首先FACS检测各种CAR的表达(见图3)，各种CAR的表达比率基本在40％左右。
然后分别按效靶比0.3:1、1:1、3:1检测这些CAR-T细胞及空载体MOCK T淋巴细胞对肝癌细胞系SK-HEP-1(GPC3-)和Huh-7(GPC3+)及PLC/PRF/5(GPC3+)的体外杀伤效果，共培养18h后，检测上清中LDH的含量。结果中可以看出表达92CAR-T的细胞特异性杀伤GPC3阳性的Huh-7及PLC/PRF/5细胞，而不杀伤GPC3阴性的SK-HEP-1细胞。在效靶比1:1的情况下共表达IFN-β的CAR-T细胞对两种GPC3阳性肝癌细胞的杀伤能力高于其它所有CAR-T细胞，见表3。
表3、各种GPC3CAR-T细胞对靶细胞的毒性杀伤检测
Figure PCTCN2017082024-appb-000009
Figure PCTCN2017082024-appb-000010
以上研究表明在CD28-Z的CAR T细胞基础上带上外源表达的IFN-beta可以增强抗肿瘤活性。并且，表达CD28Z的CAR-T共表达IFN-beta后，在某些效靶比上比表达CD28Z-41BBL的免疫细胞具有更好的肿瘤杀伤能力。
实施例3、含有IFN和不含IFN的GPC3CAR-T细胞体外诱导细胞因子释放试验
分别检测未转染的T细胞、92-28Z T细胞和92-28Z-IFN T细胞释放的细胞因子。收集慢病毒感染后1-2周内生长状态良好的上述三种T细胞，接种5×104/200μL(阳性细胞数)于24孔板，再同样方法接种5×104/200μL/24孔huh7细胞，与CAR T细胞共孵育24小时后收集上清，检测IFN-β、IFN-γ、及IL-2的浓度，结果如图4A-4C所示。
根据图4A，只有GPC3-28Z-IFN T与Huh7细胞共孵育时有IFNβ的表达，说明GPC3-28Z-IFN T细胞在被靶抗原激活后，IFNβ能够被成功诱导表达，并分泌至细胞外。根据图4B和4C中对体外细胞因子的检测，结果说明在多种GPC3阳性的细胞中，如Huh7、PLC\PRF\5、Hep-3B等细胞中，GPC3-28Z-IFN T细胞能更有效的被激活。
实施例4、含有IFN和不含IFN的CLD18A2CAR-T细胞体外诱导细胞因子释放试验
嵌合抗原受体85-28Z及85-2-28Z质粒的构建：
以PRRLSIN-cPPT.EF-1α为载体，分别构建了表达抗体85及85-2的二代嵌合抗原受体85-28Z(SEQ ID NO:55)和85-2-28Z(SEQ ID NO:54)的慢病毒质粒。85-28Z序列由CD8α信号肽、85scFV、CD8hinge、CD28跨膜区和胞内信号传导结构域区以及CD3的胞内段CD3ξ组成；85-2-28Z的序列由CD8α信号肽、hu8E5-2IscFV、CD8铰链区、CD28跨膜区和胞内信号传导结构域区以及CD3的胞内段CD3ξ组成。
嵌合抗原受体85-28Z-IFN及85-2-28Z-IFN质粒的构建：
在85-28Z及85-2-28Z的基础上构建了表达IFNb细胞因子的85-28Z-IFNb CAR(编码核苷酸序列如SEQ ID NO:58所示)，在85-2-28Z CAR的基础上构建了可以表达IFNb细胞因子的85-2-28Z-IFNb CAR(编码核苷酸序列如SEQ ID NO:59所示)。
为了验证构建的85-28Z T细胞和85-28Z-IFN T细胞同样能够在靶细胞刺激下被有效激活，我们检测了在与靶细胞共孵育后，85-28Z T、85-28Z-IFN T细胞因子的分泌。
分别检测转染空载后的T细胞(Mock)、85-28Z T细胞和85-28Z-IFN T细胞释放的细胞因子。收集慢病毒感染后1-2周内生长状态良好的上述三种T细胞，接种5×104/200μL(阳性细胞数)于24孔板，按效靶比1:1分别接种5×104/200μL/24孔靶细胞。靶细胞包括293T-A1、293T-A2、AGS、AGS-A2、BGC-823以及BGC-823-A2细胞。共培养24小时后收集上清。采用夹心ELISA的方法检测上清中CAR T淋巴细胞与靶细胞共同培养过程中释放的IFN-γ细胞因子。
实验结果如图5所示，IFN的存在导致85-28Z CAR T细胞与靶细胞共孵育时IFN-γ细胞因子分泌增多。
实施例5、含有IFN和不含IFN的GPC3CAR-T(92-28Z)细胞的杀伤活性
SK-HEP-1为GPC3阴性的人肝细胞肝癌细胞系，PLC/PRF/5为GPC3阳性的人肝细胞肝癌细胞系，HepG2为GPC3阳性的人肝细胞肝癌细胞系，Hep3B为GPC3阳性的人肝细胞肝癌细胞系，均购自美国模式培养物保藏所(ATCC)；Huh-7(又称Huh7)为GPC3阳性的人肝细胞肝癌细胞系，购自日本RIKEN细胞库。
检测方法：使用CytoTox 96非放射性细胞毒性检测试剂盒(Promega公司)进行检测(具体方法可参照CytoTox 96非放射性细胞毒性检测试剂盒说明书)，检测了CAR T淋巴细胞对Huh 7、Hep 3B、PLC/PRF/5、Hep G2及SK-HEP-1肝癌细胞的体外毒性杀伤作用。
分别用表达未转染的T细胞、92-28Z T细胞和92-28Z-IFN T细胞与肿瘤细胞以效靶比1:3、1:1和3:1，共培养18h。各实验组与各对照组的设置如下：
实验组设置：各靶细胞+表达不同的嵌合抗原受体的T淋巴细胞；
对照组1：效应细胞自发LDH释放；
对照组2：靶细胞自发LDH释放；
对照组3：靶细胞最大LDH释放
对照组4：体积校正对照；
对照组5：培养基背景对照。
实验结果的计算：将所有实验组、靶细胞自发LDH释放组和效应细胞自发LDH释放组的吸光度减去培养基背景吸光值的均值；将靶细胞最大LDH释放对照的吸光值减去体积校正对照吸光值的均值；将上述步骤获得的经过校正的值代入下面公式，计算每个效靶比所产生的细胞毒性(％)
计算公式：％细胞毒性＝(实验组-效应细胞自发组-靶细胞自发组/靶细胞最大-靶细胞自发)*100
结果如图6A-6E所示，以上数据表明，表达有IFN的CAR-GPC3T不仅能够特异性的杀伤GPC3阳性的细胞，表达有IFN的CAR-T细胞(GPC3-28Z-IFN)的杀伤活性还能够提高。
实施例6、含有IFN和不含IFN的CLD18A2CAR-T细胞的杀伤活性
293T-A1和293T-A2细胞为体外构建的稳定表达CLD18A1和CLD18A2的人肾上皮细胞系细胞系。AGS、BGC-823为人胃癌细胞系，并在此基础上构建了稳定表达CLD18A2的AGS-A2、BGC-823-A2细胞系。
检测方法：使用CytoTox 96非放射性细胞毒性检测试剂盒(Promega公司)进行检测(具体方法可参照CytoTox 96非放射性细胞毒性检测试剂盒说明书)，检测了CAR T淋巴细胞对293T-A1、293T-A2、AGS、AGS-A2、BGC-823、BGC-823-A2细胞的体外毒性杀伤作用。
为了比较85-2-28Z和85-2-28Z-IFN T细胞对靶细胞的杀伤作用，我们
分别按照效靶比1:3、1:1和3:1将表达空载T细胞(Mock)、85-28Z T细胞和85-28Z-IFN T细胞与CLD18A2阳性的293T-A2、AGS-A2和BGS-823A2细胞18小时，CLD18A2阴性的293T-A1、AGS和BGC-823细胞作为对照。
实验组：各靶细胞+表达不同嵌合抗原受体的CAR T；
①效应细胞自发LDH释放：校正效应细胞自发释放出来的LDH；
②靶细胞自发LDH释放：校正靶细胞自发释放出来的LDH；
③靶细胞最大LDH释放：计算时需要该对照确定100％的LDH释放；
④体积校正对照：校正由于加入裂解液(10×)引起的体积变化；
⑤培养基背景对照：校正由培养基中血清产生的LDH活性以及酚红造成 的背景吸收。
计算公式：细胞毒性％＝[(实验组–效应细胞对照–靶细胞对照)/(靶细胞最大裂解量–靶细胞对照)]×100。在计算前，效应细胞对照，靶细胞对照，实验组减掉培养基对照；靶细胞最大裂解量减掉体积对照。
体外毒性实验分别用表达Mock、85-2-28Z以及85-2-28Z-IFN的CAR T淋巴细胞与肿瘤细胞以效靶比1:3、1:1和3:1，共培养18h，两种CART细胞对CLD18A2阳性的靶细胞的杀伤活性结果如图7所示。
实施例7、GPC3CAR-T细胞体内存活时间测定
参照实施例3中步骤1)～3)的实验操作，通过尾静脉输注CAR-T(GPC3-28ZT细胞或GPC3-28Z-IFN T细胞)细胞7天后，检测CAR-T细胞(GPC3-28Z T细胞或GPC3-28Z-IFN T细胞)在体内存活情况。结果如表4所示，GPC3-28Z-IFN T细胞组每μl外周血中含有的T细胞(CD3+)数及CAR-T细胞数均高于GPC3-28ZT细胞组和Mock组。
表4、外周血中T细胞存活数量
  CD3+(个/μL) CAR-T(个/μL)
Mock 193.1453 0
GPC3-28Z 375.802 232.2
GPC3-28Z-IFN 1034.315 439.5
实施例8、CLD18A2 CAR-T细胞体内存活时间测定
胃癌PDX模型的建立：
接种约2×2×2mm大小的胃癌PDX瘤块于NOD/SCID小鼠的右侧腋部皮下，在肿瘤细胞接种之日记为D0天。
过继转移T细胞：
在肿瘤体积为100mm3时，腹腔注射100mg/kg的环磷酰胺，注射24小时后通过尾静脉输注1.0×107CAR-T细胞(85-2-28Z T细胞或85-2-28Z-IFN T细胞)，同时以Mock T细胞组作为对照。
分别在CAR-T细胞输注后的D5，D7和D10天，经小鼠隐静脉抽取外周血，检测CAR-T细胞(空载T细胞(Mock)，85-2-28Z T细胞或85-2-28Z-IFN T细胞)在体内的存活情况。
结果如图8A-8C所示，85-2-28Z-IFN T细胞治疗组的T细胞存活数量明显多于85-2-28Z T细胞治疗组。
实施例9、含有IFN和不含IFN的GPC3CAR-T(92-28Z)细胞的体内杀伤活性
测定未转染的T细胞(Mock)、GPC3-28Z T细胞和GPC3-28Z-IFN T细胞对Huh7皮下移植瘤的抗肿瘤治疗实验。
1)实验分组：NOD-SCID小鼠21只，6-8周龄随机分为3组，每组7只，分为未转染的T细胞组、GPC3-28Z T细胞组和GPC3-28Z-IFN T细胞组。
2)皮下移植瘤的接种：收集处于对数生长期并且生长状态良好的Huh7细胞使用生理盐水调整密度为1×107/mL，接种NOD-SCID小鼠制作小鼠模型，注射体积约为200μL(2×106/只)，在肿瘤细胞接种之日记为第0天。
3)过继转移T细胞：在肿瘤体积为200-300mm3时，腹腔注射200mg/kg的环磷酰胺，注射24小时后通过尾静脉输注1.4×107CAR-T细胞(GPC3-28Z T细胞或GPC3-28Z-IFN T细胞)，同时以未转染的T细胞组作为对照，观察测量皮下移植瘤的生长(图9A)，待对照组小鼠肿瘤大小达到2000mm3时，即将实验处死，分离肿瘤后进行拍照(图9B)。
结果如图9A和9B所示，GPC3-28Z-IFN T细胞能够显著抑制肿瘤细胞生长，在CART细胞回输后第13天时，GPC3-28Z CART细胞的抑瘤率为66.5％，GPC3-28Z-IFN CART细胞的抑瘤率为82.3％，表明GPC3-28Z-IFN T细胞进一步增强了CAR-T细胞抑制肿瘤生长的能力。
实施例10、含有IFN和不含IFN的CLD18A2CAR-T细胞的体内杀伤活性
测定未转染的T细胞(Mock)、85-28Z T细胞和85-2-28Z-IFN T细胞对BGC-823-A2细胞皮下移植瘤的抗肿瘤治疗实验。
1)BGC-823-A2皮下移植瘤的接种：收集处于对数生长期并且生长状态良好的BGC-823-A2细胞使用生理盐水调整密度为2.5×107/mL，注射细胞悬液体积200μL(5×106/只)于小鼠右侧腋部皮下。在肿瘤细胞接种之日记为第0天。
2)实验分组：肿瘤接种第11天，测量BGC-823-A2移植瘤体积，将NOD-SCID小鼠随机分为4组，每组6只。分别为未转染的T细胞组、85-28Z T细胞组、85-2-28Z细胞组和85-2-28Z-IFN T细胞组。
3)过继转移T细胞：在肿瘤体积为100-150mm3时(第11天)，腹腔注射100mg/kg的环磷酰胺，注射24小时后通过尾静脉输注1×107CAR T细胞(Mock细胞、85-28Z T、85-2-28Z T细胞或85-2-28Z-IFN细胞)，同时以未转染的T细胞组(Mock组)作为对照，观察测量皮下移植瘤的生长。
动物实验结果如图10A和图10B所示，结果表明85-2-28Z-IFN CAR T细胞对BGC-823-A2移植瘤的治疗效果要优于85-2-28Z CAR T细胞。
实施例11、含有IFN和不含IFN的CLD18A2CAR-T细胞在胃癌PDX模型皮下移植瘤中的抗肿瘤试验
观察未转染的T细胞(UTD)、85-2-28Z T细胞和85-2-28Z-IFN T细胞对胃癌PDX模型皮下移植瘤的抗肿瘤治疗实验。
1)胃癌PDX模型的建立：接种约2×2×2mm大小的胃癌PDX瘤块于6-8周龄的雌性NOD/SCID小鼠的右侧腋部皮下，在肿瘤细胞接种之日记为D0天。
2)实验分组：肿瘤接种D15天，将NOD-SCID小鼠随机分为3组，每组7只，分为未转染的T细胞组、85-2-28Z T细胞组和85-2-28Z-IFN T细胞组。
3)过继转移T细胞：在肿瘤体积为30mm3时，腹腔注射100mg/kg的环磷酰胺，注射24小时后通过尾静脉输注1.0×107CAR-T细胞(85-2-28Z T细胞或85-2-28Z-IFN T细胞)，同时以未转染的T细胞组作为对照。观察测量胃癌PDX皮下移植瘤的生长。
结果如图11所示，85-2-28Z-IFN治疗组7只小鼠有1只小鼠肿瘤完全消退。
实施例12、含有IFN和不含IFN的GPC3CAR-T(92-28Z)细胞在体内对肿瘤浸润的影响
参照实施例9中建立的动物模型，回输GPC3-28Z及GPC3-28Z-IFN两种CAR-T细胞14天后，取肿瘤组织，组化检测CD3+细胞，结果如图12A和12B所示：每个样品取4-7个视野，统计CD3阳性T细胞数量。结果表明，对照组肿瘤组织中无明显浸润的CD3+细胞，INFβ-CAR-T治疗组中CD3+T细胞数量高于28ZCART组。
实施例13、含有IFN和不含IFN的CLD18A2 CAR-T细胞在体内对肿瘤浸润的影响
参照实施例10中建立的动物模型，回输Mock、85-28Z、85-2-28Z以及85-2-28Z-IFN细胞17天后，取肿瘤组织，组化检测CD3+细胞。
结果如图13所示，Mock T细胞在肿瘤组织周围几乎观察不到T细胞的浸润，85-28Z和85-2-28Z CAR T细胞在肿瘤组织的边缘可以看到，而85-2-28Z-IFN T细胞在肿瘤组织内部可以观察到一定的浸润。
实施例14、含有IFN和不含IFN的EGFR CAR的构建
EGFR-CAR(806-28Z,SEQ ID NO:56)和EGFR-CAR-IFN(由SEQ ID NO:60所示的核酸编码)结构如图14中所示。
采用逆转录病毒包装体系包装成逆转录病毒后感染小鼠T淋巴细胞，分别为EGFR-CAR和EGFR-CAR-IFN，感染阳性率分别为65.1％和35.2％(图15)。
实施例15、含有IFN和不含IFN的EGFR CAR(806-28Z)T细胞体外分泌mIFNβ能力的测定
为了检测EGFR-CAR-IFN诱导分泌mIFNβ的功能，我们将CAR-T细胞与靶细胞CT26-VIII按1:1和3:1共培养24h，取上清，ELISA检测mIFNβ的表达。同时，以没有靶点的CT26细胞作为阴性对照，刀豆蛋白A(ConA)作为阳性对照。结果显示，经过靶细胞刺激后，mCAR-806-mIFNβ能够被成功激活，并诱导表达mIFNβ，对照组则未检出有mIFNβ的表达(图16)。
实施例16、含有IFN和不含IFN的EGFR CAR(806-28Z)-T细胞的细胞因子的释放
为了验证构建的EGFRCAR和EGFR-CAR-IFN同样能够在靶细胞刺激下被有效激活，我们检测了在与靶细胞共孵育后muCAR-T细胞因子的分泌。将未经转染的UT细胞、EGFR-CAR和EGFR-CAR-IFN分别与靶点阳性的CT26-VIII细胞按照1:1比例共孵育24小时，取培养上清检测细胞因子mIL-2，mIFN-γ，mTNF-α分泌情况，靶点阴性的CT26细胞作为对照。结果显示，EGFR-CAR和egfr-CAR-IFN与靶细胞共孵育后均有较高浓度的mIL-2，mIFN-γ，mTNF-α分泌(图17A、17B、17C)，说明EGFR-CAR和EGFR-CAR-IFN在靶抗原的刺激下，两种muCAR-T细 胞均能被有效激活。
实施例17、含有IFN和不含IFN的EGFR CAR(806-28Z)-T细胞的体外毒性试验
为了比较EGFR-CAR和EGFR-CAR-IFN对靶细胞的体外杀伤活性，我们分别按照1:3、1:1或3:1的比例将EGFR-CAR和EGFR-CAR-IFN与EGFR阳性的CT26VIII细胞共孵育18小时，未经转染的小鼠UT细胞作为同型对照，CT26作为EGFR阴性对照。
结果显示，EGFR-CAR和EGFR-CAR-IFN与UT细胞相比，对靶点阳性的CT26VIII细胞具有强效的杀伤作用，差异有显著意义(***P＜0.001)，杀伤百分比呈剂量依赖性，而未经转染的UT细胞对CT26及CT26VIII均没有杀伤作用，EGFR-CAR和EGFR-CAR-IFN两种CAR-T细胞对靶点阴性的CT26细胞没有杀伤作用(图18)。
实施例18、含有IFN和不含IFN的EGFR CAR(806-28Z)-T细胞的体内毒性试验
使用稳定转染人源EGFRvIII-806位点的小鼠结肠癌CT26细胞系，在Babl/c小鼠体内接种皮下移植瘤，然后给予EGFR-CAR T细胞的治疗，结果如图19所示，EGFR-CAR-T细胞对对肿瘤大小与对照组基本一致，未观察到抑制作用，而EGFR-CAR-IFN细胞回输后，在第7天开始出现抑制肿瘤生长的现象，抑瘤率为5.9％，第10天时达到最强，为18.5％，到第17天时，抑瘤率仍可达到12.4％，明显优于EGFR-CAR-T细胞组。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
表5、本文中所用的序列
Figure PCTCN2017082024-appb-000011
Figure PCTCN2017082024-appb-000012
Figure PCTCN2017082024-appb-000013
Figure PCTCN2017082024-appb-000014
Figure PCTCN2017082024-appb-000015
Figure PCTCN2017082024-appb-000016
Figure PCTCN2017082024-appb-000017
Figure PCTCN2017082024-appb-000018
Figure PCTCN2017082024-appb-000019
Figure PCTCN2017082024-appb-000020
Figure PCTCN2017082024-appb-000021
Figure PCTCN2017082024-appb-000022
Figure PCTCN2017082024-appb-000023
Figure PCTCN2017082024-appb-000024
Figure PCTCN2017082024-appb-000025
Figure PCTCN2017082024-appb-000026
Figure PCTCN2017082024-appb-000027
Figure PCTCN2017082024-appb-000028
Figure PCTCN2017082024-appb-000029
Figure PCTCN2017082024-appb-000030
Figure PCTCN2017082024-appb-000031

Claims (29)

  1. 一种免疫应答细胞，其特征在于，该细胞表达结合抗原的受体；和外源性I型干扰素。
  2. 如权利要求1所述的免疫应答细胞，其特征在于，所述的免疫应答细胞包括：T细胞、自然杀伤细胞、细胞毒性T淋巴细胞、自然杀伤T细胞、DNT细胞、和/或调节性T细胞。
  3. 如前述权利要求之一所述的免疫应答细胞，其特征在于，所述的抗原是肿瘤抗原或病原体抗原。
  4. 如前述权利要求之一所述的免疫应答细胞，其特征在于，所述的外源性I型干扰素是组成性表达或诱导性表达；较佳地，用于表达所述的I型干扰素的启动子包括：免疫细胞诱导型启动子；较佳地，所述的免疫细胞诱导型启动子是NFAT6启动子。
  5. 如前述权利要求之一所述的免疫应答细胞，其特征在于，该细胞表达内源性或重组的结合抗原的受体；较佳地，所述结合抗原的受体包括顺序连接的：特异性结合所述抗原的抗体，跨膜区和胞内信号区。
  6. 如前述权利要求之一所述的免疫应答细胞，其特征在于，所述免疫应答细胞的胞内信号区含有T细胞刺激信号分子或T细胞刺激信号分子与T细胞激活共刺激分子的组合；较佳地，所述T细胞刺激信号分子选自：CD3ζ或FcεRIγ；更佳地为CD3ζ；或所述的T细胞激活共刺激分子选自：CD27，CD28，CD137，CD134，ICOS蛋白的胞内信号区，或其组合。
  7. 如前述权利要求之一所述的免疫应答细胞，其特征在于，所述结合抗原的受体的氨基酸序列与以下序列中的一个具有至少90％的同一性：
    SEQ ID NO:49；SEQ ID NO:50；SEQ ID NO:51；SEQ ID NO:54；SEQ ID NO:55；SEQ ID NO:56；SEQ ID NO:61；SEQ ID NO:62；SEQ ID NO:63；SEQ ID NO: 64；SEQ ID NO:65；SEQ ID NO:66；SEQ ID NO:67；SEQ ID NO:68；SEQ ID NO:69；SEQ ID NO:70；SEQ ID NO:71；SEQ ID NO:72；SEQ ID NO:73；SEQ ID NO:74；SEQ ID NO:75；以及SEQ ID NO:77。
  8. 如权利要求7所述的免疫应答细胞，其特征在于，所述的特异性结合抗原的受体以及所述的外源性I型干扰素由与SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60或SEQ ID NO:76具有至少90％同一性的核苷酸序列编码。
  9. 如前述权利要求之一所述的免疫应答细胞，其特征在于，所述的免疫应答细胞不包含外源的共刺激配体。
  10. 如前述权利要求之一所述的免疫应答细胞，其特征在于，所述的I型干扰素包括：IFNα或IFNβ。
  11. 如前述权利要求之一所述的免疫应答细胞，其特征在于，所述的结合抗原的受体和/或I型干扰素组成性或诱导性表达在免疫应答细胞的表面。
  12. 如前述权利要求之一所述的免疫应答细胞，其特征在于，所述的免疫应答细胞中包含表达构建物，该表达构建物包括：所述结合抗原的受体的表达盒；和所述I型干扰素的表达盒。
  13. 如前述权利要求之一所述的免疫应答细胞，其特征在于，所述的结合抗原的受体和/或I型干扰素利用病毒载体表达；较佳地，所述的病毒载体是逆转录病毒载体；更佳地，所述的病毒载体包括：慢病毒载体，逆转录病毒载体或腺病毒载体。
  14. 如前述权利要求之一所述的免疫应答细胞，其中所述结合抗原的受体识别并结合病原微生物。
  15. 如权利要求14所述的免疫应答细胞，其中所述病原微生物包括病毒、细菌、真菌、原生动物或寄生虫；更佳地，所述病原微生物为病毒；或者更佳地，所 述病原体微生物选自巨细胞病毒、爱泼斯坦-巴尔病毒、人类免疫缺陷病毒及流感病毒。
  16. 如前述权利要求之一所述的免疫应答细胞，其特征在于，所述的抗原包括：前列腺特异性膜抗原(PSMA)、癌胚抗原(CEA)、IL13Ralpha、HER-2、CD19、NY-ESO-1、HIV-1Gag、Lewis Y、MART-1、gp100、酪氨酸酶、WT-I、hTERT、间皮素、EGFR、EGFRvIII、磷脂酰肌醇蛋白聚糖3、EphA2、HER3、EpCAM、MUC1、MUC16、CLDN18.2、叶酸受体、CLDN6、CD30、CD138、ASGPR1、CDH16、GD2、5T4、8H9、αvβ6整合素、B细胞成熟抗原(BCMA)、B7-H3、B7-H6、CAIX、CA9、CD20、CD22、κ轻链(kappa light chain)、CD33、CD38、CD44、CD44v6、CD44v7/8、CD70、CD123、CD171、CSPG4、EGP2、EGP40、ERBB3、ERBB4、ErbB3/4、FAP、FAR、FBP、胚胎型AchR、GD2、GD3、HLA-AI MAGE A1、MAGE3、HLA-A2、IL11Ra、KDR、Lambda、MCSP、NCAM、NKG2D配体、PRAME、PSCA、PSC1、ROR1、Sp17、SURVIVIN、TAG72、TEM1、TEM8、VEGRR2、HMW-MAA、VEGF受体、和/或纤连蛋白、腱生蛋白或肿瘤坏死区的癌胚变体。
  17. 一种表达构建物，其特征在于，该表达构建物包括顺序连接的：结合抗原的受体的表达盒；和I型干扰素的表达盒；其中所述结合抗原的受体和I型干扰素如前述权利要求之一所定义。
  18. 一种提高向个体给予的免疫应答细胞活力的方法，其特征在于，所述免疫应答细胞表达如权利要求1至16之一所述的结合抗原的受体，并且其中所述方法包括向所述个体给予所述免疫应答细胞以及有效量的外源性I型干扰素。
  19. 如权利要求18所述的方法，其中所述外源性I型干扰素与所述表达结合抗原的受体的免疫应答细胞顺序给予或者同时给予。
  20. 如权利要求18或19所述的方法，其中所述外源性I型干扰素通过在免疫应答细胞中共表达，与所述免疫应答细胞同时向患者给予。
  21. 如权利要求18至20之一所述的方法，其中所述免疫应答细胞包括T细胞、 自然杀伤细胞、细胞毒性T淋巴细胞、自然杀伤T细胞、DNT细胞、和/或调节性T细胞。
  22. 如权利要求18至21之一所述的方法，其中所述方法使得在向所述个体给予所述免疫应答细胞后，与不存在所述外源性I型干扰素的情况相比，所述个体外周血中细胞毒性T细胞和辅助T细胞的数量之和提高至少50％。
  23. 如权利要求18至22之一所述的方法，其中所述方法使得在向所述个体给予所述免疫应答细胞约5天后，所述个体外周血中细胞毒性T细胞和辅助T细胞的数量之和大于15,000个/μL；给予所述免疫应答细胞约7天后，所述个体外周血中细胞毒性T细胞和辅助T细胞的数量之和大于500个/μL；或者给予所述免疫应答细胞约10天后，所述个体外周血中细胞毒性T细胞和辅助T细胞的数量之和大于50个/μL。
  24. 权利要求1-16之一所述的免疫应答细胞在制备用于治疗有此需要的个体的肿瘤、病原体感染、或增强个体免疫耐受能力的药物组合物中的用途。
  25. 如权利要求24所述的用途，其特征在于，所述的肿瘤包括：胰腺癌、肝癌、肺癌、胃癌、头颈部鳞状细胞癌、前列腺癌、结肠癌、乳腺癌、淋巴瘤、胆囊癌、肾癌、白血病、骨髓瘤、卵巢癌、宫颈癌、卵巢癌、宫颈癌或胶质瘤；或
    所述的病原体包括：病毒、细菌、真菌、原生动物或寄生虫；较佳地，所述的病毒包括：巨细胞病毒、爱泼斯坦-巴尔病毒、人类免疫缺陷病毒或流感病毒。
  26. 如权利要求24或25所述的用途，其中根据计算机断层扫描测量，所述药物使得肿瘤减小至少30％。
  27. 如权利要求24或25所述的用途，其中根据计算机断层扫描测量，所述药物使得肿瘤完全消失。
  28. 药物组合物，其特征在于，所述的药物组合物包括：
    权利要求1-16之一所述的免疫应答细胞；以及
    药学上可接受的载体或赋形剂。
  29. 试剂盒，其包含：
    如权利要求1-16之一所述的免疫应答细胞；以及
    指导如何向个体给予所述免疫应答细胞的说明书。
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EP17823694.9A EP3483182B1 (en) 2016-07-08 2017-07-10 Antibody for anti-claudin 18a2 and use thereof
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SG11201900171QA SG11201900171QA (en) 2016-07-08 2017-07-10 Antibody for anti-claudin 18a2 and use thereof
ES17823694T ES2979068T3 (es) 2016-07-08 2017-07-10 Anticuerpos para anti-claudina 18A2 y utilización de los mismos
CA3030257A CA3030257A1 (en) 2016-07-08 2017-07-10 Antibody for anti-claudin 18a2 and use thereof
AU2017294276A AU2017294276B2 (en) 2016-07-08 2017-07-10 Antibody for anti-claudin 18A2 and use thereof
RU2019101430A RU2793445C2 (ru) 2016-07-08 2017-07-10 Антитело против клаудина 18а2 и его применение
US16/316,331 US11111295B2 (en) 2016-07-08 2017-07-10 Antibody for anti-claudin 18A2 and use thereof
KR1020197003874A KR20190038564A (ko) 2016-07-08 2017-07-10 항클라우딘 18a2의 항체 및 이의 응용
CN201780042611.4A CN109790222B (zh) 2016-07-08 2017-07-10 抗密蛋白18a2的抗体及其应用
IL264144A IL264144B2 (en) 2016-07-08 2017-07-10 Antibody 18A2 against CLAUDIN and its use
PCT/CN2017/092381 WO2018006882A1 (zh) 2016-07-08 2017-07-10 抗密蛋白18a2的抗体及其应用
BR112019000327A BR112019000327A8 (pt) 2016-07-08 2017-07-10 Anticorpo para anticlaudina 18a2 e uso do mesmo
CL2019000061A CL2019000061A1 (es) 2016-07-08 2019-01-08 Anticuerpo para anti-claudin 18a2 y su utilización.
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