RU2813668C2 - Method of production of t-cells - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the methods of the production of T-cells, according to which the stages of activation, stimulation, transduction using a viral vector containing a polynucleotide encoding a chimeric antigen receptor (CAR) to the B-cell maturation antigen (BCMA), as well as Cultivation of transduced T-cells for proliferation is performed in the presence of a phosphatidyl-inositol 3-kinase (PI3K) inhibitor. The use of this inhibitor increases the expression of CD62L.

EFFECT: T-cells obtained in this way can be used in adaptive therapy.

15 cl, 6 dwg, 6 ex

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC § 119(e) to US Provisional Patent Application No. 62/008957, filed June 6, 2014, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Technical field

The present invention relates to improved T cell-based compositions and methods for producing T cells. More specifically, the present invention relates to methods for producing T cells that lead to adoptive T cell immunotherapy agents with improved survival, expansion and persistence in vivo.

Description of the Prior Art

Adaptive immunotherapy is the transfer of T lymphocytes to a subject to treat a disease. The potential of adaptive immunotherapy to treat a wide range of diseases such as cancer, infectious disease, autoimmune disease, inflammatory disease and immunodeficiency has yet to be realized. However, most, if not all, adaptive immunotherapy strategies require T cell activation and expansion steps to produce a clinically effective therapeutic dose of T cells. Current technologies for producing therapeutic doses of T cells, including engineered T cells, remain limited by cumbersome manufacturing processes for obtaining T cells. For example, expansion of T cells often requires labor-intensive and expensive cloning and/or multiple rounds of activation/expansion to achieve therapeutically relevant numbers of T cells. In addition, existing methods of T cell activation/expansion typically involve significant T cell differentiation and typically result in short-term effects, including short-term survival and lack of persistence and in vivo expansion of transferred T cells. Thus, current T cell production processes produce a low-quality T cell product that is susceptible to depletion and loss of function of effector immune cells.

To date, the clinical effectiveness of adaptive immunotherapies based on developed T cells is limited due to poor proliferation and persistence of T cells after infusion into patients. Thus, such therapies are not suitable for widespread clinical use. Accordingly, there is a continuing, unmet need for improvements in the production of T cells and T cell therapeutic compositions that are capable of surviving, proliferating and persisting in vivo.

SHORT DESCRIPTION

The present invention generally provides adaptive T-cell immunotherapy agents comprising persistent and highly active T-cell-based antitumor compositions and methods for their preparation.

The present invention relates to improved T cell-based compositions and methods for producing T cells. More specifically, the present invention relates to methods for producing T cells that result in improved survival, expansion and persistence in vivo.

In various embodiments, a method of producing T cells is provided, comprising: (a) isolating a population of T cells, such as tumor-infiltrating cytotoxic T lymphocytes (TILs), from a subject; (b) activating the T cell population and stimulating the T cell population to proliferate, where the activation and stimulation steps are performed in the presence of an AKT/mTOR pathway inhibitor; (c) culturing T cells for proliferation; wherein the activation and stimulation steps performed in the presence of a PI3K/AKT/mTOR pathway inhibitor result in the maintenance of T cell proliferation compared to the proliferation of T cells that were activated and stimulated in the absence of a PI3K/AKT/mTOR pathway inhibitor.

In various embodiments, a method of producing T cells is provided, comprising: (a) activating a population of T cells and stimulating the population of T cells to proliferate, wherein the activation and stimulation steps are performed in the presence of an AKT/mTOR pathway inhibitor; (b) transducing T cells with a viral vector containing a designed T cell receptor (TCR) or chimeric antigen receptor (CAR); (c) culturing the transduced T cells for proliferation; wherein the activation and stimulation steps performed in the presence of a PI3K/AKT/mTOR pathway inhibitor result in the maintenance of proliferation of transduced T cells compared to the proliferation of transduced T cells that were activated and stimulated in the absence of a PI3K/AKT/mTOR pathway inhibitor.

In specific embodiments, the methods discussed herein involve isolating peripheral blood mononuclear cells as a source of T cells.

In certain embodiments, activation of T cells includes bringing the T cells into contact with an anti-CD3 antibody or a CD3-binding fragment thereof.

In additional embodiments, T cell stimulation comprises contacting the T cells with an anti-CD28 antibody or a CD28-binding fragment thereof, B7-1 or a CD28-binding fragment thereof, or B7-2 or a CD28-binding fragment thereof.

In some embodiments, the cells are transduced with a viral vector prior to T cell proliferation.

In certain embodiments, the cells are transduced with a viral vector after T cell proliferation.

In specific embodiments, the vector is a retroviral vector.

In additional embodiments, the vector is a lentiviral vector.

In other specific embodiments, the cells comprise a chimeric antigen receptor (CAR).

In specific embodiments, the CAR comprises an extracellular domain that binds an antigen selected from the group consisting of folate receptor alpha, 5T4, αyβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD 19, CD20, CD22, CD30, CD33 , CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, CMV, EBV, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AChR, FRα, GD2, GD3, glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, HPV, IL-11Rα, IL-13Rα2, lambda chain, Lewis-Y, kappa chain, mesothelin, Muc1, Muc16, NCAM, ligands NKG2D, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM and VEGFR2; a transmembrane domain derived from a polypeptide selected from the group consisting of CD8α; CD4, CD28, CD45, PD-1 and CD152; one or more intracellular costimulatory signal transduction domains selected from the group consisting of CD28, CD54 (ICAM), CD134 (OX40), CD137 (41BB), CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), and CD278 (ICOS); and CD3ζ signaling domain.

In additional embodiments, the extracellular domain comprises an antibody or antigen-binding fragment that binds an antigen.

In certain embodiments, the transmembrane domain is derived from CD8α or CD28.

In additional embodiments, one or more costimulatory signal transduction domains are selected from the group consisting of CD28, CD134, and CD137.

In additional embodiments, the CAR comprises a hinge region polypeptide.

In specific embodiments, the hinge region polypeptide comprises an IgG1 or CD8α hinge region.

In certain embodiments, the CAR comprises a signal peptide.

In some embodiments, the signal peptide comprises an IgG1 heavy chain signal polypeptide or a CD8α signal polypeptide.

In some embodiments, the PI3K/AKT/mTOR pathway inhibitor is selected from the group consisting of BEZ235, LY294002, GDC-0941, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, IPI-145, MK-2206, GSK690693, GDC- 0068, A-674563, SST128930, AZD8055, INK128, rapamycin, PF-04691502, everolimus, BI-D1870, H89, PF-4708671, FMK, AT7867, NU7441, PI-103, NU7026, PIK-75, ZSTK474 and PP- 121.

In specific embodiments, the PI3K/AKT/mTOR pathway inhibitor is a general PI3K inhibitor selected from the group consisting of BEZ235, LY294002, and GDC-0941.

In other specific embodiments, the PI3K/AKT/mTOR pathway inhibitor is a selective PI3K inhibitor selected from the group consisting of BYL719, GSK2636771, TGX-221, AS25242, CAL-101, and IPI-145.

In other specific embodiments, the PI3K/AKT/mTOR pathway inhibitor is a PI3K inhibitor, ZSTK474.

In specific embodiments, the PI3K/AKT/mTOR pathway inhibitor is a general AKT inhibitor selected from the group consisting of MK-2206, GSK690693, and GDC-0068.

In additional embodiments, the PI3K/AKT/mTOR pathway inhibitor is a selective AKT1 inhibitor, A-674563.

In certain embodiments, the PI3K/AKT/mTOR pathway inhibitor is a selective AKT2 inhibitor, CCT128930.

In certain embodiments, a PI3K/AKT/mTOR pathway inhibitor inhibits activation of AKT by DNA-PK.

In additional embodiments, a PI3K/AKT/mTOR pathway inhibitor inhibits AKT activation by PDK-1.

In specific embodiments, a PI3K/AKT/mTOR pathway inhibitor inhibits activation of AKT by mTORC2.

In additional embodiments, a PI3K/AKT/mTOR pathway inhibitor inhibits AKT activation by HSP.

In other specific embodiments, the PI3K/AKT/mTOR pathway inhibitor is a general mTOR inhibitor selected from the group consisting of AZD8055, INK128, and rapamycin.

In certain embodiments, the PI3K/AKT/mTOR pathway inhibitor is a selective mTORCl inhibitor selected from the group consisting of PF-04691502 and everolimus.

In specific embodiments, the PI3K/AKT/mTOR pathway inhibitor is an s6-kinase inhibitor selected from the group consisting of BI-D1870, H89, PF-4708671, FMK, and AT7867.

In specific embodiments, the PI3K/AKT/mTOR pathway inhibitor is a DNA-PK inhibitor selected from the group consisting of NU7441, PI-103, NU7026, PIK-75, and PP-121.

In some embodiments, the T cell population that has been activated and stimulated in the presence of a PI3K/AKT/mTOR pathway inhibitor is characterized by an increased number of T cells expressing one or more markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD122 and CD127, compared to a population of T cells that were activated and stimulated in the absence of a PI3K/AKT/mTOR pathway inhibitor.

In further embodiments, the T cell population that is activated and stimulated in the presence of a PI3K/AKT/mTOR pathway inhibitor does not express CD57 or KLRG1 or expresses less CD57 or KLRG1 compared to the T cell population that is activated and stimulated in the absence of the inhibitor PI3K/AKT/mTOR pathways.

In various embodiments, a method is provided for maintaining proliferation and reducing differentiation of re-stimulated T cells expressing a engineered TCR or CAR, comprising (a) contacting all or a portion of a population of proliferating T cells containing the engineered TCR or CAR with an anti-CD3 antibody, or its CD3 binding fragment and an anti-CD28 antibody or CD28-binding fragment thereof, which stimulates the CD28 accessory molecule on the surface of T cells, thereby re-stimulating activated T cells to proliferate; wherein the re-stimulated T cells exhibit sustained proliferation and reduced differentiation compared to the proliferation of T cells that have been stimulated or re-stimulated in the absence of a PI3K/AKT/mTOR pathway inhibitor.

In certain embodiments, the cells contain a designed TCR.

In certain embodiments, the cells contain CAR.

In other specific embodiments, the cells contain a viral vector encoding the designed TCR or CAR.

In additional embodiments, the vector is a retroviral vector.

In additional embodiments, the vector is a lentiviral vector.

In specific embodiments, the CAR contains an extracellular domain that binds an antigen selected from the group consisting of folate receptor alpha, 5T4, α _v β6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM , FAP, fetal AChR, FRα, GD2, GD3, 'glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA- A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Rα, IL-13Ra2, lambda chain, Lewis-Y, kappa chain, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY -ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM and VEGFR2; a transmembrane domain derived from a polypeptide selected from the group consisting of CD8α; CD4, CD28, CD45, PD-1 and CD 152; one or more intracellular costimulatory signal transduction domains selected from the group consisting of CD28, CD54 (ICAM), CD 134 (OX40), CD137 (41 BB), CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1 ) and CD278 (ICOS); and CD3ζ signaling domain.

In some embodiments, the extracellular domain comprises an antibody or antigen-binding fragment that binds an antigen.

In certain embodiments, the transmembrane domain is derived from CD8α or CD28.

In additional embodiments, one or more costimulatory signal transduction domains are selected from the group consisting of CD28, CD134, and CD137.

In certain embodiments, the CAR comprises a hinge region polypeptide.

In additional embodiments, the hinge region polypeptide comprises an IgG1 or CD8α hinge region.

In additional embodiments, the CAR comprises a signal peptide.

In other specific embodiments, the signal peptide comprises an IgG1 heavy chain signal polypeptide or a CD8α signal polypeptide.

In specific embodiments, the PI3K/AKT/mTOR pathway inhibitor is selected from the group consisting of BEZ235, LY294002, GDC-0941, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, IPI-145, MK-2206, GSK690693, GDC- 0068, A-674563, SST128930, AZD8055, INK128, rapamycin, PF-04691502, everolimus, BI-D1870, H89, PF-4708671, FMK, AT7867, NU7441, PI-103, NU7026, PIK-75, Z STK474 and PP- 121.

In additional embodiments, the PI3K/AKT/mTOR pathway inhibitor is a general PI3K inhibitor selected from the group consisting of BEZ235, LY294002, and GDC-0941.

In other specific embodiments, the PI3K/AKT/mTOR pathway inhibitor is a selective PI3K inhibitor selected from the group consisting of BYL719, GSK2636771, TGX-221, AS25242, CAL-101, and IPI-145.

In other specific embodiments, the PI3K/AKT/mTOR pathway inhibitor is a PI3K inhibitor, ZSTK474.

In certain embodiments, the PI3K/AKT/mTOR pathway inhibitor is a general AKT inhibitor selected from the group consisting of MK-2206, GSK690693, and GDC-0068.

In specific embodiments, the PI3K/AKT/mTOR pathway inhibitor is a selective AKT1 inhibitor, A-674563.

In additional embodiments, the PI3K/AKT/mTOR pathway inhibitor is a selective AKT2 inhibitor, CCT128930.

In some embodiments, a PI3K/AKT/mTOR pathway inhibitor inhibits activation of AKT by DNA-PK.

In specific embodiments, the PI3K/AKT/mTOR pathway inhibitor inhibits AKT activation by PDK-1.

In certain embodiments, a PI3K/AKT/mTOR pathway inhibitor inhibits activation of AKT by mTORC2.

In specific embodiments, a PI3K/AKT/mTOR pathway inhibitor inhibits AKT activation by HSP.

In additional embodiments, the PI3K/AKT/mTOR pathway inhibitor is a general mTOR inhibitor selected from the group consisting of AZD8055, INK128, and rapamycin.

In additional embodiments, the PI3K/AKT/mTOR pathway inhibitor is a selective mTORC1 inhibitor selected from the group consisting of PF-04691502 and everolimus.

In some embodiments, the PI3K/AKT/mTOR pathway inhibitor is an s6-kinase inhibitor selected from the group consisting of BI-D1870, H89, PF-4708671, FMK, and AT7867.

In other specific embodiments, the PI3K/AKT/mTOR pathway inhibitor is a DNA-PK inhibitor selected from the group consisting of NU7441, PI-103, NU7026, PIK-75, and PP-121.

In specific embodiments, the population of activated T cells that have been restimulated in the presence of a PI3K/AKT/mTOR pathway inhibitor is characterized by an increased number of T cells expressing one or more markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD122 and CD127, compared to a population of T cells that were activated and stimulated in the absence of a PI3K/AKT/mTOR pathway inhibitor.

In further embodiments, the activated T cell population that is restimulated in the presence of a PI3K/AKT/mTOR pathway inhibitor does not express CD57 or KLRG1 or expresses less CD57 or KLRG1 compared to the T cell population that is activated and stimulated in the absence of the inhibitor PI3K/AKT/mTOR pathways.

In various embodiments, a population of T cells is provided containing a vector containing a designed TCR or CAR, where the cells have been activated and stimulated to proliferate in the presence of a PI3K/AKT/mTOR pathway inhibitor.

In various specific embodiments, a population of T cells is provided containing a vector containing a designed TCR or CAR, wherein the cells have been activated and stimulated to proliferate in the presence of a PI3K/AKT/mTOR pathway inhibitor and have been restimulated by bringing all or part of the population of proliferating immune effectors cells into contact with an anti-CD3 antibody or a CD3-binding fragment thereof and an anti-CD28 antibody or a CD28-binding fragment thereof, which stimulates the CD28 accessory molecule on the surface of immune effector cells.

In certain embodiments, immune effector cells include T cells.

In one embodiment, the immune effector cells are TILs.

In various embodiments, a composition is provided comprising a population of immune effector cells discussed herein and a physiologically acceptable excipient.

In various specific embodiments, a method of treating cancer in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of a T cell-based composition disclosed herein.

In specific embodiments, the cancer is selected from the group consisting of Wilms tumor, Ewing's sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary system cancer, cervical cancer, endometrial cancer, esophageal cancer, stomach cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma and bladder cancer.

In one embodiment, the cancer is associated with or caused by a viral infection, e.g., an infection due to CMV, HPV or EBV.

In additional embodiments, the cancer is pancreatic cancer, and the extracellular binding domain binds an epitope of PSCA or MUC1.

In some embodiments, the cancer is bladder cancer, and the extracellular binding domain binds an epitope of PSCA or MUC1.

In additional embodiments, the cancer is glioblastoma multiforme, and the extracellular binding domain binds an epitope of EHA2, EGFRvIII, or CSPG4.

In specific embodiments, the cancer is lung cancer, and the extracellular binding domain binds a PSCA or GD2 epitope.

In certain embodiments, the cancer is breast cancer, and the extracellular binding domain binds an epitope of CSPG4 or HER2.

In additional embodiments, the cancer is melanoma, and the extracellular binding domain binds an epitope of CSPG4 or GD2.

In specific embodiments, the cancer is a B cell neoplasm and the binding domain binds a BCMA epitope.

In various embodiments, a method of treating hematologic malignancy in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of a T cell-based composition disclosed herein.

In certain embodiments, the hematologic malignancy is a B cell neoplasm selected from the group consisting of multiple myeloma (MM), chronic lymphocytic leukemia (CLL), or non-Hodgkin's lymphoma (NHL).

In specific embodiments, the MM is selected from the group consisting of clinical multiple myeloma, smoldering multiple myeloma, plasmacytic leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma, and extramedullary plasmacytoma.

In certain embodiments, NHL is selected from the group consisting of Burkitt's lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), diffuse large B cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, lymphoblastic B cell progenitor lymphoma, and mantle zone.

BRIEF DESCRIPTION OF SEVERAL ASPECTS OF THE GRAPHIC MATERIALS

Figure 1 shows a typical example of the maintenance of T cell proliferation in T cells treated with an AKT inhibitor. The T cells were cultured with varying concentrations of the AKT inhibitor, MK-22067, for up to seven days. The rightmost panels show the percentage of dividing T cells from T cell cultures initiated from six PBMC samples from normal donors. Each symbol in the far right panel represents a unique culture that was grown in parallel with a titrated dose of MK-2206. The leftmost panels show a typical example from these experiments. A) After three days of culture with MK-2206, T cell proliferation was only slightly reduced compared to the untreated control. B) After 7 days of culture with MK-2206, T cell proliferation was virtually no different compared to the untreated control.

Figure 2 shows a typical example of CD62L expression in T cells treated with an AKT inhibitor. The T cells were cultured with varying concentrations of the AKT inhibitor, MK-22067, for up to seven days. The rightmost panels show the percentage of T cells expressing CD62L from T cell cultures initiated from six PBMC samples from normal donors. Each symbol in the far right panel represents a unique culture that was grown in parallel with a titrated dose of MK-2206. The leftmost panels show a typical example from these experiments. A) After three days of culture with MK-2206, CD62L expression on T cells treated with MK-2206 was virtually no different compared to untreated controls. B) After 7 days of culture, T cells treated with MK-2206 showed significantly higher levels of CD62L expression compared to untreated controls.

Figure 3 shows CD62L expression on BCMA CAR T cells assessed by flow cytometry at the end of culture with MK-2206, TCN, or ZSTK474. CAR T cell cultures treated with MK-2206 and ZSTK474 had significantly higher levels of CD62L expression compared to CAR T cell cultures treated with IL-2 alone or with TCN.

Figure 4 shows the average tumor volume for multiple myeloma tumors in mice that were treated with BCMA CAR T cells cultured with IL-2, IL-7 and IL-15, MK-2206, ZST747 or TCN. Anti-BCMA CAR T cells cultured with IL-7 and IL-15, MK-2206, or ZST747 showed similar levels of antitumor activity compared to anti-BCMA CAR T cells cultured with standard IL-2. Anti-BCMA CAR T cells cultured with TCN did not demonstrate an antitumor response.

Figure 5 shows the antitumor activity of anti-BCMA CAR T cells treated with IL-2, IL-7/15, MK-2206, TCN or ZSTK474 in the Daudi tumor model. The progression of Daudi tumors was not affected by subsequent treatment with anti-BCMA CAR T cells cultured with IL-2 or IL7/15. Anti-BCMA CAR T cells cultured with either MK-2206 or ZST474 induced complete tumor regression.

Figure 6 shows the persistence of anti-BCMA CAR T cells cultured with ZSTK474 in animals treated with anti-BCMA CAR T cells cultured with IL-2, MK-2206, or ZSTK474 that have 100 mm ³ tumors. RPMI-8226 has been completely regressed. Animals were re-injected with RPMI-8226 after 13 days in the other side. CAR T cells cultured with IL-2 treated with animals were not able to prevent tumor growth. Animals treated with anti-BCMA CAR T cells cultured with ZSTK474 showed no evidence of tumor engraftment.

DETAILED DESCRIPTION

A. Review

The present invention generally relates to improved methods for the production of T cell-based compositions. Without wishing to be bound by any particular theory, the methods of the present invention discussed herein uncouple proliferation and differentiation of T cells to produce T cells with improved properties, such as increased survival, expansion and persistence in vivo, with concomitant reduced differentiation compared to existing T cell-based compositions known in the art. Accordingly, the T cell-based compositions discussed herein contain highly active T cells that have the characteristics of young or naïve T cell populations that are capable of multiple rounds of expansion with limited differentiation. Moreover, the expanded cells are capable of subsequent differentiation and providing the functions of immune effector cells.

In various embodiments, a method of producing T cells is provided that maintains or minimally reduces T cell proliferation and reduces, reduces, or decreases the degree of T cell differentiation during T cell expansion. In specific preferred embodiments, the T cell-based composition is produced using the methods discussed herein, which can further enhance the effectiveness of the adaptive T cell immunotherapy agent. The manufactured T cell-based compositions discussed herein are useful for the treatment or prevention of a variety of conditions, including, but not limited to, cancer, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency. Without wishing to be bound by any particular theory, the inventor has surprisingly and unexpectedly discovered that modulation of cell signaling pathways in T cells, pathways that are normally associated with proliferation in cancer cells, results in virtually maintained or little decreased T cell proliferation and decreased T cell differentiation during T cell proliferation compared to T cells in which cell signaling pathways are not modulated.

In one embodiment, a method for producing engineered T cells includes contacting the T cells with an agent that inhibits the PI3K/AKT/mTOR pathway in the cells. Cells can be brought into contact before, during and/or after activation and proliferation. Engineered T cell compositions retain sufficient T cell activity such that they can undergo multiple rounds of expansion without significantly increasing differentiation.

Accordingly, the methods and compositions discussed herein represent a significant improvement over existing adaptive cell immunotherapies.

In carrying out the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology and cell biology, within the competence of those skilled in the art, will be used, many of which are described below for purposes of illustration. Such techniques are fully explained in the literature. See, for example, Sambrook, et al., Molecular Cloning. A Laboratory Manual ( ^3rd Edition, 2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual ( ^2nd Edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998) Current Protocols in Immunology QE Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; as well as monographs in journals such as Advances in Immunology.

All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

B. Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly known to those skilled in the art to which the present invention relates. Although any methods and materials similar or equivalent to those described herein may be used in practicing or testing the present invention, preferred embodiments of the compositions, methods and materials are described herein. For the purposes of the present invention, the following terms are defined below.

The singular and plural forms used herein denote one or more (ie, at least one) grammatical entities of the subject. By way of example, "element" means one element or more elements.

As used herein, the term "about" or "about" refers to an amount, level, value, number, frequency, percentage, measurement, size, magnitude, weight or length that varies up to 30, 25, 20, 25, 10 , 9, 8, 7, 6, 5, 4, 3, 2 or 1% relative to a reference amount, level, value, number, frequency, percentage, measurement, size, magnitude, weight or length. In specific embodiments, the terms “about” or “about,” when used before a numerical value, indicate a value plus or minus a range of 15%, 10%, 5%, or 1%.

As used herein, the term “significantly” refers to an amount, level, value, number, frequency, percentage, measurement, size, magnitude, weight or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of a reference amount, level, value, number, frequency, percentage, measurement, size, magnitude, weight or length. In one embodiment, "substantially the same" refers to the amount, level, value, number, frequency, percentage, measurement, size, magnitude, weight, or length that has an effect, such as a physiological effect, that is approximately the same as that of reference quantity, level, value, number, frequency, percentage, measurement, size, magnitude, weight or length.

Throughout this specification, unless the context otherwise requires, the words “comprise,” “comprises,” and “comprising” are to be understood to mean the inclusion of a specified step or element or group of steps or elements, but not the exclusion of any other step or element, or groups of stages or elements. By “consisting of” is meant to include and be limited to what follows the phrase “consisting of.” Thus, the phrase "consisting of" indicates that the listed elements are necessary or obligatory, and that no other elements may be present. By “substantially consisting of” is meant to include any of the elements listed after this phrase and be limited to other elements that do not interfere with or facilitate the activities or actions set forth in this disclosure with respect to the listed elements. Thus, the phrase "essentially consisting of" indicates that the listed elements are necessary or required, but other elements are optional and may be present or absent depending on whether they affect the activity or effect of the listed elements.

Reference throughout this specification to “one embodiment,” “an embodiment,” “a specific embodiment,” “a similar embodiment,” “a specific embodiment,” “an additional embodiment,” or “another embodiment,” or combinations thereof means that the particular feature, structure, or characteristic described in connection with this embodiment is included in at least one embodiment of the present invention. Thus, the occurrence of all of the above phrases at various places throughout the present description does not necessarily refer to a single embodiment. Moreover, specific features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

As used herein, the terms “T cell production” or “T cell production methods” or similar terms refer to the process of producing a T cell therapeutic composition, wherein the production methods may include one or more or all of the following stages: collection, stimulation, activation and reproduction.

The terms “T cell” or “T lymphocyte” are used in the art to include thymocytes, naïve T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cell may be a T helper cell (Th), such as a T helper cell type 1 (Th1) or a T helper cell type 2 (Th2). The T cell may be a helper T cell (HTL; CD4 ⁺ T cell), a CD4 ⁺ T cell, a cytotoxic T cell (CTL; CD8 ⁺ T cell), a tumor infiltrating cytotoxic T cell (TIL; CD8 ⁺ T cell), CD4 ⁺ CD8 ⁺ T cell, CD4 ^- CD8 ^- T cell, or any other T cell subset. Other exemplary T cell populations suitable for use in particular embodiments include naïve T cells and memory T cells.

“Highly active T cells” and “young T cells” are used interchangeably in particular embodiments and refer to T cell phenotypes in which the T cell is capable of proliferation and there is a concomitant decrease in differentiation. In specific embodiments, the young T cell has a "naïve T cell" phenotype. In various embodiments, the production methods discussed herein produce young T cells; cells in which T cell proliferation was uncoupled from T cell differentiation during T cell stimulation, activation, and expansion. Without wishing to be bound by any particular theory, highly active T cells produced using the subject compositions and methods have greater antitumor efficacy after adoptive transfer. In specific embodiments, the young T cells contain one or more or all of the following biological markers: CD62L, CCR7, CD28, CD27, CD122, and CD127. In one embodiment, young T cells contain one or more or all of the following biological markers: CD62L, CCR7, CD28, CD27, CD122 and CD127 and lack expression of CD57, CD244, CD160, PD-1, CTLA4, TIM3 and LAG3 .

As used herein, the term “proliferation” refers to an increase in cell division, both symmetrical and asymmetrical cell division. In specific embodiments, “proliferation” refers to the symmetrical or asymmetrical division of T cells. "Increased proliferation" is observed when there is an increase in the number of cells in the treated sample compared to the number of cells in the untreated sample.

As used herein, the term “differentiation” refers to a method of reducing the activity or proliferation of a cell, or of placing a cell into a state of limited developmental activity. In specific embodiments, differentiated T cells acquire the functions of immune effector cells.

An "immune effector cell" is any cell of the immune system that is characterized by one or more effector functions (eg, cytolytic activity of a cytotoxic cell, secretion of cytokines, induction of ADCC and/or CDC). Exemplary immune effector cells discussed herein are T lymphocytes, particularly cytotoxic T cells (CTL; CD8 ⁺ T cells), TILs, and helper T cells (HTL; CD4 ⁺ T cells).

“Modified T cells” refers to T cells that have been modified by introducing a polynucleotide encoding the designed TCR or CAR discussed herein. Modified T cells include both genetic and non-genetic modifications {eg, episomal or extrachromosomal).

As used herein, the term “genetically engineered” or “genetically modified” refers to the addition of additional genetic material in the form of DNA or RNA to the overall genetic material of a cell.

The terms "genetically modified cells", "modified cells" and "redirected cells" are used interchangeably.

As used herein, the term “gene therapy” refers to the introduction of additional genetic material in the form of DNA or RNA into the general genetic material of a cell that restores, corrects, or modifies gene expression or serves to express a therapeutic polypeptide, such as a TCR or CAR, and/or one or more cytokines. In specific embodiments, T cells are modified to express a designed TCR or CAR without modifying the genome of the cells, for example, by introducing an episomal vector that expresses the TCR or CAR in the cell.

The term "ex vivo" generally refers to activities performed outside the body, such as research and measurements, performed in or on living tissue under artificial conditions outside the body, preferably with minimal differences from natural conditions. In specific embodiments, ex vivo procedures involve living cells or tissues taken from the body and cultured or modulated using laboratory equipment, typically under sterile conditions and typically over a period of several hours or up to about 24 hours, but including up to 48 or 72 hours, depending on the circumstances. In certain embodiments, such tissues or cells can be collected and frozen and later thawed for ex vivo processing. Tissue culture experiments or procedures lasting longer than a few days using living cells or tissues are generally considered to be “in vitro,” although in certain embodiments the term may be used interchangeably with ex vivo.

The term "in vivo" generally refers to activities occurring within an organism, such as cell self-renewal and cell proliferation. In one embodiment, the term "in vivo proliferation" refers to the ability of a population of cells to increase in number in vivo.

The term “stimulation” refers to the primary response induced by the binding of a stimulatory molecule (eg, the TCR/CD3 complex) to its cognate ligand, thereby mediating a signaling event, including without limiting signaling through the TCR/CD3 complex.

“Stimulatory molecule” refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand.

As used herein, a “stimulatory ligand” means a ligand that, when present on an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell, etc.) can specifically bind to a cognate binding partner (referred to herein as document "stimulating molecule") on a T cell, thereby mediating the primary response of the T cell, including without limitation activation, initiation of an immune response, proliferation, etc. Stimulatory ligands include, but are not limited to, CD3 ligands, eg anti-CD3 antibody, and CD2 ligands, eg anti-CD2 antibody, and peptides, eg CMV, HPV, EBV peptides.

The term "activation" refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. In certain embodiments, activation may also be associated with induced cytokine production and detectable effector functions. The term "activated T cells" refers, among other things, to T cells that proliferate. Signals generated by the TCR alone are insufficient to fully activate the T cell, and one or more secondary or co-stimulatory signals are required. Thus, T cell activation involves a primary stimulatory signal through the TCR/CD3 complex and one or more secondary co-stimulatory signals. Costimulation may be indicated by proliferation and/or cytokine production by T cells that have received a primary activation signal, such as stimulation through the CD3/TCR complex and through CD2.

“Co-stimulatory signal” refers to a signal that, in combination with a primary signal, such as TCR/CD3 binding, results in T cell proliferation, cytokine production, and/or up- or down-regulation of certain molecules (eg, CD28).

"Co-stimulatory ligand" refers to a molecule that binds a costimulatory molecule. The costimulatory ligand may be soluble or surface-exposed. “Costimulatory molecule” refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand (eg, an anti-CD28 antibody).

"Autologous" as used herein refers to cells from the same subject.

"Allogeneic" as used herein refers to cells of the same species that are genetically different when compared to a given cell.

"Syngeneic" as used herein refers to cells from another subject that are genetically identical when compared to a given cell.

"Xenogeneic" as used herein refers to cells of a different species when compared to a given cell. In preferred embodiments, the cells of the present invention are allogeneic.

As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal exhibiting a cancer symptom that can be treated with gene therapy vectors, cell-based therapeutics, and methods disclosed elsewhere. of this document. Suitable subjects (eg, patients) include laboratory animals (such as a mouse, rat, rabbit, or guinea pig), farm animals, and pets or pets (such as a cat or dog). Non-human primates and preferably human patients are included. Typical subjects are human patients who have cancer, have been diagnosed with cancer, or are at risk of developing cancer.

As used herein, the term “patient” refers to a subject who has been diagnosed with a specific indication that can be treated with gene therapy vectors, cell-based therapeutics, and methods disclosed elsewhere herein.

As used herein, “treating” or “providing a treatment” includes any beneficial or desired effect on the symptoms or pathological signs of a disease or condition, and may include even a small reduction in one or more measurable markers of the disease or condition being treated, such as cancer . Treatment may not necessarily involve either reducing or ameliorating the symptoms of a disease or condition or delaying the progression of a disease or condition. “Treatment” does not necessarily mean complete elimination or cure of a disease or condition, or associated symptoms.

As used herein, “prevent” and similar words such as “prevented,” “warning,” etc. denote an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition, such as cancer. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, “prevention” and similar words also include reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to manifestation or recurrence of the disease or condition.

As used herein, the term “amount” refers to an “amount effective for” or an “effective amount” of a genetically modified therapeutic cell, such as a T cell, to achieve a useful or desired prophylactic or therapeutic result, including clinical results.

A “prophylactically effective amount” refers to an amount of a genetically modified therapeutic cell effective to achieve the desired prophylactic result. Typically, but not necessarily, since the prophylactic dose is administered to subjects before the onset of the disease or at an early stage thereof, the prophylactically effective amount is less than the therapeutically effective amount.

The "therapeutically effective amount" of a genetically modified therapeutic cell may vary depending on factors such as the stage of the disease, the age, sex and weight of the individual, as well as the ability of the T cells to elicit the desired response in the individual. A therapeutically effective amount is also an amount at which any toxic or harmful effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount that is effective to "treat" a subject (eg, a patient). If a therapeutic amount is indicated, the exact amount of the compositions of the present invention to be administered may be determined by the attending physician, taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject).

As used herein, the term “cancer” refers generally to a class of diseases or conditions in which abnormal cells divide uncontrollably and are capable of invading nearby tissue.

As used herein, the term “malignant” refers to a cancer in which a group of tumor cells exhibits one or more of uncontrolled growth (i.e., dividing beyond normal limits), invasion (i.e., invading and destroying adjacent tissues), and metastasis (i.e. spread to other parts of the body through lymph or blood). As used herein, the term “metastasize” refers to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a “metastatic tumor” or “metastasis.” A metastatic tumor consists of cells similar to the cells of the original (primary) tumor.

As used herein, the term “benign” or “non-cancerous” refers to tumors that may increase in size but do not spread to other parts of the body. Benign tumors are self-limiting and usually do not invade or metastasize.

"Cancer cell" or "tumor cell" refers to an individual cell of a cancerous tumor or tissue. A tumor, in general, is a swelling or pathological change formed due to abnormal cell growth, which can be benign, precancerous or malignant. Most forms of cancer form tumors, but some, such as leukemia, do not necessarily form tumors. For those forms of cancer that form tumors, the terms cancerous (cell) and neoplastic (cell) are used interchangeably. The amount of tumor in an individual represents the “tumor load,” which can be measured as the number, volume, or weight of the tumor.

“Infectious disease” refers to a disease that can be transmitted from person to person or from organism to organism, and which is caused by a microbial agent (for example, an upper respiratory tract infection). Infectious diseases are known in the art and include, for example, hepatitis, sexually transmitted diseases (eg, chlamydia, gonorrhea), tuberculosis, HIV/AIDS, diphtheria, hepatitis B, hepatitis C, cholera and influenza.

“Autoimmune disease” refers to a disease in which the body develops an immunogenic reaction (i.e., immune system response) against some constituent of its own tissue. In other words, the immune system loses its ability to recognize any tissue or system within the body as “self” and targets and attacks it as if it were foreign. Autoimmune diseases can be classified into those that primarily affect one organ (eg, hemolytic anemia and autoimmune thyroiditis) and those in which the autoimmune disease pathology affects multiple tissues (eg, systemic lupus erythematosus). For example, multiple sclerosis is thought to be caused by T cells that attack the sheaths of nerve fibers in the brain and spinal cord. This leads to loss of coordination, weakness and blurred vision. Autoimmune diseases are known in the art and include, for example, Hashimoto's thyroiditis, Graves' disease, lupus, multiple sclerosis, rheumatoid arthritis, hemolytic anemia, autoimmune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, etc. P.

“Immunodeficiency” means a condition of a patient whose immune system has been weakened due to disease or the administration of chemicals. This condition creates a systemic deficiency in the number and types of blood cells needed to protect against foreign substances. Immunodeficiency conditions or diseases are known in the art and include, for example, AIDS (acquired immunodeficiency syndrome), SCID (severe combined immunodeficiency), isolated IgA deficiency, variable unclassified immunodeficiency, X-linked agammaglobulinemia, chronic granulomatosis, ranep- IgM syndrome and diabetes.

“Enhance” or “promote” or “increase” or “enhance” generally refers to the ability of a composition contemplated herein to produce, promote, or cause a greater physiological response (i.e., downstream effects) than a reaction caused by either the vehicle or the control molecule/composition. The measured physiological response may include increased proliferation, activation, persistence of T cells and/or increased ability to induce cytolysis of cancer cells, among other things apparent from the understanding in the prior art and the description herein. An "increased" or "increased" amount is generally a "statistically significant" amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all whole numbers and decimal places between them greater than 1, e.g., 1.5, 1.6, 1.7 , 1.8, etc.) exceeds the response due to the vehicle or control composition.

“Reduce” or “lower” or “facilitate” or “lower” or “attenuate” generally refers to the ability of a composition contemplated herein to produce, promote, or cause a lesser physiological response (i.e., downstream effects ) compared to the response caused by either the vehicle or the control molecule/composition. A "reduced" or "reduced" amount is generally a "statistically significant" amount, and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all whole numbers and decimal places between them greater than 1, e.g., 1.5, 1.6, 1.7 , 1.8, etc.) is less compared to the reaction (reference reaction) caused by the vehicle, control composition, or reaction in a particular cell line.

“Maintain” or “maintain” or “maintenance” or “no change” or “no significant change” or “no significant reduction” generally refers to the ability of a composition contemplated herein to produce, promote or cause more a weak physiological response (ie, downstream effects) in the cell compared to the response due to either the vehicle or control molecule/composition, or the response in a particular cell line. A commensurate reaction is a reaction that is not significantly different or measurably different from the reference reaction.

The terms "specific binding affinity" or "specifically binds" or "specifically bound" or "specific binding" or "specifically targets" as used herein describe the binding of one molecule to another with greater binding affinity than background binding . A binding domain (or a CAR containing a binding domain, or a fusion protein containing a binding domain) "specifically binds" to a target molecule if it binds or associates with the target molecule with affinity or Ka (i.e., the equilibrium association constant of a particular binding interaction in units of 1/M), for example, greater than or equal to approximately 10 ⁵ M ^-1 . In certain embodiments, the binding domain (or a fusion protein based thereon) binds to a target with a Ka greater than or equal to about 10 ⁶ M ^-1 , 10 ⁷ M ^-1 , 10 ⁸ M ^-1 , 10 ⁹ M ^-1 , 10 ¹⁰ M ^-11 , M ^-1 , 10 ¹² M ^-1 or 10 ¹³ M ^-1 . Binding domains (or single-chain fusion proteins based on them) with “high affinity” are those binding domains with a K _a of at least 10 M ⁷ , at least 10 M ^-1 , at least 10 ⁹ M ^-1 , at least at least 10 ¹⁰ M ^-1 , at least 10 ¹¹ M ^-1 , at least 10 ¹² M ^-1 , at least 10 ¹³ M ^-1 or more.

Alternatively, affinity can be defined as the equilibrium dissociation constant (K _d ) of a particular binding interaction in units of M (eg, 10 ^-5 M to 10 ^-13 M or less). The affinities of the binding domain polypeptides and CAR proteins of the present disclosure can be readily determined using conventional techniques, for example, competitive ELISA, or binding or displacement association assays using labeled ligands, or using a surface plasmon resonance device , such as the Biacore T100, available from Biacore, Inc., Pisectaway, NJ, or optical biosensor technology, such as the EPIC or En Spire system, available from Corning and Perkin Elmer, respectively (see also, for example, Scatchard et al (1949) Ann NY Acad Sci 51:660; US Patent Nos. 5,283,173; 5,468,614 or the like).

In one embodiment, the specific binding affinity is approximately 2 times the background binding affinity, approximately 5 times the background binding affinity, approximately 10 times the background binding affinity, approximately 20 times the background binding affinity, approximately 50 times the background binding affinity binding is approximately 100 times the background binding affinity or approximately 1000 times the background binding affinity or greater.

"Antigen (Ag)" refers to a compound, composition or substance that can stimulate antibody production or a T cell response in an animal, including compositions (such as a composition containing a tumor-specific protein) that are injected into the animal or absorbed into the animal. The antigen reacts with products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as disclosed antigens. A “target antigen” or “target antigen of interest” is the antigen that the binding domain of the CAR discussed herein is designed to bind.

"Epitope" or "antigenic determinant" refers to the region of the antigen to which the binding agent binds.

"Isolated peptide" or "isolated polypeptide" and the like as used herein refer to the in vitro isolation and/or purification of a peptide or polypeptide molecule from the cellular environment, as well as from association with other cellular components, i.e. it is not significantly associated with in vivo substances. Likewise, the term “isolated cell” refers to a cell that has been obtained from an in vivo tissue or organ and contains substantially no extracellular matrix.

As used herein, “isolated polynucleotide” refers to a polynucleotide that has been cleared of sequences that flank it in a naturally occurring state, for example, a DNA fragment that has been cleared of sequences that are normally located adjacent to the fragment. “Isolated polynucleotide” also refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotide that does not exist in nature and that is man-made.

C. Methods for producing T cells

T cells produced using the methods discussed herein provide improved compositions for adaptive immunotherapy. Without wishing to be bound by any particular theory, it is believed that T cell-based compositions produced using the methods discussed herein are endowed with improved properties, including increased survival, expansion in the relative absence of differentiation, and persistence in vivo . In one embodiment, a method of producing T cells includes contacting the cells with one or more agents that modulate the PI3K/Akt/mTOR cell signaling pathway. In various embodiments, T cells can be obtained from any source and brought into contact with the agent during the activation and/or expansion phases of the manufacturing process. The resulting T cell-based compositions are enriched in highly developmentally active T cells that have the ability to proliferate and express one or more of the following biomarkers: CD62L, CCR7, CD28, CD27, CD122, and CD127.

In one embodiment, modified T cells are produced to maintain levels of proliferation and reduced differentiation. In a specific embodiment, T cells are produced by stimulating T cells to enter an activated state and proliferate in the presence of one or more stimulatory signals and agents that is an inhibitor of the PI3K/Akt/mTOR cell signaling pathway.

The T cells can then be modified to express one or more of the designed TCRs or CARs. In one embodiment, T cells are modified by transducing the T cells with a viral vector containing a designed TCR or CAR. In a certain embodiment, T cells are modified prior to stimulation and activation in the presence of an inhibitor of the PI3K/Akt/mTOR cell signaling pathway. In another embodiment, T cells are modified after stimulation and activation in the presence of an inhibitor of the PI3K/Akt/mTOR cell signaling pathway. In a specific embodiment, the T cells are modified within 12 hours, 24 hours, 36 hours, or 48 hours after stimulation and activation in the presence of an inhibitor of the PI3K/Akt/mTOR cell signaling pathway.

After T cell activation, the cells are cultured to proliferate. T cells can be cultured for at least 1, 2, 3, 4, 5, 6 or 7 days, at least 2 weeks, at least 1, 2, 3, 4, 5 or 6 months or more with 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more breeding rounds.

In various embodiments, T cell-based compositions are produced in the presence of one or more PI3K/AKT/mTOR pathway inhibitors. Inhibitors can specifically target one or more activities in a pathway or a single activity. Without wishing to be bound by any particular theory, it is believed that treating or exposing T cells to one or more inhibitors of the PI3K/AKT/mTOR pathway during the stimulation, activation and/or expansion phases of the production process preferentially increases the number of young T cells -cells, thereby obtaining more advanced therapeutic compositions based on T cells.

In a specific embodiment, a method is provided for increasing the proliferation of T cells expressing a designed T cell receptor. Such methods may include, for example, collecting a source of T cells from a subject, stimulating and activating the T cells in the presence of one or more PI3K/AKT/mTOR pathway inhibitors, modifying the T cells to express a designed TCR or CAR, and expanding the T cells in culture.

In a certain embodiment, a method is provided for obtaining T cell populations enriched for the expression of one or more of the following biomarkers: CD62L, CCR7, CD28, CD27, CD 122 and CD127. In a similar embodiment, a method is provided for increasing the number of T cells expressing CD62L, CCR7, CD28, CD27, CD122 and CD127 and not expressing or expressing low levels of CD57, CD244, CD160, PD-1, CTLA4, TIM3 and LAG3. As discussed elsewhere in this document, the expression levels of biomarkers of young T cells are compared with the expression levels of such markers in more differentiated T cells or immune effector cell populations.

In one embodiment, peripheral blood mononuclear cells (PBMCs) are used as a source of T cells in the T cell production methods discussed herein. PBMC comprise a heterogeneous population of T lymphocytes, which may be CD4 ⁺ , CD8 ⁺ , or CD4 ⁺ and CD8 ⁺ , and they may include other mononuclear cells such as monocytes, B cells, NK cells, and NKT cells. An expression vector containing a polynucleotide encoding a designed TCR or CAR discussed herein can be introduced into a population of human donor T cells, NK cells or NKT cells. Successfully transduced T cells that carry the expression vector can be sorted using flow cytometry to isolate CD3-positive T cells and then further expanded to increase the number of modified T cells in addition to cell activation using anti-CD3 antibodies and/or antibodies to CD28 and IL-2, IL-7 and/or IL-15 or any other methods known in the art described elsewhere in this document.

The manufacturing methods discussed herein may further include cryopreservation of the modified T cells for storage and/or preparation for use in a human subject. T cells are cryopreserved so that the cells remain viable after thawing. If necessary, cryopreserved transformed immune effector cells can be thawed, grown and expanded to increase the number of such cells. As used herein, “cryopreservation” refers to the preservation of cells by cooling to temperatures below freezing, such as (typically) 77 K or -196°C (the boiling point of liquid nitrogen). In order to prevent the destruction of stored cells during freezing at low temperatures or during heating to room temperature, cryoprotective agents are often used. Cryoprotectants and optimal cooling rates can protect cells from damage. Cryoprotectants that may be used include, but are not limited to, dimethyl sulfoxide (DMSO) (Lovelock and Bishop, Nature, 1959; 183:1394-1395; Ashwood-Smith, Nature, 1961; 190:1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, Ann. N.Y. Acad. Sci., 1960; 85: 576) and polyethylene glycol (Sloviter and Ravdin, Nature, 1962; 196: 48). The preferred cooling rate is 1°-3°C/minute. After at least two hours, the T cells reach a temperature of -80°C and can be placed directly into liquid nitrogen (-196°C) for permanent storage, such as in a cryogenic vessel for long-term storage.

1. T cells

The present invention contemplates the production of improved T cell-based compositions. T cells can be autologous/autogeneic (“self”) or non-autologous (“non-self”, for example, allogeneic, syngeneic or xenogeneic). In preferred embodiments, the T cells are obtained from a mammalian subject. In a more preferred embodiment, the T cells are obtained from a primate subject. In the most preferred embodiment, the T cells are obtained from a human subject.

T cells can be obtained from a variety of sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any of a variety of techniques known to one of ordinary skill in the art, such as precipitation, such as FICOLL™ separation. In one embodiment, cells from the circulating blood of an individual are obtained through apheresis. The apheresis product typically contains 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 may be washed to remove the plasma fraction and to place the cells in a suitable buffer or medium for subsequent processing. Cells can be washed with PBS or other suitable solution that is free of calcium, magnesium, and most if not all other divalent cations. As will be appreciated by those of ordinary skill in the art, the washing step can be performed using methods known to those skilled in the art, such as using a semi-automated flow centrifuge. For example, Cobe 2991 Cell Processor, Baxter CytoMate, etc. After washing, cells can be resuspended in various biocompatible buffers or other saline solutions, with or without buffer. In certain embodiments, unwanted components from the apheresis sample can be removed in the culture medium to directly resuspend the cells.

In specific embodiments, a population of cells containing T cells, such as PBMC, is used in the production methods discussed herein. In other embodiments, the production methods discussed herein utilize an isolated or purified population of T cells. Cells can be isolated from peripheral blood mononuclear cells (PBMCs) by erythrocyte lysis and monocyte depletion, for example, by PERCOLL™ gradient centrifugation. In some embodiments, after isolation of PBMCs, both cytotoxic and helper T lymphocytes can be sorted into subpopulations of naïve T cells, memory T cells, and effector T cells, either before or after activation, expansion, and/or genetic modification.

Specific T cell subsets expressing one or more of the following markers: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD 127 and HLA-DR can be further isolated using positive or negative selection techniques. In one embodiment, a specific subpopulation of T cells expressing one or more markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD 122 and CD 127 is further isolated using positive or negative selection techniques. In various embodiments, the produced T cell compositions do not express, or substantially do not express, one or more of the following markers: CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3.

In one embodiment, the expression of one or more markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD 122 and CD 127 is increased by at least 1.5-fold, at least 2-fold, by at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 25 times or more compared to a T cell population that was activated and expanded without a PI3K/AKT/mTOR inhibitor.

In one embodiment, the expression of one or more markers selected from the group consisting of CD57, CD244, CD160, PD-1, CTLA4, TIM3 and LAG3 is reduced by at least 1.5-fold, at least 2-fold, by at least at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10-fold, at least 25-fold or more compared to the T cell population that was activated and expanded with a PI3K/AKT/mTOR inhibitor.

In one embodiment, the production methods discussed herein increase the number of T cells containing one or more markers of naive T cells or T cells with high developmental activity. Without wishing to be bound by any particular theory, the present inventor believes that treating a population of cells containing T cells with one or more PI3K/AKT/mTOR inhibitors uncouples T cell proliferation and differentiation signals, thereby leading to enhancing the expansion of T cells with high developmental activity and provides a more reliable and effective means of adaptive immunotherapy than existing T-cell therapies.

Illustrative examples of naïve or developmentally active T cell markers elevated in T cells produced using the methods discussed herein include, but are not limited to, CD62L, CCR7, CD28, CD27, CD95, CD122, and CD127 .. In specific embodiments, naïve T cells do not express or essentially do not express one or more of the following markers: CD57, CD244, CD160, PD-1, BTLA, CD45RA, CTLA4, TIM3, and LAG3.

In the case of T cells, T cell populations generated by the various expansion techniques discussed herein may exhibit a range of specific phenotypic properties, depending on the conditions used. In various embodiments, the expanded T cell populations contain one or more of the following phenotypic markers: CCR7, CD3, CD4, CD8, CD27, CD28, CD62L, CD95, CD122, CD127, and HLA-DR.

In one embodiment, such phenotypic markers include increased expression of one or more or all of CD62L, CCR7, CD28, CD27, CD122 and CD127. In specific embodiments, CD8 ⁺ T lymphocytes are expanded that are characterized by the expression of naïve T cell phenotypic markers, including CD62L, CCR7, CD28, CD27, CD 122, and CD 127.

In specific embodiments, T cells are expanded that express phenotypic markers of central memory T cells, including CD45RO, CD62L, CCR7, CD28, CD27, CD122, and CD127, and are granzyme B negative. In some embodiments, the central T cells are memory cells are CD45RO ⁺ , CD62L ⁺ , CD8 ⁺ T cells.

In certain embodiments, CD4 ⁺ T lymphocytes are expanded that are characterized by the expression of phenotypic markers of naïve CD4 ⁺ cells, including CD62L, and are negative for the expression of CD45RA and/or CD45RO. In some embodiments, the CD4 ⁺ cells are characterized by the expression of phenotypic markers of central memory CD4 + cells, including CD62L, and CD45RO-positive. In some embodiments, the effector CD4 ⁺ cells are CD62L-positive and CD45RO-negative.

In certain embodiments, T cells are isolated from an individual and modified without further manipulation ex vivo or in vitro. Such cells can then be directly reintroduced to the individual. In additional embodiments, T cells are first activated and stimulated to proliferate in vitro before being genetically modified to express a designed TCR or CAR. In this regard, T cells can be cultured before and/or after they are genetically modified (ie, transduced or transfected to express the designed TCR or CAR discussed herein).

2. Activation and reproduction

To achieve sufficient therapeutic doses of T cell-based compositions, one or more rounds of T cell stimulation, activation and/or expansion are often performed. Typically, T cells can be activated and expanded using methods described, for example, in US patents 6352694; 6534055; 6905680; 6692964; 5858358; 6887466; 6905681; 7144575; 7067318; 7172869; 7232566; 7175843; 5883223; 6905874; 6797514 and 6867041, each of which is incorporated herein by reference in its entirety. T cells modified to express a designed TCR or CAR can be activated and expanded before and/or after T cell modification. In addition, T cells can be brought into contact with one or more agents that modulate the PI3K/AKT/mTOR cell signaling pathway before, during and/or after activation and/or expansion. In one embodiment, T cells produced using the methods discussed herein are subjected to one, two, three, four, or five or more rounds of activation and expansion, each of which may include one or more agents that modulate the signal transduction pathway in the PI3K/AKT/mTOR cell.

In one embodiment, a costimulatory ligand is present on an antigen presenting cell (e.g., aAPC, dendritic cell, B cell, etc.) that specifically binds a cognate costimulatory molecule on the T cell, thereby providing a signal that, in addition to the primary signal provided, for example, by the binding of the TCR/CD3 complex, mediates the desired T cell response. Suitable costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L 1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule ( ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin receptor beta, ILT3, ILT4, agonist or antibody that binds Toll-like receptor, and ligand that specifically binds B7- H3.

In a specific embodiment, the costimulatory ligand includes an antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule present on a T cell, including but not limited to CD27, CD28,4-IBB, OX40, CD30, CD40, PD-1, 1COS, lymphocyte functional antigen-1 (LFA-1), CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds CD83.

Suitable costimulatory ligands further include target antigens that may be in soluble form or expressed on APCs or aAPCs that bind engineered TCRs or CARs expressed on engineered T cells.

In various embodiments, a method for producing T cells discussed herein includes activating a population of cells including the T cells and expanding the population of T cells. Activation of T cells can be accomplished by providing a primary stimulatory signal through the TCR/CD3 T cell complex or by stimulating the surface protein CD2 and by providing a secondary co-stimulatory signal through an accessory molecule, such as CD28.

The TCR/CD3 complex can be stimulated by contacting the T cell with a suitable CD3 binding agent, for example, a CD3 ligand or an anti-CD3 monoclonal antibody. Illustrative examples of anti-CD3 antibodies include, but are not limited to, OKTZ, G19-4, BC3, and 64.1.

In another embodiment, a CO2 binding agent may be used to provide a primary stimulatory signal to T cells. Illustrative examples of CO2-binding agents include, but are not limited to, CD2 ligands and anti-CD2 antibodies, for example, antibody T11.3 in combination with antibody T11.1 or T11.2 (Meuer, S.C. et al. (1984) Cell 36:897 -906) and antibody 9.6 (which recognizes the same epitope as TI 1.1) in combination with antibody 9-1 (Yang, S. Y. et al. (1986) J Immunol. 137:1097-1100). Other antibodies that bind to the same epitopes as any of the antibodies described above can also be used. Additional antibodies or combinations of antibodies can be prepared and identified using standard techniques disclosed elsewhere in this document.

In addition to the primary stimulatory signal provided through the TCR/CD3 complex or through CD2, a secondary, co-stimulatory signal is required to induce T cell responses. In specific embodiments, a CD28 binding agent may be used to provide a co-stimulatory signal. Illustrative examples of CD28 binding agents include, but are not limited to: natural CD28 ligands, e.g., a natural ligand for CD28 (e.g., a member of the B7 family of proteins, such as B7-1(CD80 ) and B7-2 (CD86); and a monoclonal antibody to CD28 or a fragment thereof capable of cross-reacting with the CD28 molecule, for example, monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8, 248.23.2 and EX5.3D10.

In one embodiment, the molecule that provides the primary stimulatory signal, for example, the molecule that provides stimulation through the TCR/CD3 and CD2 complex, and the co-stimulatory molecule are attached to the same surface.

In certain embodiments, the binding agents that provide stimulatory and co-stimulatory signals are localized on the cell surface. This can be accomplished by transfecting or transducing the cell with nucleic acid encoding the binding agent in a form suitable for expression on the cell surface, or alternatively by attaching the binding agent to the cell surface.

In another embodiment, the molecule that provides the primary stimulatory signal, for example, the molecule that provides stimulation through the TCR/CD3 and CD2 complex, and the co-stimulatory molecule are presented on antigen presenting cells.

In one embodiment, the molecule that provides the primary stimulatory signal, for example, the molecule that provides stimulation through the TCR7CD3 and CD2 complex, and the costimulatory molecule are located on separate surfaces.

In a certain embodiment, one of the binding agents that provide the stimulatory and costimulatory signals is soluble (provided in the form of a solution) and the other agent(s) are present on one or more surfaces.

In a specific embodiment, both binding agents that provide the stimulatory and co-stimulatory signals are in soluble form (provided as a solution).

In various embodiments, methods for producing T cells discussed herein include activating T cells with an anti-CD3 antibody and an anti-CD28 antibody.

Compositions based on T cells produced using the methods discussed herein contain T cells activated and/or expanded in the presence of one or more agents that inhibit the PI3K/AKT/mTOR cell signaling pathway. T cells modified to express a designed TCR or CAR can be activated and expanded before and/or after T cell modification. In specific embodiments, a population of T cells is activated, modified to express a designed TCR or CAR, and then cultured for expansion.

In one embodiment, T cells produced using the methods discussed herein contain an increased number of T cells expressing markers indicative of high proliferative potential and self-renewal capacity, but which do not express or express virtually undetectable amounts of T differentiation markers -cells. These T cells can be repeatedly activated and expanded in a reliable manner, thereby providing an improved T cell therapeutic composition.

In one embodiment, the population of T cells that have been activated and expanded in the presence of one or more agents that inhibit the PI3K/AKT/mTOR cell signaling pathway increases by at least 1.5-fold, at least 2-fold, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, according to at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 250 times, at least 500 times, at least 1000 times or more compared to a T cell population that was activated and expanded without a PI3K/AKT/mTOR inhibitor.

In one embodiment, a population of T cells characterized by the expression of markers of young T cells that have been activated and expanded in the presence of one or more agents that inhibit the PI3K/AKT/mTOR cell signaling pathway is increased by at least 1.5-fold , at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 250 times, at least 500 times, according to at least 1000-fold or more compared to a T cell population that was activated and expanded without a PI3K/AKT/mTOR inhibitor.

In one embodiment, expansion of T cells activated using the methods discussed herein further comprises culturing a population of cells containing the T cells for several hours (about 3 hours) to about 7 days, to about 28 days, or any integer number of hours between them. In another embodiment, the T cell-based composition can be cultured for 14 days. In a specific embodiment, the T cells are cultured for approximately 21 days. In another embodiment, the T cell-based compositions are cultured for approximately 2-3 days. Multiple cycles of stimulation/activation/expansion may also be required, so T cell culture times may be 60 days or more.

In specific embodiments, conditions suitable for culturing T cells include a suitable medium (e.g., minimal essential medium, or RPMI 1640 medium, or X-vivo 15 (Lonza)) and one or more factors necessary for proliferation and viability, in including, without limitation, serum (eg, fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGFβ or TNF-α, or any other additives required for cell growth known to one skilled in the art.

Additional illustrative examples of cell culture media include, but are not limited to, RPMI 1640, Click medium, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1 5 and X-Vivo 20, Optimizer, with added amino acids, pyruvate sodium and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a specific set of hormones and/or an amount of cytokine(s) sufficient for the growth and proliferation of T cells.

Illustrative examples of other T cell expansion additives include, but are not limited to, surfactant, Plasmanate, pH buffers such as HEPES, and reducing agents such as N-acetylcysteine and 2-mercaptoethane.

Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cell cultures that are intended to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, at an appropriate temperature (eg, 37°C) and atmosphere (eg, air with 5% CO2).

In specific embodiments, PBMCs or isolated T cells are contacted with a stimulatory agent and a co-stimulatory agent, such as antibodies to CD3 and CD28, typically attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL- 2, IL-7 and/or IL-15.

In other embodiments, artificial APCs (aAPCs) are produced by engineering K562, U937, 721.221, T2, and C1R cells that are designed to stably express and secrete a variety of co-stimulatory molecules and cytokines. In a specific embodiment, aAPC in the form of K32 or U32 is used to expose one or more antibody-based stimulatory molecules to the cell surface of the AAPC. T cell populations can be expanded using aAPCs expressing various co-stimulatory molecules, including but not limited to CD137L (4-1BBL), CD134L (OX40L) and/or CD80 or CD86. Finally, aAPCs provide an effective platform for the expansion of genetically modified T cells and for maintaining CD28 expression on CD8 T cells. The aAPCs provided for in WO 03/057171 and US2003/0147869 are hereby incorporated by reference in their entirety.

3. Means

In various embodiments, a method of producing T cells is provided in which undifferentiated or highly developmentally active T cells are expanded, comprising contacting the T cells with an agent that modulates the PI3K/AKT/mTOR pathway in the cells. Cells can be brought into contact before, during and/or after activation and proliferation. T cell-based compositions retain sufficient T cell activity such that they can undergo multiple rounds of expansion without significantly increasing differentiation.

As used herein, the terms “modulate,” “modulator,” or “modulatory agent,” or similar term, refer to the ability of an agent to cause a change in a signal transduction pathway in a cell. A modulator can increase or decrease the amount or activity of a pathway component, or increase or decrease the desired effect or output of a signaling pathway in a cell. In one embodiment, the modulator is an inhibitor. In another embodiment, the modulator is an activator.

“Agent” refers to a compound, small molecule, e.g., small organic molecule, nucleic acid, polypeptide or fragment, isoform, variant, analog or derivative thereof, useful in modulating the PI3K/AKT/mTOR pathway.

“Small molecule” refers to a composition that has a molecular weight of less than about 5 kDa, less than about 4 kDa, less than about 3 kDa, less than about 2 kDa, less than about 1 kDa, or less than about 0.5 kDa. Small molecules may include nucleic acids, peptides, polypeptides, peptidomimetics, peptoids, carbohydrates, lipids, components thereof, or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial or algal extracts, are known in the art and can be screened using any of the assays of the present invention. Examples of methods for synthesizing molecular libraries can be found in (Carell et al., 1994a; Carell et al., 1994b; Cho et al., 1993; DeWitt et al., 1993; Gallop et al., 1994; Zuckermann et al., 1994 ).

"Analogue" refers to a small organic compound, nucleotide, protein or polypeptide that has similar or identical activity or function(s) as the compound, nucleotide, protein or polypeptide, or a compound with the activity required by the present invention, optionally containing a sequence or structure that is similar or identical to the sequence or structure of the preferred embodiment.

"Derivative" refers to any compound, protein or polypeptide that contains the amino acid sequence of the original protein or polypeptide that has been modified by the introduction of substitution, deletion or addition of an amino acid residue, or a nucleic acid or nucleotide that has been modified by any introduction of nucleotide substitutions or deletions, additions or mutations. A derived nucleic acid, nucleotide, protein, or polypeptide has a similar or identical function to that of the parent polypeptide.

In various embodiments, an agent that modulates the PI3K/AKT/mTOR pathway activates a component of the pathway. "Activator" or "agonist" refers to an agent that stimulates, enhances, or induces one or more activities of a molecule in the PI3K/AKT/mTOR pathway, including but not limited to a molecule that inhibits one or more activities of PI3K, Akt, or mTOR (or complex mTORC1, mTORC2).

In various embodiments, an agent that modulates the PI3K/AKT/mTOR pathway inhibits a component of the pathway. “Inhibitor” or “antagonist” refers to an agent that inhibits, reduces or reduces one or more activities of a molecule in the PI3K/AKT/mTOR pathway, including but not limited to PI3K, Akt or mTOR (or the mTORC1, mTORC2 complex). In one embodiment, the inhibitor is a two-molecule inhibitor. In one embodiment, the inhibitor prevents the formation of protein complexes, such as the mTORC1 complex or similar complexes. In a particular embodiment, an inhibitor may inhibit a class of molecules that have the same or substantially similar activity (general inhibitor), or may specifically inhibit the activity of a molecule (selective or specific inhibitor). Inhibition may also be irreversible or reversible.

In one embodiment, the inhibitor has an IC ₅₀ of at least 1 nM, at least 2 nM, at least 5 nM, at least 10 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 500 nM, at least 1 µM, at least 10 µM, at least 50 µM or at least 100 µM. The determination of IC ₅₀ can be carried out using any conventional techniques known in the art. For example, the IC ₅₀ can be determined by measuring the activity of a given enzyme in the presence of a range of concentrations of the inhibitor of interest. The experimentally obtained enzyme activity values are then plotted against the inhibitor concentrations used. The inhibitor concentration at which 50% of the enzyme activity occurs (compared to the activity in the absence of any inhibitor) is taken as the “IC ₅₀ ” value. Likewise, other inhibitory concentrations can be determined by appropriate activity determination.

In various embodiments, T cells are contacted or treated or cultured with one or more PI3K/AKT/mTOR pathway modulators at a concentration of at least 1 nM, at least 2 nM, at least 5 nM, at least 10 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 500 nM, at least 1 µM, at least 10 µM, at least 50 µM, at least 100 µM or at least 1 M.

In specific embodiments, T cells can be contacted or treated or cultured with one or more PI3K/AKT/mTOR pathway modulators for at least 12 hours, 18 hours, at least 1,2, 3, 4, 5, 6 or 7 days, at least 2 weeks, at least 1, 2, 3, 4, 5 or 6 months or more with 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 or more rounds of breeding.

A. PI3K/AKT/mTOR pathway

The phosphatidyl-inositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway serves as an intermediary for the integration of growth factor signaling with cell proliferation, differentiation, metabolism, and survival. PI3Ks are a family of highly conserved intracellular lipid kinases. Class IA PI3Ks are activated by growth factor receptor tyrosine kinases (RTKs), either directly or through interaction with the insulin receptor family of adapter substrate molecules. This activity leads to the formation of phosphatidyl-inositol 3,4,5-trisphosphate (PIP3), a regulator of the serine/threonine kinase Akt. mTOR acts through the canonical PI3K pathway through 2 distinct complexes, each characterized by distinct binding partners that mediate different activities. mTORC1 (mTOR complexed with PRAS40, raptor, and mLST8/GbL) acts as a downstream effector of PI3K/Akt signaling by coupling growth factor signals to protein translation, cell growth, proliferation, and survival. mTORC2 (mTOR in complex with rictor, mSINl, protor and mLST8) act as an upstream activator of Akt.

Following growth factor-mediated activation of PI3K, Akt is recruited to the membrane through the interaction of its plestrin homology domain with PIP3, thereby opening its activation loop and allowing phosphorylation at threonine 308 (Thr308) by constitutively active phosphoinositide-dependent protein kinase 1 (PDK1). For maximal activation, Akt is also phosphorylated by mTORC2 at serine 473 (Ser473) of its C-terminal hydrophobic motif. DNA-PK and HSP have also been shown to be important in regulating Akt activity. Akt activates mTORC1 through inhibitory phosphorylation of TSC2, which, together with TSC1, produces negative regulation of mTORC1 by inhibiting the GTPase Rheb, a positive regulator of mTORC1. mTORC1 has 2 well-defined substrates, p70S6K (referred to hereinafter as S6K1) and 4E-BP1, both of which are critical for the regulation of protein synthesis. Thus, mTORC1 is an important downstream effector of PI3K linking growth factor signaling to protein translation and cell proliferation.

b. mTOR inhibitors

The terms “mTOR inhibitor” or “agent that inhibits mTOR” refer to a nucleic acid, peptide, compound or small organic molecule that inhibits at least one mTOR protein activity, such as, for example, serine/threonine protein kinase activity towards at least one of its substrates (eg, p70S6 kinase 1, 4E-BP1, AKT/PKB and eEF2). mTOR inhibitors are capable of directly binding to and inhibiting mTORC1, mTORC2, or both mTORC1 and mTORC2.

Inhibition of mTORC1 and/or mTORC2 activity can be detected by decreased signaling in the PI3K/Akt/mTOR pathway. A number of indicators can be used to determine the reduction in the outcome of such a signal transduction pathway. Some non-limiting examples of indicators include (1) decreased phosphorylation of Akt at residues, including but not limited to 5473 and T308; (2) decreased activation of Akt, as evidenced, for example, by decreased phosphorylation of Akt substrates, including without limitation Fox01/O3a T24/32, GSK3a/β; S21/9 and TSC2 T1462; (3) decreased phosphorylation of signal transduction molecules located downstream of mTOR, including but not limited to ribosomal S6 S240/244, 70S6K T389, and 4EBP1 T37/46; and (4) inhibition of cancer cell proliferation.

In one embodiment, mTOR inhibitors are active site inhibitors. They are mTOR inhibitors that bind to the ATP binding site (also called the ATP binding pocket) of mTOR and inhibit the catalytic activity of both mTORC1 and mTORC2. One class of active site inhibitors useful in the T cell production methods discussed herein are dual-specificity inhibitors that target and directly inhibit both PI3K and mTOR. Dual specificity inhibitors bind to both ATP binding sites in mTOR and PI3K. Illustrative examples of such inhibitors include, but are not limited to: imidazoquinazolines, wortmannin, LY294002, PI-103 (Cayman Chemical), SF1126 (Semafore), BGT226 (Novartis), XL765 (Exelixis) and NVP-BEZ235 (Novartis).

Another class of mTOR active site inhibitors useful in the methods discussed herein selectively inhibit the activity of mTORC1 and mTORC2 over one or more type I phosphatidylinositol 3-kinases, such as PB kinase α, β, γ, or δ. These active site inhibitors bind to the active site of mTOR but not PI3K. Illustrative examples of such inhibitors include, but are not limited to: pyrazolopyrimidines, Torinl (Guertin and Sabatini), PP242 (2-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indole -5-ol), PP30, Ku-0063794, WAY-600 (Wyeth), WAY-687 (Wyeth), WAY-354 (Wyeth) and AZD8055 (Liu et al., Nature Review, 8, 627-644, 2009 ). I

In one embodiment, a selective mTOR inhibitor refers to an agent that exhibits a 50% inhibitory concentration (IC50) on mTORCl and/or mTORC2 that is lower than the IC50 of the inhibitor on one, two, three or more type I PI3 kinases or all Type I PI3-kinases at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more.

Another class of mTOR inhibitors for use in the present invention are referred to herein as "rapalogues". As used herein, the term “rapalogues” refers to compounds that specifically bind to the FRB domain of mTOR (FKBP-rapamycin binding domain), are structurally related to rapamycin, and retain mTOR inhibitory properties. The term rapalogue excludes rapamycin. Rapalogues include rapamycin esters, ethers, oximes, hydrazones and hydroxylamines, as well as compounds in which functional groups on the rapamycin backbone have been modified, for example by reduction or oxidation. Pharmaceutically acceptable salts of such compounds are also considered rapamycin derivatives. Illustrative examples of rapalogues suitable for use in the methods discussed herein include, but are not limited to, temsirolimus (CC1779), everolimus (RAD001), deforolimus (AP23573), AZD8055 (AstraZeneca) and OSI-027 (OSI).

In one embodiment, the agent is a mTOR inhibitor of rapamycin (sirolimus).

In a specific embodiment, examples of mTOR inhibitors for use in the present invention inhibit one of mTORC1, mTORC2, or both mTORC1 and mTORC2 with an IC ₅₀ (concentration at which 50% of activity is inhibited) of about 200 nM or less, preferably about 100 nM or less , even more preferably about 60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 μM, 50 μM, 25 μM, 10 μM, 1 μM or less. In one aspect, an mTOR inhibitor for use in the present invention inhibits one of mTORC1, mTORC2, or both mTORC1 and mTORC2 with an IC ₅₀ of from about 2 nM to about 100 nM, more preferably from about 2 nM to about 50 nM, even more preferably from about 2 nM nM to approximately 15 nM.

In one embodiment, examples of mTOR inhibitors inhibit either PI3K and mTORC1 or mTORC2, or both mTORC1 and mTORC2 and PI3K with an IC ₅₀ (the concentration at which 50% of the activity is inhibited) of about 200 nM or less, preferably about 100 nM or less, even more preferably about 60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 μM, 50 μM, 25 μM, 10 μM, 1 μM or less. In one aspect, an mTOR inhibitor for use in the present invention inhibits PI3K and mTORC1 or mTORC2 or both mTORC1 and mTORC2 and PI3K with an IC ₅₀ of from about 2 nM to about 100 nM, more preferably from about 2 nM to about 50 nM, even more preferably from approximately 2 nM to approximately 15 nM.

Additional illustrative examples of mTOR inhibitors suitable for use in the specific embodiments discussed herein include, but are not limited to, AZD8055, INK 128, rapamycin, PF-04691502, and everolimus.

mTOR has been shown to exhibit robust and specific catalytic activity toward physiological substrate proteins, p70 S6 ribosomal protein kinase I (p70S6Kl) and eIP4E-binding protein 1 (4EBP1), as measured by phosphorus-specific antibodies in Western blotting.

In one embodiment, the PI3K/AKT/mTOR pathway inhibitor is a ββ kinase inhibitor selected from the group consisting of BI-D1870, H89, PF-4708671, FMK, and AT7867.

With. PI3K inhibitors

As used herein, the term “ROK inhibitor” refers to a nucleic acid, peptide, compound or small organic molecule that binds to and inhibits at least one PI3K activity. PI3K proteins can be divided into three classes, PI3K class 1, PI3K class 2 and PI3K class 3. Class 1 PI3Ks exist as heterodimers consisting of one of the four p110 catalytic subunits (p110α, p110β, p110δ and p110γ) and one of two families regulatory subunits. The PI3K inhibitor of the present invention preferably targets class 1 PI3K inhibitors. In one embodiment, the PI3K inhibitor will exhibit selectivity for one or more class 1 PI3K inhibitor isoforms (i.e., selectivity for p110α, p110β, p110δ and p110γ or one or more of p110α, p110β, p110δ and p110γ). In another aspect, a PI3K inhibitor will not exhibit isoform selectivity and will be considered a “general PI3K inhibitor.” In one embodiment, a PI3K inhibitor will compete for ATP binding with the PI3K catalytic domain.

In certain embodiments, a PI3K inhibitor, for example, can target PI3K as well as additional proteins in the PI3K-AKT-mTOR pathway. In specific embodiments, a PI3K inhibitor that targets both mTOR and PI3K can be referred to as either an mTOR inhibitor or a PI3K inhibitor. A PI3K inhibitor that targets only PI3K can be referred to as a selective PI3K inhibitor. In one embodiment, a selective PI3K inhibitor may be meant to refer to an agent that exhibits a 50% inhibitory concentration on PI3K that is at least 10-fold lower than the IC50 of the inhibitor on mTOR and/or other proteins in the pathway. 20 times, at least 30 times, at least 50 times, at least 100 times, at least 1000 times or more.

In a specific embodiment, examples of PI3K inhibitors inhibit PI3K with an IC50 (concentration at which 50% of activity is inhibited) of about 200 nM or less, preferably about 100 nM or less, even more preferably about 60 nM or less, about 25 nM, about 10 nM, approximately 5 nM, approximately 1 nM, 100 μM, 50 μM, 25 μM, 10 μM, 1 μM or less. In one embodiment, the PI3K inhibitor inhibits PI3K with an IC50 of from about 2 nM to about 100 nM, more preferably from about 2 nM to about 50 nM, even more preferably from about 2 nM to about 15 nM.

Illustrative examples of PI3K inhibitors suitable for use in the T cell production methods discussed herein include, but are not limited to, BKM120 (class 1 PI3K inhibitor, Novartis), XL147 (class 1 PI3K inhibitor, Exelixis), (general PI3K inhibitor, GlaxoSmithKline) and PX-866 (class 1 PI3K inhibitor; p110α, p110β and p110γ isoforms, Oncothyreon).

Other illustrative examples of selective PI3K inhibitors include, but are not limited to, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, ZSTK474, and IPI-145.

Additional illustrative examples of general PI3K inhibitors include, but are not limited to, BEZ235, LY294002, GSK1059615, and GDC-0941.

d. ACT inhibitors

As used herein, the term “AKT inhibitor” refers to a nucleic acid, peptide, compound or small organic molecule that inhibits at least one AKT activity. AKT inhibitors can be grouped into several classes, including lipid-based inhibitors (eg, inhibitors that target the pleckstrin homologous domain of AKT, which prevents AKT localization to plasma membranes), ATP-competitive inhibitors, and allosteric inhibitors. In one embodiment, AKT inhibitors act by binding to the AKT catalytic site. In a specific embodiment, Akt inhibitors act by inhibiting phosphorylation of downstream targets of Akt, such as mTOR. In another embodiment, AKT activity is inhibited by inhibiting input signals to Akt by inhibiting, for example, AKT activation by DNA-PK, AKT activation by PDK-1, and/or Akt activation by mTORC2.

AKT inhibitors can target all three AKT isoforms, AKT1, AKT2, AKT3, or can be isoform selective and target only one or two of the AKT isoforms. In one embodiment, an AKT inhibitor may target AKT as well as additional proteins in the PI3K-AKT-mTOR pathway. An AKT inhibitor that targets only AKT can be referred to as a selective AKT inhibitor. In one embodiment, a selective AKT inhibitor may be meant to refer to an agent that exhibits a 50% inhibitory concentration on AKT that is at least 10-fold, at least 20-fold lower than the IC ₅₀ of the inhibitor on other proteins in the pathway, at least 30 times, at least 50 times, at least 100 times, at least 1000 times or more.

In a specific embodiment, examples of AKT inhibitors inhibit AKT with an _IC50 (concentration at which 50% of the activity is inhibited) of about 200 nM or less, preferably about 100 nM or less, even more preferably about 60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 μM, 50 μM, 25 μM, 10 μM, 1 μM or less. In one embodiment, AKT inhibits AKT with an IC ₅₀ of about 2 nM to about 100 nM, more preferably from about 2 nM to about 50 nM, even more preferably from about 2 nM to about 15 nM.

Illustrative examples of AKT inhibitors for use in combination with auristatin-based antibody-drug conjugates include, for example, perifosine (Keryx), MK2206 (Merck), VQD-002 (VioQuest), XL418 (Exelixis), GSK690693, GDC-0068 and PX316 (PROLX Pharmaceuticals).

An illustrative non-limiting example of a selective Aktl inhibitor is A-674563.

An illustrative non-limiting example of a selective Akt2 inhibitor is CCT128930.

In specific embodiments, an Akt inhibitor inhibits Akt activation by DNA-PK, Akt activation by PDK-1, Akt activation by mTORC2, and Akt activation by HSP.

Illustrative examples of DNA-PK inhibitors include, but are not limited to, NU7441, PI-103, NU7026, PIK-75, and PP-121.

D. Engineered T-cell receptors and chimeric antigen receptors

The disclosed T cell production methods are particularly useful for expanding T cells modified to express high affinity T cell receptors (TCR engineered) or chimeric antigen receptors (CARs) without concomitantly increasing the differentiation of these modified T cells. In one embodiment, the T cell is genetically modified to express one or more engineered TCRs or CARs. T cells used herein that are modified to express the engineered TCRs or CARs discussed herein may be referred to as "antigen-specific redirected T cells."

1. Designed by TCR

Naturally occurring T cell receptors contain two subunits, an α subunit and a β subunit, each of which is a unique protein formed through an act of recombination in the genome of each T cell. TCR libraries can be screened for their selectivity for specific target antigens. Thus, natural TCRs that are characterized by high avidity and reactivity towards target antigens can be selected, cloned and then introduced into the T cell population used for adaptive immunotherapy.

In one embodiment, T cells are modified by introducing a polynucleotide encoding a TCR subunit that is capable of forming TCRs that provide the T cells with specificity for tumor cells expressing the target antigen. In specific embodiments, the subunits are characterized by one or more amino acid substitutions, deletions, insertions, or modifications relative to the naturally occurring subunits, provided that the subunits retain the ability to form TCRs conferring on the transfected T cells the ability to target cells and participate in immunogenic cytokine signaling . The engineered TCRs preferably also bind target cells bearing the corresponding tumor-associated peptide with high avidity and optionally mediate efficient cytolysis of target cells bearing the corresponding peptide in vivo.

The nucleic acids encoding the designed TCRs are preferably isolated from their natural context on the (naturally occurring) T cell chromosome and can be inserted into suitable vectors as described elsewhere herein. Both nucleic acids and vectors containing them can be efficiently transferred into a cell, which is preferably a T cell. The modified T cells then become capable of expressing one or more TCR chains (and preferably two chains) encoded by the transduced nucleic acid or nucleic acids. In preferred embodiments, the designed TCR is an exogenous TCR in that it is introduced into T cells that do not normally express the particular TCR. An essential aspect of engineered TCRs is that they are characterized by high avidity for tumor antigen presented by a major histocompatibility complex (MHC) molecule or similar immunological component. Unlike engineered TCRs, CARs are designed to bind target antigens in an MHC-independent manner.

The protein encoded by the nucleic acids of the present invention can be expressed with additional polypeptides attached to the amino-terminal or carboxy-terminal portion of the α chain or β chain of the TCR of the present invention, provided that the attached additional polypeptide does not interfere with the ability of the α chain or β-chains form a functional T-cell receptor and mediate MHC-dependent antigen recognition.

Antigens that are recognized by the engineered TCRs discussed herein include, but are not limited to, cancer antigens, including antigens from both hematopoietic cancers and solid tumors. Exemplary antigens include, but are not limited to, folate receptor alpha _, 5T4, _αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EPCAM, FAP, fetal AChR, FRα, GD2, GD3, glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY -ESO-1, IL-11Rα, IL-13Rα2, lambda chain, Lewis-Y, kappa chain, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM and VEGFR2.

2. Chimeric antigen receptors (CARs)

Methods for producing T cells discussed herein include modifying T cells to express one or more of the CARs discussed herein. In various embodiments, the present invention provides T cells that are genetically engineered using vectors designed to express CARs that redirect cytotoxicity to tumor cells. CARs are molecules that combine the specificity of an antibody for a target antigen (eg, tumor antigen) with the intracellular activating domain of a T cell receptor to produce a chimeric protein that exhibits specific antitumor cellular immune activity. As used herein, the term “chimeric” means composed of parts of different proteins or DNA from different sources.

The CARs discussed herein contain an extracellular domain that binds to a specific target antigen (also called a binding domain or antigen-specific binding domain), a transmembrane domain, and an intracellular signal transduction domain. The main characteristic of CARs is their ability to redirect the specificity of an immune effector cell, thereby triggering proliferation, cytokine production, phagocytosis, or the production of molecules that can mediate cell death of a cell expressing the target antigen, in a major histocompatibility complex (MHC)-independent manner, using specific regarding the cell targeting abilities of monoclonal antibodies, soluble ligands or cell-specific coreceptors.

In specific embodiments, the CAR comprises an extracellular binding domain, including, but not limited to, an antibody or an antigen-binding fragment thereof, a bound ligand, or a coreceptor extracellular domain that specifically binds a target antigen selected from the group consisting of folate receptor alpha, 5T4, α _v β6 -integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR , EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AChR, FRα, GD2, GD3, glypican-3 (GPC3), HLA-A1+MAGE1, HLA- A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL11Rα, IL-13Rα2, lambda chain, Lewis-Y, kappa chain, mesothelin, Muc1, Muc16, NCAM, ligands NKG2D, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM and VEGFR2; one or more hinge domains or spacer domains; a transmembrane domain, including, but not limited to, the transmembrane domains from CD8α, CD4, CD45, PD-1, and CD 152; one or more intracellular costimulatory signal transduction domains, including but not limited to the intracellular costimulatory signal transduction domains of CD28, CD54 (ICAM), CD134 (OX40), CD137 (411BB), CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1) and CD278 (ICOS); and primary signal transduction domains from CD3ξ or FcRγ.

A. Binding domain

In specific embodiments, the CARs discussed herein comprise an extracellular binding domain that specifically binds to a target polypeptide, for example, a target antigen expressed on a tumor cell. As used herein, the terms "binding domain", "extracellular domain", "extracellular binding domain", "antigen-specific binding domain" and "extracellular antigen-specific binding domain" are used interchangeably and refer to a CAR with the ability to specifically bind to a target antigen. of interest. The binding domain may comprise any protein, polypeptide, oligopeptide, or peptide that has the ability to specifically recognize and bind a biological molecule (eg, a cell surface receptor or tumor protein, lipid, polysaccharide, or other target cell surface molecule or component thereof). A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule of interest.

In specific embodiments, the extracellular binding domain of the CAR comprises an antibody or an antigen-binding fragment thereof. The term "antibody" refers to a binding agent that is a polypeptide containing at least an immunoglobulin light chain or heavy chain variable region that specifically recognizes and binds an epitope of a target antigen, such as a peptide, lipid, polysaccharide or nucleic acid containing an antigenic determinant recognized by the immune cell. Antibodies include their antigen-binding fragments. The term also includes genetically engineered forms such as chimeric antibodies (eg, humanized mouse antibodies), heteroconjugate antibodies (such as bispecific antibodies) and antigen-binding fragments thereof. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, ^3rd Ed., W. H. Freeman & Co., New York, 1997.

In specific embodiments, the target antigen is an epitope of a folate receptor polypeptide alpha, 5T4, α _v β ₆ integrin, BCMA, B7-H3, B7-H6, CALX, CD 19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR families, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AChR, FRα, GD2, GD3, glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1 , HLA-A3+NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chain, Lewis-Y, kappa chain, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGFR2.

The light and heavy chain variable regions contain a “framework region” interrupted by hypervariable regions, also called “complementarity determining regions” or “CDRs”. CDRs can be determined or identified using traditional methods, for example, using the sequence according to Kabat et al. (Wu, TT and Kabat, E. A., J Exp Med. 132(2):211-50, (1970); Borden , P. and Kabat E. A, PNAS, 84: 2440-2443 (1987); (See Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is hereby incorporated by references), or using the structure according to Choithia, C. and Lesk, A.M., J Mol. Biol., 196(4): 901-917 (1987), Choithia, C. et al., Nature, 342:877-883 (1989)).

The framework sequences of the various light or heavy chains are relatively conserved within a species, such as humans. The antibody framework, which is the combined framework regions of the antibody's light and heavy chains, serves to regulate the position and alignment of the CDRs in three-dimensional space. First of all, CDRs are responsible for binding to the epitope of the antigen. The CDRs of each chain are typically called CDR1, CDR2, and CDR3, numbered sequentially starting at the N-terminus, and are also typically identified by the strand on which the particular CDR is found. Thus, the CDRs located in the variable domain of the heavy chain of an antibody are called CDRH1, CDRH2 and CDRH3, while the CDRs located in the variable domain of the light chain of an antibody are called CDRL1, CDRL2 and CDRL3. Antibodies with different specificities (i.e. different paratopes for different antigens) have different CDRs. Although CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Such positions within the CDR are called specificity-determining residues (SDRs).

References to “VH” or “VH” refer to the variable region of an immunoglobulin heavy chain, including that of an antibody, Fv, ScFv, dsFv, Fab or other antibody fragment disclosed herein. References to “VL” or “VL” refer to the variable region of an immunoglobulin light chain, including that of an antibody, Fv, ScFv, dsFv, Fab or other antibody fragment disclosed herein.

A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced using methods known to those skilled in the art, for example, by creating antibody-producing hybrid cells by fusing myeloma cells with spleen immune cells. Monoclonal antibodies include humanized monoclonal antibodies.

A "chimeric antibody" contains framework residues from one species, such as a human, and CDRs (which generally mediate antigen binding) from another species, such as a mouse. In specific preferred embodiments, the CAR disclosed herein comprises an antigen-specific binding domain that is a chimeric antibody or an antigen-binding fragment thereof.

In some preferred embodiments, the antibody is a humanized antibody (such as a humanized monoclonal antibody) that specifically binds to a tumor cell surface protein. A "humanized" antibody is an immunoglobulin comprising a human framework region and one or more CDRs from a non-human immunoglobulin (eg, murine, rat, or synthetic). Humanized antibodies can be constructed using genetic engineering techniques (see, for example, US Pat. No. 5,585,089).

In specific embodiments, the extracellular binding domain of the CAR comprises an antibody or an antigen-binding fragment thereof, including, but not limited to, camelid Ig (camelid (VHH) antibody), IgNAR, Fab fragments, Fab' fragments, F(ab)'2- fragments, F(ab)'3-fragments, Fv, single chain Fv antibody (“scFv”), bis-scFv, (scFv) ₂ , minibody, diabody, tribody, tetrabody, disulfide-stabilized Fv protein (“dsFv” ) and single domain antibody (sdAb, nanobody).

"Camel Ig" or "camelid VHH antibody" as used herein refers to the smallest known antigen-binding unit of a heavy chain-only antibody (Koch-Nolte, et al., FASEB J., 21: 3490- 3498 (2007)). The terms “heavy chain-only antibody” or “camelid antibody” refer to an antibody that contains two VH domains and no light chains (Riechmann L. et al., J. Immunol. Methods 231:25-38 (1999 ); WO 94/04678; WO 94/25591; US patent No. 6005079).

"IgNAR" or "immunoglobulin antigen receptor" refers to a class of antibodies from the shark immune spectrum that consist of homodimers of one novel antigen receptor (VNAR) variable domain and five novel antigen receptor (CNAR) constant domains.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with one antigen-binding site, and the remaining "Fc" fragment, the name of which reflects its ability to readily crystallize. The Fab fragment contains the heavy and light chain variable domains, and also contains the light chain constant domain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteine residues from the antibody hinge region. Fab'-SH as used herein refers to Fab' in which the cysteine residue(s) of the constant domains have a free thiol group. F(ab') ₂ antibody fragments were originally produced as pairs of Fab' fragments that share cysteine residues in the hinge region. Other means for chemically coupling antibody fragments are also known.

"Fv" is the minimal antibody fragment that contains the complete antigen-binding site. In a single-chain Fv (scFv) molecule, one heavy chain variable domain and one light chain variable domain can be covalently linked through a flexible peptide linker such that the light and heavy chains can be combined into a “dimeric” structure similar to that of a double-chain Fv molecule.

The term "diabodies" refers to antibody fragments with two antigen-binding sites, the fragments containing a heavy chain variable domain (VH) linked to a light chain variable domain (VL) into a single polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between two domains on one chain, the domains are forced to pair with complementary domains on the other chain and create two antigen-binding sites. Diabodies can be bivalent or bispecific. Diabodies are described more fully, for example, in EP 404097; WO 1993/01161; Hudson et al, Nat. Med. 9:129-134 (2003); and Hollinger et al., PNAS USA 90: 6444-6448 (1993). Tribodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134(2003).

“Single domain antibody” or “sdAb” or “nanobody” refers to an antibody fragment that consists of an antibody heavy chain variable region (VH domain) or an antibody light chain variable region (VL domain) (Holt, L., et al. al., Trends in Biotechnology, 21(11): 484-490). "Single chain Fv" or "scFv" antibody fragments contain the VH and VL domains of the antibody, where these domains are present on the same polypeptide chain and in any orientation (eg, VL-VH or VH-VL). Typically, the scFv polypeptide additionally contains a polypeptide linker between the VH and VL domains, which allows the scFv to form the required structure for antigen binding. For a review of scFv, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp.269-315.

In preferred embodiments, the CAR disclosed herein comprises an antigen-specific binding domain that is a scFv (mouse, human, or humanized scFv) that binds an antigen expressed on a cancer cell. In a certain embodiment, the scFv binds folate receptor alpha, 5T4, avp6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD 19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a , CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AChR, FRα, GD2, GD3, ' glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY -ESO-1, IL11Rα, IL-13Rα2, lambda chain, Lewis-Y, kappa chain, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM and VEGFR2.

b. Linkers

In certain embodiments, the CARs discussed herein may contain linker residues between different domains, for example, between the V _H and V _L domains, added to appropriately separate them in space and provide conformation of the molecule. The CARs discussed herein may contain one, two, three, four or five or more linkers. In specific embodiments, the linker length is from about 1 to about 25 amino acids, from about 5 to about 20 amino acids, or from about 10 to about 20 amino acids, or any other length of amino acids in between. In some embodiments, the linker has a length of 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 or more amino acids.

Illustrative examples of linkers include glycine polymers (G) _n ; glycine-serine polymers (G _1-5 S _1-5 ) _n , where n is an integer of at least one, two, three, four or five; glycine-alanine polymers; alanine-serine polymers and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore can serve as neutral linkers between the domains of fusion proteins such as CARs described herein. Glycine provides significantly more phi-psi space than alanine and is much less restricted than longer chain residues (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). One skilled in the art will appreciate that the CAR structure in particular embodiments may include linkers that are fully or partially flexible, such that the linker may include a flexible linker as well as one or more portions that provide a less flexible structure to provide the desired CAR structure.

Other examples of linkers include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ ID NO: X); TGEKP (SEQ ID NO: X) (see, for example, Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: X) (Pomerantz et al. 1995, supra); (GGGGS _n where = 1, 2, 3, 4 or 5 (SEQ ID NO: X) (Kim et al., PNAS 93, 1156-1160 (1996.); EGKSSGSGSESKVD (SEQ ID NO: X) (Chaudhary et al ., 1990, Proc. Natl. Acad. Sci. USA 87:1066-1070); X); LRQRDGERP (SEQ ID NO:X); LRQKDGGGSERP (SEQ ID NO:X); LRQKd(GGGS) ₂ ERP (SEQ ID NO:X). Alternatively, flexible linkers can be designed by rational design methods using a computer program , capable of modeling both DNA-binding sites and peptides themselves (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91:11099-11103 (1994), or using phage display methods.

In certain embodiments, the CAR comprises a scFV that further comprises a variable region linker sequence. A "variable region linker sequence" is an amino acid sequence that connects a heavy chain variable region to a light chain variable region and provides a spacer function compatible with the interaction of the two binding subdomains such that the resulting polypeptide retains a specific binding affinity for the same target molecule as an antibody that contains the same light and heavy chain variable regions. In one embodiment, the variable region linker sequence has a length of 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 or more amino acids. In a specific embodiment, the variable region linker sequence comprises a glycine-serine polymer (G _1-5 S _1-5 ) _n , where n is an integer of at least 1, 2, 3, 4, or 5. In another embodiment, the linker sequence the variable region sequence contains an amino acid linker (G ₄ S) ₃ .

With. Spacer domain

In specific embodiments, the CAR binding domain is followed by one or more “spacer domains,” which refers to the region that extends the antigen-binding domain away from the surface of the effector cell to ensure proper cell/cell contact, antigen binding, and activation (Patel et al., Gene Therapy , 1999;6:412-419). The spacer domain can be obtained from a natural, synthetic, semi-synthetic or recombinant source. In certain embodiments, a spacer domain is a portion of an immunoglobulin including, without limitation, one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain may include an amino acid sequence from a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region.

In one embodiment, the spacer domain comprises CH2 and CH3 from IgG1.

d. Hinge domain

The CAR binding domain is typically followed by one or more “hinge domains” that play a role in regulating the position of the antigen binding domain, moving it away from the surface of the effector cell to ensure proper cell/cell contact, antigen binding and activation. A CAR typically contains one or more hinge domains between the binding domain and the transmembrane (TM) domain. The hinge domain can be obtained from a natural, synthetic, semi-synthetic or recombinant source. The hinge domain may comprise an amino acid sequence from a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region.

Exemplary hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8α, CD4, CD28, and CD7, which may be wild-type hinge regions from these molecules or may be changed. In another embodiment, the hinge domain comprises a CD8α hinge region.

e. Transmembrane domain (TM)

The "transmembrane domain" is the part of the CAR that combines the extracellular binding portion and the intracellular signal transduction domain, and anchors the CAR to the plasma membrane of the immune effector cell. The TM domain can be obtained from a natural, synthetic, semi-synthetic or recombinant source.

Exemplary TM domains may be derived from (i.e., contain at least transmembrane region(s)) T cell receptor alpha, beta, or zeta chain, epsilon-CO3, zetaCD3, CD4, CD5, CD9, CD16, CD22 , CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152 and CD154.

In one embodiment, the CAR discussed herein contains a TM domain derived from CD8α. In another embodiment, the CAR disclosed herein comprises a CD8α-derived TM domain and a short oligo- or polypeptide linker of preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, which links the TM domain and the intracellular signal transduction domain of CAR. A glycine-serine linker is a particularly suitable linker.

f. Intracellular signal transduction domain

In specific embodiments, the CARs discussed herein comprise an intracellular signal transduction domain. The “intracellular signal transduction domain” refers to the portion of a CAR that is involved in communicating the effective binding of the CAR to a target antigen to the interior of an immune effector cell to trigger the cell's effector function, such as activation, cytokine production, proliferation, and cytotoxic activity, including release cytotoxic factors into the CAR-bound target cell, or other cellular responses caused by the binding of antigen to the extracellular domain of the CAR.

The term "effector function" refers to a specialized function of a cell. The effector function of a T cell, for example, may be a cytolytic activity, or an auxiliary activity, or an activity involving cytokine secretion. Thus, the term "intracellular signal transduction domain" refers to the part of the protein that transmits the signal to initiate effector function and that causes the cell to perform a specialized function. Although it is generally possible to use the entire intracellular signal transduction domain, in many cases it is not necessary to use the entire domain. In cases where a truncated portion of an intracellular signal transduction domain is used, the truncated portion may be used in place of the entire domain as long as it transmits a signal to initiate effector function. The term intracellular signal transduction domain is intended to include any truncated portion of the intracellular signal transduction domain sufficient to transmit a signal to initiate effector function.

It is known that signals generated by the TCR alone are insufficient to fully activate a T cell, and that a repeat or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two different classes of intracellular signaling domains: primary signaling domains, which initiate antigen-dependent primary activation via the TCR (e.g., the TCR/CD3 complex), and co-stimulatory signaling domains, which act in an antigen-independent manner to provide a secondary or co-stimulatory signal. In preferred embodiments, the CAR discussed herein comprises an intracellular signal transduction domain that contains one or more “costimulatory signal transduction domains” and a “primary signal transduction domain.”

Primary signal transduction domains respond to primary activation of the TCR complex by either stimulation or inhibition. Primary signal transduction domains that act in a stimulatory manner may contain signal transduction motifs that are known as immunoreceptor tyrosine activating motifs or IT AMs.

Illustrative examples of ITAM-containing primary signal transduction domains that are particularly useful in the present invention include those derived from TCRξ FcRγ, FcRβ, CD3γ, CD38, CD3δ, CD22, CD79a, CD79b and CD66d. In particular preferred embodiments, the CAR comprises a CD3ξ primary signaling domain and one or more co-stimulatory signaling domains. The intracellular primary signal transduction and co-stimulatory signal transduction domains can be attached in any order sequentially to the carboxyl terminus of the transmembrane domain.

The CARs discussed herein contain one or more co-stimulatory signal transduction domains to enhance the efficacy and expansion of T cells expressing CAR receptors. As used herein, the term “costimulatory signal transduction domain” or “costimulation domain” refers to the intracellular signal transduction domain of a costimulatory molecule.

Illustrative examples of such co-stimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), CTLA4, LFA-1, CD2, CD7, LIGHT, TRIM, LCK3 , SLAM, DAP10, LAG3, HVEM and NKD2C, as well as CD83. In one embodiment, the CAR comprises one or more co-stimulatory signaling domains selected from the group consisting of CD28, CD137 and CD134, as well as a CD3ξ primary signaling domain.

In one embodiment, the CAR contains a scFv that binds folate receptor polypeptide alpha, 5T4, α _v β ₆ integrin, BCMA, B7-H3, B7-H6, CALX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbP32 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AChR, FRα, GD2, GD3, glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1 , HLA-A3+NY-ESO-1, IL-URa, IL-13Rα2, lambda chain, Lewis-Y, kappa chain, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGFR2; a transmembrane domain derived from a polypeptide selected from the group consisting of CD8α; CD4, CD45, PD-1 and CD 152; one or more intracellular costimulatory signal transduction domains selected from the group consisting of CD28, CD54, CD 134, CD137, CD 152, CD273, CD274 and CD278; and the CD3ξ primary signaling domain.

In another embodiment, the CAR contains a scFv that binds folate receptor alpha, 5T4, α _v β6 integrin, BCMA, B7-H3, B7-H6, CALX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/ 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AChR, FRα, GD2, GD3, glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA -A3+NY-ESO-1, IL11Rα, IL-13Rα2, lambda chain, Lewis-Y, kappa chain, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGFR2; a hinge domain selected from the group consisting of IgG1 and CD8α hinge/CH2/CH3, as well as CD8α; a transmembrane domain derived from a polypeptide selected from the group consisting of CD8α; CD4, CD45, PD-1 and CD152; one or more intracellular costimulatory signal transduction domains selected from the group consisting of CD28, CD134 and CD137; as well as the CD3ξ primary signaling domain.

In yet another embodiment, the CAR comprises a scFv further comprising a linker that binds a folate receptor polypeptide alpha, 5T4, α _v β6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR families, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP , fetal AChR, FRα, GD2, GD3, glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-Al+NY-ESO-1, HLA-A2+NY -ESO-1, HLA-A3+NY-ESO-1, IL11Rα, IL-13Rα2, lambda chain, Lewis-Y, kappa chain, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGFR2; a hinge domain selected from the group consisting of IgG1 and CD8α hinge/CH2/CH3, as well as CD8α; a transmembrane domain containing a TM domain derived from a polypeptide selected from the group consisting of CD8α; CD4, CD45, PD-1 and CD152, as well as a short oligo- or polypeptide linker of preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length, which links the TM domain to the intracellular transmission domain CAR signal; and one or more intracellular costimulatory signal transduction domains selected from the group consisting of CD28, CD134 and CD137; as well as the CD3ξ primary signaling domain.

In a specific embodiment, the CAR contains a scFv that binds folate receptor polypeptide alpha, 5T4, α _v β6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD 19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AChR, FRα, GD2, GD3, glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-Al+NY-ESO-1, HLA-A2+NY-ESO-1 , HLA-A3+NY-ESO-1, IL11Rα, IL-13Rα2, lambda chain, Lewis-Y, kappa chain, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM and VEGFR2; a hinge domain containing a CD8α polypeptide; a CD8α transmembrane domain containing a polypeptide linker of approximately 3 amino acids; one or more intracellular costimulatory signal transduction domains selected from the group consisting of CD28, CD134 and CD137; as well as the CD3ξ primary signaling domain.

E. Polypeptides

The present invention contemplates, in particular, engineered TCR and CAR polypeptides or fragments thereof, cells and compositions containing them, as well as vectors that express the polypeptides. "Polypeptide", "polypeptide fragment", "peptide" and "white") are used interchangeably unless otherwise stated and in accordance with their generally accepted meaning, i.e. sequence of amino acids. Polypeptides are not limited to a specific length, for example, they may contain a full-length protein sequence or a full-length protein fragment, and may include post-translational modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, etc., as well as other modifications known in the art , both occurring and not occurring in nature. In various embodiments, the polypeptides discussed herein contain a signal (or leader) sequence at the N-terminal end of the protein that co-translationally or post-translationally directs protein transfer. Illustrative examples of suitable signal sequences useful herein include, but are not limited to, an IgG1 heavy chain signal sequence and a CD8α signal sequence. Polypeptides can be produced using any of a variety of well-known recombination and/or synthesis techniques. The polypeptides contemplated herein particularly include CARs of the present disclosure or sequences that have deletions, additions, and/or substitutions of one or more amino acids relative to the polypeptide contemplated herein.

Polypeptides include "polypeptide variants". Polypeptide variants may differ from the naturally occurring polypeptide by one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be produced synthetically, for example by modifying one or more of the polypeptide sequences mentioned above. For example, in certain embodiments, it may be necessary to improve the binding affinity and/or other biological properties of the designed TCR or CAR by introducing one or more substitutions, deletions, additions and/or insertions. Preferably, the polypeptides of the present invention include polypeptides having at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% amino acid identity therewith.

Polypeptides include "polypeptide fragments". Polypeptide fragments refer to a polypeptide, which may be monomeric or multimeric, characterized by an amino-terminal deletion, a carboxy-terminal deletion, and/or an internal deletion or substitution relative to a naturally occurring or recombinantly produced polypeptide. In certain embodiments, the polypeptide fragment may comprise an amino acid chain having a length of at least 5 to about 500 amino acids. It should be appreciated that in certain embodiments the fragments have a length of at least 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, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400 or 450 amino acids.

The polypeptide may also be fused in frame or conjugated to a linker or other sequence to facilitate synthesis, purification or identification of the polypeptide (eg, poly-His), or to enhance binding of the polypeptide to a solid support.

As noted above, the polypeptides of the present invention can be modified in a variety of ways, including amino acid substitutions, deletions, truncations and insertions. Methods for such manipulations are well known from the prior art. For example, variants of the amino acid sequence of a reference polypeptide can be generated through DNA mutations. Methods for mutagenesis and changes in nucleotide sequence are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad Sci. USA. 82: 488-492), Kunkel et al., (1987, Methods in Enzymol, 154: 367-382), US patent No. 4873192, Watson , J.D. et al., (Molecular Biology of the Gene, Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987) and referenced documents cited therein. Guidance regarding suitable amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.).

In certain embodiments, the variant will contain conservative substitutions. A "conservative substitution" is one in which an amino acid is replaced by another amino acid that has similar properties such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Modifications can be made to the structure of the polynucleotides and polypeptides of the present invention and still provide a functional molecule that encodes a variant or derivative polypeptide with the desired characteristics.

Polypeptide variants further include glycosylated forms, conjugates formed by aggregation with other molecules, and conjugates formed by covalent bonding with unrelated chemical moieties (eg, PEGylated molecules). Covalently linked variants can be prepared by linking functional groups to groups that are found in the amino acid chain or to an N- or C-terminal residue, as is known in the art. Variants also include allelic variants, species variants and muteins. Truncations or deletions of regions that do not affect the functional activity of proteins also result in the formation of variants.

In one embodiment where expression of one or more polypeptides is required, the polynucleotide sequences encoding them can be separated using an IRES sequence, as discussed elsewhere herein. In another embodiment, two or more polypeptides may be expressed as a fusion protein that contains one or more self-cleaving polypeptide sequences.

The polypeptides of the present invention include fusion polypeptides. In preferred embodiments, fusion polypeptides and polynucleotides encoding fusion polypeptides are provided. Fusion polypeptides and fusion proteins refer to a polypeptide that has at least two, three, four, five, six, seven, eight, nine or ten, or more polypeptide segments. Fusion polypeptides are typically C-terminal to N-terminal linked, although they can also be C-terminally to C-terminally linked, N-terminally to N-terminally linked, or N-terminally to C-terminally linked. The polypeptides in a fusion protein can be in any order or in a specific order. Fusion polypeptides or fusion proteins may also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and cross-species homologs, as long as the desired transcriptional activity of the fusion polypeptide is retained. Fusion polypeptides can be produced using chemical synthesis methods or by chemically linking two fragments, or generally can be produced using other standard techniques. The ligated DNA sequences encoding the fusion polypeptide are operably linked to suitable transcriptional or translational control elements, as discussed elsewhere herein.

In one embodiment, the fusion partner contains a sequence that promotes expression of the protein (an expression enhancer), resulting in higher yields compared to the native recombinant protein. Other fusion partners may be selected to increase the solubility of the protein, or to allow targeting of the protein to desired intracellular compartments, or to facilitate transport of the fusion protein across the cell membrane.

Fusion polypeptides may further comprise a polypeptide cleavage signal between each of the polypeptide domains described herein. In addition, the polypeptide cleavage site may be placed within the sequence of any linker peptide. Examples of peptide cleavage signals include polypeptide cleavage recognition sites, such as protease cleavage sites, nuclease cleavage sites (e.g., low-cleaving enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5(8); 616-26).

Suitable protease cleavage sites and self-cleaving peptides are well known to one skilled in the art (see, for example, Ryan et al., 1997. J. Gen. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Examples of protease cleavage sites include, but are not limited to, cleavage sites for potyvirus NIa proteases (e.g., tobacco etch virus proteases), potyvirus HC proteases, potyvirus P1(P35) proteases, bimovirus NIa proteases, RNA-2-encoded bimovirus proteases, Afthovirus L-protease, Enterovirus 2A-protease, Rhinovirus 2A-protease, Picornavirus 3C-protease, Comovirus 24K-protease, Nepovirus 24K-protease, RTSV 3C-like protease (rice tungro spherical virus), PYVF 3C-like protease (yellow tungro virus) parsnip spot), heparin, thrombin, factor Xa and enterokinase. In one embodiment, due to high cleavage fidelity, cleavage sites for TEV (tobacco etch virus) protease, e.g., EXXYXQ(G/S) (SEQ ID NO:), e.g., ENLYFQG (SEQ ID NO:) and ENLYFQS (SEQ ID NO:), where X is any amino acid (cleavage by TEV occurs between Q and G or Q and S).

In a specific embodiment, the self-cleaving peptides include polypeptide sequences derived from the 2A peptides of potyvirus and cardiovirus, FMDV (foot-and-mouth disease virus), equine rhinitis virus type A, Thosea asigna virus, and porcine teschovirus.

In certain embodiments, the self-cleaving polypeptide site includes a 2A site or 2A-like site sequence or domain (Donnelly et al., 2001. J. Gen. Virol. 82:1027-1041).

F. Polynucleotides

In specific embodiments, polynucleotides are provided that encode one or more TCR or CAR designed polypeptides discussed herein. As used herein, the terms “polynucleotide” or “nucleic acid” refer to messenger RNA (mRNA), RNA, genomic RNA (gRNA), plus-strand RNA (RNA(+)), minus-strand RNA (RNA(-)) , genomic DNA (gDNA), complementary DNA (cDNA) or recombinant DNA. Polynucleotides include single- and double-stranded polynucleotides. Preferably, the polynucleotides of the present invention include polynucleotides or variants characterized by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, 99%, or 100% sequence identity to any of the reference sequences described herein (see, e.g., Sequence Listing), and typically the variant retains at least one biological activity of the reference sequence. Various illustrative embodiments of the present invention provide, in particular, polynucleotides containing expression vectors, viral vectors and vector plasmids, as well as compositions and cells containing them.

In particular embodiments of the present invention, polynucleotides are provided that encode at least about 5, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 500, 1000, 1250, 1500, 1750, or 2000 or more contiguous amino acid residues of the polypeptide of the present invention, as well as all polypeptides of intermediate length. It is easy to understand that "intermediate length" in this context means any length between the given values, for example, 6, 7, 8, 9, etc., 101, 102, 103, etc.; 151, 152, 153, etc.; 201,202, 203, etc.

As used herein, the terms "polynucleotide variant" and "variant" and the like. refer to polynucleotides that exhibit substantial sequence identity to a reference polynucleotide sequence, or polynucleotides that hybridize to a reference sequence under stringent conditions as defined below herein. These terms include polynucleotides in which one or more nucleotides have been added or deleted, or replaced by different nucleotides compared to the reference polynucleotide. However, it is well known in the art that certain changes, including mutations, additions, deletions and substitutions, can be made to a reference polynucleotide as long as the modified polynucleotide retains the biological function or activity of the reference polynucleotide.

Listings of “sequence identity” or containing, for example, “sequence 50% identical,” as used herein, refer to the extent to which sequence data is identical in a pairwise nucleotide comparison or pairwise amino acid comparison in a comparison window. Thus, "percentage sequence identity" can be calculated by comparing two sequences subjected to optimal alignment in a comparison window, determining the number of positions at which an identical nucleic acid base (e.g., A, T, C, G, I) or identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occur in both sequences to give number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percent sequence identity. Nucleotides and polynucleotides are provided having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 % or 100% sequence identity to any of the reference sequences described herein, generally provided that the variant polypeptide retains at least one biological activity of the reference polypeptide.

Terms used to describe the sequence relationship of two or more polynucleotides or polypeptides include the following: “reference sequence”, “window of comparison”, “sequence identity”, “percentage sequence identity” and “substantial identity”. The length of the "reference sequence" is at least 12, but more often 15-18 and often at least 25 monomer units, including nucleotides and amino acid residues. Because each of two polynucleotides may contain (1) a sequence (i.e., only part of the complete polynucleotide sequence) that is similar in the two polynucleotides, and (2) a sequence that is different in the two polynucleotides, comparing the sequence of two (or more) polynucleotides, typically performed by comparing the sequences of two polynucleotides in a “window of comparison” to identify and compare local regions of sequence similarity. A “comparison window” refers to a speculative segment of at least 6 contiguous positions, typically from about 50 to about 100, more commonly from about 100 to about 150, in which the sequence is compared to a reference sequence of the same number of contiguous positions after two sequences were subjected to optimal alignment. The comparison window may contain approximately 20% or less additions or deletions (ie, gaps) compared to the reference sequence (which contains no additions or deletions) for optimal alignment of the two sequences. Optimal sequence alignments for comparison window alignments can be performed using computerized implementation of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package version 7.0, Genetics Computer Group, 575 Science Drive, Madison, WI, USA) or by viewing the best alignment (i.e., resulting in the highest percentage homology value in the comparison window) obtained using any of the various selected methods. Mention may also be made of the BLAST family of programs, for example those disclosed in Altschul et al., 1997, Nucl. Acids Res. 25:3389. A detailed discussion of sequence analysis can be found in section 19.3 in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15.

The polynucleotides of the present invention, regardless of the length of the coding sequence per se, can be combined with other DNA sequences, such as promoters and/or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites {e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, as described elsewhere herein or as known from prior art, so that their overall length may vary significantly. It is therefore contemplated that a polynucleotide fragment of virtually any length may be used, with the overall length preferably limited by ease of preparation and use in the intended recombinant DNA protocol.

Polynucleotides can be produced, manipulated and/or expressed using any of a variety of well established techniques known and available in the art. To express the desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector. Examples of vectors are plasmids, autonomously replicating sequences, and mobile genetic elements. Additional examples of vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC), bacteriophages such as phage lambda or phage M13 , as well as animal viruses. Examples of categories of animal viruses useful as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (eg, herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (eg, SV40). Examples of expression vectors are pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DEST™, pLenti6/V5-DESTTM, and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In specific embodiments, the coding sequences of the chimeric proteins disclosed herein can be introduced into such expression vectors to express the chimeric protein in mammalian cells.

The "control elements" or "regulatory sequences" present in an expression vector are the untranslated regions of the vector - origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence), introns, polyadenylation sequence , 5' and 3' untranslated regions - which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in strength and specificity. Depending on the vector system and host used, any number of suitable transcription and translation elements can be used, including universal promoters and inducible promoters.

In specific embodiments, a vector for use in the practice of the present invention, including, without limitation, expression vectors and viral vectors, will include exogenous, endogenous, or heterologous control sequences, such as promoters and/or enhancers. An "endogenous" control sequence is a sequence that is naturally associated with a given gene in the genome. An "exogenous" control sequence is a sequence that is placed in close proximity to a gene by gene manipulation (ie, molecular biology techniques) such that transcription of the gene is driven by the associated enhancer/promoter. A “heterologous” control sequence is an exogenous sequence that originates from a species other than the cell being genetically manipulated.

The term "promoter" as used herein refers to the recognition site of a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes polynucleotides operably linked to a promoter. In specific embodiments, promoters active in mammalian cells comprise an AT-rich region located approximately 25-30 bases upstream of the transcription start site, and/or another sequence located 70-80 bases upstream of the transcription start site, the CNCAAT region, where N may be any nucleotide.

The term "enhancer" refers to a segment of DNA that contains sequences capable of promoting enhanced transcription and, in some cases, capable of functioning independently of their orientation relative to another control sequence. An enhancer may function cooperatively or additively with promoters and/or other enhancer elements. The term "promoter/enhancer" refers to a segment of DNA that contains sequences capable of providing both promoter and enhancer functions.

The term "operably coupled" refers to the immediate proximity in which the described components are in a relationship that allows them to function in their intended manner. In one embodiment, the term refers to a functional relationship between a nucleic acid expression control sequence (such as a promoter and/or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide of interest, wherein the expression control sequence enables transcription of a nucleic acid corresponding to the second sequence.

As used herein, the term “constitutive expression control sequence” refers to a promoter, enhancer, or promoter/enhancer that continuously or permanently enables the transcription of an operably linked sequence. The constitutive expression control sequence may be a “universal” promoter, enhancer, or promoter/enhancer that allows expression in a wide variety of cell types and tissues, or “cell specific,” “cell type specific,” “relation specific.” cell line" or "tissue specific" promoter, enhancer, or promoter/enhancer that allows expression in a limited number of cell types and tissues, respectively.

Exemplary generic expression control sequences suitable for use in particular embodiments of the present invention include, but are not limited to, the cytomegalovirus (CMV) immediate-early promoter, the simian virus 40 (SV40) viral promoter (e.g., early or late), the murine leukemia virus LTR promoter Moloney (MoMLV), Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) promoter (thymidine kinase), H5, P7.5, and vaccinia virus P11 promoters, elongation factor 1-alpha (EF1a) promoter, factor early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), 70 kDa heat shock protein 5 (HSPA5), 90 kDa heat shock protein beta representative 1 (HSP90B1), 70 kDa heat shock protein (HSP70), β-kinesin (β-HRP), human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)) , ubiquitin C (UBC) promoter, phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer/chicken β-actin promoter (CAG) promoter, β-actin promoter and myeloproliferative sarcoma virus enhancer (MND) promoter, with negative control region removed , with the primer binding site replaced by the sequence from d1587rev (Challita et al., J Virol. 69(2):748-55 (1995)).

In a particular embodiment, it may be necessary to express the polynucleotide containing the designed TCR or CAR from a promoter that allows stable and long-term expression in T cells and at a level sufficient to redirect the T cells to cells expressing the target antigen. In a preferred embodiment, the promoter is an EF1α promoter or an MND promoter.

As used herein, “conditional expression” can refer to any type of conditional expression, including but not limited to inducible expression; repressed expression; expression in cells or tissues characterized by a specific physiological, biological or disease state, etc. This definition is not intended to exclude cell type or tissue specific expression. In certain embodiments of the present invention, conditional expression of a polynucleotide of interest is provided, e.g., expression is controlled by exposing a cell, tissue, organism, etc., to a treatment or condition that causes expression of the polynucleotide or that causes an increase or decrease in the level of expression of the polynucleotide encoded by the polynucleotide of interest.

Illustrative examples of inducible promoters/systems include, but are not limited to, steroid-inducible promoters such as promoters of genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the appropriate hormone), metallothionein promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible interferon), mifepristone-regulated "GeneSwitch" system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switch (WO 2002/088346), tetracycline-dependent regulatory systems, etc.

Conditional expression can also be achieved using site-specific DNA recombinase. In accordance with certain embodiments of the present invention, the vector contains at least one (usually two) site(s) for recombination mediated by a site-specific recombinase. As used herein, the terms “recombinase” or “site-specific recombinase” include excision or integration proteins, enzymes, cofactors, or associated proteins that are involved in recombination reactions involving one or more recombination sites [e.g., two, three, four, five , seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)) or their mutant derivatives (eg, fusion proteins containing recombination protein sequences or fragments thereof), fragments and variants. Illustrative examples of recombinases suitable for use in particular embodiments of the present invention include, but are not limited to, Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, PS1, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc , Gin, SpCCEl and ParA.

G. Viral vectors

In specific embodiments, a cell (eg, a T cell) is transduced with a retroviral vector, such as a lentiviral vector encoding a designed TCR or CAR discussed herein. Transduced T cells induce stable, long-term, and durable T cell responses.

As used herein, the term “retrovirus” refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates its genomic DNA into the host genome. Exemplary retroviruses suitable for use in particular embodiments include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney mouse sarcoma virus (MoMSV), Harvey mouse sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Freud's murine leukemia virus, murine stem cell virus (MSCV), Rous sarcoma virus (RSV) and lentivirus.

As used herein, the term “lentivirus” refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visnα-maedi virus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FrV); bovine immunodeficiency virus (BIV) and simian immunodeficiency virus (SIV). In one embodiment, HIV-based vector backbones (ie, cis-acting HIV sequence elements) are preferred.

As used herein, the term “vector” refers to a nucleic acid molecule capable of carrying or transporting another nucleic acid molecule. The transferred nucleic acid is typically associated with, for example inserted into, a vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication in the cell, or may contain sequences sufficient to ensure integration into the DNA of the host cell. Useful vectors include, for example, plasmids (eg, DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-defective retroviruses and lentiviruses.

As will be apparent to one skilled in the art, the term “viral vector” is widely used to refer to either a nucleic acid molecule (eg, a transfer plasmid) that includes elements derived from the viral nucleic acid that typically facilitate the transfer of the nucleic acid molecule or its integration into the genome of a cell or viral particle that mediates the transfer of nucleic acid. Viral particles will typically include various viral components and sometimes also host cell components in addition to nucleic acid(s).

The term "viral vector" can refer either to a virus or viral particle capable of carrying a nucleic acid into a cell, or to the transferred nucleic acid itself. Viral vectors and vector plasmids contain structural and/or functional genetic elements that are predominantly derived from the virus. The term "retroviral vector" refers to a viral vector or plasmid containing structural or functional genetic elements or parts thereof that are derived primarily from a retrovirus.

The term "lentiviral vector" refers to a viral vector or plasmid containing structural or functional genetic elements or parts thereof, including LTRs, that are derived primarily from a lentivirus. The term “hybrid vector” refers to a vector, LTR, or other nucleic acid containing both retroviral, eg, lentiviral, and non-lentiviral viral sequences. In one embodiment, a hybrid vector refers to a vector or transfer plasmid containing retroviral, eg, lentiviral, sequences for reverse transcription, replication, integration and/or packaging.

In specific embodiments, the terms “lentiviral vector”, “lentiviral expression vector” may be used to refer to lentiviral vector plasmids and/or infectious lentiviral particles. When reference is made herein to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it is understood that the sequences of such elements are in RNA form in the lentiviral particles of the present invention and are in DNA form in DNA plasmids according to the present invention.

At each end of the provirus are structures called "long terminal repeats" or "LTRs". The term “long terminal repeat (LTR)” refers to base-pair domains located at the ends of retroviral DNAs that, in the context of their natural sequences, are direct repeats and contain the U3, R, and U5 regions. LTRs typically provide functions essential for retroviral gene expression (eg, induction, initiation, and polyadenylation of gene transcripts) and for viral replication. The LTR contains many regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences required for viral genome replication and integration. The viral LTR is divided into three regions called U3, R and U5. The U3 region contains enhancer and promoter elements. The U5 region is the sequence between the primer binding site and the R region and contains the polyadenylation sequence. The R (repeat) region is flanked by the U3 and U5 regions. The LTR consists of the U3, R, and U5 regions and occurs at both the 5' and 3 ends of the viral genome. Near the 5' LTR there are sequences necessary for reverse transcription of the genome (tRNA primer binding site) and for efficient packaging of viral RNA into particles (Psi site).

As used herein, the term “packaging signal” or “packaging sequence” refers to sequences located within the retroviral genome that are required for insertion of viral RNA into the viral capsid or particle, see, for example, Clever et al., 1995. J. of Virology, Vol.69, No. 4; pp. 2101-2109. Some retroviral vectors use a minimal packaging signal (also called the psi ψ sequence) required to enclose the viral genome within the capsid. Thus, as used herein, the terms “packaging sequence,” “packaging signal,” “psi,” and the symbol “ψ” are used to refer to the non-coding sequence required to enclose the retroviral RNA strands within the capsid during viral particle assembly.

In various embodiments, the vectors contain modified 5' LTRs and/or 3' LTRs. Each or both LTRs may contain one or more modifications, including, without limitation, one or more deletions, insertions or substitutions. Often, modifications to the 3' LTR are made to improve the safety of lentiviral or retroviral systems by making the viruses replication defective. As used herein, the term “replication-defective” refers to a virus that is incapable of full, efficient replication such that infectious virions are not produced (eg, replication-defective lentiviral progeny). The term “replication competent” refers to a wild-type or mutant virus that is capable of replication such that viral replication of the virus results in the formation of infectious virions (eg, replication competent lentiviral progeny).

The term “self-inactivating” (SIN) vectors refers to replication-defective vectors, such as retroviral or lentiviral vectors, in which the enhancer-promoter region of the right (3') LTR, known as the III region, has been modified (eg, by deletion or substitution ) to prevent viral transcription after the first round of viral replication. This occurs because during viral replication, the U3 region of the right (3') LTR is used as a template for the U3 region of the left (5') LTR, and thus the viral transcript cannot be produced without the promoter enhancer in U3. In a further embodiment of the present invention, the 3' LTR is modified such that the U5 region is replaced, for example, by an ideal poly(A) sequence. It should be noted that modifications of the LTR, such as modifications of the 3' LTR, 5' LTR, or both the 3' and 5' LTR, are also included within the scope of the present invention.

An additional increase in safety is provided by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during the formation of viral particles. Examples of heterologous promoters that may be used include, for example, simian virus 40 (SV40) viral promoter (e.g., early or late), cytomegalovirus (CMV) promoter (e.g., immediate-early), Moloney murine leukemia virus (MoMLV) promoter , Rous sarcoma virus (RSV) promoter and herpes simplex virus (HSV) promoter (thymidine kinase). Typical promoters are capable of driving high levels of transcription in a Tat-independent manner. This substitution reduces the possibility of recombination to produce replication-competent viruses, since the virus production system lacks the complete U3 sequence. In certain embodiments, a heterologous promoter has the additional advantage of controlling the manner in which the viral genome is transcribed. For example, a heterologous promoter may be inducible such that transcription of all or part of the viral genome will occur only in the presence of inducing factors. Inducing factors include, but are not limited to, one or more chemical compounds or physiological conditions, such as temperature or pH, under which the host cells are cultured.

In some embodiments, viral vectors contain a TAR element. The term "TAR" refers to a genetic "transactivation response" element located in the R region of lentiviral (eg, HIV) LTRs. This element interacts with the lentiviral transactivator (tat) genetic element to enhance viral replication. However, this element is not necessary in embodiments where the U3 region of the 5' LTR is replaced by a heterologous promoter.

"R region" refers to the region within the retroviral LTRs starting from the beginning of the capping group (ie, the transcription start point) and ending just before the start of the poly-A tract. The R region is also defined as flanked by the U3 and U5 regions. The R region plays an important role during reverse transcription, allowing the growing strand of DNA to move from one end of the genome to the other.

As used herein, the term “FLAP element” refers to a nucleic acid whose sequence contains the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, for example, HIV-1 or HIV-2. Suitable FLAP elements are described in US Pat. No. 6,682,907 and Zennou, et al., 2000, Cell, 101:173. During HIV-1 reverse transcription, central initiation of plus-strand DNA in the central polypurine tract (cPPT) and central termination in the central termination sequence (CTS) results in the formation of a three-stranded DNA structure: the HIV-1 central DNA flap. Without wishing to be bound by theory, DNA flap may act as a cis-active determinant of nuclear import of the lentiviral genome and/or may increase virus titer. In specific embodiments, the retroviral or lentiviral vector backbone comprises one or more FLAP elements located in the vectors upstream or downstream of heterologous genes of interest. For example, in certain embodiments, the transfer plasmid contains a FLAP element. In one embodiment, the vector of the present invention contains a FLAP element isolated from HIV-1.

In one embodiment, retroviral or lentiviral vectors contain one or more export elements. The term "export element" refers to a cis-acting post-transcriptional regulatory element that regulates the transport of RNA transcript from the nucleus to the cytoplasm of the cell. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see, e.g., Cullen et al., 1991. J. Virol 65: 1053; and Cullen et al., 1991. Cell 58: 423) and hepatitis B virus posttranscriptional regulatory element (HPRE). Typically, the RNA export element is located within the 3' UTR of the gene and can be inserted in single or multiple copies.

In specific embodiments, the expression of heterologous sequences in viral vectors is increased by introducing post-transcriptional regulatory elements, effective polyadenylation sites, and, optionally, transcription termination signals into the vectors. A variety of posttranscriptional regulatory elements can enhance the expression of heterologous nucleic acid into protein, for example, woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol, 73:2886); hepatitis B virus post-transcriptional regulatory element (HPRE) (Huang et al., Mol Cell Biol, 5:3864); and the like (Liu et al, 1995, Genes Dev., 9:1766). In specific embodiments, the vectors of the present invention contain a post-transcriptional regulatory element, such as WPRE or HPRE.

In specific embodiments, the vectors of the present invention are devoid of or do not contain a post-transcriptional regulatory element, such as WPRE or HPRE, because in some cases such elements increase the risk of cellular transformation and/or do not substantially or significantly increase the amount of mRNA transcript or increase stability mRNA. Therefore, in some embodiments, the vectors of the present invention are devoid of or do not contain WPRE or HPRE as an additional security measure.

Elements that control efficient termination or polyadenylation of heterologous nucleic acid transcripts increase the expression of heterologous genes. Transcription termination signals are usually located downstream of the polyadenylation signal. In specific embodiments, the vectors contain a polyadenylation sequence beyond the 3' end of the polynucleotide encoding the polypeptide to be expressed. As used herein, the term “poly(A) site” or “poly(A) sequence” describes a DNA sequence that directs both termination and polyadenylation of the growing strand of an RNA transcript by RNA polymerase P. Polyadenylation sequences can enhance stability mRNA by adding a poly(A) tail to the 3' end of the coding sequence, thereby helping to increase translation efficiency. Efficient polyadenylation of a recombinant transcript is necessary because transcripts lacking a poly(A) tail are unstable and easily degraded. Illustrative examples of poly(A) signals that can be used in a vector of the present invention include an ideal poly(A) sequence (e.g., AATAAAA, ATTAAA, AGTAAA), a bovine growth hormone poly(A) sequence (BGHpA), rabbit β-globin poly(A) sequence (rβgpA) or other suitable heterologous or endogenous poly(A) sequence known in the art.

In various embodiments, the vectors of the present invention contain a promoter operably linked to a polynucleotide encoding a polypeptide of the designed TCR or CAR. Vectors may contain one or more LTRs, with each LTR containing one or more modifications, such as one or more nucleotide substitutions, additions or deletions. Vectors may further contain one or more auxiliary elements to increase transduction efficiency (e.g., cPPT/FLAP), viral packaging (e.g., Psi(ψ) packaging signal, RRE), and/or other elements that enhance therapeutic gene expression (e.g., poly (A)-sequences), and may optionally contain WPRE or HPRE. One skilled in the art will appreciate that many other different embodiments can be modeled on existing embodiments of the present invention.

“Host cell” includes cells that have been transfected, infected, or transduced in vivo, ex vivo, or in vitro with a recombinant vector or polynucleotide of the present invention. Host cells may include packaging cells, production cells, and cells infected with viral vectors. In specific embodiments, host cells infected with a viral vector of the present invention are administered to a subject in need of therapy. In certain embodiments, the term “target cell” is used interchangeably with the term “host cell α” and refers to the desired cell type that has been transfected, infected, or transduced. In preferred embodiments, the target cell is a T cell.

To obtain an acceptable virus titer, large-scale production of virus particles is often necessary. Viral particles are produced by transfecting a vector vector into a packaging cell line that contains viral structural and/or accessory genes, for example gag, pol, env, tat, rev, vif, vpr, vpu, vpx or nef genes or other retroviral genes.

As used herein, the term “packaging vector” refers to an expression vector or viral vector that lacks a packaging signal and contains a polynucleotide encoding one, two, three, four or more viral structural and/or accessory genes. Typically, packaging vectors are included in a packaging cell and are introduced into the cell through transfection, transduction, and infection. Methods of transfection, transduction and infection are well known to those skilled in the art. The retroviral/lentiviral vectoβ transporter of the present invention can be introduced into a packaging cell line through transfection, transduction and infection to obtain a production cell or cell line. The packaging vectors of the present invention can be introduced into human cells or cell lines using standard methods, including, for example, calcium phosphate transfection, lipofection, or electroporation. In some embodiments, packaging vectors are introduced into cells together with a dominant selectable marker, such as neomycin, hygromycin, puromycin, blastocidin, zeocin, thymidine kinase, DHFR, glutamine synthetase, or ADA, followed by selection in the presence of the appropriate drug and isolation of clones. The selectable marker gene may be physically linked to genes encoded in the packaging vector, for example, through IRES or self-cleaving viral peptides.

Viral envelope (env) proteins define the range of host cells that can ultimately be infected and transformed by recombinant retroviruses derived from cell lines. In the case of lentiviruses such as HIV-1, HIV-2, SIV, FIV and EIV, the env proteins include gp41 and gp120. Preferably, the viral env proteins expressed by the packaging cells of the present invention are encoded in a separate vector from the viral gag and pol genes, as previously described.

Illustrative examples of retrovirus-derived env genes that can be used in the present invention include, but are not limited to, the MLV envelope, 10A1 envelope, BAEV envelope, FeLV-B, RD114, SSAV, Ebola virus, Sendai virus, FPV (fowl distemper virus) and influenza virus. Similarly, genes encoding the envelopes of RNA viruses (for example, RNA viruses from the families Picornaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Birnaviridae, Retroviridae) can be used. as well as DNA viruses (families Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxviridae and Iridoviridae). Common examples include FeLV, VEE, HFVW, WDSV, SFV, rabies virus, ALV, BIV, BLV, EBV, CAEV, SNV, ChTLV, STLV, MPMV, SMRV, RAV, FuSV, MH2, AEV, AMV, CT10, and EIAV.

In other embodiments, the envelope proteins for pseudotyping the virus of the present invention include, but are not limited to, proteins from any of the following viruses: influenza A virus such as H1N1, H1N2, H3N2 and H5N1 (avian influenza), influenza B virus, influenza C virus, hepatitis virus A, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rotavirus, any virus from the group of noroviruses, enteric adenoviruses, parvovirus, dengue fever virus, monkeypox virus, viruses of the order Mononegavirales, lyssavirus such as rabies virus, Lagos Island bat virus, Mokola virus, Duvenhag virus, European bat virus types 1 and 2 and Australian bat virus, ephemeral virus, vesiculovirus, vesicular stomatitis virus (VSV), herpesviruses such as herpes simplex virus types 1 and 2, virus chickenpox, cytomegalovirus, Epstein α-Barr virus (EBV), human herpes virus (HHV), human herpes virus types 6 and 8, human immunodeficiency virus (HIV), papilloma virus, murine gammaherpesvirus, arenaviruses such as Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia virus associated with hemorrhagic fever, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, lymphocytic choriomeningitis virus (LCMV), Bunyaviridiae such as Crimean-Congo hemorrhagic fever virus, hantivirus, virus causing hemorrhagic fever with renal syndrome, Rift Valley fever virus, Filoviridae (filovirus), including Ebola hemorrhagic fever virus and Marburg hemorrhagic fever, Flaviviridae, including Kiasanur forest disease virus, Omsk hemorrhagic fever virus, tick-borne encephalitis virus, and Paramyxoviridae , such as Hendra virus and Nipah virus, Variola major and Variola minor viruses (variola viruses), alphaviruses such as Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, SARS-associated coronavirus (SARS-CoV), West Nile virus and any virus that causes encephalitis.

In one embodiment, the present invention provides packaging cells that produce a recombinant retrovirus, for example, a lentivirus pseudotyped with the VSV-G glycoprotein.

As used herein, the terms “pseudotyping” or “pseudotyping” refer to a virus whose envelope proteins have been replaced by those of another virus having preferred characteristics. For example, HIV can be pseudotyped using the envelope proteins vesicular stomatitis virus G protein (VSV-G), which allows HIV to infect a wider range of cells because HIV envelope proteins (encoded by the env gene) normally target the virus to CD4+ cells. In a preferred embodiment of the present invention, the lentivirus envelope proteins are pseudopted with VSV-G. In one embodiment, the present invention provides packaging cells that produce a recombinant retrovirus, for example, a lentivirus pseudotyped with the VSV-G envelope glycoprotein.

As used herein, the term “packaging cell lines” is used to refer to cell lines that do not contain a packaging signal but stably or transiently express viral structural proteins and replication enzymes (eg, gag, pol, and env) that are required for proper packaging of viral particles. Any suitable cell line can be used to produce the packaging cells of the present invention. Typically, said cells are mammalian cells. In a specific embodiment, the cells used to obtain the packaging cell line are human cells. Suitable cell lines that can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PAZ17 cells, WEHI cells, COS cells, BSC cells 1, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells and 211A cells. In preferred embodiments, the packaging cells are 293 cells, 293T cells, or A549 cells.

As used herein, the term “producing cell line” refers to a cell line that is capable of producing recombinant retroviral particles containing a packaging cell line and a vector vector construct containing a packaging signal. The production of infectious virus particles and virus stock solutions can be accomplished using traditional techniques. Methods for preparing virus stock solutions are known in the art and are illustrated, for example, in Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633, and N. R. Landau et al. (1992) J. Virol. 66:5110–5113. Infectious viral particles can be collected from packaging cells using traditional techniques. For example, infectious particles can be collected by cell lysis and collection of cell culture supernatant, as is known in the art. If necessary, the collected virus particles may not necessarily be purified. Suitable cleaning techniques are well known to those skilled in the art.

Delivery of gene(s) or other polynucleotide sequence using a retroviral or lentiviral vector through viral infection rather than transfection is called “transduction.” In one embodiment, retroviral vectors are transduced into a cell through infection and integration of a provirus. In certain embodiments, a target cell, such as a T cell, is “transduced” if it contains a gene or other polynucleotide sequence delivered into the cell through infection with a viral or retroviral vector. In specific embodiments, the transduced cell contains one or more genes or other polynucleotide sequences delivered into the cell genome using a retroviral or lentiviral vector.

In specific embodiments, host cells transduced with a viral vector of the present invention that expresses one or more polypeptides are administered to a subject for the treatment and/or prevention of a B cell neoplasm. Other methods associated with the use of viral vectors in gene therapy that can be used in accordance with some embodiments of the present invention are described, for example, in Kay, M. A. (1997) Chest 111(6 Supp.): 138S-142S ; Ferry, N. and Heard, J. M. (1998) Hum. Gene Ther. 9:1975-81; Shiratory, Y. et al. (1999) Liver 19:265-74; Oka, K. et al. (2000) Cum Opin. Lipidol. 11:179-86; Thule, P. M. and Liu, J. M. (2000) Gene Ther. 7:1744-52; Yang, N. S. (1992) Crit. Rev. Biotechnol. 12:335-56; Alt, M. (1995) J. Hepatol. 23:746-58; Brody, S. L. and Crystal, R. G. (1994) Ann. N.Y. Acad Sci. 716:90-101; Strayer, D. S. (1999) Expert Opinion. Investig. Drugs 8:2159–2172; Smith-Arica, J. R. and Bartlett, J. S. (2001) Curr. Cardiol Rep. 3:43-49; and Lee, H. C. et al. (2H) Nature 408:483-8.

H. Compositions and formulations

The compositions discussed herein may contain one or more of the polypeptides, polynucleotides, vectors containing them, and T cell-based compositions discussed herein. The compositions include, without limitation, pharmaceutical compositions. "Pharmaceutical composition" means a composition formulated in pharmaceutically acceptable or physiologically acceptable solutions for administration to a cell or animal, either alone or in combination with one or more other therapies. It should also be understood that, if desired, the compositions of the present invention can also be administered in combination with other agents, such as, for example, cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or others. various pharmaceutically active agents. There are virtually no restrictions on other components that may also be included in the compositions, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapeutic agent.

The phrase “pharmaceutically acceptable” is used herein to denote those compounds, materials, compositions and/or finished dosage forms that, within medical indications, are suitable for use in contact with human and animal tissues without causing excessive toxicity or irritation. , allergic reaction or other problem or complication commensurate with an acceptable benefit/risk ratio.

As used herein, "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, adjuvant, carrier, excipient, lubricant, sweetener, diluent, preservative, coloring agent, flavor enhancer, surfactant, wetting agent, dispersant an agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier that has been approved by the U.S. Food and Drug Administration for use in humans or pets. Examples of pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyhydric alcohols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline solution; Ringer's solution; ethanol; phosphate buffer solutions and any other compatible substances used in pharmaceutical formulations.

In specific embodiments, the compositions of the present invention contain a number of modified T cells obtained using the methods discussed herein. In preferred embodiments, T cell-based pharmaceutical compositions contain highly active T cells with one or more or all of the following markers: CD62L, CCR7, CD28, CD27, CD122 and CD127.

In general, it can be stated that a pharmaceutical composition containing T cells produced using the methods discussed herein can be administered at dosages of 10 ² -10 ¹⁰ cells/kg body weight, 10 ⁵ -10 ⁹ cells/kg body weight, 10 ⁵ -10 ⁸ cells/kg body weight, 10 ⁵ -10 ⁷ cells/kg body weight, 10 ⁷ -10 ⁹ cells/kg body weight or 10 ⁷ -10 ⁸ cells/kg body weight, including all integer values within these ranges. The number of cells will depend on the end use for which the composition is intended, as well as the type of cells included therein. For the uses provided herein, the number of cells per liter volume or less may be 500 ml or less, even 250 ml or 100 ml or less. Therefore, the cell density required is typically greater than 10 ⁶ cells/ml and typically greater than 10 ⁷ cells/ml, typically 10 ⁸ cells/ml or greater. A clinically significant number of immune cells can be distributed over multiple infusions that collectively equal or exceed 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ or 10 ¹² cells. In some aspects of the present invention, particularly if all cells administered will be redirected to a specific target antigen {eg, BCMA), reduced numbers of cells in the range of 10 ⁶ /kg (10 ⁶ -10 ¹¹ per patient) can be administered. T cells modified to express a designed TCR or CAR can be administered multiple times at dosages within these ranges. The cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient being treated. If necessary, to enhance engraftment and function of infused T cells, treatment may also include administration of mitogens {eg, RIA) or lymphokines, cytokines and/or chemokines {eg, IFN-γ, IL-2, IL-7, IL-15, IL -12, TNF-alpha, IL-18 and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MlP1α, etc.) described herein.

In general, compositions containing cells activated and expanded as described herein can be used in the treatment and prevention of diseases that occur in immunocompromised individuals. In particular, compositions containing modified T cells obtained using the methods discussed herein are used in the treatment of cancer.

The modified T cells of the present invention can be administered either alone or as a pharmaceutical composition in combination with carriers, diluents, excipients and/or other components such as IL-2, IL-7 and/or IL-15, or others cytokines or cell populations. In specific embodiments, the pharmaceutical compositions discussed herein contain a number of genetically modified T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

Pharmaceutical compositions containing modified T cells discussed herein may further contain buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg aluminum hydroxide) and preservatives. The compositions of the present invention are preferably formulated for parenteral administration, for example, intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.

Liquid pharmaceutical compositions, whether in solutions, suspensions or other similar forms, may include one or more of the following: sterile diluents such as water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride solution, fixed oils, such as synthetic mono- or diglycerides, which can serve as a solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and tonicity agents such as sodium chloride or dextrose. The drug for parenteral administration may be contained in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic. The injectable pharmaceutical composition is preferably sterile.

In a specific embodiment, the compositions disclosed herein comprise an effective amount of an expanded modified T cell composition alone or in combination with one or more therapeutic agents. Thus, T cell-based compositions can be administered alone or in combination with other known cancer treatments such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormonal therapy, photodynamic therapy, etc. The compositions can also be administered in combination with antibiotics. Such therapeutic agents may be accepted in the art as standard treatment for a particular disease state described herein, such as a particular form of cancer. Examples of contemplated therapeutic agents include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiotherapy agents, therapeutic antibodies, or other active and adjuvant agents.

In certain embodiments, compositions containing T cells discussed herein can be administered in combination with any number of chemotherapeutic agents. Illustrative examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylene imines and methyl melamines, including altretamine, triethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide and trimethylol melamine resin; nitrogen mustards such as chlorambucil, chlornaphasine, chlorphosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorosotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycin, actinomycin, anthramycin, azaserin, bleomycin, cactinomycin, calichemycin, carabicin, carminomycin, carcinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, ezorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogs such as ancesitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepithiostane, testolactone; drugs that inhibit the functions of the adrenal cortex, such as aminoglutethimide, mitotane, trilostane; a folate supplement such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; demecolcine; diaziquon; elformitine; elliptinium acetate; ethoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamole; nitracrine; pentostatin; phenomet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sisofiran; spirogermanium; tenuazonic acid; triaziquon; 2, 2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustin; mitobronitol; mitolactol; pipobromance; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepu; taxoids, for example, paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (TAXOTERE®., Rhne-Poulenc Rorer, Anthony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; Novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; SRT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™ (aliretinoin); ONTAK™ (denileukin diftitox); esperamycins; capecitabine; as well as pharmaceutically acceptable salts, acids and derivatives of any of the foregoing. Also included in this definition are antihormonal agents that regulate or inhibit the action of hormones on tumors, such as antiestrogens including, for example, tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; as well as pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

A variety of other therapeutic agents can be used in combination with the compositions described herein. In one embodiment, the composition containing T cells is administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), non-steroidal anti-inflammatory drugs (NSAIDS), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide and mycophenolate.

Other examples of NSAIDs are selected from the group including ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as VIOXX® (rofecoxib) and CELEBREX® (celecoxib) and sialylates. Examples of pain relievers are selected from the group consisting of acetaminophen, oxycodone, tramadol, or propoxyphene hydrochloride. Examples of glucocorticoids are selected from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone or prednisone. Examples of biological response modifiers include molecules directed against cell surface markers (e.g. CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g. etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE ®)), chemokine inhibitors and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies as well as recombinant forms of the molecules. Examples of DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofine) and intramuscular), and minocycline.

Illustrative examples of therapeutic antibodies suitable for combination with CAR-modified T cells discussed herein include, but are not limited to, abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, citutuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drositumab, duligotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, ercomeximab, elotuzumab, ensituximab, ertumaxomab , etaracizumab, farietuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzuma b, minretumomab, mitumomab, moxetumomab, narnatumab, naptumomab, necitumumab, nimotuzumab, nofetumomab, ocaratuzumab, ofatumumab, olaratumab, onartuzumab, oportuzumab, oregovomab, panitumumab, parsatuzumab, partitumab, pemtumomab, pertuzumab, pintumomab, pritumumab, racotumomab, radretumab, rilotumumab, rituximab , robatumumab, satumomab, sibrotuzumab, siltuximab, simtuzumab, solitomab, takatuzumab, taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab, vorsetuzumab, votumumab, zalutumumab, CC49 and 3F8.

In certain embodiments, the compositions described herein are administered in combination with a cytokine. As used herein, “cytokine” is a general term for proteins released by one population of cells that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, chemokines and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and luteinizing hormone (LH); hepatocyte growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; Mullerian inhibitory substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet growth factor; transforming growth factors (TGFs), such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinducing factors; interferons such as interferon-alpha, -beta and -gamma; colony-stimulating factors (CSF), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF) and granulocyte-CSF (G-CSF); interleukins (IL) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL-11, IL-12; IL-15, tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term “cytokine” includes proteins from natural sources or from recombinant cell culture, as well as biologically active equivalents of native sequence cytokines.

I. Target cells and antigens

The present invention provides, in particular, genetically modified immune effector cells redirected to a target cell, such as a tumor or cancer cell, and which contain engineered T cell receptors or CARs with a binding domain that binds to target antigens on the cells. Cancer cells can also spread to other parts of the body through the circulatory and lymphatic systems. There are several main types of cancer. Carcinoma is cancer that begins in the skin or in the tissues lining or covering internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat tissue, muscle tissue, blood vessels, or other connective or supporting tissue. Leukemia is a cancer that starts in blood-forming tissue, such as bone marrow, and causes large numbers of abnormal blood cells to appear and enter the bloodstream. Lymphoma and multiple myeloma are forms of cancer that begin in the cells of the immune system. Central nervous system cancers are forms of cancer that begin in the tissues of the brain and spinal cord.

In one embodiment, the target cell expresses an antigen, for example, a target antigen that is not substantially found on the surface of other normal (desired) cells. In one embodiment, the target cell is a pancreatic parenchyma cell, pancreatic duct cell, liver cell, myocardial cell, skeletal muscle cell, osteoblast, skeletal myoblast, neuron, vascular endothelial cell, pigment cell, smooth muscle cell, glial cell, fat cell , osteocyte, chondrocyte, pancreatic islet cell, CNS cell, PNS cell, liver cell, lipocyte, kidney cell, lung cell, skin cell, ovarian cell, follicular cell, epithelial cell, immune cell or endothelial cell.

In certain embodiments, the target cell is a portion of pancreatic tissue, neural tissue, heart tissue, bone marrow, muscle tissue, bone tissue, skin tissue, liver tissue, hair follicle, vascular tissue, adipose tissue, lung tissue, and kidney tissue.

In a specific embodiment, the target cell is a tumor cell. In another specific embodiment, the target cell is a cancer cell, such as a cell from a patient with cancer. Examples of cells that can be killed using the disclosed methods include cells from the following tumors: hematological malignancies, such as leukemia, including acute leukemia (such as acute lymphocytic leukemia, acute myelocytic leukemia, as well as myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia) , chronic leukemias (such as chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease).

In another embodiment, the cell is a solid tumor cell, such as forms of sarcoma or carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma and other sarcomas, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer , breast cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, lung cancer, colorectal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (eg adenocarcinoma of the pancreas, colon, ovary, lung, breast, stomach, prostate , cervix or esophagus), sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumor, bladder carcinoma, central nervous system tumors (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma).

In one embodiment, the cancer is selected from the group consisting of the Method of claim 1, wherein the cancer is selected from the group consisting of Wilms tumor, Ewing's sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, cancer rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary system cancer, cervical cancer, endometrial cancer, esophageal cancer, stomach cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myeogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma and bladder cancer.

In one embodiment, the target cell is a malignant cell of the liver, pancreas, lung, breast, bladder, brain, bone, thyroid, kidney, skin, and hematopoietic system. In another embodiment, the target cell is a cell in liver cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, brain cancer, bone cancer, thyroid cancer, kidney cancer, skin cancer, or hematopoietic cancer.

In one embodiment, the target cell is a cell, such as a cancer cell, infected with a virus, including but not limited to CMV, HPV, and EBV.

In one embodiment, the target antigen is a folate receptor epitope alpha, 5T4, avp6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, CMV, EBV, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AChR, FRα, GD2, GD3, glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA -A3+NY-ESO-1, HPV, IL-11Rα, IL-13Rα2, lambda chain, Lewis-Y, kappa chain, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGFR2.

J. Methods of therapy

Modified T cells produced using the methods discussed herein provide improved adaptive immunotherapy for use in the treatment of various conditions, including but not limited to cancer, infectious disease, autoimmune disease, inflammatory disease and immunodeficiency. In specific embodiments, the specificity of the primary T cell is redirected to tumor or cancer cells by genetically modifying the primary T cell with an engineered TCR or CAR discussed herein. In one embodiment, the present invention provides a type of cell therapy in which T cells are modified to express a designed TCR or CAR that targets cancer cells expressing the target antigen, and the modified T cells are administered to a recipient in need thereof. The injected cells are capable of destroying tumor cells in the recipient. Unlike antibody-based therapies, T cells modified with an engineered TCR or CAR are able to replicate in vivo, resulting in long-term persistence that can provide long-term cancer therapy.

In one embodiment, T cells engineered with the TCR and CAR of the present invention can undergo active in vivo T cell expansion and can persist for an extended period of time. In another embodiment, T cells engineered with a TCR or CAR of the present invention are converted into specific memory T cells that can be reactivated to inhibit any additional tumor formation or growth.

In specific embodiments, compositions comprising an immune effector cell genetically modified with a vector containing a promoter operably linked to a polynucleotide encoding a CAR are used in the treatment of solid tumors or forms of cancer, including but not limited to liver cancer, pancreatic cancer, cancer lung, breast cancer, bladder cancer, brain cancer, bone cancer, thyroid cancer, kidney cancer or skin cancer.

In specific embodiments, compositions comprising an immune effector cell genetically modified with a vector containing a promoter operably linked to a polynucleotide encoding a designed TCR or CAR that contains an antigen-specific binding domain that binds a PSCA or MUC1 epitope are used in the treatment of various forms of cancer, including but not limited to the pancreas, bladder and lung.

In specific embodiments, compositions comprising an immune effector cell genetically modified with a vector containing a promoter operably linked to a polynucleotide encoding a designed TCR or CAR are used in the treatment of hematologic malignancies, including but not limited to leukemia, including acute leukemia (e.g. , ALL, AML and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (eg, CLL, SLL, CML, HCL), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenström's macroglobulinemia and heavy chain disease .

In specific embodiments, compositions comprising an immune effector cell genetically modified with a vector containing a promoter operably linked to a polynucleotide encoding a designed TCR or CAR are used for the treatment of B cell neoplasms, including but not limited to multiple myeloma (MM), non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL).

Multiple myeloma is a B-cell neoplasm with the morphology of mature plasma cells, characterized by malignant transformation of a single clone of these cell types. These plasma cells proliferate in the VM and can invade adjacent bone and sometimes blood. Variant forms of multiple myeloma include clinical multiple myeloma, smoldering multiple myeloma, plasmacytic leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma (see, for example, Braunwald, et al. (eds), Harrison's Principles of Internal Medicine, ^15th Edition (McGraw-Hill 2001)).

Non-Hodgkin's lymphoma covers a large group of cancers of lymphocytes (white blood cells). Non-Hodgkin lymphomas can occur at any age and are often characterized by enlarged lymph nodes, fever, and weight loss. There are many types of non-Hodgkin lymphoma. For example, non-Hodgkin's lymphoma can be divided into aggressive (fast-growing) and indolent (slow-growing) types. Although non-Hodgkin's lymphomas can originate from B cells and T cells, the terms "non-Hodgkin's lymphoma" and "B-cell non-Hodgkin's lymphoma" are used interchangeably as used herein. B-cell non-Hodgkin's lymphomas (NHL) include Burkitt's lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, lymphoblastic B-cell progenitor lymphoma, and mantle cell lymphoma zones. Lymphomas that occur after bone marrow or stem cell transplantation are typically B-cell non-Hodgkin lymphomas.

Chronic lymphocytic leukemia (CLL) is an indolent (slow-growing) cancer that causes a slow increase in the number of immature white blood cells called B lymphocytes or B cells. Cancer cells spread through the blood and bone marrow and can also affect lymph nodes or other organs such as the liver and spleen. CLL eventually causes damage to the bone marrow. Sometimes in later stages of the disease the disease is called small cell lymphocytic lymphoma.

In specific embodiments, methods are provided that include administering a therapeutically effective amount of the modified T cells discussed herein, or compositions containing them, to a patient in need thereof, alone or in combination with one or more therapeutic agents. In certain embodiments, the cells of the present invention are used in the treatment of patients at risk of developing cancer. Thus, the present invention provides methods for treating or preventing cancer comprising administering to a subject in need thereof a therapeutically effective amount of the modified T cells of the present invention.

In one embodiment, a method of treating cancer in a subject in need thereof comprises administering an effective amount, such as a therapeutically effective amount, of a composition comprising genetically modified immune effector cells discussed herein. The amount and frequency of administration will be determined depending on factors such as the patient's condition and the type and severity of the patient's disease, although appropriate dosages can be determined through clinical trials.

In one embodiment, the number of T cells in the composition administered to a subject is at least 0.1 x 10 ⁵ cells, at least 0.5 x 10 ⁵ cells, at least 1 x 10 ⁵ cells, at least 5 × 10 ⁵ cells, at least 1 × 10 ⁶ cells, at least 0.5 × 10 ⁷ cells, at least 1 × 10 ⁷ cells, at least 0.5 × 10 ⁸ cells, at least 1 × 10 ⁸ cells, at least 0.5 × 10 ⁹ cells, at least 1 × 10 ⁹ cells, at least 2 × 10 ⁹ cells, at least 3 × 10 ⁹ cells, at least 4 × 10 ⁹ cells , at least 5 × 10 ⁹ cells or at least 1 × 10 ¹⁰ cells. In specific embodiments, the subject is administered from about 1 x 10 ⁹ CAR T cells to about 1 x 10 ¹⁰ CAR T cells, from about 2 x 10 ⁷ CAR T cells to about 0.9 x 10 CAR T cells, from approximately 3 x 10 ⁷ CAR T cells to approximately 0.8 x 10 ⁹ CAR T cells, approximately 4 x 10 ⁷ CAR T cells to approximately 0.7 x 10 ⁹ CAR T cells, approximately 5 x 10 CAR T cells to approximately 0.6 x 10 CAR T cells or from approximately 5 x 10 ⁷ CAR T cells to approximately 0.5 x 10 ⁹ CAR T cells.

In one embodiment, the number of T cells in the composition administered to the subject is at least 0.1 x 10 ⁴ cells/kg body weight, at least 0.5 x 10 ⁴ cells/kg body weight, at least 1 x 10 ⁴ cells/kg body weight, at least 5 × 10 ⁴ cells/kg body weight, at least 1 × 10 ⁵ cells/kg body weight, at least 0.5 × 10 ⁶ cells/kg body weight, according to at least 1 × 10 ⁶ cells/kg body weight, at least 0.5 × 10 ⁷ cells/kg body weight, at least 1 × 10 ⁷ cells/kg body weight, at least 0.5 × 10 ⁸ cells /kg body weight, at least 1 × 10 ⁸ cells/kg body weight, at least 2 × 10 ⁸ cells/kg body weight, at least 3 × 10 ⁸ cells/kg body weight, at least 4 × 10 ⁸ cells/kg body weight, at least 5×10 ⁸ cells/kg body weight or at least 1×10 ⁹ cells/kg body weight. In specific embodiments, the subject is administered from about 1 x 10 ⁶ CAR T cells/kg body weight to about 1 x 10 ⁸ CAR T cells/kg body weight, from about 2 x 10 ⁶ CAR T cells/kg body weight to approximately 0.9 x 10 ⁸ CAR T cells/kg body weight, from approximately 3 x 10 ⁶ CAR T cells/kg body weight to approximately 0.8 x 10 ⁸ CAR T cells/kg body weight, from approximately 4 × 10 ⁶ CAR T cells/kg body weight to approximately 0.7 × 10 ⁸ CAR T cells/kg body weight, from approximately 5 × 10 ⁶ CAR T cells/kg body weight to approximately 0.6 × 10 ⁸ CAR T cells/kg body weight or from approximately 5 x 10 ⁶ CAR T cells/kg body weight to approximately 0.5 x 10 ⁸ CAR T cells/kg body weight.

One of ordinary skill in the art will appreciate that multiple administrations of the compositions of the present invention may be required to provide the desired therapy. For example, the composition may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more times over 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months , 5 months, 6 months, 1 year, 2 years, 5 years, 10 years or more.

In certain embodiments, it may be necessary to administer activated T cells to a subject, and then sequentially collect blood (or perform apheresis), activate the resulting T cells in accordance with the present invention, and reinfuse those activated and expanded T cells into the patient . This process can be repeated many times every few weeks. In certain embodiments, T cells can be activated from blood collected from 10 ^cc to 400 ^cc . In certain embodiments, T cells are activated from blood collected at 20 cc ^, 30 ^cc , 40 ^cc , 50 ^cc , 60 ^cc , 70 ^cc , 80 ^cc , 90 ^cc , 100 ^cc , 150 cm ³ , 200 cm ³ , 250 cm ³ , 300 cm ³ , 350 cm ³ or 400 cm ³ or more. Without being bound by any theory, the use of such a multiple blood draw/multiple reinfusion protocol may serve to select specific T cell populations.

Administration of the compositions discussed herein can be accomplished by any convenient route, including aerosol inhalation, injection, oral administration, transfusion, implantation, or transplantation. In a preferred embodiment, the compositions are administered parenterally. As used herein, the phrases “parenteral administration” and “parenterally administered” refer to routes of administration other than enteral and topical administration, which are typically by injection and which include, but are not limited to, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrathoracic injection and infusion. In one embodiment, the compositions discussed herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

In one embodiment, an effective amount of a composition is administered to a subject in need thereof to enhance a cellular immune response to cancer in the subject. The immune response may include cellular immune responses mediated by cytotoxic T cells, which can lead to cytolysis of infected cells, regulatory T cells, as well as reactions with helper T cells. Humoral immune responses can also be induced, mediated primarily by helper T cells that can activate B cells, resulting in the production of antibodies. A variety of techniques can be used to analyze the type of immune responses induced by the compositions of the present invention, which are well described in the art; for example, Current Protocols in Immunology, ed. John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, N.Y.

In T cell-mediated cell cytolysis, CAR ligand binding initiates CAR signaling to the T cell, which results in the activation of a variety of signal transduction pathways in the T cell, which induces the T cell to produce or release proteins capable of inducing apoptosis of the target cell through various mechanisms. Such T cell-mediated mechanisms include (but are not limited to) the transfer of intracellular cytotoxic granules from the T cell to the target cell, T cell secretion of anti-inflammatory cytokines that can induce cell cytolysis directly (or indirectly by recruiting killer effector cells), and increased expression of ligands death receptors (eg, FasL) on the surface of T cells that induce apoptosis of the target cell after binding to its cognate death receptor (eg, Fas) on the target cell.

In one embodiment, the present invention provides a method of treating a subject who has been diagnosed with cancer, comprising removing immune effector cells from the subject, genetically modifying said immune effector cells with a vector containing a nucleic acid encoding a designed TCR or CAR discussed herein, thereby obtaining a population of modified immune effector cells, and administering the modified immune effector cells to the same subject. In a preferred embodiment, the immune effector cells include T cells.

Certain embodiments of the present invention also provide methods for stimulating an immune effector cell-mediated immunomodulatory response to a population of target cells in a subject, comprising the steps of administering to the subject a population of immune effector cells expressing a nucleic acid construct encoding a designed TCR or CAR molecule.

Methods of administering the cell compositions described herein include any method that is effective for either reintroducing ex vivo genetically modified immune effector cells that directly express an engineered TCR or CAR into a subject, or reintroducing genetically modified immune effector cell precursors, which, when administered to a subject, differentiate into mature immune effector cells that express the engineered TCR or CAR. One method involves transducing peripheral blood T cells ex vivo with a nucleic acid construct in accordance with the present invention and returning the transduced cells to the subject.

All publications, patent applications and issued patents cited herein are incorporated herein by reference as if each individual publication, patent application or issued patent were specifically and individually identified as being incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to one of ordinary skill in the art in light of the teachings of the present invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily be able to identify a number of minor parameters that could be changed or modified to obtain substantially the same results.

EXAMPLES

EXAMPLE 1

Proliferation of T cells cultured with AKT inhibitor

The purpose of this experiment was to determine the effect of Akt inhibitors on T cell proliferation. The effect of MK-2206 (Selleckchem) on T cell proliferation was assessed by measuring T cell division.

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous cell population containing T cells. PBMCs were collected from normal donors and labeled with a fluorescent dye (CellTrace® Violet, Molecular Probes), the intensity of which gradually decreased by half with each cell division. Labeled PBMCs were used as a cell source for T cell expansion. T cells were activated and expanded by culturing labeled PBMCs with anti-CD3 antibody and anti-CD28 antibody (Miltenyi Biotec) in medium containing IL-2 (CellGenix).

The effects of MK-2206 on cell division were assessed by assessing the decrease in CellTrace Violet fluorescence by flow cytometry following the addition of 0.025 μM, 0.074 μM, 0.222 μM, 0.67 μM, or 2.0 μM MK-2206 to T cell cultures on day 0. Fresh XVIVO-15 culture medium containing IL-2 and MK-2206 was added to the T cell cultures every two to three days for a total of seven days to promote T cell proliferation. Treatment with MK-2206 did not significantly reduce T cell division three days after culture initiation (Figure 1A). Additionally, T cells cultured in the presence of MK-2206 for seven days showed no statistically significant differences in T cell division compared to medium or untreated controls (Figure 1B). A paired t test was performed for all seven day-old cultures; (each concentration of MK-2206 was compared with 0 µM MK-2206: cultures were not statistically significantly different at the p≤0.05 level).

EXAMPLE 2

Expression of CD62L on T cells treated with MK-2206

The purpose of this experiment was to determine the effect of Akt inhibitors on T cell markers that determine T cell activity. The effect of MK-2206 (Selleckchem) on T cell activity was assessed by measuring CD62L expression in T cell cultures prepared in Example 1.

In mouse models, CD62L expression has been shown to indicate highly active T cells; T cells with greater antitumor efficacy after adoptive transfer. However, T cells cultured with IL-2 in vitro have been shown to have decreased CD62L expression and, taking this into account, this correlates with decreased antitumor activity of tumor-specific T cells. Surprisingly, the present author found that the AKT inhibitor, MK-2206, significantly increased CD62L expression at all concentrations of MK-2206 tested after seven days of culture and in the presence of IL-2. Figure 2. Paired t-test was performed for all seven-day cultures; (each concentration of MK-2206 was compared with 0 μM MK-2206: *p<0.05, **p<0.01).

EXAMPLE 3

Expression of CD62L on CAR T cells treated with Mk-2206 or ZSTK474

CAR T cells were cultured with either MK-2206, TCN (AKT inhibitors) or ZSTK474 (PI3K inhibitor). B-cell maturation antigen (BCMA)-specific CAR T cells were used in this series of experiments. CAR T cell culture was performed using a system that is directly scalable to large clinical production processes. Briefly, peripheral blood mononuclear cells (PBMCs) were cultured in stationary culture flasks in medium containing IL-2 (CellGenix) and antibodies specific for CD3 and CD28 (Miltenyi Biotec). 2x10 ⁸ transduction units of lentivirus encoding the CAR to BCMA were added one day after culture initiation. Anti-BCMA CAR T cells were maintained in logarithmic growth phase by the addition of fresh medium containing IL-2 and an optimized dose of inhibitor for a total of ten days of culture. At the end of culture, T cells with CAR to BCMA were examined for their phenotype.

CD62L expression on BCMA CAR T cells was assessed by flow cytometry at the end of culture. Anti-BCMA CAR T cells were specifically detected using recombinant human BCMA-Fc IgG conjugated to PE. CD62L expression was determined by co-staining with an antibody specific for CD62L. Anti-BCMA CAR T cells from three normal donors cultured with IL-2 and MK2206 had significantly higher expression of CD62L compared to cultures treated with IL-2 alone. Transduction of lentivirus to express CAR to BCMA did not reduce the improved phenotype caused by MK2206. A similar improvement was observed with ZST474 applied during culture, but not with TCN. (Figure 3).

EXAMPLE 4

CAR T cells treated with Mk-2206 or ZSTK474 demonstrate improved therapeutic activity

Anti-BCMA CAR T cells treated with MK-2206, TCN, or ZSTK474 as described in Example 3 were tested to evaluate whether increased CD62L expression was associated with increased antitumor activity. Anti-BCMA CAR-expressing T cells were obtained after culture with IL-2 and one of MK2206, TCN, or ZSTK474. An aliquot of CAR T cells was also cultured in medium supplemented with IL-7 and IL-15 at doses previously shown to enhance the therapeutic efficacy of CAR T cells. Animals bearing 100 mm subcutaneous multiple myeloma tumors (RPMI-8226) were infused with equivalent doses of CAR T cells (1×10 ⁶ CAR-positive cells).

Anti-BCMA CAR T cells cultured with IL-2 were sufficient to induce complete tumor regression. Anti-BCMA CAR T cells cultured with IL-7 and IL-15, MK-2206, or ZST747 showed similar levels of antitumor activity compared to anti-BCMA CAR T cells cultured with standard IL-2 in this study. animal model of multiple myeloma. In contrast, anti-BCMA CAR T cells cultured with TCN completely abrogated any antitumor responses. The results of these experiments are shown in Figure 4.

EXAMPLE 5

CAR T cells treated with Mk-2206 or ZSTK474 demonstrate improved therapeutic activity in an aggressive tumor model

A more aggressive and intractable tumor model was used to determine the antitumor activity of CAR T cells treated with IL-2, MK-2206, TCN, ZSTK474, IL7/15, or media. Daudi tumor cells express BCMA protein at low levels and can be effectively treated with BCMA CAR T cells. The progression of Daudi tumors was not affected by subsequent treatment with anti-BCMA CAR T cells cultured with IL-2 or IL7/15. Strikingly, anti-BCMA CAR T cells cultured with either MK-2206 or ZST474 induced complete tumor regression. The results of these experiments are shown in Figure 5.

EXAMPLE 6

CAR T cells treated with Mk-2206 or ZSTK474 demonstrate improved persistence

Clinical data from patients treated with CAR T cells suggest that persistence is associated with objective tumor response to treatment. The persistence of anti-BCMA CAR T cells was assessed by reintroducing tumors into mice that showed complete regression. Animals that were treated with anti-BCMA CAR T cells cultured with IL-2, MK-2206, or ZSTK474 and had completely regressed 100 mm ³ RPMI-8226 tumors were reintroduced with RPMI-8226 after 13 days. the other side. CAR T cells cultured with IL-2 treated with animals were not able to prevent tumor growth. In contrast, none of the animals treated with anti-BCMA CAR T cells cultured with ZSTK474 showed any evidence of tumor engraftment. These data demonstrate that therapeutically active anti-BCMA CAR T cells are retained in anti-BCMA CAR T cells cultured with ZSTK474. The results of these experiments are shown in Figure 6.

In general, in the following claims, the terms used are not to be construed as limiting the claims to the specific embodiments disclosed in the present specification and claims, but are to be construed to include all possible embodiments together with the full scope of equivalents that such claims cover. Accordingly, the claims are not limited to the present disclosure.

Claims (29)

1. A method for producing T cells, comprising: (a) activating the T cell population and stimulating the T cell population to proliferate; (b) transducing T cells with a viral vector containing a polynucleotide encoding a chimeric antigen receptor (CAR) to B-cell maturation antigen (BCMA); (c) culturing the transduced T cells for proliferation; wherein steps a) to c) are performed in the presence of a phosphatidylinositol 3-kinase (PI3K) inhibitor, and wherein the level of T cell differentiation is reduced in the case of T cells produced in accordance with steps a) to c) performed in the presence of a PI3K inhibitor, compared to the level of T cell differentiation in the case of T cells produced in accordance with steps a)-c), performed in the absence of a PI3K inhibitor. 2. The method of claim 1, wherein the CAR comprises an extracellular domain comprising an antibody or antigen-binding fragment that binds BCMA, a hinge domain, a transmembrane domain, one or more co-stimulatory domains, and a primary signal transduction domain. 3. A method for producing T cells, comprising: (a) activating the T cell population and stimulating the T cell population to proliferate; (b) transducing the T cells with a viral vector containing a polynucleotide encoding a CAR to BCMA, wherein the CAR contains an antibody or antigen binding fragment that binds BCMA , IgG1, CD28, or CD8α hinge domain, CD28 or CD8α transmembrane domain, CD28, OX40, or 4-1BB co-stimulatory domains, and CD3ζ primary signal transduction domain; (c) culturing the transduced T cells for proliferation; wherein steps a) to c) are performed in the presence of a phosphatidylinositol 3-kinase (PI3K) inhibitor, and wherein the level of T cell differentiation is reduced in the case of T cells produced in accordance with steps a) to c) performed in the presence of a PI3K inhibitor, compared to the level of T cell differentiation in the case of T cells produced in accordance with steps a)-c), performed in the absence of a PI3K inhibitor. 4. The method according to claim 2 or 3, where the antibody or antigen-binding fragment thereof is selected from the group consisting of camel Ig (camelid antibody (VHH)), Ig NAR, Fab fragments, Fab' fragments, F(ab) '2-fragment, F(ab)'3-fragment, Fv, single chain Fv antibody ("scFv"), bis-scFv, (scFv)2, minibody, diabody, tribody, tetrabody, disulfide-stabilized Fv protein ( "dsFv") and single domain antibodies (sdAb, nanobodies). 5. Method according to any one of paragraphs. 1-4, wherein the PI3K inhibitor is a general PI3K inhibitor selected from the group consisting of BEZ235, LY294002 and GDC-0941. 6. Method according to any one of paragraphs. 1-4, wherein the PI3K inhibitor is a selective PI3K inhibitor selected from the group consisting of BYL719, GSK2636771, TGX-221, AS25242, CAL-101 and IPI-145. 7. Method according to any one of paragraphs. 1-4, wherein the PI3K inhibitor is the PI3-K inhibitor ZSTK474. 8. A method for producing T cells, comprising: (a) activating the T cell population and stimulating the T cell population to proliferate; (b) transducing T cells with a viral vector containing a polynucleotide encoding a CAR to BCMA, wherein the CAR contains a scFv that binds BCMA, a CD8α hinge domain, a CD8α transmembrane domain, 4-1BB co-stimulatory domains, and a CD3ζ primary signal transduction domain; (c) culturing the transduced T cells for proliferation; wherein steps a) to c) are performed in the presence of a phosphatidylinositol 3-kinase (PI3K) inhibitor, and wherein the level of T cell differentiation is reduced in the case of T cells produced in accordance with steps a) to c) performed in the presence of a PI3K inhibitor, compared to the level of T cell differentiation in the case of T cells produced in accordance with steps a) to c) performed in the absence of a PI3K inhibitor, wherein the PI3K inhibitor is ZSTK4. 9. Method according to any one of paragraphs. 1-8, wherein the anti-BCMA CAR further comprises a signal peptide. 10. The method of claim 9, wherein the signal peptide comprises an IgG1 heavy chain signal polypeptide or a CD8α signal polypeptide. 11. Method according to any one of paragraphs. 1-10, where the method includes isolating peripheral blood mononuclear cells as a source of T cells. 12. Method according to any one of paragraphs. 1-11, where activation of T cells includes bringing the T cells into contact with an anti-CD3 antibody or a CD3-binding fragment thereof. 13. Method according to any one of paragraphs. 1-12, wherein stimulating the T cells comprises contacting the T cells with an anti-CD28 antibody or a CD28-binding fragment thereof, B7-1 or a CD28-binding fragment thereof, or B7-2 or a CD28-binding fragment thereof. 14. Method according to any one of paragraphs. 1-13, wherein the viral vector is a retroviral vector. 15. Method according to any one of paragraphs. 1-13, wherein the viral vector is a lentiviral vector.