WO2017186121A1 - Method for improving function of immune response cell - Google Patents

Abstract

Provided are a method for improving the function of an immune response cell and an immune response cell which expresses at least one receptor capable of binding to an antigen and type I interferon. The cell has a significant ability to kill tumours or pathogens and can be used for treating tumours and infectious diseases.

Description

Method for improving immune response cell function

cross reference

The present application claims the benefit of the Chinese patent application No. 201610265614. filed on Apr. 26, 2016, and the Chinese patent application No. The entire content is incorporated herein.

Technical field

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.

Background technique

Chimeric antigen receptor (CAR) 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. In recent years, the use of CAR-modified T-cell (CAR-T) cells targeting CD19 in clinical trials has been a great success in the treatment of B-cell leukemia. However, many leukemia patients have recurrence. And not all blood tumors are very effective. In addition, the treatment of solid tumors with CAR-T cells is also less effective. Therefore, how to improve existing CAR-T cell technology and increase its anti-tumor activity is still very important.

Type I interferons were discovered more than half a century ago. Type I interferons contain IFNα protein (a class of identical proteins encoded by 13 human genes from IFNA1 to IFNA13), IFNβ (encoded by a single individual and mouse gene IFNB1), and other less studied interferons such as IFNε, IFNκ and IFNω (2. Trinchieri, G. Type Iinterferon: friend or foe? J. Exp. Med. 207, 2053-2063 (2010). 3. Kaur, S. & Platanias, LCIFN-β-specific signaling via a unique IFNAR1 interaction .Nat.Immunol. 14, 884-885 (2013)). 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 IFNα/β receptor 1 (IFNAR1), which has a particularly high affinity for IFNβ, or IFNAR1-IFNAR2 heterodimer (which binds to all type I interferons) signal. Activation of these receptors leads to transcriptional upregulation of IFN-stimulated genes (ISGs), triggering many 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-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, 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-α 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. 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)), such serious side effects severely limit its application.

Summary of the invention

The present invention overcomes the aforementioned problems and has additional advantages.

According to an aspect of the present invention, the present invention provides an immune response cell characterized in that the cell expresses an antigen-binding receptor; and an exogenous type I interferon.

In some embodiments, 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.

In some embodiments, 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). In some embodiments, examples of the ITAM comprising a cytoplasmic signaling sequence include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

In some embodiments, the intracellular signaling region of the antigen binding receptor comprises one or more costimulatory domains. In some embodiments, the costimulatory domain is selected from one or more of those listed in Table 1. In some embodiments, 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. In some embodiments, 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 .

In some embodiments, 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 One has at least 90% identity.

In some embodiments, 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.

In some embodiments, the immune response cell does not comprise an exogenous co-stimulatory ligand.

In some embodiments, the antigen capable of being bound by the antigen-binding receptor comprises a tumor antigen or a pathogen antigen. In some embodiments, 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, αvβ6 integrin, B cell mature antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD20, CD22, kappa light chain (kappa Light chain), CD33, CD38, CD44, CD44v6, CD44v7/8, CD70, CD123, CD171, CSPG4, EGP2, EGP40, ERBB3, ERBB4, ErbB3/4, FAP, FAR, FBP, embryonic AchR, GD2, GD3, HLA-AI MAGE A1, MAGE3, HLA-A2, IL11Ra, KDR, Lambda, MCSP, NCAM, NKG2D ligand, PRAME, PSCA, PSC1, ROR1, Sp17, SURVIVIN, TAG72, TEM1, TEM8, VEGRR2, HMW-MAA, Carcinoembryonic variants of VEGF receptors, and/or fibronectin, tenascin, or tumor necrotic regions.

In some embodiments the pathogen antigen comprises a viral antigen, a bacterial antigen, a fungal antigen, or a parasite antigen. In some embodiments, the pathogen is a virus. The viruses include cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, and influenza virus.

In some embodiments, 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: IFNα or IFN β.

According to an aspect of the present invention, 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. In some embodiments, 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 expression of the type I interferon is inducible expression and the expression for expressing the type I interferon is an inducible promoter. In some embodiments, the inducible promoter for expression of the type I interferon is the NFAT6 promoter. In some embodiments, the NFAT6 promoter comprises the nucleic acid sequence set forth in SEQ ID NO:78.

According to one aspect of the invention, the invention provides a vector which expresses an antigen binding receptor of the invention and/or a type I interferon. In some embodiments, the viral vector is a lentiviral vector, a retroviral vector, or an adenoviral vector. In some embodiments, the viral vector is a retroviral vector.

According to one aspect of the invention, 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. In some embodiments, the exogenous type I interferon is administered sequentially or simultaneously with the immune response cell expressing the antigen binding receptor. In some embodiments, the exogenous type I interferon is administered to a patient simultaneously with the immune response cell by co-expression in an immune response cell.

According to one aspect of the invention, 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.

In some embodiments, 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%. In some embodiments, 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/μL; 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 / μL; 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/μL.

In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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.

According to one aspect of the invention, the invention provides a pharmaceutical composition comprising an immune response cell of the invention and a pharmaceutically acceptable carrier or excipient.

According to one aspect of the invention, 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.

Incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the extent Into the general.

DRAWINGS

The drawings further illustrate the new features disclosed in this specification. The features and advantages of the present disclosure will be better understood from the following description of the accompanying drawings. The scope of the limit is limited.

Figure 1 shows the structure of the recombinant lentiviral vector pRRL-EF-1α-92-CAR (Fig. 1A) and the construction of the 92-28Z-NFAT6-IFN-β 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-β 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-β 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-γ-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 IFNβ 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 IFNβ can be activated more efficiently.

Figure 6 shows GPC3-28Z CAR T cells containing IFNβ and GPC3 CAR T cells not containing IFNβ 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.

Figure 8 shows that CLD18A2 CAR-T cells containing IFNβ and CLD18A2 CAR-T cells not containing IFNβ 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 IFNβ was significantly higher than that of the CLD18A2 CAR-T cell group not containing IFNβ at all time points.

Figure 9 is a comparison of the effect of GPC3-28Z CAR T cells containing IFNβ and GPC3-28Z CAR T cells not containing IFNβ 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 IFNβ were able to more significantly reduce tumor volume than GPC3-28Z CAR T cells not containing IFNβ and the control group.

Figure 10 is a graph showing the effect of CLD18A2 CAR-T cells containing IFNβ and CLD18A2 CAR-T cells not containing IFNβ 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 IFNβ were able to more significantly reduce tumor volume than CLD18A2 CAR-T cells not containing IFNβ 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, and 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 INFβ-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 mIFNβ in vitro. The results showed that mCAR-806-mIFNβ was successfully activated and induced to express mIFNβ after stimulation by target cells, and no expression of mIFNβ 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-γ; Figure 17C: mTNF-α) 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. However, untransfected UT cells had no killing effect on CT26 and CT26VIII, and 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.

detailed description

The detailed description below discloses the embodiments disclosed herein in detail. It should be understood that the description is not intended to be limited to the particular embodiments disclosed herein, but may vary. It will be appreciated by those skilled in the art that the present invention may be susceptible to various changes and modifications. Each embodiment can be combined arbitrarily with any other embodiment unless otherwise stated.

Certain embodiments disclosed herein are intended to encompass a range of values, and some aspects of the invention may be described in a range. The scope of the invention is to be understood as being limited by the scope of the invention Therefore, the description of the scope of the invention should be construed as being specifically described as all possible sub-ranges and all possible specific numerical points within the range, as these sub-ranges and numerical points are explicitly recited herein. For example, the description of the range from 1 to 6 should be considered to specifically disclose sub-ranges from 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and within these ranges Specific numerical points, such as 1, 2, 3, 4, 5, 6. Regardless of the breadth and width of the stated values, the above principles are equally applicable. When a range description is used, the range includes the endpoints of the range.

In order to overcome the defects in the prior art, 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. On the basis of this, 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. Disease and other diseases have significantly superior ability to kill tumors or pathogens.

The terms "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. For example, the term "activation" can refer to the process by which T cells are gradually activated. For example, 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). In vitro, anti-CD3 can simulate the first signal and anti-CD28 can simulate the second signal. For example, engineered T cells can be activated by the expressed CAR. As used herein, 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.

The term "co-stimulatory ligand" as used herein 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. Antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and ligands that specifically bind to CD83.

The term "costimulatory molecule" as used herein, 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.

As used herein, "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.

The term "antigen binding unit" as used herein 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"). Also included in the term "antigen-binding unit" are 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. The term "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. ".

As used herein, "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.

The term "immunoglobulin" or "Ig" as used herein 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.

The term "self" as used herein and its grammatical other forms may refer to from the same source. For example, a sample (eg, a cell) can be removed, processed, and administered to the same individual (eg, a patient) at a later time. The autologous process differs from the allogeneic process in which the donor and recipient are different individuals.

As used herein, "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.

As used herein, "allografting" and other forms of grammar thereof (eg, 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.

As used herein, "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.

The term "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.

The term "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. For example, 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.

The term "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. The term "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.

The term "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.

The term "transfection" as used herein 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.

The term "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. The term "stable transfectant" refers to a cell that stably integrates foreign DNA into genomic DNA.

As used herein, the terms "nucleic acid molecule encoding", "coding DNA sequence" and "coding DNA" refer 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.

The term "peripheral blood lymphocytes" (PBL) 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.

The term "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.

The term "T cell" and its grammatical other forms as used herein may refer to T cells of any origin. For example, 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.

The term "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.

The term "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.

The term "effective amount" as used herein refers to an amount that provides a therapeutic or prophylactic benefit.

The term "expression vector" as used herein, 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).

The term "lentivirus" as used herein 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.

The term "operably linked" as used herein, refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the latter. For example, 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. For example, a promoter is operably linked to a coding sequence if the promoter affects transcription or expression of the coding sequence. Typically, the operably linked DNA sequences are contiguous and, where necessary, ligated two protein coding regions in the same reading frame.

The term "promoter" as used herein, 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.

The term "vector," as used herein, 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. A number of vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. Thus, the term "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. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

The term sequence "identity" as used herein, 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 %). 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.

The term "type I interferon" as used herein includes IFNα, IFNβ, and IFN-ε, IFN-κ, and IFN-ω 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 IFNα or IFNβ. In some embodiments, the term "type I interferon" as used herein is IFNβ. In some embodiments, a type I interferon as used herein includes human, mouse, or synthetic type I. Interferon. In some embodiments, 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. In some embodiments, the term "interferon beta" (INF-β) 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.

In some embodiments, 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. For example, recombinant elements or type I interferons can be produced according to conventional genetic engineering recombination techniques. Preferably, 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.

The bioactive fragments of the respective elements or polypeptides of type I interferon can be used in the present invention. As used herein, the meaning of 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. Typically, the biologically active fragment retains at least 50% of the activity of the full length polypeptide. Under more preferred conditions, 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. A modified or modified polypeptide. That is, any variation that does not affect the biological activity of the polypeptide can be used in the present invention.

The term "disease" or "condition" or "disorder" as used herein, refers to any alteration or disorder that impairs or interferes with the normal function of a cell, tissue or organ. For example, the term "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).

The term "tumor" as used herein 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, endothelial sarcoma, lymphangisarcoma, angiosarcoma, lymphatic endothelial sarcoma, synovial vioma , mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary Cancer, papillary adenocarcinoma, cancer, bronchial carcinoma, medullary carcinoma, renal cell carcinoma, liver cancer, Nile tube cancer, choriocarcinoma, fine Tumor, embryonic carcinoma, nephroblastoma, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, Ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannomas, meningioma, melanoma, neuroblastoma, retinoblastoma), esophageal cancer, gallbladder carcinoma , kidney cancer, multiple myeloma. Preferably, 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. When the immune system is immature and unable to respond, 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. .

Non-limiting examples of 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. Other large, protein-based antigens include 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-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, 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 binding protein\Cyclophilin C-related protein, TAAL6, TAG72 , TLP, and TPS.

In some embodiments, 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, αvβ6 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, CD171, CSPG4, EGP2, EGP40, ERBB3, ERBB4, ErbB3/4, FAP, FAR, FBP, embryonic AchR, GD2, GD3, HLA-AI MAGE A1, MAGE3, HLA-A2, IL11Ra, KDR, Lambda, MCSP, NCAM, NKG2D ligand, PRAME, PSCA, PSC1, ROR1, Sp17, SURVIVIN, TAG72, TEM1, TEM8, VEGRR2, HMW-MAA, VEGF receptor, and/or carcinoembryonic variants of fibronectin, tenascin, or tumor necrotic regions.

The term "pathogen" as used herein refers to protozoa that are capable of causing a disease, including: viruses, bacteria, fungi or parasites. The "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 (such as influenza virus); subviral (such as Hantavirus, partial virus, white peony and Neurovirus); stage virus family (hemorrhagic fever virus); reoviridae (such as reovirus, circovirus and rotavirus); ribonucleic acid virus; liver virus (hepatitis B virus); Branch (parvovirus); papovavirus family (papilloma virus, polyoma virus); adenoviridae (most adenovirus); herpes virus (herpes simplex virus (HSV) 1 and 2, chickenpox-band Herpes virus, cytomegalovirus (CMV), herpes simplex virus; poxvirus (variola virus, vaccinia virus, poxvirus); iridescent virus family (such as African swine fever virus); and unclassified virus (such as delta hepatitis agent) Is a defective satellite TE hepatitis B), non-A, non-B hepatitis drugs, (Grade 1 = internal delivery; 232 non-intestinal transmission (ie hepatitis C); Norwalk and related viruses and Astrovirus).

Typical bacteria include, but are not limited to, Pasteurella, Staphylococcus aureus, Streptococcus, Escherichia coli, Salmonella, and Pseudomonas aeruginosa. Specific examples of infectious bacteria include, but are not limited to, Helicobacter pylori, spirochetes, Legionella pneumophila, Mycobacterium sp. (S. tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis, M. Kansaii, M. gordonae) , Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (group A streptococci), Streptococcus agalactiae (group B streptococcus streptococci) Group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic SPS), Streptococcus pneumoniae, Campylobacter, Enterococcus, Haemophilus influenzae, Bacillus anthracis, Diphtheria, Corynebacterium, Clostridium erysipelas Filaria, Clostridium perfringens, tetanus, Enterobacter cloacae, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides, Fusobacterium nucleatum, Rosary chain, Treponema pallidum, Yarrow , Leptospira, Rickettsia, Israel actinomycetes.

In some embodiments, 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. In some embodiments, 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 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 costaricensi s; 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; Trombulidae; Pulex irritans; Ixodidae and Argasidae.

The term "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. Examples of 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, vitiligo, myxedema, pernicious anemia, ulcerative colitis, etc.

"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 &quot;regulatory&quot; 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. . In adults, 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.

Immunological recognition of non-self antigens often complicates the transplantation and transplantation of foreign tissues from organisms of the same species (allogeneic allografts), resulting in transplant rejection. Lymphocytes, particularly T lymphocytes, play a key role in allograft rejection, graft failure, and GVHD. There are usually two cases where allogeneic transplantation is acceptable. One is when a cell or tissue is transplanted into an immune-immunity site (such as in the eye or testis) isolated from an immune surveillance system, or has a strong molecular signal to prevent dangerous inflammation (as in the brain). 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).

The term "enhancing immune response cell function" as used herein includes, for example, enhancing T cell function. Taking T cells as an example, 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. Examples of 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. In one embodiment, 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.

The term "T cell dysfunctional disease" as used herein includes a condition or disorder of a T cell characterized by a decrease in antigenic stimulatory reactivity. In some embodiments, the T cell dysfunctional disorder is a disorder associated with inappropriately increased signaling specificity by PD-1. In some embodiments, 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.

The term "exogenous" as used herein, 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. Thus, "exogenous" includes recombinant nucleic acid molecules or polypeptides expressed in a cell, such as exogenous, heterologous and overexpressed nucleic acid molecules and polypeptides.

The term "receptor" as used herein, refers to a polypeptide, or a portion thereof, that selectively binds one or more ligands on a cell membrane.

In some embodiments, an antigen binding receptor of the invention specifically binds to an antigen to which it binds.

In some embodiments, the antigen binding receptor of the invention is a chimeric antigen receptor. The term "Chimeric Antigen Receptor (CAR)" as used herein refers to a tumor antigen binding domain fused to an intracellular signal transduction domain that activates T cells. Typically, 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. In some embodiments, 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.

A variety of 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. In some embodiments, 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. Examples of 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. Alternatively, extracellular antigen binding regions can be obtained from whole-out cleavage of tumor cells or primary cells, such as tumor infiltrating lymphocytes (TIL).

In some embodiments, 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. In many cases, 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. For example, 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.

In certain aspects of any of the embodiments disclosed herein, the extracellular antigen binding region, eg, the scFv, 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). In some embodiments, 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. In some embodiments, 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. In some examples, a set of CDRs present on a common light chain can be collectively referred to as a light chain CDR.

In certain aspects of any of the embodiments disclosed herein, the extracellular antigen binding region, eg, the scFv, 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). In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In other cases, 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.

In some embodiments, 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. In some embodiments, 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. For example, 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.

In some embodiments, 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. In other cases, the natural transmembrane portion of CD8α 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. In some embodiments, 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. Thus, the term 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.

In some embodiments, the intracellular signaling region can contain a known signal motif for an immunoreceptor tyrosine activation motif (ITAM). Examples of ITAMs containing cytoplasmic signaling sequences include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. However, in a preferred embodiment, the intracellular signal domain is derived from a CD3 ζ chain.

An example of a T cell signaling domain containing one or more ITAM motifs is the CD3ζ domain, also known as the T cell receptor T3 ζ 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. As used herein, CD3ζ primarily refers to human CD3ζ and its isoforms, as known from the Swissprot entry P20963, including proteins having substantially the same sequence. As part of the chimeric antigen receptor, it is reiterated that the full T cell receptor T3 ζ chain is not required and that any derivative of the signal domain comprising the T cell receptor T3 ζ chain is suitable, including any functional equivalent thereof. .

The intracellular signaling domain can be selected from any one of the domains of Table 1. In some embodiments, 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 ζ chain (first generation CAR) or it is with CD28 or 4-1BB (second generation CAR). In other examples, the intracellular signaling region can comprise two costimulatory domains, such as CD28/OX40 or CD28/4-1BB (third generation).

Together with intracellular signaling domains such as CD8, these costimulatory domains can generate downstream activation of the kinase pathway, thereby supporting gene transcription and functional cellular responses. The co-stimulatory domain of CAR can activate activation of CD28 (phosphatidylinositol-4,5-diphosphate 3-kinase) or 4-1BB/OX40 (TNF-receptor-associated factor adapter protein) pathways as well as MAPK and Akt activation. Proximal signal protein.

In some cases, signals generated by the CAR may be combined with an auxiliary or costimulatory signal. For costimulatory signaling domains, chimeric antigen receptor-like complexes can be designed to contain several possible costimulatory signal domains. As is well known in the art, in naive T cells, the individual engagement of T cell receptors is not sufficient to induce Complete activation of T cells is cytotoxic T cells. A second co-stimulatory signal is required for complete productive T cell activation. Several 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.

In some embodiments, the addition of a costimulatory domain to a chimeric antigen receptor-like complex can enhance the efficacy and durability of engineered cells. In another embodiment, the T cell signal domain and the costimulatory domain are fused to each other to form a signaling region.

Table 1. Costimulatory domains

The chimeric antigen receptor binds to the target antigen. When T cell activation is measured in vitro or ex vivo, 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. In some embodiments, 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.

The target antigen can also be derived from or isolated from the pathogenic bacteria described herein. In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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. For example, in some embodiments, a chimeric antigen receptor of the invention recognizes and binds to a tumor antigen comprising TSA and TAA as described herein before.

The term "modulation" as used herein refers to a positive or negative change. Modification examples include 1%, 2%, 10%, 25%, 50%, 75%, or 100% variation.

The term "treatment" as used herein 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.

The term "immune dysfunction" as used herein 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.

The term "constitutive expression" as used herein refers to expression under all physiological conditions.

The term "induced expression" as used herein refers to expression under conditions such as when T cells bind to an antigen. One skilled in the art how to perform conventional "induced expression".

In some embodiments, the invention provides an immune response cell that expresses an antigen binding receptor and an exogenous type I interferon.

In some embodiments, 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. ,Annexin II,ART-4,ARTC1,BAGE,BAGE-1,BCLX(L),BCMA,BING-4,BRACHYURY(IVS7T/C polymorphism),BRACHYURY(TIVS7-2,polymorphism),BRACHYURY, B-RAF, CAMEL, CAR-ABL fusion protein (b3a2), CASP-5, CASP-8, Cdc27, CDC27/m, CDK4, CDK-4/m, CDKN2A, CEA, COA-1, CPSF, Cyp-B , DAM-6, -10, dek-can fusion protein, DKK1, ETFUD2, EGFR, ELF2M, ENAH (hMena), EP-CAM, EphA3, ESO-1/LAGE-2, ETV6-AML1 fusion protein, 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-fucosyltransferase fusion protein, 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, MUC 1 (VNTR more State) -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 and TEL/AML1pml-RARα fusion protein, 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- or -SSX2 fusion protein, TBRACHYURY, T, TAG-1, TAG-2, TGF-βRII, TPI/mbcr-abl, TLAG-3, TRP-2, TRP -1, TRP-1/gp75, TRP-2, TRP-2/INT2, TRP2-INT2g, VEGF and/or WT1, XAGE-1b, α-actinin-4, β-catenin, β-catenin Protein/m, triphosphate isomerase, mammaglobin-A, human papillomavirus (HPV), human epidermal growth factor receptor 2 (HER2/neu), human epidermal growth factor receptor 3 (HER3), elongation Factor 2, prostate specific antigen (PSA), survivin, new PAP, kallikrein 4, alpha-fetoprotein, telomerase, mucin, cyclin D1, myosin/m, myosin I, Intestinal carboxylesterase, caspase-8/m, tyrosinase.

Furthermore, the immune response cells of the invention can target tumor associated antigens. 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. In some cases, the antigen can be a neo-antigen. The new antigen can be derived from somatic mutations in cancer cells. For example, 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.

In certain instances, 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. For example, 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). In some cases, the target antigen can be expressed on the surface of cancer cells. In some cases, 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. In some cases, 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.

In some embodiments, the antigen binding receptor comprises an antigen binding domain (extracellular binding region) and an intracellular signal domain capable of activating immune response cells. Preferably, a transmembrane region is also included between the antigen binding domain and the intracellular signal domain (intracellular signal region). In one embodiment, 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.

In some embodiments, 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.

In some embodiments, an antigen binding domain of the invention is a binding domain that binds to a tumor antigen. In some embodiments, 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 Such as 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; and viral antigens such as Epstein Barr virus antigen EBVA and human papilloma Virus (HPV) antigens E6 and E7, etc. Other large, protein-based antigens include 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-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, 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 binding protein\Cyclophilin C-related protein, TAAL6, TAG72 One or more of TLP, TLP, and TPS. In some embodiments, 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, αvβ6 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, CD171, CSPG4, EGP2, EGP40, ERBB3, ERBB4, ErbB3/4, FAP, FAR, FBP, embryonic AchR, GD2, GD3, HLA-AIMAGE A1, MAGE3, HLA- A2, IL11Ra, KDR, Lambda, MCSP, NCAM, NKG2D ligand, PRAME, PSCA, PSC1, ROR1, Sp17, SURVIVIN, TAG72, TEM1, TEM8, VEGRR2, HMW-MAA, VEGF receptor, and/or fibronectin One or more of carcinoembryonic variants of neoplastic or tumor necrotic regions. In some embodiments, 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.

In some embodiments, 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, αβ or γδ. In some embodiments, the transmembrane region is selected from the transmembrane region of CD8 alpha or CD28. In addition, the CD8α 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 CD3ζ, FcεRIγ, CD28 costimulatory signal domain, CD137 costimulatory signal domain, and combinations thereof. The CD3 molecule consists of five subunits, of which the CD3ζ subunit (also known as CD3zeta, abbreviated as Z) contains three ITAM motifs, which are important signal transduction regions in the TCR-CD3 complex. Furthermore, as previously described, 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-γ secreted by immune response cells, and improve the survival cycle and anti-tumor effect of CAR immune response cells in vivo. In some embodiments, the intracellular signal transduction domain is a combination of a CD3ζ signal domain or a CD3ζ signal domain with other costimulatory signals such as CD28.

In some embodiments, 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, CD3ζ, and in opposition to the aforementioned elements Linked NFAT6, type I interferon expression unit. Preferably, the antibody and the CD28 costimulatory signal domain are joined by a CD8 alpha transmembrane region and a CD8 alpha hinge region.

In some embodiments, 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. Based on this consideration, 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).

According to one aspect of the invention, 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. In a specific embodiment, 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. 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. 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. Thus, those skilled in the art will appreciate that the use of these 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.

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. Those skilled in the art will appreciate that competitively blocking the interaction of PD-L1 with its receptor PD-1 facilitates the recovery of anti-tumor T cell responses, thereby inhibiting tumor growth.

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.

In some embodiments, 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. For example, a transgene can be incorporated into an immune response cell, such as a T cell. When inserted into a 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.

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. For example, 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.

In some embodiments, 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. In some embodiments 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.

In some embodiments, the transgene can encode a suicide gene. As evidenced by many effective treatments for cancer patients, CAR immune response cells cause tumor regression but can be associated with toxicity. In some embodiments, 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"). In other cases, a systemic disturbance of the immune system, called cytokine release syndrome (CRS), can occur. The 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. In most cases, 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.

In some embodiments, 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. In addition, 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.

One or more of the transgenes used herein may be from different species. For example, 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. For example, 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.

As described above, the transgene can be inserted into the genome of the immunoreactive cell in a random or site-specific manner. For example, 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. For example, a transgene can encode its own promoter or can be inserted into a position controlled by its internal promoter. Alternatively, 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.

In some embodiments, 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. Alternatively, 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.

In some embodiments, 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.

In some embodiments, a polynucleic acid encoding a receptor of interest can be designed to be delivered to a cell by non-viral techniques. In some cases, the polynucleic acid can be a Good Manufacturing Practice (GMP) compatible reagent.

Expression of a polynucleic acid encoding a receptor that binds to an antigen or a CAR can be controlled by one or more promoters. 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. For example, one or more transgenes can be inserted adjacent to or proximate to the endogenous or exogenous ROSA26 promoter. Furthermore, the promoter may be specific for immunoreactive cells. For example, 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. For example, 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.

The term "inducible promoter" as used herein 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. For example, in some embodiments, 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. In some embodiments, an inducible promoter for use herein includes Hypoxia-Inducible Transcription factor-1α (HIF-1α). In some embodiments, 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. A gene operably linked thereto, and only when the immune response cell contacts the antigen or the immune response cell is activated, drives the gene operably linked to a high level of expression or is expressed under conditions such as hypoxia. In some embodiments, the "immune cell-inducible promoter" comprises a NFAT (activated T cell nuclear factor) type promoter.

As used herein, "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). 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.

Based on the role of 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. An operatively linked gene.

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). As used herein, "NFAT-type promoter" refers to one or more NFAT response elements linked to the minimal promoter of any gene expressed by a T cell. Preferably, 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. In some embodiments, more than one NFAT binding motif can be included in a "NFAT-type promoter" as described herein. For example, the "NFAT-type promoter" can include 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NFAT binding motifs. In some embodiments, the "NFAT-type promoter" includes up to 12 NFAT binding motifs. In some embodiments, 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. In some embodiments, the NFAT type promoters described herein comprise six NFAT binding motifs, designated (NFAT) ₆ . For convenience purposes, the (NFAT) ₆ is also referred to as NFAT6. In some embodiments, the NFAT6 also represents a 6-repeat NFAT binding motif (SEQ ID NO: 78) in the NFAT-type promoter.

Furthermore, although not essential for expression, 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.

In some embodiments, 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. In some embodiments, the transgene is inserted into the PD1 and/or CTLA-4 locus. In other cases, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, a transgene can be introduced into an immunoreactive cell using a retroviral vector (gamma-retroviral or lentiviral vector). For example, 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.

Many virus-based systems have been developed for the transfer of genes into mammalian cells. For example, 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. In some embodiments, 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. In some embodiments, the amount of nucleic acid (eg, double stranded DNA) required for optimal transfection efficiency and/or cell viability varies depending on the cell type. In some embodiments, 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.

In some embodiments, the immunoreactive cell can be a dry memory consisting of CD45RO(-), CCR7(+), CD45RA(+), CD62L+ (L-selectin), CD27+, CD28+, and/or IL-7Rα+ T _SCM cells, which also express CD95, IL-2Rβ, CXCR3, and/or LFA-1, and exhibit many different functional properties from the stem memory cells. Alternatively, 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.

Delivery of 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. Alternatively, 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. In some cases, 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. In some cases, 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. In one embodiment, 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. In one embodiment, cells collected by apheresis collection can be washed to remove plasma fractions and placed in a suitable buffer or medium for subsequent processing steps.

Alternatively, cells can be derived from a healthy donor, from a patient diagnosed with cancer, or a patient diagnosed with an infection. In some embodiments, 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. Alternatively, the cells can be used for allogeneic transplantation. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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. It is also possible to select T cells from a large population, for example to select T cells from whole blood. T cells can also be expanded from a large population. T cells may also be inclined to specific populations and phenotypes. For example, a T cell can be tilted to a phenotype comprising CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+), and/or IL-7Rα(+). Suitable cells may be selected from one or more of the following list: CD45RO (-), CCR7 (+), CD45RA (+), CD62L (+), CD27 (+), CD28 (+) and/or IL-7Rα(+). 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. 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) ). In some embodiments, 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. In In some embodiments, 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. In some embodiments, 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. In some embodiments, determining the amount of therapeutically effective cells required can include determining a function associated with changes in cells over time. In some embodiments, 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). In some embodiments, 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. In some embodiments, 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%.

In some embodiments, 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. For example, in some cases, 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. For example, in some embodiments, 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. The term "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.

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。Specific examples of some substances which can be used as pharmaceutically acceptable carriers or components thereof are 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.

The 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. Thus, a therapeutically effective population of immunoreactive cells can be administered to an individual. Typically, administration contain from about 1 × 10 ⁴ to about 1 × 10 ¹⁰ cells reactive immunization formulation. In most cases, 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. However, depending on the location, source, identity, extent and severity of the cancer, the age and physical condition of the individual to be treated, and the like, the number of CAR immunoreactive cells administered to the individual will vary from wide range. The doctor will finalize the appropriate dose to use.

In some embodiments, a chimeric antigen receptor is used to stimulate an immune cell mediated immune response. For example, 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. In another embodiment, 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.

In certain instances, 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. As described above, 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. As described herein, since 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.

In some embodiments, an immune response cell of the invention can be administered in combination with another therapeutic agent. In some embodiments, 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, chlorambucil, briquetazine, uracil mustard, cloprofen and dacarbazine; antimetabolites, including cytarabine, fluorouracil, methotrexate, Anthraquinone, azathioprine and procarbazine; antibiotics including but not limited to doxorubicin, bleomycin, dactinomycin, daunorubicin, mycinmycin, mitomycin, sarcoma C And daunorubicin; and other chemotherapeutic drugs, including but not limited to anti-tumor antibodies, Dacaba , Azacytidine, Emushakang, melphalan, ifosfamide and mitoxantrone.

In some embodiments, 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.

In some embodiments, 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. If desired, 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.

The invention also provides 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. In one embodiment, 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. In some embodiments, 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. In some embodiments, immunoreactive cells, such as CAR T cells, can be provided, as well as 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. Instructions will generally include information regarding the composition used to treat or prevent cancer, pathogen infection, immune disease, or allogeneic transplantation. In some embodiments, the kit can include from about 1 x 10 ⁴ cells to about 1 x 10 ⁶ cells. In some embodiments, 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 × 10 9 cells, at least about 1 × 10 ¹⁰ cells, at least about 2 x 10 ¹⁰ cells, at least about 3 x 10 ¹⁰ cells, at least about 4 x 10 ¹⁰ cells, at least about 5 x 10 ¹⁰ cells, at least about 6 x 10 ¹⁰ cells, at least ab at least about 9 x 10 ¹⁰ cells, at least about 9 × 10 ¹⁰ cells At least about 1 x 10 ¹¹ cells, at least about 2 x 10 ¹¹ cells, at least about 3 x 10 ¹¹ cells, at least about 4 x 10 ¹¹ cells, at least about 5 x 10 ¹¹ cells, at least about 8 x 10 ¹¹ cells, at least about 9 x 10 ¹¹ cells, or at least about 1 x 10 ¹² cells. For example, you can include about 5 × ¹⁰ 10 cells in the kit. In another example, the kit may comprise 3 × 10 ⁶ cells; cells may be expanded to about 5 × ¹⁰ 10 cells and administered to the subject.

In some embodiments, the kit can include allogeneic cells. In some embodiments, a kit can include cells that can include genomic modifications. In some embodiments, the kit can comprise "off the shelf" cells. In some embodiments, the kit can include cells that can be expanded for clinical use. In some cases, the kit may contain content for research purposes.

In some embodiments, 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. In some embodiments, 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. In some cases, the instructions provide methods of administering an immunoreactive cell of the invention before, after or simultaneously with the administration of a chemotherapeutic agent.

According to one aspect of the invention, 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. In some embodiments, 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. In some embodiments, the methods comprise administering to the individual in need thereof an antigen binding receptor of the invention and an exogenous type I interferon. In some embodiments, the exogenous type I interferon is administered sequentially or simultaneously with the immune response cell expressing the antigen binding receptor. In some embodiments, 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. In some embodiments, the exogenous type I interferon is administered sequentially or simultaneously with the immune response cell expressing the antigen binding receptor. In some embodiments, the exogenous type I interferon is administered to a patient simultaneously with the immune response cell by co-expression in an immune response cell.

In some embodiments, it is because of the increased viability of the immune response cell of the present invention that the exogenous type I interferon is not co-expressed with the exogenous type I interferon or the immune response cell. In contrast, the immune response cells of the invention can be administered at lower doses and/or lower frequencies.

In some embodiments, 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%. In some embodiments, 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%. Alternatively, 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. In the case of an immune response cell, 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%.

In some embodiments, 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%. In some embodiments, 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/μL, 10,000/μL, 15,000/μL, 20,000/μL, 25,000/μL; 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/μL, 200/μL, 300/μL, 400/μL, 500/μL, 600/μL, 700 / μL, 800 / μL, 900 / μL, 1,000 / μL, 1,500 / μL, 2,000 / μL, 2,500 / μL, 3,000 / μL, 3,500 / μL, 4,000 / μL, 4,500 / μL, or 5,000 / μL; or about 10 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 10 / μL, 20 / μL 30 / μL, 40 / μL, 50 / μL, 60 / μL, 70 / μL, 80 / μL, 90 / μL, or 100 / μL.

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. Preferably, 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) can be collected from patients in need of treatment, and T cells can be activated and expanded using methods described herein and known in the art and then injected into a patient. In other cases, allogeneic cells can be used to treat a patient.

The methods disclosed herein can include transplantation. Transplantation can refer to adoptive transplantation of cellular products. The transplant can be autograft, allogeneic, xenograft or any other transplant. For example, the transplant can be a xenograft. Transplantation can also be allogeneic transplantation.

In some embodiments, 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. For example, 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. In some embodiments, the immunoreactive cells that can be administered to the subject are from about 10 to about 14 or about 20 days of age. In some embodiments, the "age" of an immunoreactive cell can be determined by the telomere length. For example, a "young" immune response cell can have a longer telomere length than "depleted" or "old" immunoreactive cells. Not subject to a particular theory, It is believed that 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. Without being bound by a particular theory, it is believed that a longer telomere length can be associated with a positive objective clinical response in a patient and persistence of cells in vivo.

Cells (eg, engineered cells or engineered primary T cells) can be functional before, after, and/or during transplantation. For example, 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. In some embodiments, the transplanted cells can function during the life of the recipient.

In addition, 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. For example, 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. For example, the transplant can be a subcapsular transplant. Transplantation can also be intramuscular transplantation. The transplant can be a portal vein transplant.

The 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. For example, 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. For example, after providing an immune response cell of the invention, 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.

In some cases, 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. In addition, 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.

For example, 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. In addition, 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. For example, 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, cyclosporin, deoxygenin, soluble complement receptor 1, cobra venom, anti-C5 antibody Eculizumab/Soliris), methylprednisolone, FTY720, everolimus, leflunomide, Anti-IL-2R-Ab, rapamycin, anti-CXCR3 antibody, anti-ICOS antibody, anti-OX40 antibody and anti-CD122 antibody. In addition, 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. In some cases, daclizumab (Zenapax) can be used for induction therapy, and tacrolimus (Prograf) and sirolimus (Rapimune) can be used to maintain treatment. 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.

Example

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually prepared according to conventional conditions such as J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the manufacturer. The suggested conditions.

In the following examples of the invention, when the antigen-binding receptor or CAR is constructed, the CD28 costimulatory signal domain is abbreviated as 28; CD3ζ is abbreviated as Z; 4-1BB or CD137 is abbreviated as BB. For example, a chimeric antigen receptor constructed as an intracellular signal domain with an scFv code of 85-2 and a CD3ζ and a CD28 costimulatory signal domain can be designated as 85-2-28Z. This is true for the construction of CARs for different antigens.

Experimental material

The 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.

 Human T-Activator CD3/CD28：Life technologies，Cat#11161D。

Human T-Activator CD3/CD28: Life technologies, Cat#11161D.

Fetal bovine serum (FCS): Gibco, Cat #10099-141.

IL-2: Shanghai Huaxin, recombinant human interleukin-2 for injection.

Goat anti-human F (ab ') ₂ antibody: Jackson ImmunoResearch, Cat # 109-066-006.

PE-Streptavidin: BD pharmingen, Cat #554061.

非放射性细胞毒性检测：Promega，Cat#G1780。 CytoTox

Non-radioactive cytotoxicity assay: Promega, Cat# G1780.

2. Experimental methods

2.1 lentiviral vector construction

2.1.1 Construction of pRRL-EF1α-92-CAR lentiviral vector

As an example, 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 and 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.

In the present system, the inventors first modified the empty vector pRRLSIN-cPPT.PGK-GFP.WPRE by a conventional molecular cloning technique, and started with elongation factor-1α (elongation factor-1α, EF-1α). The promoter replaces the promoter of the original vector and adds a MluI cleavage site between the promoter and the CD8αsp signal peptide. Specifically, 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-1α-EGFP.WPRE.

An antibody 92 that is humanized and capable of specifically recognizing human GPC3 protein is described in Chinese Patent 201510481235.1. For the construction of the 92-CAR lentiviral plasmid, the plasmid containing the fragment of the 92 heavy chain variable region (SEQ ID NO: 80 in patent 201510481235.1) was used as a template, and the upstream primer 5'-ctccacgccgccaggccggaggtgcagctggtgcag-3' (SEQ ID NO: 1) was used. And 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) As a template, 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 CD8α 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.

Using the upstream primer 5'-gcaggggaaagaatagtagaca-3' (SEQ ID NO: 6) and the downstream primer 5'-CGGCCTGGCGGCGTGGAG-3' (SEQ ID NO: 7), the vector plasmid pRRLSIN-cPPT.EF- constructed in this example was used. 1α-EGFP.WPRE was used as a template to amplify the EF-1α promoter (SEQ ID NO: 8) containing the CD8α 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. -F2A-GPC3-28Z and pWPT-eGFP-F2A-GPC3-28BBZ as templates (specifically refer to Chinese patent CN 104140974A), amplifying fragment 3 containing Hinge-BBZ (SEQ ID NO: 11), Hinge-28Z (SEQ Fragment 4 of ID NO: 12) and fragment 5 of Hinge-28BBZ (SEQ ID NO: 13) (both containing the Sal I restriction site) were 694 bp, 703 bp and 829 bp, 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 extension 68 ° C, 10 min. The theoretical size of the DNA fragment (SEQ ID NO: 14) obtained by amplification of 92-BBZ was 1865 bp, respectively. The amplified product was confirmed by agarose gel electrophoresis to be in agreement with the theoretical size.

An equimolar amount of about 50 ng of fragment 2, fragment 1 and fragment 4 was subjected to splicing PCR, and the splicing reaction conditions were the same, and the DNA fragment of the 92-28Z (SEQ ID NO: 15) was amplified to have a theoretical size of 1874 bp. The amplified product was confirmed by agarose gel electrophoresis to be in agreement with the theoretical size.

An equimolar amount of about 50 ng of fragment 2, fragment 1 and fragment 5 was subjected to splicing PCR, and the splicing reaction conditions were the same, and the DNA fragment of 92-28BBZ (SEQ ID NO: 16) was amplified to have a theoretical size of 2000 bp, respectively. The amplified product was confirmed by agarose gel electrophoresis to be in agreement with the theoretical size.

The above vector plasmids pRRLSIN-cPPT.EF-1α-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-1α-92-BBZ, pRRL-EF- 1α-92-28Z and pRRL-EF-1α-92-28BBZ.

2.1.2 Construction of 92-CAR lentiviral vector co-expressing 4-1BBL

In addition, to construct a plasmid co-expressing 92-28Z and 41BBL, using the pRRL-EF-1α-92-28Z plasmid constructed above as a template, 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). In order to obtain the F2A-41BBL fragment, 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'-ctgccacccgcctcctccgaggctcggaa-3' (SEQ ID NO: 20) and the downstream primer 5'-TGATTGTCGACTTATTCCGACCTCGGTGAAGGGA-3' (SEQ ID NO: 21), fragment 8 was amplified by PCR, and fragments 7 and 8 were spliced in equal molars, and primer pairs (SEQ ID NO: 18 and SEQ ID NO: 21) were subjected to PCR amplification to obtain fragment 9. Finally, 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-1α-EGFP.WPRE by the same method as above, and confirmed by sequencing to obtain plasmid pRRL-EF-1α-92. -28Z-F2A-41BBL.

2.1.3 Construction of 92-CAR lentiviral vector capable of regulating IFN expression

To construct a plasmid that can co-express 92-28Z and IFN beta (while inserting a NFAT element in front of the IFN beta for controllable expression), 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. The repeat vector pRRLSIN-NFAT3-EGFP-PA2. With this pRRLSIN-NFAT3-EGFP-PA2 as a template, fragment 12 was amplified with primer pairs (SEQ ID NO: 39 and SEQ ID NO: 40) and primer pairs (SEQ ID NO: 41 and SEQ ID NO: 42), respectively. And 13, and then by duplex PCR, primer pairs (SEQ ID NO: 39 and SEQ ID NO: 42) were amplified to obtain fragment 14, which was subsequently digested with Mlu I and SalI, ligated to the same digested pRRLSIN- In NFAT3-EGFP-PA2, it was confirmed by sequencing that a vector pRRLSIN-NFAT6-EGFP-PA2 containing six NFAT repeats was obtained. Fragment 15 was amplified by primer pair (SEQ ID NO: 43 and SEQ ID NO: 44) using the vector pGMT-IFN-β (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) as a template. Using this fragment 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-huIFNβ-PA2 plasmid. Using the constructed pRRLSIN-NFAT6-huIFNβ-PA2 plasmid as a template, 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 -1α-EGFP.WPRE, it was verified by sequencing that the plasmid pRRLSIN-EF1α-EGFP-NFAT6-huIFNβ-PA2 was obtained. Finally, the plasmid pRRL-EF-1α-92-28Z was digested with MluI and SalI to obtain a 92-28Z fragment, which was ligated into the same double-digested pRRLSIN-EF1α-EGFP-NFAT6-huIFNβ-PA2 vector. The correct plasmid pRRLSIN-EF1α-92-28Z-NFAT6-hu IFNβ-PA2 was sequenced.

The above five plasmids pRRL-EF-1α-92-BBZ, pRRL-EF-1α-92-28Z, pRRL-EF-1α-92-28BBZ, pRRL-EF-1α-92-28Z-F2A-41BBL and pRRL- EF-1α-92-28Z-NFAT6-huIFNβ-PA2 is commonly referred to as pRRL-EF-1α-92-CAR (Fig. 1). The 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-β 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.

2.2 virus preparation

1) 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;

2) The lentiviral shuttle vector pRRL-92-28Z-NFAT6-IFN-β5.2ug and packaging plasmid pRsv-REV 6.2μg, pRRE-PMDLg 6.2μg, VSVg 2.4μg dissolved in 800μL serum-free DMEM culture medium, and mixed;

3) 60 μg of PEI (1 μg / μl) was dissolved in 800 μl of serum-free DMEM medium, gently mixed (or vortexed at 1000 rpm for 5 seconds), incubated for 5 min at room temperature;

4) Formation of transfection complex: 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;

5) 1.6 ml of the transfection complex was added dropwise to a 10 cm culture dish containing 11 ml of DMEM medium, and after 4-5 hours, the fresh medium was replaced;

6) After 72 h, the viral supernatant was collected.

2.3 virus concentration

1) Preparation of 5X PEG8000NaCl: Weigh 8.766g of NaCl, 50g of PEG8000 dissolved in 200ml of Milli-Q pure water, sterilize by damp heat for 30min at 121°C for 30min, and then store at room temperature and store in 4°C refrigerator.

2) The collected virus supernatant was filtered using a 0.45 μm filter, and 1/4 ml of a 5× PEG-8000 NaCl mother liquor was added, and the mixture was mixed upside down;

3) Mix once every 20 to 30 minutes, a total of 3-5 times;

4) placed at 4 ° C overnight;

5) 4 ° C, 4000 g, centrifugation for 60 min;

6) After removing the supernatant, add appropriate amount of AIM V medium (containing 2% AB serum) to dissolve the resuspended virus precipitate;

7) The concentrated lentiviral suspension is divided into 50μl 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. .

2.4 lentivirus titer determination

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;

After incubating at 37 ° C, 5% CO ₂ overnight, fresh medium was replaced;

After 72 hours of infection, 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;

Centrifuge at 400g for 5min, discard the supernatant, and wash once with PBS + 2% FBS solution;

6) Take appropriate amount of cells, add Biotin-labeled goat anti-human Fab antibody in a 1:50 dilution ratio, and incubate on ice for 30 min;

7) After washing once with 1 mL of PBS + 2% FBS solution, add PE-labeled streptavidin at a dilution ratio of 1:50, and incubate on ice for 30 min;

8) After washing twice with PBS+2% FBS solution, resuspend the cells by adding an appropriate volume of PBS + 2% FCS solution, and transfer to a flow tube;

9) After flow cytometry detection, it is advisable to take a cell sample with a positive rate of 5-20%, and calculate the titer (TU/mL) = cell number (10 ⁵ ) × positive rate / virus volume (mL).

2.5 Preparation of Lentiviral Transduction T Lymphocytes-CAR-T Lymphocytes

1) 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;

2) Retronectin coated 48-well plates: 160 μl of retronectn solution (5 μg/mL) was added to each well and incubated overnight at 4 °C;

3) Discard the Retronectin solution in a 48-well plate and wash twice with 1 ml of PBS;

4) The cells were seeded in a Retronectin-coated 48-well plate, the number of cells per well was 3×10 ⁵ , and the lentivirus was added according to MOI=10, and the medium was supplemented to 300 μL;

5) 32 ° C, 1800 rpm, after centrifugation for 40 min, transferred to a cell culture incubator for 24 h;

6) Replace the fresh medium, adjust the cell density to 5 × 10 ⁵ /mL, and pass through every 2-3 days.

2.6 T lymphocyte chimeric antigen receptor expression

1) After 7 days of infection, take 4×10 ⁵ T cells, 4° C., 400 g, centrifuge for 5 min, discard the supernatant, and wash once with PBS+2% FCS;

2) Resuspend the cells with 50 μL PBS + 2% FCS, add 1 μL of Biotin-labeled goat anti-human Fab antibody, and incubate for 30 min on ice;

3) After washing twice with PBS+2% FCS, resuspend the cells by adding 50 μL PBS + 2% FCS, add 1 μL of PE-labeled Streptavidin, and incubate for 30 min on ice;

4) After washing twice with PBS+2% FCS, the cells were resuspended by adding 400 μL of PBS+2% FCS, transferred to a flow tube, and the infection efficiency was detected by flow cytometry.

2.7 in vitro toxicity test

Target cell

The target cells corresponding to 92-CAR are SK-HEP-1 (GPC3-) and Huh-7 (GPC3+);

Adjustment of target cells at a concentration of 1 × 10 ⁶ / mL, 100μL were seeded into 96well plate taken;

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;

Five replicate wells were set for each group, and the average of five replicate wells was taken.

Each experimental group and each control group are as follows:

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;

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。Detection method: Effector cells were co-cultured with target cells for 18 hours, and then 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.

The cytotoxicity calculation formula is:

3, the results

Example 1. Expression of 92-CAR on human T lymphocytes

Human T lymphocytes were stimulated with anti-CD3 and CD28 antibody-coated magnetic beads for 48 h and then infected with a high titer of lentivirus at MOI=10. The positive rate of Lentivirus-infected T lymphocytes was detected by flow cytometry on the 7th day after infection. 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-β 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.

Example 2. In vitro antitumor activity of 92-CAR T cells

92-CAR T after detection of infection positive rate was detected as T lymphocytes expressing 92-28Z-NFAT6-IFN-β, 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 results showed that 92-28Z CAR-T cells specifically kill GPC3-positive Huh-7 and PLC/PRF/5 cells without killing GPC3-negative SK-HEP-1 cells; CARs co-expressing huIFN-β The killing ability of T cells was higher than that of 92-28Z CAR-T cells with the same target ratio, as shown in Table 2.

Table 2, 92-CAR T cell toxicity detection of target cells

The present inventors further integrated 92-28Z-NFAT6-IFN-β 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. First, FACS detects the expression of various CARs (see Figure 3). The expression ratio of various CARs is about 40%.

Then, these CAR-T cells and empty vector MOCK T lymphocytes were tested for hepatoma cell lines SK-HEP-1 (GPC3 ^- ) and Huh-7 (GPC3 ⁺ ) according to the target ratio of 0.3:1, 1:1, and 3:1 ^. And the in vitro killing effect of PLC/PRF/5 (GPC3 ⁺ ), after 18 hours of co-culture, the content of LDH in the supernatant was detected. As a result, it was observed that cells expressing 92CAR-T specifically killed GPC3-positive Huh-7 and PLC/PRF/5 cells without killing GPC3-negative SK-HEP-1 cells. CAR-T cells co-expressing IFN-β at a target-to-target ratio of 1:1 had higher killing ability against both GPC3-positive liver cancer cells than all other CAR-T cells, as shown in Table 3.

Table 3. Toxicity killing of various GPC3CAR-T cells to target cells

The above studies indicate that exogenously expressed IFN-beta can enhance antitumor activity based on CAR T cells of CD28-Z. Moreover, when CAR-T expressing CD28Z co-expressed IFN-beta, it had better tumor killing ability than some immune cells expressing CD28Z-41BBL at some effective target ratios.

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 μL (positive cell number) in 24-well plates, and inoculate 5×10 ⁴ /200 μL/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-β, IFN-γ, and IL-2, and the results are shown in Figures 4A-4C.

According to Fig. 4A, only GPC3-28Z-IFN T was incubated with Huh7 cells for IFNβ 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. According to the detection of in vitro cytokines in Figures 4B and 4C, 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

Construction of chimeric antigen receptors 85-28Z and 85-2-28Z plasmids:

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-1α as a vector. 54) Lentiviral plasmid. The 85-28Z sequence consists of the CD8α signal peptide, 85scFV, CD8hinge, CD28 transmembrane and intracellular signaling domain regions, and the intracellular CD3ξ of CD3; the sequence of 85-2-28Z is composed of CD8α signal peptide, hu8E5-2IscFV, The CD8 hinge region, the CD28 transmembrane region and the intracellular signaling domain region, and the intracellular CD3ξ of CD3 are composed.

Construction of chimeric antigen receptor 85-28Z-IFN and 85-2-28Z-IFN plasmid:

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.

To verify that the constructed 85-28Z T cells and 85-28Z-IFN T cells were also efficiently activated under target cell stimulation, we tested 85-28Z T, 85-28Z-IFN T after co-incubation with target cells. Secretion of 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 μL (positive cell number) in 24-well plates, and inoculate 5×104/200 μL 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-γ cytokine released during the co-culture of CAR T lymphocytes with target cells in the supernatant was detected by sandwich ELISA.

The experimental results are shown in Figure 5. The presence of IFN resulted in increased secretion of IFN-γ cytokines when 85-28Z CAR T cells were co-incubated with target cells.

Example 5, Killing activity of GPC3CAR-T (92-28Z) cells containing IFN and no IFN

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, and 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.

Detection method: 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.

Calculation of experimental results: the absorbance of the spontaneous LDH release group and the spontaneous LDH release group of the effector cells were subtracted from the absorbance of the medium background; the absorbance of the target LDH release control was subtracted from the volume-corrected control. The mean value of the absorbance; the corrected value obtained in the above step is substituted into the following formula to calculate the cytotoxicity (%) produced by each target ratio.

Calculation formula: % cytotoxicity = (experimental group - effector cell spontaneous group - target cell spontaneous group / target cell maximum - target cell spontaneous) * 100

The results are shown in Figures 6A-6E. The above data indicate that CAR-GPC3T expressing IFN can not only specifically kill GPC3-positive cells, but also the cytotoxic activity of CAR-T cells expressing IFN (GPC3-28Z-IFN). Can improve.

Example 6. Killing activity of CLD18A2 CAR-T cells containing IFN and no IFN

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.

Detection method: 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.

To compare the killing effects of 85-2-28Z and 85-2-28Z-IFN T cells on target cells, we

Expression of empty T cells (Mock), 85-28Z T cells and 85-28Z-IFN T cells and CLD18A2 positive 293T-A2, AGS-A2 were performed according to the target ratios of 1:3, 1:1 and 3:1, respectively. CLD18A2-negative 293T-A1, AGS and BGC-823 cells were used as controls for 18 hours with BGS-823A2 cells.

Experimental group: each target cell + CAR T expressing different chimeric antigen receptors;

1 effector cells spontaneous LDH release: correcting the spontaneous release of LDH by effector cells;

2 target cell spontaneous LDH release: correcting the spontaneous release of LDH from target cells;

3 target cell maximum LDH release: the control is required to determine 100% LDH release;

4 volume correction control: correct volume change due to the addition of lysate (10×);

5 medium background control: correction of LDH activity produced by serum in the medium and phenol red The background is absorbed.

Calculation formula: % cytotoxicity = [(experimental group - effector cell control - target cell control) / (maximum amount of target cell lysis - target cell control)] x 100. Before the calculation, 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.

In vitro toxicity experiments were performed with CAR T lymphocytes expressing Mock, 85-2-28Z and 85-2-28Z-IFN, and tumor cells at a ratio of 1:3, 1:1 and 3:1 for 18 h, respectively. The killing activity of CART cells against CLD18A2-positive target cells is shown in FIG.

Example 7, Determination of in vivo survival time of GPC3 CAR-T cells

Referring to the experimental procedure of steps 1) to 3) in Example 3, CAR-T (GPC3-28ZT cells or GPC3-28Z-IFN T cells) cells were infused through the tail vein for 7 days, and then CAR-T cells (GPC3-28Z) were detected. T cells or GPC3-28Z-IFN T cells) survive in vivo. The results are shown in Table 4. The number of T cells (CD3+) and CAR-T cells per μl 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.

Table 4. Number of T cell survival in peripheral blood

CD3+ (pieces / μL) CAR-T (pieces / μL) 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

Establishment of a gastric cancer PDX model:

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.

Adoptive transfer of T cells:

At a tumor volume of 100 mm ³ , 100 mg/kg of cyclophosphamide was intraperitoneally injected, and 1.0×10 ⁷ CAR-T cells (85-2-28Z T cells or 85-2-28Z-) were infused through the tail vein 24 hours after the injection. IFN T cells), with the Mock T cell group as a control.

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.

Results As shown in Figures 8A-8C, the number of T cells survived in the 85-2-28Z-IFN T cell treated group was significantly greater than in the 85-2-28 Z T cell treated group.

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.

1) Experimental group: 21 NOD-SCID mice were randomly divided into 3 groups at 6-8 weeks old, with 7 rats in each group, divided into untransfected T cell group, GPC3-28Z T cell group and GPC3-28Z- IFN T cell group.

2) Inoculation of subcutaneous xenografts: 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 μL. (2×10 ⁶ /only), the day of tumor cell inoculation is the 0th day.

3) Adoptive transfer of T cells: 200 mg/kg of cyclophosphamide was intraperitoneally injected at a tumor volume of 200-300 mm ³ , and 1.4×10 ⁷ CAR-T cells (GPC3-28Z T cells) were infused through the tail vein 24 hours after the injection. Or GPC3-28Z-IFN T cells), and the untransfected T cell group was used as a control to observe the growth of subcutaneous xenografts (Fig. 9A). When the tumor size of the control mice reached 2000 mm ³ , the experiment was sacrificed. Photographs were taken after tumor isolation (Fig. 9B).

Results As shown in Figures 9A and 9B, GPC3-28Z-IFN T cells were able to significantly inhibit tumor cell growth. On day 13 after CART cell reinfusion, the inhibition rate of GPC3-28Z CART cells was 66.5%, GPC3-28Z- The tumor inhibition rate of IFN CART cells was 82.3%, indicating that GPC3-28Z-IFN T cells further enhanced the ability of CAR-T cells to inhibit tumor growth.

Example 10, In vivo killing activity of CLD18A2 CAR-T cells containing IFN and no IFN

Anti-tumor treatment experiments of untransfected T cells (Mock), 85-28Z T cells and 85-2-28Z-IFN T cells on subcutaneous xenografts of BGC-823-A2 cells were determined.

1) Inoculation of BGC-823-A2 subcutaneous xenografts: 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 μL ( 5 × 10 ⁶ / only) subcutaneously in the right axilla of mice. The day of tumor cell inoculation is recorded as day 0.

2) Experimental grouping: On the 11th day of tumor inoculation, the volume of BGC-823-A2 tumor was measured, and the NOD-SCID mice were randomly divided into 4 groups, 6 in each group. They were untransfected T cell group, 85-28Z T cell group, 85-2-28Z cell group and 85-2-28Z-IFN T cell group, respectively.

3) 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.

The results of animal experiments are shown in Figures 10A and 10B. The results show that 85-2-28Z-IFN CAR T cells are superior to 85-2-28Z CAR T cells in the treatment of BGC-823-A2 xenografts.

Example 11. Anti-tumor test of CLD18A2 CAR-T cells containing IFN and no IFN in subcutaneous xenografts of gastric cancer PDX model

Anti-tumor treatment experiments of untransfected T cells (UTD), 85-2-28Z T cells and 85-2-28Z-IFN T cells on subcutaneous xenografts of gastric cancer PDX model were observed.

1) Establishment of PDX model of gastric cancer: Inoculate a gastric cancer PDX tumor of about 2×2×2 mm in the right axillary area of female NOD/SCID mice of 6-8 weeks old, and record the day of tumor cell inoculation as D0 days. .

2) Experimental grouping: Tumor inoculation D15 days, NOD-SCID mice were randomly divided into 3 groups, 7 in each group, divided into untransfected T cell group, 85-2-28Z T cell group and 85-2- 28Z-IFN T cell group.

3) Adoptive transfer of 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.

As a result, as shown in Fig. 11, one mouse of the 7 mice in the 85-2-28Z-IFN treatment group completely disappeared.

Example 12, Effect of GPC3CAR-T (92-28Z) cells containing IFN and no IFN on tumor invasion in vivo

Referring to the animal model established in Example 9, after returning GPC3-28Z and GPC3-28Z-IFN two CAR-T cells for 14 days, tumor tissues were taken and histologically examined for CD3+ cells, and the results are shown in Figures 12A and 12B: One sample took 4-7 fields and the number of CD3 positive T cells was counted. The results showed that there was no significant infiltration of CD3+ cells in the control tissue, and the number of CD3+ T cells in the INFβ-CAR-T treatment group was higher than that in the 28ZCART group.

Example 13. Effect of CLD18A2 CAR-T cells containing IFN and no IFN on tumor invasion in vivo

Referring to the animal model established in Example 10, after returning Mock, 85-28Z, 85-2-28Z and 85-2-28Z-IFN cells for 17 days, tumor tissues were taken and CD3+ cells were detected by histochemistry.

The results are shown in Figure 13. 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.

Example 14, Construction of EGFR CAR containing IFN and no IFN

The structure of EGFR-CAR (806-28Z, SEQ ID NO: 56) and EGFR-CAR-IFN (encoded by the nucleic acid set forth in SEQ ID NO: 60) is shown in Figure 14.

The T lymphocytes of 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 mIFNβ by EGFR CAR (806-28Z) T cells containing IFN and no IFN

In order to detect the function of EGFR-CAR-IFN-induced secretion of mIFNβ, we co-cultured CAR-T cells with target cells CT26-VIII for 1:1 and 3:1 for 24 h, and supernatanted. The expression of mIFNβ was detected by ELISA. At the same time, concanavalin A (ConA) was used as a positive control with CT26 cells without a target as a negative control. The results showed that mCAR-806-mIFNβ was successfully activated and induced to express mIFNβ after stimulation by target cells, and no expression of mIFNβ was detected in the control group ( FIG. 16 ).

Example 16. Release of cytokines from EGFR CAR (806-28Z)-T cells containing IFN and no IFN

To verify that the constructed EGFRCAR and EGFR-CAR-IFN were also efficiently activated under target cell stimulation, we examined the secretion of muCAR-T cytokines after co-incubation with target cells. Untransfected UT cells, EGFR-CAR and EGFR-CAR-IFN were incubated with target-positive CT26-VIII cells in a 1:1 ratio for 24 hours, and the culture supernatant was taken to detect cytokine mIL-2, mIFN. - γ, mTNF-α secretion, target-negative CT26 cells served as controls. The results showed that EGFR-CAR and egfr-CAR-IFN were co-incubated with target cells and had higher concentrations of mIL-2, mIFN-γ, and mTNF-α secretion (Fig. 17A, 17B, 17C), indicating EGFR-CAR and EGFR-CAR-IFN stimulates the target antigen, two muCAR-T Cells can be activated effectively.

Example 17, in vitro toxicity test of EGFR CAR (806-28Z)-T cells containing IFN and no IFN

To compare the in vitro killing activity of EGFR-CAR and EGFR-CAR-IFN on target cells, we performed EGFR-CAR and EGFR-CAR-IFN with EGFR-positive CT26VIII at a ratio of 1:3, 1:1 or 3:1, respectively. Cells were incubated for 18 hours, untransfected mouse UT cells served as isotype controls, and CT26 served as EGFR negative controls.

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. However, untransfected UT cells had no killing effect on CT26 and CT26VIII, and EGFR-CAR and EGFR-CAR-IFN two CAR-T cells had no killing effect on target-negative CT26 cells (Fig. 18).

Example 18, in vivo toxicity test of EGFR CAR (806-28Z)-T cells containing IFN and no IFN

The mouse colon cancer CT26 cell line stably transfected with the human EGFRvIII-806 site was used to inoculate a subcutaneous xenograft in Babl/c mice, and then treated with EGFR-CAR T cells. The results are shown in Figure 19, EGFR. -The number of tumors in the CAR-T cells was basically the same as that in the control group, and no inhibition was observed. After the EGFR-CAR-IFN cells were reinfused, the tumor growth inhibition began on the 7th day, and the tumor inhibition rate was 5.9%. On the 10th day, the strongest was 18.5%. By the 17th day, the tumor inhibition rate was still 12.4%, which was significantly better than the EGFR-CAR-T cell group.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Table 5, the sequences used in this article

Claims (29)

An immune response cell characterized in that the cell expresses an antigen-binding receptor; and an exogenous type I interferon. The immune response cell according to claim 1, wherein said immune response cell comprises: T cell, natural killer cell, cytotoxic T lymphocyte, natural killer T cell, DNT cell, and/or regulatory T. cell. The immune response cell according to any one of the preceding claims, wherein the antigen is a tumor antigen or a pathogen antigen. The immune response cell according to any one of the preceding claims, wherein the exogenous type I interferon is constitutively or inducibly expressed; preferably, for expressing the type I interferon The promoter includes: an immune cell-inducible promoter; preferably, the immune cell-inducible promoter is a NFAT6 promoter. The immune response cell according to any one of the preceding claims, wherein the cell expresses an endogenous or recombinant antigen-binding receptor; preferably, the antigen-binding receptor comprises a sequence-connected: specificity An antibody that binds to the antigen, a transmembrane region and an intracellular signal region. The immune response cell according to any one of the preceding claims, wherein the intracellular signal region of the immune response cell comprises a T cell stimulation signal molecule or a combination of a T cell stimulation signal molecule and a T cell activation costimulatory molecule; Preferably, the T cell stimulation signal molecule is selected from the group consisting of: CD3ζ or FcεRIγ; more preferably CD3ζ; or the T cell activation costimulatory molecule is selected from the group consisting of: CD27, CD28, CD137, CD134, intracellular signal of ICOS protein District, or a combination thereof. The immune response cell according to any one of the preceding claims, wherein the amino acid sequence of the antigen-binding receptor has at least 90% identity to one of the following sequences: 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; and SEQ ID NO:77. The immune response cell according to claim 7, wherein said receptor for specifically binding antigen and said exogenous type I interferon are composed of SEQ ID NO: 57 and SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 or SEQ ID NO: 76 is encoded by a nucleotide sequence having at least 90% identity. The immune response cell of any of the preceding claims, wherein said immune response cell does not comprise an exogenous co-stimulatory ligand. The immune response cell according to any one of the preceding claims, wherein the type I interferon comprises: IFNα or IFNβ. The immune response cell according to any one of the preceding claims, wherein the antigen-binding receptor and/or type I interferon is constitutively or inducibly expressed on the surface of the immune response cell. The immune response cell according to any one of the preceding claims, wherein the immune response cell comprises an expression construct comprising: an expression cassette of the antigen-binding receptor; and the I Type of interferon expression cassette. The immune response cell according to any one of the preceding claims, wherein the antigen-binding receptor and/or type I interferon is expressed using a viral vector; preferably, the viral vector is a retrovirus The vector; more preferably, the viral vector comprises a lentiviral vector, a retroviral vector or an adenoviral vector. An immune response cell according to any one of the preceding claims, wherein the antigen binding receptor recognizes and binds to a pathogenic microorganism. The immune response cell according to claim 14, wherein the pathogenic microorganism comprises a virus, a bacterium, a fungus, a protozoa or a parasite; more preferably, the pathogenic microorganism is a virus; or more preferably, The pathogenic microorganisms are selected from the group consisting of cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus and influenza virus. The immune response cell according to any one of the preceding claims, wherein the antigen comprises: 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, αvβ6 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, CD171, CSPG4, EGP2, EGP40, ERBB3, ERBB4, ErbB3/4, FAP, FAR , FBP, embryonic AchR, GD2, GD3, HLA-AI MAGE A1, MAGE3, HLA-A2, IL11Ra, KDR, Lambda, MCSP, NCAM, NKG2D ligand, PRAME, PSCA, PSC1, ROR1, Sp17, SURVIVIN, TAG72 , TEM1, TEM8, VEGRR2, HMW-MAA, VEGF receptor, and/or carcinoembryonic variants of fibronectin, tenascin, or tumor necrotic regions. An expression construct comprising: an expression cassette comprising: an antigen-binding receptor; and an expression cassette of type I interferon; wherein the antigen-binding receptor and type I interferon As defined in one of the preceding claims. 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 according to any one of claims 1 to 16, and wherein the method comprises The individual is administered the immune response cell as well as an effective amount of exogenous type I interferon. The method according to claim 18, wherein the exogenous type I interferon and the immune response cell expressing the antigen-binding receptor are administered sequentially or simultaneously. The method according to claim 18 or 19, wherein said exogenous type I interferon is administered to a patient simultaneously with said immune response cell by co-expression in an immune response cell. The method according to any one of claims 18 to 20, wherein said immune response cells comprise T cells, Natural killer cells, cytotoxic T lymphocytes, natural killer T cells, DNT cells, and/or regulatory T cells. The method according to any one of claims 18 to 21, wherein said method is such that, after administration of said immune response cells to said individual, said said compared to said absence of said exogenous type I interferon The sum of the number of cytotoxic T cells and helper T cells in the peripheral blood of the individual is increased by at least 50%. The method according to any one of claims 18 to 22, wherein said method causes the number of cytotoxic T cells and helper T cells in said peripheral blood of said individual after about 5 days of administration of said immune response cells to said individual And greater than 15,000/μL; after about 7 days of 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 500/μL; or about 10 of the immune response cells are administered. After the day, the sum of the number of cytotoxic T cells and helper T cells in the peripheral blood was greater than 50/μL. Use of an immune response cell according to any one of claims 1 to 16 for the preparation of a pharmaceutical composition for the treatment of a tumor, a pathogen infection, or an enhancement of an individual's immune tolerance in an individual in need thereof. The use according to claim 24, wherein 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. , kidney cancer, leukemia, myeloma, ovarian cancer, cervical cancer, ovarian cancer, cervical cancer or glioma; or The pathogen includes: 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 an influenza virus. The use according to claim 24 or 25, wherein the drug reduces the tumor by at least 30% as measured by computed tomography. The use according to claim 24 or 25, wherein the drug causes the tumor to completely disappear as measured by computed tomography. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises: An immune response cell according to any one of claims 1-16; A pharmaceutically acceptable carrier or excipient. A kit comprising: An immune response cell according to any one of claims 1-16; Instructions for how to administer the immune response cells to an individual are directed.

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