Subset-optimized chimeric antigen receptor-containing cells

Description

[0001] This application claims priority to U.S. Serial No. 62 / 031,699 filed July 31, 2014 .SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format . Said ASCII copy, created on July 28, 2015, is named N2067-70660WO_SL.txt and is 174,225 bytes in size.FIELD OF THE INVENTION

[0003] The present disclosure relates generally to the use of T cells engineered to express a Chimeric Antigen Receptor (CAR) to treat a disease, e.g., a disease associated with the expression of a tumor antigen, e.g., wherein the CAR is optimized for T cells of the T cell subset in which is is provided.BACKGROUND OF THE INVENTION

[0004] The development of T cells which are genetically modified to express a chimeric antigen receptor (CAR) has opened the door for many new potential therapies for diseases, e.g., cancers. Generally, CARs comprise an extracellular antigen binding domain and an intracellular domain. The exact composition of the intracellular domain can provide unique characteristics to the CAR and to the cell population expression the CAR.

[0005] T cells redirected to express CARs have shown remarkable efficacy in treating some B cell malignancies. Targeting of different cancers using redirected T cells has shown some promising anti-tumor activity, but the activity was limited by poor persistence of the infused CAR T cell product. Thus, there is a need for methods and compositions for increasing the persistence and the anti-tumor activity of the redirected CAR T cells for treatment of disease. Moeller et al. Blood (2005); 106(9):2995-3003 relates to adoptive transfer of gene-engineered CD4 +< helper T cells with induction of tumor rejection.SUMMARY OF THE INVENTION

[0006] The invention provides a subset-optimized CART cell combination for use in a method of treating a subject having cancer, the method comprising administering to the subject an effective amount of: (a) a CD4+ T cell comprising a CAR (the CAR CD4+< ) comprising: an antigen binding domain; a transmembrane domain; an ICOS costimulatory domain; and an intracellular signaling domain comprising a CD3 zeta (CD3Z) domain; (b) a CD8+ T cell comprising a CAR (the CAR CD8+< ) comprising: an antigen binding domain; a transmembrane domain; a costimulatory domain selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICAM-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, GITR, BAFFR, HVEM (LIGHTR), SLAMf7, NKP80 (KLRF1), CD160 (BY55), CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, C49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (C244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, C100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, and PAG / Cbp; and an intracellular signaling domain comprising a CD3 zeta domain (CD3Z); and (c) a second CD8+ T cell comprising a CAR (the second CAR CD8+< ) comprising: an antigen binding domain; a transmembrane domain; a costimulatory domain selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICAM-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, GITR, BAFFR, HVEM (LIGHTR), SLAMf7, NKP80 (KLRF1), CD160 (BY55), CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, C49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (C244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, C100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, and PAG / Cbp; and an intracellular signaling domain comprising a CD3 zeta domain (CD3Z); wherein the subset-optimized CART cell combination enhances CAR T cells in vivo persistence.

[0007] The invention also provides a kit comprising: (a) a CD4+ T cell comprising said CAR CD4+< ; (b) a CD8+ T cell comprising said CAR CD8+< ; and (c) a CD8+ T cell comprising said second CAR CD8+< . DETAILED DESCRIPTION

[0008] The technical information set out below may in some respects go beyond the scope of the invention, which is defined exclusively by the appended claims. The additional technical information is provided to place the actual invention in a broader technical context and to illustrate possible related technical developments. In addition, incidental references to methods for treatment of the human or animal body by surgery or therapy and diagnostic methods practised on the human or animal body are not to be construed as claiming protection for such method as such, but are instead to be construed as referring to products, in particular substances or compositions, for use in any of these methods.

[0009] The present disclosure is based on the surprising discovery that when subsets of T cells, e.g., CD4 +< and CD8 +< T cells, are engineered to express CARs containing different intracellular signaling domains, the persistence and anti-tumor activity of the infused CAR-expressing T cells can be modulated. Accordingly, the present disclosure describes methods and compositions of CD4 +< and CD8 +< T cells that express CARs containing specific combinations of intracellular signaling domains can be used to increase persistence and anti-tumor activity of the infused CAR-expressing T cells for treating a subject having a disease, e.g., a cancer.

[0010] Described herein is a CD4 +< T cell for use in the treatment of a subject having cancer, wherein the CD4 +< T cell comprises a CAR (the CAR CD4+< ) comprising: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain; wherein the subject has received, is receiving or is about to receive: a CD8 +< T cell comprising a CAR (the CAR CD8+< ) comprising: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain; wherein the CAR CD4+< and the CAR CD8+< differ from one another.

[0011] Also described herein is a CD8 +< T cell for use in the treatment of a subject having cancer, wherein the CD8 +< T cell comprises a CAR (the CAR CD8+< ) comprising: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain; wherein the subject has received, is receiving or is about to receive: a CD4 +< T cell comprising a CAR (the CAR CD4+< ) comprising: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain; wherein the CAR CD4+< and the CAR CD8+< differ from one another.

[0012] Also described herein are products for use in a method of treating a subject having cancer, comprising, administering to said subject, an effective amount of: 1) a CD4 +< T cell comprising a CAR (the CAR CD4+< )comprising: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain; and 2) a CD8 +< T cell comprising a CAR (the CAR CD8+< ) comprising: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain; wherein the CAR CD4+< and the CAR CD8+< differ from one another. The intracellular signaling domain of the CAR CD4+< may differ from the CAR CD8+< intracellular signaling domain. The CAR CD4+< may comprise a first costimulatory signaling domain not present on the the CAR CD8+< . The CAR CD4+< may comprise a first costimulatory signaling domain and the CAR CD8+< may comprise a second costimulatory signaling domain. The CAR CD4+< may comprise a first costimulatory signaling domain not present on the CAR CD8+< and the CAR CD8+< may comprise a second costimulatory signaling domain not present on the CAR CD4+< . The antigen binding domain of the CAR CD4+< may differ from the CAR CD8+< antigen binding domain, e.g., they are different antigen binding domains, to the same, or to different antigens. The method of treating a subject having cancer may further comprise administering to the subject, 3) a second CD8 +< T cell comprising a CAR (the second CAR CD8+< ) comprising: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain, wherein the CARCD4+ , the CARCD8+ , and the second CARCD8+ differ from one another.

[0013] Accordingly, the present invention provides a subset-optimized CART cell composition for use in a method of treating a subject having cancer, which utilises a CAR CD4+< , a CAR CD8+< and a second CAR CD8+< as defined in the claims.

[0014] In an embodiment, the intracellular signaling domain of the CAR CD8+< differs from the second CAR CD8+< intracellular signaling domain. In an embodiment, the CAR CD8+< comprises a first costimulatory signaling domain not present on the second CAR CD8+< . In an embodiment, the CAR CD8+< comprises a first costimulatory signaling domain and the second CAR CD8+< comprises a second costimulatory signaling domain. In an embodiment, the CAR CD4+< comprises a first costimulatory signaling domain not present on the CAR CD8+< and the CAR CD8+< comprises a second costimulatory signaling domain not present on the CAR CD4+< . In an embodiment, the antigen binding domain of the CAR CD8+< differs from the second CAR CD8+< antigen binding domain, e.g., they are different antigen binding domains, to the same, or to different antigens. In an embodiment, the intracellular signaling domain of the CAR CD4+< , the intracellular signaling domain of the CAR CD8+< , and the intracellular signaling domain of the second CAR CD8+< , differ from one another. In an embodiment, each of the CAR CD4+< , CAR CD8+< , and second CAR CD8< , comprises a costimulatory signaling domain not present in either of the others. In the invention, the CAR CD4+< comprises an ICOS domain. In an embodiment, the ICOS domain comprises the sequence of SEQ ID NO: 46.

[0015] Also described herein are products for use in a method of treating a subject having cancer, comprising, administering to said subject, an effective amount of: 1) a CD4 +< T cell comprising a CAR (the CAR CD4+< ) comprising: an antigen binding domain; a transmembrane domain; and an ICOS domain; and 2) a CD8 +< T cell comprising a CAR (the CAR CD8+< ) comprising: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain which differ from an intracellualar signaling domain in CAR CD4+< , and, optionally, wherein the CAR CD8+< does not comprise an ICOS domain; and optionally, 3) a second CD8 +< T cell comprising a CAR (the second CAR CD8+< ) comprising: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain, wherein the second CARCD8+ comprises an intracellular signaling domain, e.g., a costimulatory signaling domain, not present on the CARCD8+, and, optionally, does not comprise an ICOS signaling domain.

[0016] The present invention provides a subset-optimized CART cell composition for use in a method of treating a subject having cancer, which utilises a CAR CD4+< , a CAR CD8+< and a second CAR CD8+< as defined in the claims.

[0017] In an embodiment, the CAR CD8+< does not comprise an ICOS domain.

[0018] In an embodiment, treating a subject having cancer comprises providing an anti-tumor immunity in the subject.

[0019] In an embodiment, treating a subject having cancer comprises stimulating a T cell-mediated immune response to a target cell population or tissue in the subject.

[0020] The method of treating a subject having cancer may further comprise administering to the subject, 3) a second CD8 +< T cell comprising a CAR (the second CAR CD8+< ) comprising: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain, wherin the second CAR CD8+< does not comprise an ICOS signaling domain and comprises an intracellular signaling domain not present on the CAR CD8+< .

[0021] The method may comprise administering: a second CD8 +< T cell comprising a CAR (the second CAR CD8+< ) comprising: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain, wherein the second CAR CD8+< comprises an intracellular signaling domain not present on the CAR CD8+< , and, optionally, does not comprise an ICOS domain.

[0022] The method of treating a subject may comprise administering a CD4 +< T cell comprising a CAR CD4+< comprising: an antigen binding domain; a transmembrane domain; and a primary intracellular signaling domain, e.g., a CD3zeta domain (and an ICOS domain); a CD8 +< T cell comprising a CAR CD8+< comprising: an antigen binding domain; a transmembrane domain; and a primary intracellular signaling domain, e.g., a CD3zeta domain, and a first costimulatory signaling domain, e.g., a 4-1BB domain; and a second CD8 +< T cell comprising a second CAR CD8+< comprising: an antigen binding domain; a transmembrane domain; and a primary intracellular signaling domain, e.g., a CD3zeta domain, and a second costimulatory signaling domain, e.g.,a CD28 domain, wherein the second CAR CD8+< does not comprise an ICOS signaling domain wherein said first and second costimulatory signaling domains are different.

[0023] The present invention provides a subset-optimized CART cell composition for use in a method of treating a subject having cancer, which utilises a CAR CD4+< , a CAR CD8+< and a second CAR CD8+< as defined in the claims.

[0024] In an embodiment, the CD4 +< T cell comprising a CAR CD4+< persists in the subject for at least 10, 15, 30, 45, 60, or 90 days after administration.

[0025] In an embodiment, the CD8+ T cell comprising a CAR CD8+< persists in the subject for at least 10, 15, 30, 45, 60, or 90 days after administration.

[0026] In an embodiment, the method comprises administering a second CD8+ T cell comprising a second CAR CD8+< , wherein the second CD8+ T cell persists in the subject for at least 10, 15, 30, 45, 60, or 90 days after administration.

[0027] In an embodiment, the method of treating a subject comprises administering a preparation of T cells to the subject, wherein at least 10, 20, 30, 40, 50, 60, 70, or 80 % of the T cells, or cells, in the preparation are CD4 +< T cells comprising a CAR CD4+< .

[0028] In an embodiment, the method of treating a subject comprises administering a preparation of T cells to the subject, wherein at least 10, 20, 30, 40, 50, 60, 70, or 80 % of the T cells, or cells, in the preparation are CD8 +< T cells comprising a CAR CD8+< .

[0029] In an embodiment, the method of treating a subject comprises administering a preparation of T cells to the subject, wherein at least 10, 20, 30, 40, 50, 60, 70, or 80 % of the T cells, or cells, in the preparation are CD4 +< T cells comprising a CAR CD4+< ; and at least 10, 20, 30, 40, 50, 60, 70, or 80% of the T cells, or cells, in the preparation are CD8 +< T cells comprising a CAR CD8+< or second CAR CD8+< (it being understood that the total cells do not exceed 100%).

[0030] In an embodiment, the method of treating a subject comprises administering a preparation of T cells to the subject, wherein 10 to 70% of the T cells, or cells, in the preparation are CD4 +< T cells comprising a CAR CD4+< ; and 10 to 70% of the T cells, or cells, in the preparation are CD8 +< T cells comprising a CAR CD8+< or second CAR CD8+< (it being understood that the total cells do not exceed 100%).

[0031] In an embodiment, the method of treating a subject comprises administering a preparation of T cells to the subject, wherein at least 10, 20, 30, 40, 50, 60, 70, or 80 % of the T cells, or cells, in the preparation are CD4 +< T cells comprising a CAR CD4+< ; at least 10, 20, 30, 40, 50, 60, 70, or 80 % of the T cells, or cells, in the preparation are CD8 +< T cells comprising a CAR CD8+< ; and at least 10, 20, 30, 40, 50, 60, 70, or 80 % of the T cells, or cells, in the preparation are CD8 +< T cells comprising a second CAR CD8+< (it being understood that the total cells do not exceed 100%).

[0032] In an embodiment, the CD4 +< T cell comprising the CAR CD4+< is an autologous T cell. In an embodiment, the CD8+ T cell comprising the CAR CD8+< is an autologous T cell. In an embodiment, the CD4 +< T cell comprising the CAR CD4+< and the CD8 +< T cell comprising the CAR CD8+< are autologous T cells. In an embodiment, administering a second CD8+ T cell comprising a second CAR CD8+< , wherein the second CD8 +< T cell is an autologous T cell.

[0033] In an embodiment, the CD4 +< T cell comprising the CAR CD4+< is an allogeneic T cell. In an embodiment, the CD8 +< T cell comprising the CAR CD8+< is an allogeneic T cell. In an embodiment, the CD4 +< T cell comprising the CAR CD4+< and the CD8 +< T cell comprising the CAR CD8+< are allogeneic T cells. In an embodiment, the method further comprises administering a second CD8 +< T cell comprising a second CAR CD8+< , wherein the second CD8 +< T cell is an allogeneic T cell.

[0034] In an embodiment, the CD4 +< T cell comprising the CAR CD4+< is an autologous Tcell and the CD8 +< T cell comprising the CAR CD8+< is an allogeneic T cell.

[0035] In an embodiment, the CD4 +< T cell comprising the CAR CD4+< is an allogeneic Tcell and the CD8 +< T cell comprising the CAR CD8+< is an autologous T cell.

[0036] In an embodiment, the method further comprises evaluating the subject for a side effect of said treatment. In an embodiment, said side effect comprises acute respiratory distress syndrome, febrile neutropenia, hypotension, encephalopathy, hepatic transaminitis, seizure, or macrophage activation syndrome.

[0037] In an embodiment, the method further comprises treating the subject having a side effect with anti-cytokine agent, e.g., a tumor necrosis factor antagonist, e.g., a TNF-Ig fusion, e.g., etanercept, an IL-6 antagonist, e.g., an IL-6 receptor antagonist, e.g., an anti-IL6 receptor antibody, e.g., tocilizumab, or a corticosteroid. In an embodiment, treating the subject having a side effect comprises administering an anti-IL6 receptor antibody to the subject.

[0038] In an embodiment, the subject is a human.

[0039] The CAR-expressing CD4 +< T cells and CD8 +< T cells described herein can be administered simultaneously, in the same or in separate compositions, or sequentially. In an embodiment, the CAR-expressing CD4 +< T cells are administered before the CAR-expressing CD8 +< T cells, and in an embodiment, the CAR-expressing CD8 +< T cells are administered before the CAR-expressing CD4 +< T cells.

[0040] The following describes any of the CAR CD4+< , or CD4+ T cell comprising the CAR CD4+< , the CAR CD8+< , or CD8+ T cell comprising the CAR CD8+< , or the second CAR CD8+< or the second CD8+ T cell comprising the second CAR CD8+< , encompassed in any of the methods, compositions, or kits disclosed herein, including the invention as defined in the claims.

[0041] In an embodiment, the CAR CD4+< does not include a 4-1BB domain.

[0042] In an embodiment, the CAR CD4+< does not include a CD28 domain.

[0043] In an embodiment, the CAR CD4+< does not include a costimulatory domain other than the ICOS domain.

[0044] In an embodiment, the CAR CD4+< comprises a primary intracellular signaling domain, e.g., a primary signal domain from Table 4, e.g., a CD3zeta domain. In an embodiment, the CAR CD4+< comprises a CD3zeta domain but does not include a costimulatory domain other than the ICOS domain.

[0045] In an embodiment, the CAR CD4+< comprises a second costimulatory signaling domain, e.g., from Table 5.

[0046] In an embodiment, the CAR CD8+< comprises a primary intracellular signaling domain, e.g., a primary signal domain from Table 4, e.g., a CD3zeta domain and a costimulatory signaling domain, as defined in the claims.

[0047] In an embodiment, the CAR CD8+< comprises a second costimulatory signaling domain, e.g., from Table 5.

[0048] In the invention, the CAR CD8+< comprises a CD3zeta domain.

[0049] In the invention, the CAR CD8+< comprises a costimulatory signaling domain other than ICOS, as defined in the claims, e.g., 4-1BB or CD28.

[0050] In an embodiment, the CAR CD8+< comprises a 4-1BB domain.

[0051] In an embodiment, the CAR CD8+< comprises a CD3zeta domain and a 4-1BB domain.

[0052] In an embodiment, the CAR CD8+< comprises a CD28 domain.

[0053] In an embodiment, the CAR CD8+< comprises a CD3zeta domain and a CD28 domain.

[0054] In an embodiment, the CAR CD8+< comprises a CD3zeta domain, a 4-1BB domain and a CD28 domain.

[0055] In an embodiment, the CAR CD4+< comprises a CD3zeta domain; and the CAR CD8+< comprises a CD3zeta domain and a 4-1BB domain.

[0056] In an embodiment, the CAR CD4+< comprises a CD3zeta domain; and the CAR CD8+< comprises a CD3zeta domain and a CD28 domain.

[0057] In an embodiment, the CAR CD4+< comprises a CD3zeta domain (and an ICOS domain); and the CAR CD8+< comprises a CD3zeta domain, a 4-1BB domain, and a CD28 domain.

[0058] In an embodiment, the CAR CD4+< comprises a CD3zeta domain (e.g., according to SEQ ID NO: 18 or SEQ ID NO: 20) and an ICOS domain (e.g., according to SEQ ID NO: 40 or SEQ ID NO: 46); and the CAR CD8+< comprises a CD3zeta domain (e.g., according to SEQ ID NO: 18 or SEQ ID NO: 20) and a 4-1BB domain (e.g., according to SEQ ID NO: 14). In the invention, the CAR CD4+< comprises a CD3zeta domain (e.g., according to SEQ ID NO: 18 or SEQ ID NO: 20); and the CAR CD8+< comprises a CD3zeta domain (e.g., according to SEQ ID NO: 18 or SEQ ID NO: 20). In an embodiment, the CAR CD4+< comprises an ICOS domain (e.g., according to SEQ ID NO: 40 or SEQ ID NO: 46); and the CAR CD8+< comprises a 4-1BB domain (e.g., according to SEQ ID NO: 14). In an embodiment, the CAR CD4+< comprises a CD3zeta domain (e.g., according to SEQ ID NO: 18 or SEQ ID NO: 20); and the CAR CD8+< comprises a CD3zeta domain (e.g., according to SEQ ID NO: 18 or SEQ ID NO: 20) and a 4-1BB domain (e.g., according to SEQ ID NO: 14). In an embodiment, the CAR CD4+< comprises an ICOS domain (e.g., according to SEQ ID NO: 40 or SEQ ID NO: 46); and the CAR CD8+< comprises a CD3zeta domain (e.g., according to SEQ ID NO: 18 or SEQ ID NO: 20) and a 4-1BB domain (e.g., according to SEQ ID NO: 14). In the invention, the CAR CD4+< comprises a CD3zeta domain (e.g., according to SEQ ID NO: 18 or SEQ ID NO: 20) and an ICOS domain (e.g., according to SEQ ID NO: 40 or SEQ ID NO: 46); and the CAR CD8+< comprises a CD3zeta domain (e.g., according to SEQ ID NO: 18 or SEQ ID NO: 20). In an embodiment, the CAR CD4+< comprises a CD3zeta domain (e.g., according to SEQ ID NO: 18 or SEQ ID NO: 20) and an ICOS domain (e.g., according to SEQ ID NO: 40 or SEQ ID NO: 46); and the CAR CD8+< comprises a 4-1BB domain (e.g., according to SEQ ID NO: 14).

[0059] In an embodiment, the second CAR CD8+< does not comprise an ICOS domain.

[0060] In an embodiment, the second CAR CD8+< comprises a primary intracellular signaling domain, e.g., a primary signal domain from Table 4, e.g., a CD3zeta domain.

[0061] In the invention, the second CAR CD8+< comprises a costimulatory signaling domain other than ICOS, as defined in the claims, e.g., a 4-1BB domain.

[0062] In the invention, the second CAR CD8+< comprises a costimulatory signaling domain other than ICOS, as defined in the claims, e.g., a CD28 domain.

[0063] In an embodiment, the second CAR CD8+< comprises a CD3zeta domain and a 4-1BB domain.

[0064] In an embodiment, the second CAR CD8+< comprises a CD3zeta domain and a CD28 domain.

[0065] In an embodiment, the CAR CD4+< comprises a CD3zeta domain (and an ICOS domain); the CAR CD8+< comprises a CD3zeta domain and a 4-1BB domain; and the second CAR CD8+< comprises a CD3zeta domain and a CD28 domain.

[0066] In an embodiment, the CD4 +< cell is a Th17 polarized cell. In an embodiment, the CD4 +< cell produces IL-17A and / or IFNγ. In an embodiment, the CD4 +< cell expresses IL-23R and / or CD161 on the cell surface.

[0067] In an embodiment, the CAR CD4+< comprises an antibody variable domain, an scFv, or a nanobody, or an antigen binding fragment thereof. In an embodiment, the antigen binding domain specific for a tumor antigen selected from CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, and any combination thereof. In an embodiment, the CAR CD4 +< comprises an antigen binding domain which binds an antigen described herein, e.g., an antigen listed in Table 2. In an embodiment, the CAR CD4+< comprises an antigen binding domain from Table 2.

[0068] In an embodiment, the CAR CD8+< comprises an antibody variable domain, an scFv, or a nanobody, or an antigen binding fragment thereof. In an embodiment, the CAR CD8+< comprises an antigen binding domain specific for a tumor antigen selected from CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, and any combination thereof. In an embodiment, the CAR CD8+< comprises an antigen binding domain which binds a cancer cell, e.g., which binds an antigen described in Table 2. In an embodiment, the CAR CD8+< comprises an antigen binding domain from Table 2.

[0069] In an embodiment, the CAR CD4+< and the CAR CD8+< each comprises an antigen binding domain which binds a cancer cell, e.g., which binds an antigen described in Table 2. In an embodiment, each of the CAR CD4+< and CAR CD8+< comprises an antigen binding domain which binds the same antigen. In an embodiment, the CAR CD4+< and the CAR CD8+< each comprises an antigen binding domain from Table 2. In an embodiment, each of the CAR CD4+< and CAR CD8+< comprises the same antigen binding domain.

[0070] In an embodiment, a second CD8+ T cell comprising a second CAR CD8+< is administered, and the second CAR CD8+< comprises an antigen binding domain which binds a cancer cell, e.g., which binds an antigen described in Table 2.

[0071] In an embodiment, a second CD8+ T cell comprising a second CAR CD8+< is administered, and the second CAR CD8+< comprises an antigen binding domain from Table 2.

[0072] In an embodiment, a second CD8+ T cell comprising a second CAR CD8+< is administered, and the CAR CD4+< , CAR CD8+< , and the second CAR CD8+< each comprises an antigen binding domain which binds a cancer cell, e.g., which binds an antigen described in Table 2.

[0073] In an embodiment, a second CD8+ T cell comprising a second CAR CD8+< is administered, and each of the CAR CD4+< , CAR CD8+< , and second CAR CD8+< comprises an antigen binding domain which binds the same antigen.

[0074] In an embodiment, the CAR CD4+< CAR CD8+< and the second CAR CD8+< each comprises an antigen binding domain from Table 2.

[0075] In an embodiment, a second CD8+ T cell comprising a second CAR CD8+< is administered, and wherein each of the CAR CD4+< CAR CD8+< , and second CAR CD8+< comprises the same antigen binding domain.

[0076] In the invention, the CAR CD4+< comprises an ICOS domain. In an embodiment, the CAR CD8+< does not comprise an ICOS domain. In an embodiment, the second CAR CD8+< does not comprise an ICOS signaling domain and comprises an intracellular signaling domain not present on the CAR CD8+< .

[0077] The intracellular signaling domain of a CAR molecule described herein may comprise a costimulatory domain. The intracellular signaling domain of a CAR molecule may comprise a primary signaling domain. The intracellular signaling domain of an isolated CAR molecule may comprise a costimulatory domain and a primary signaling domain.

[0078] A costimulatory domain may comprise a functional signaling domain of a protein selected from the group consisting of MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, and a ligand that specifically binds with CD83. A costimulatory domain may comprise 4-1BB, CD27, CD28 or ICOS. In the invention, the CAR molecules comprise a costimulatory domain and an intracellular signalling domain as defined in the claims.

[0079] In one embodiment, the 4-1BB costimulatory domain comprises a sequence of SEQ ID NO: 14. In one embodiment, the 4-1BB costimulatory domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 14, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO:14. In one embodiment, the 4-1BB costimulatory domain is encoded by a nucleic acid sequence of SEQ ID NO:15, or a sequence with with 95-99% identity thereof.

[0080] In one embodiment, the CD27 costimulatory domain comprises a sequence of SEQ ID NO: 16. In one embodiment, the CD27 costimulatory domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 16, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO:16. In one embodiment, the CD27 costimulatory domain is encoded by a nucleic acid sequence of SEQ ID NO:17, or a sequence with with 95-99% identity thereof.

[0081] In one embodiment, the CD28 costimulatory domain comprises a sequence of SEQ ID NO: 44. In one embodiment, the CD28 costimulatory domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 44, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO:44. In one embodiment, the CD28 costimulatory domain is encoded by a nucleic acid sequence of SEQ ID NO:45, or a sequence with with 95-99% identity thereof.

[0082] In one embodiment, the wild-type ICOS costimulatory domain comprises a sequence of SEQ ID NO: 40. In one embodiment, the wild-type ICOS costimulatory domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 40, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO:40. In one embodiment, the wild-type ICOS costimulatory domain is encoded by a nucleic acid sequence of SEQ ID NO:41, or a sequence with with 95-99% identity thereof.

[0083] In one embodiment, the Y to F mutant ICOS costimulatory domain comprises a sequence of SEQ ID NO: 46. In one embodiment, the Y to F mutant ICOS costimulatory domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 46, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO:46. In one embodiment, the Y to F mutant ICOS costimulatory domain is encoded by a nucleic acid sequence with 95-99% identity to a nucleic acid sequence of SEQ ID NO:41 (wherein SEQ ID NO: 41 encodes wild-type ICOS).

[0084] In embodiments, the primary signaling domain comprises a functional signaling domain of CD3 zeta. In embodiments, the functional signaling domain of CD3 zeta comprises SEQ ID NO: 18 (mutant CD3 zeta) or SEQ ID NO: 20 (wild-type human CD3 zeta).

[0085] In one embodiment, the intracellular signaling domain of a CAR, e.g., a CAR CD4+< or a CAR CD8+< , comprises a functional signaling domain of CD27 and a functional signaling domain of CD3 zeta. In one embodiment, the intracellular signaling domain of a CAR, e.g., a CAR CD4+< or a CAR CD8+< , comprises a functional signaling domain of CD28 and a functional signaling domain of CD3 zeta. In the invention, the intracellular signaling domain of a CAR CD4+< comprises a functional signaling domain of ICOS and a functional signaling domain of CD3 zeta. In one embodiment, the intracellular signaling domain of a CAR, e.g., a CAR CD4+< or a CAR CD8+< , comprises a functional signaling domain of 4-1BB and a functional signaling domain of CD3 zeta.

[0086] In one embodiment, a CART cell displays an enhanced persistence compared to a control T cell, e.g., a T cell of a different type (e.g., CD8+ or CD4+) expressing the same CAR. In some embodiments, a CD4+ T cell comprises a CAR described herein, which CAR comprises an intracellular signaling domain suitable for (e.g., optimized for, e.g., leading to enhanced persistence in) a CD4+ T cell, e.g., an ICOS domain.

[0087] In the invention, the CD4+ T cell comprises a CAR comprising an ICOS costimulatory domain.

[0088] In some embodiments, a CD8+ T cell comprises a CAR in accordance with the claims, which CAR comprises an intracellular signaling domain suitable for (e.g., optimized for, e.g., leading to enhanced persistence of) a CD8+ T cell, e.g., a 4-1BB domain, a CD28 domain, or another costimulatory domain other than an ICOS domain.

[0089] In the invention, the CD8+ T cell comprises a CAR comprising a costimulatory domain and an intracellular signaling domain as defined in the claims.

[0090] Also disclosed herein is a composition, e.g., a pharmaceutically acceptable composition, or set of compositions, e.g., a set of pharmaceutically acceptable compositions, comprising one or more of the following components: 1) a CD4 +< T cell comprising a CAR (the CAR CD4+< ) comprising: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain; and 2) a CD8 +< T cell comprising a CAR (the CAR CD8+< ) comprising: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain; and, wherein the the CAR CD4+< and the CAR CD8+< differ from one another.

[0091] The composition, or set of compositions, may further comprise: 3) a second CD8 +< T cell comprising a CAR (the second CAR CD8+< ) comprising: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain, wherein the second CARCD8+ differs from the CARCD4+ and the the CARCD8+

[0092] The composition, or set of compositions, may comprise a plurality of components 1), 2), and 3).

[0093] The composition, or set of compositions, may comprise a composition comprising component 1).

[0094] The composition, or set of compositions, may comprise a composition comprising component 2).

[0095] The composition, or set of compositions, may comprise a composition comprising component 3).

[0096] The composition, or set of compositions, may comprise a composition comprising components 1) and 2).

[0097] The composition, or set of compositions, may comprise a composition comprising components 1) and 3).

[0098] The composition, or set of compositions, may comprise a composition comprising 2) and 3).

[0099] The composition, or set of compositions, may comprise a composition comprising components 1), 2) and 3).

[0100] The composition, or set of compositions, may comprise a set of compositions, the set comprising a composition comprising component 1) and a composition comprising component 2).

[0101] The composition, or set of compositions, may comprise a set of compositions, the set comprising a composition comprising component 1) and a composition comprising component 3).

[0102] The composition, or set of compositions, may comprise a set of compositions, the set comprising a composition comprising component 2), and a composition comprising component 3).

[0103] The composition, or set of compositions, may comprise a set of compositions, the set comprising a composition comprising component 1), a composition comprising component 2), and a composition comprising component 3).

[0104] The composition may be a pharmaceutically acceptable composition.

[0105] The set of compositions may comprise one or more (e.g., all) pharmaceutically acceptable compositions.

[0106] Also disclosed herein is a composition, e.g., a pharmaceutically acceptable composition, or set of compositions, e.g., a set of pharmaceutically acceptable compositions, comprising one, or more, or all of a CD4 +< T cell comprising a CAR CD4+< , a CD8 +< T cell comprising a CAR CD8+< , and a second CD8 +< T cell comprising a second CAR CD8+< , for use as a medicament.

[0107] Also disclosed herein is a composition, e.g., a pharmaceutically acceptable composition, or set of compositions, e.g., a set of pharmaceutically acceptable compositions, comprising one, or more, or all of a CD4 +< T cell comprising a CAR CD4+< , a CD8 +< T cell comprising a CAR CD8+< , and a second CD8 +< T cell comprising a second CAR CD8+< , for use as a medicament in the treatment of a disorder described herein, e.g., cancer.

[0108] Any of the CAR CD4+< , CAR CD8+< , or second CAR CD8 +< disclosed herein can be used in the compositions or set of compositions described herein.

[0109] The present invention provides a subset-optimized CART cell composition for use in a method of treating a subject having cancer, which utilises a CAR CD4+< , a CAR CD8+< and a second CAR CD8+< as defined in the claims.

[0110] Also disclosed herein is a kit comprising, one, or more, or all of the following components: 1) a nucleic acid comprising sequence encoding a CAR CD4+< comprising: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain; and 2) a nucleic acid comprising sequence encoding a CAR CD8+< comprising: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain; and, wherein the the CAR CD4+< and the CAR CD8+< differ from one another.

[0111] The CAR CD4+< may comprise an ICOS domain. The CAR CD8+< may not comprise an ICOS domain.

[0112] The kit may further comprise : 3) a nucleic acid comprising sequence encoding a second CAR CD8+< comprising: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain, wherein the second CARCD8+ differs from the CARCD4+ and the the CARCD8+.

[0113] The second CAR CD8+< may not comprise an ICOS signaling domain and comprises an intracellular signaling domain not present on the CAR CD8+< .

[0114] The kit may comprise component 1).

[0115] The kit may comprise component 2).

[0116] The kit may comprise component 3).

[0117] The kit may comprise a plurality of components 1), 2), and 3).

[0118] The kit may comprise components 1) and 2).

[0119] The kit may comprise 1) and 3).

[0120] The kit may comprise 1), 2) and 3).

[0121] The kit may comprise a plurality of components 1), 2) and 3) and each component of the plurality is disposed in a separate container.

[0122] The kit may further comprise a buffer.

[0123] The kit may further comprise a reagent useful for introducing one of the components into a T cell, e.g., a buffer or transfection reagent.

[0124] Component 1) may comprise a viral vector, e.g., a lenti viral vector.

[0125] Component 2) may comprise a viral vector, e.g., a lenti viral vector.

[0126] Component 3) may comprise a viral vector, e.g., a lenti viral vector.

[0127] The kit disclosed herein may comprise one, or more, or all of the following components: 1) a CD4 +< T cell, or preparation thereof, comprising a CAR (the CAR CD4+< comprising: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain; and 2) a CD8 +< T cell, or preparation thereof, comprising a CAR (the CAR CD8+< ) comprising: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain; and, wherein the CAR CD4+< and the CAR CD8+< differ from one another. The CAR CD4+< may comprise an ICOS domain. The CAR CD8+< may not comprise an ICOS domain.

[0128] The kit may further comprise 3) a second CD8 +< T cell, or preparation thereof, comprising a CAR (the second CAR CD8+< ) comprising: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain, wherein the second CARCD8+ differs from the CARCD4+ and the the CARCD8+.

[0129] The second CAR CD8+< may not comprise an ICOS signaling domain and may comprise an intracellular signaling domain not present on the CAR CD8+<

[0130] The CD4 +< T cell, or preparation thereof, and the CD8 +< T cell, or preparation thereof, may be disposed in a container.

[0131] The CD4 +< T cell, or preparation thereof, may be disposed in a first conatainer and the CD8 +< T cell, or preparation thereof, may be disposed in a second container.

[0132] The kit may comprise component 1).

[0133] The kit may comprise component2).

[0134] The kit may comprise component 3).

[0135] The kit may comprise a plurality of components 1), 2) and 3).

[0136] The kit may comprise components 1) and 2).

[0137] The kit may comprise components 1) and 3).

[0138] The kit may comprise components 1), 2) and 3).

[0139] The invention provides a kit comprising: (a) a CD4+ T cell comprising a CAR CD4< as defined in the claims; (b) a CD8+ T cell comprising a CAR CD8+< as defined in the claims; and (c) a CD8+ T cell comprising a second CAR CD8+< as defined in the claims.

[0140] In an embodiment, each component of the kit of the invention is disposed in a separate container.

[0141] Also disclosed herein is a kit comprising, one, or more, or all of, a nucleic acid comprising sequence encoding a CAR CD4+< a nucleic acid comprising sequence encoding a CAR CD8+< , and a nucleic acid comprising sequence encoding a second CAR CD8+< , for use as a medicament.

[0142] Also disclosed herein is a kit comprising, one, or more, or all of, a nucleic acid comprising sequence encoding a CAR CD4+< , a nucleic acid comprising sequence encoding a CAR CD8+< , and a nucleic acid comprising sequence encoding a second CAR CD8+< , for use as a medicament in the treatment of a disorder described herein, e.g., cancer.

[0143] Also disclosed herein is a kit comprising one, or more, or all of a CD4 +< T cell, or preparation thereof, comprising a CAR CD4+< , a CD8 +< T cell, or preparation thereof, comprising a CAR CD8+< , and a second CD8 +< T cell, or preparation thereof, comprising a second CAR CD8+< , for use as a medicament.

[0144] Also disclosed herein is a kit comprising one, or more, or all of a CD4 +< T cell, or preparation thereof, comprising a CAR CD4+< , a CD8 +< T cell, or preparation thereof, comprising a CAR CD8+< , and a second CD8 +< T cell, or preparation thereof, comprising a second CAR CD8+< , for use as a medicament in the treatment of a disorder described herein, e.g., cancer.

[0145] Any of the CAR CD4+< , CAR CD8+< , or second CAR CD8 +< disclosed herein can be used in the kit, or the components of the kit, described herein.

[0146] In an embodiment, at least 10, 20, 30, 40, 50, 60, 70, or 80 % of the T cells, or a preparation thereof, in the combination or kit of the invention are CD4 +< T cells comprising a CAR CD4+< .

[0147] In an embodiment, at least 10, 20, 30, 40, 50, 60, 70, or 80 % of the T cells, or a prepration thereof, in the combination or kit of the invention are CD8 +< T cells comprising a CAR CD8+< .

[0148] In an embodiment, at least 10, 20, 30, 40, 50, 60, 70, or 80 % of the T cells , or a preparation thereof, in the combination or kit of the invention are CD4 +< T cells comprising a CAR CD4+< ; and at least 10, 20, 30, 40, 50, 60, 70, or 80% of the T cells, or a preparation thereof, in the combination or a kit of the invention are CD8 +< T cells comprising a CAR CD8+< or second CAR CD8+< (it being understood that the total cells do not exceed 100%).

[0149] In an embodiment, at least 10 to 70% of the T cells, or a preparation thereof, in the combination or kit of the invention are CD4 +< T cells comprising a CAR CD4+< ; and at least 10 to 70% of the T cells, or a preparation thereof, in the combination or kit of the invention are CD8 +< T cells comprising a CAR CD8+< or second CAR CD8+< (it being understood that the total cells do not exceed 100%).

[0150] In an embodiment, at least 10, 20, 30, 40, 50, 60, 70, or 80 % of the T cells, or a preparation thereof, in the combination or kit of the invention are CD4 +< T cells comprising a CAR CD4+< ; at least 10, 20, 30, 40, 50, 60, 70, or 80 % of the T cells, or a preparation thereof, in the combination or kit of the invention are CD8 +< T cells comprising a CAR CD8+< ; and at least 10, 20, 30, 40, 50, 60, 70, or 80 % of the T cells, or a preparation thereof, in the combination or kit of the invention are CD8 +< T cells comprising a second CAR CD8+< (it being understood that the total cells do not exceed 100%).

[0151] Also disclosed herein is a method of making a CD4 +< T cell comprising a CAR (a CAR CD4+< ) and a CD8 +< T cell comprising a CAR (a CAR CD8+< ) (or a kit or composition comprising the same), the method comprising: providing a CD4 +< T cell comprising a CAR CD4+< , e.g., by introducing a nucleic acid sequence that encodes a CAR CD4+< into a CD4 +< T cell; providing a CD8 +< T cell comprising a CAR CD8+< , e.g., by introducing a nucleic acid sequence that encodes a CAR CD8+< into a CD8+ T cell; wherein, the CAR CD4+< comprises: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain; and the CAR CD8+< comprises: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain, wherein the CAR CD4+< and the CAR CD4+< are different. The CAR CD4+< may comprise an ICOS domain. The CAR CD8+< may not comprise an ICOS domain.

[0152] The method of making a CD4 +< T cell comprising a CAR (a CAR CD4+< ) and a CD8 +< T cell comprising a CAR (a CAR CD8+< ) (or a kit or composition comprising the same) may comprise: a) providing a CD4 +< T cell; b) introducing a nucleic acid sequence that encodes a CAR CD4+< into the CD4 +< T cell to provide a CD4 +< T cell comprising a CAR CD4+< ; c) providing a CD8 +< T cell; d) introducing a nucleic acid sequence that encodes a CAR CD8+< into the CD8+ T cell to provide a CD8 +< T cell comprising a CAR CD8+< . c) may be performed before a). Step a) may comprise selecting a CD4 +< T cell, e.g., by negative selection or by cell sorting. Step c) may comprise selecting a CD8 +< T cell, e.g., by negative selection or by cell sorting. a) and c) may be performed simultaneously.

[0153] The method of making a CD4 +< T cell comprising a CAR (a CAR CD4+< ) and a CD8 +< T cell comprising a CAR (a CAR CD8+< ) (or a kit or composition comprising the same) may further comprise e) Th 17 polarizing the CD4 +< T cell or the CD4 +< T cell comprising a CAR CD4+< .

[0154] The method may further comprise expanding the CD4 +< T cell to provide progeny CD4 +< T cells. The method may further comprise expanding the CD4 +< T cell comprising a CAR CD4+< to provide progeny CD4 +< T cells comprising a CAR CD4+< .

[0155] The method may further comprise expanding the Th 17 polarized CD4 +< T cell to provide progeny Th17 polarized CD4 +< T cells. The method may further comprise expanding the Th 17 polarized CD4 +< T cell comprising a CAR CD4+< to provide Th 17 polarized progeny CD4 +< T cells comprising a CAR CD4+< .

[0156] The method may further comprise expanding the CD8+ T cell to provide progeny CD8 +< T cells. The method may further comprise expanding the CD8 +< T cell comprising a CAR CD8+< to provide progeny CD8 +< T cells comprising a CAR CD8+< .

[0157] The method may further comprise providing a second CD8 +< T cell comprising a second CAR CD8+< , e.g., by introducing a nucleic acid sequence that encodes a CAR CD8+< into a CD8 +< T cell; wherein, the second CAR CD8+< comprises: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain, wherein the second CAR CD8+< differs from the CAR CD4+< .

[0158] The second CAR CD8+< may not comprise an ICOS signaling domain and comprises an intracellular signaling domain not present on the CAR CD8+< .

[0159] The method may further comprise f) providing a second CD8 +< T cell; g) introducing a nucleic acid sequence that encodes a second CAR CD8+< into the CD8+ T cell to provide a CD8 +< T cell comprising a CAR CD8+< . f) may be performed before a). f) may comprise selecting a second CD8 +< T cell, e.g., by negative selection or by cell sorting.

[0160] The method may further comprise expanding the second CD8 +< T cell to provide progeny second CD8 +< T cells. The method may further comprise expanding the second CD8 +< T cell comprising a CAR CD8+< to provide progeny second CD8 +< T cells comprising a secondCAR CD8+< .

[0161] The methosd of making disclosed herein may further comprise contacting the population of CD4+ cells, CD8+ cells, or both CD4+ and CD8+ cells, with a nucleic acid encoding a telomerase subunit, e.g., hTERT. The nucleic acid encoding the telomerase subunit can be DNA.

[0162] The method of making disclosed herein may further comprise culturing the population of of CD4+ cells, CD8+ cells, or both CD4+ and CD8+ cells, in serum comprising 2% hAB serum.

[0163] Headings, sub-headings or numbered or lettered elements, e.g., (a), (b), (i) etc, are presented merely for ease of reading. The use of headings or numbered or lettered elements in this document does not require the steps or elements be performed in alphabetical order or that the steps or elements are necessarily discrete from one another.BRIEF DESCRIPTION OF THE DRAWINGS

[0164] Figures 1A and 1B are schematic representations of ICOS-based CARs. Figure 1A shows a panel of chimeric receptors that contain the SS1 single chain fragment that binds to mesothelin and differ in the intracellular domain. Figure 1B shows a panel of chimeric receptors that contain the Mov19 single chain fragment that binds to Folate Receptor-α and differ in the intracellular domain. Figures 2A and 2B show tumor cell killing by CD4 +< and CD8 +< T cells redirected with SS1-CARs. CD4 +< (Fig. 2A) and CD8 +< T cells (Fig. 2B) were cocultured with firefly Luciferase (fLuc)-expressing L55 target cells for 18 hours at the indicated effector-target (E:T) ratios. Specific cytolysis was determined using a bioluminescence assay. Figures 3A, 3B, 3C, 3D, and 3E show the cytokine release by redirected CD4 +< and CD8 +< T cells after antigen recognition in tumor cells. CD4 +< T cells (Figs. 3A, 3B, and 3C) or CD8 +< T cells (Fig. 3D and 3E) (4 x 10 5< , 60% chimeric receptor positive) were cocultured with 2 x 10 5< tumor cells in culture media. Supernatants were obtained 24 hours after coculture, and TNF-α, IL-2 and IFN-γ were analyzed by ELISA. Error bars indicate standard deviation (SD) in duplicate samples. Figures 4A, 4B and 4C show that ICOS intracellular domain enhanced the in vivo persistence of CAR-expressing CD4 +< T cells, an effect that is independent of the CAR intracellular domain used to redirect CD8 +< T cells. NSG mice bearing subcutaneous non-small cell lung tumors (L55) were treated 30 days after tumor implantation with two doses CD4 +< and CD8 +< T cells redirected with SS1-CARs. The concentration of CD4 +< T cells was determined in the blood of treated animals 22 days after T cell injection. Error bars represent SEM (n=7-10) Figure 4A shows that CD4 +< and CD8 +< T cells were redirected with the same construct (z, 28z, BBz or ICOSz). Figure 4B shows that CD8 +< T cells were redirected with SS1-BBz and CD4 +< T cells were left untransduced (UTD-BBz) or redirected with 28z (28z-BBz), BBz (BBz-BBz) or ICOSz (ICOSz-BBz). Figure 4C shows that CD4 +< T cells were redirected with ICOSz and CD8 +< T cells were redirected with 28z, BBz or ICOSz. Figures 5A, 5B, and 5C show CD4 +< T cells expressing an ICOS-based CAR significantly increased the persistence of CD8 +< T cells expressing either CD28- or 4-1BB-based SS1-CARs. NSG mice bearing subcutaneous non-small cell lung tumors (L55) were treated 30 days after tumor implantation with two doses CD4 +< and CD8 +< T cells redirected with SS1-CARs. The concentration of CD8 +< T cells was determined in the blood of treated animals 22 days after T cell injection. Error bars represent SEM (n=7-10). In Figure 5A, both CD4 +< and CD8 +< T cells were redirected with the same construct (z, 28z, BBz or ICOSz). In Figure 5B, CD8 +< T cells were redirected with SS1-BBz and CD4 +< T cells were left untransduced (UTD-BBz) or redirected with 28z (28z-BBz), BBz (BBz-BBz) or ICOSz (ICOSz-BBz). In Figure 5C, CD8 +< T cells were redirected with 28z and CD4 +< T cells were redirected with 28z or ICOSz. Figures 6A and 6B show T H 17-polarized CD4 +< T cells expressing an ICOS-based CAR significantly increased the circulatory persistence of bulk CD8 +< T cells expressing 4-1BB-based CARs. NSG mice bearing subcutaneous non-small cell lung tumors (L55) were treated 30 days after tumor implantation with two doses CD4 +< and CD8 +< T cells redirected with SS1-CARs. CD8 +< T cells were redirected with BBz and CD4 +< T cells were left untransduced or redirected with ICOSz. ICOSz redirected CD4 +< T cells were cultured with or without T H 17 polarizing conditions. The concentration of CD4 +< T cells (Fig. 6A) and CD8 +< T cells (Fig. 6B) was determined in the blood of treated animals 22 days after T cell injection. Error bars represent SEM (n=7-10). Figure 7 shows the antitumor effect and T cell persistence of CD4 +< and CD8 +< T cells redirected with SS1-CARs in mice with non-small cell lung tumors. NSG mice bearing subcutaneous ovarian tumors (L55) were treated 15 days after tumor implantation with two doses CD4 +< and CD8 +< T cells redirected with SS1-CARs. CD8 +< T cells were redirected with 28z and CD4 +< T cells were left untransduced or redirected with 28z, BBz or ICOSz. Figure 7 depicts tumor volume was analyzed 13 days following T cell injection. Results are expressed as a mean tumor volume (+ / - SE) with n=7-8 mice per group. Figure 8 shows CD4 +< T cell infiltration in tumors treated with redirected T cells. NSG mice bearing subcutaneous non-small cell lung tumors (L55) were treated 15 days after tumor implantation with two doses CD4 +< and CD8 +< T cells redirected with SS1-CARs. CD8 +< T cells were redirected with 28z and CD4 +< T cells were left untransduced or redirected with 28z, BBz or ICOSz. ICOSz redirected CD4 +< T cells were cultured with or without T H 17 polarizing conditions. The immune infiltrate was evaluated by immunohistochemistry for human CD4 +< T cells at day 27 after treatment. The percentage of nuclei that stained positive for CD4 +< T cells in intact areas of tumor was quantified with Aperio ImageScope software. Box plots show median (line) and 25th-75th percentile (box). The end of the whiskers represents the minimum and the maximum of all of the data. * p > 0.05 vs all groups, ** p > 0.05 vs UTD-28z and BBz-28z. Figures 9A, 9B, and 9C show the antitumor effect and T cell persistence of CD4 +< and CD8 +< T cells redirected with Mov19-CARs in mice with ovarian tumors. NSG mice bearing subcutaneous ovarian tumors (SKOV3) were treated 30 days after tumor implantation with two doses CD4 +< and CD8 +< T cells redirected with Mov19-CARs. CD8 +< T cells were redirected with BBz and CD4 +< T cells were left untransduced or redirected with z, 28z, BBz or ICOSz. Figure 8A depicts tumor volume was analyzed at indicated time points. Results are expressed as a mean tumor volume (+ / - SE) with n=6-8 mice per group. The concentration of CD4 +< (Fig. 9B) and CD8 +< T cells (Fig. 9C) were determined in the blood of treated animals 27 days after T cell injection. Error bars represent SEM (n=6-8). Figures 10A, 10B, 10C, and 10D show combination therapy using CD4 +< T cells redirected with an ICOS-based CAR and CD8 +< T cells redirected with 28z and BBz CARs. NSG mice bearing ovarian (SKOV3), pancreatic (Capan-2) or lung (L55) subcutaneous tumors were treated 15 days after tumor implantation with two doses CD4 +< and CD8 +< T cells redirected with SS1-CARs. CD4 +< T cells were redirected with ICOSz and CD8 +< T cells were redirected with a mix of 28z and BBz CARs. Tumor volume was analyzed at indicated time points for the ovarian tumor model (SKOV3) (Fig. 10A), pancreatic tumor model (Capan-2) (Fig. 10B), and lung tumor (L55) (Fig. 10C). Results are expressed as a mean tumor volume (+ / - SE) with n=5 mice per group. White circles indicate the control population and black squares indicate the treated population. In Figure 10D, the concentrations of CD4 +< (white bars) and CD8 +< T cells (hatched bars) were determined in the blood of treated animals 33 days after T cell injection. Error bars represent SEM (n=5). Figures 11A, 11B, 11C and 11D show the antitumor effect and T cell persistence of CD4 +< T cells redirected with an ICOS-mutant-CAR in mice with pancreatic tumors. Figure 11A shows the amino acid sequences of the intracellular domain of ICOS used in chimeric antigen receptors. The FMFM (SEQ ID NO: 47) mutations are underlined and the signaling interaction with PI3K is indicated. NSG mice bearing subcutaneous pancreatic tumors (Capan-2) were treated 15 days after tumor implantation with two doses CD4 +< and CD8 +< T cells redirected with SS1-CARs. CD8 +< T cells were redirected with BBz and CD4 +< T cells were redirected with delz, BBz, ICOSz or ICOS(FMFM)z (Fig. 11B) ("FMFM" disclosed as SEQ ID NO: 47). Tumor volume was analyzed at indicated time points. Results are expressed as a mean tumor volume (+ / - SE) with n=6-8 mice per group. The concentration of CD4 +< (Fig. 11C) and CD8 +< T cells (Fig. 12D) were determined in the blood of treated animals 21 days after T cell injection. Error bars represent SEM (n=6-8). Figure 12 shows that the proliferation of CAR-expressing, transduced T cells is enhanced by low doses of RAD001 in a cell culture system. CARTs were co-cultured with NALM6 (Nalm-6) cells in the presence of different concentrations of RAD001 (nM). The number of CAR-positive CD3-positive T cells (black) and total T cells (white) was assessed after 4 days of co-culture. Figure 13 depicts tumor growth measurements of NALM6-luc cells with daily RAD001 dosing at 0.3, 1, 3, and 10 mg / kg (mpk) or vehicle dosing. Circles denote the vehicle; squares denote the 10 mg / kg dose of RAD001; triangles denote the 3 mg / kg dose of RAD001, inverted triangles denote the 1 mg / kg dose of RAD001; and diamonds denote the 0.3 mg / kg dose of RAD001. Figures 14A and 14B show pharmacokinetic curves showing the amount of RAD001 in the blood of NSG mice with NALM6 tumors. FIG. 14A shows day 0 PK following the first dose of RAD001. FIG. 14B shows Day 14 PK following the final RAD001 dose. Diamonds denote the 10 mg / kg dose of RAD001; squares denote the 1 mg / kg dose of RAD001; triangles denote the 3 mg / kg dose of RAD001; and x's denote the 10 mg / kg dose of RAD001. Figures 15A and 15B show in vivo proliferation of humanized CD19 CART cells with and without RAD001 dosing. Low doses of RAD001 (0.003 mg / kg) daily lead to an enhancement in CAR T cell proliferation, above the normal level of huCAR19 proliferation. Figures 15A shows CD4+ CAR T cells; FIG. 15B shows CD8+ CAR T cells. Circles denote PBS; squares denote huCTL019; triangles denote huCTL019 with 3 mg / kg RAD001; inverted triangles denote huCTL019 with 0.3 mg / kg RAD001; diamonds denote huCTL019 with 0.03 mg / kg RAD001; and circles denote huCTL019 with 0.003 mg / kg RAD001. FURTHER DETAILED DESCRIPTION DEFINITIONS

[0165] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

[0166] "A" and "an" as the term is used herein, refers to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0167] "About" as the term is used herein, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or in some embodiments ±10%, or in some embodiments ±5%, or in some embodiments ±1%, or in some embodiments ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0168] The phrase "about to receive", when used herein in the context of a patient receiving a first therapeutic who is about to receive a second therapeutic, refers to a situation where the patient is receiving or has received the first therapeutic for a disorder (e.g., a cancer), wherein the patient receives or will receive the second therapeutic in the course of treatment for that disorder.

[0169] An "antigen binding domain" as the term is used herein, refers to a molecule that has affinity for a target antigen, typically an antigen on a target cell, e.g., a cancer cell. An exemplary antigen binding domain comprises a polypeptide, e.g., an antibody molecule (which includes an antibody, and antigen binding fragments thereof, e.g., a immunoglobulin, single domain antibody (sdAb, e.g., a nanobody, and an scFv), or a non-antibody scaffold, e.g., a fibronectin, and the like. In embodiments, the antigen binding domain is a single polypeptide. In embodiments, the antigen binding domain comprises, one, two, or more, polypeptides. In embodiments the antigen binding domain comprises a fragment of an antibody that is sufficient to confer recognition and specific binding to the target antigen. Examples of an antibody fragment include, but are not limited to, an Fab, Fab', F(ab') 2 , or Fv fragment, an scFv antibody fragment, a linear antibody, single domain antibody such as an sdAb, e.g., a nanobody, (either VL or VH), a camelid VHH domain, and multi-specific antibodies formed from antibody fragments. In an embodiment, the antigen binding domain is a "scFv," which can comprise a fusion protein comprising a VL chain and a VH chain of an antibody, where the VH and VL are linked via a short flexible polypeptide linker. The scFv is capable of being expressed as a single chain polypeptide and retains the specificity of the intact antibody from which it is derived. Moreover, the VL and VH variable chains can be linked in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL. In embodiments, the antigen binding domain comprises a non antibody scaffold, e.g., a fibronectin, ankyrin, domain antibody, e.g., a nanobody, lipocalin, small modular immuno-pharmaceutical, maxybody, Protein A, or affilin. The non antibody scaffold has the ability to bind to target antigen on a cell. In embodiments, the antigen binding domain is a polypeptide or fragment thereof of a naturally occurring protein expressed on a cell. In an embodiment, the antigen binding domain binds a growth factor or hormone receptor. While not wishing to be bound by theory, the antigen binding domain serves to provide specificity for target cells, and in embodiments, optimize and immune effector function by coupling antigen binding to generation of a signal by an intracellular signaling domain on an intracellular signaling member. Extracellular domains that bind a counter ligand, e.g., on target cells, are also within the definition of antigen binding domain.

[0170] As used herein, the term "antibody molecule" refers to a protein, e.g., an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. In an embodiment, an antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In an embodiment, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

[0171] The term "antibody," as used herein, refers to an immunoglobulin molecule which specifically binds with a target antigen. An antibody can be intact immunoglobulin derived from natural sources or from recombinant sources and can be an immunoreactive portion of intact immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. The antibody molecule described herein may exist in a variety of forms where the antigen binding portion of the antibody is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) and a humanized or human antibody, e.g., as described herein.

[0172] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, a single chain domain antibody (sdAb), Fab, Fab', F(ab')2, Fv fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CH1 domains, linear antibodies, scFv antibodies, a linear antibody, single domain antibody such as an sdAb (either VL or VH), a camelid VHH domain, multispecific molecules formed from antibody fragments such as a bivalent fragment comprising two or more, e.g., two, Fab fragments linked by a disulfide bridge at the hinge region, or two or more, e.g., two isolated CDR or other epitope binding fragments of an antibody linked. An antibody fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antibody fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3)(see U.S. Patent No.: 6,703,199, which describes fibronectin polypeptide minibodies).

[0173] An "antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0174] An "antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. κ and λ light chains refer to the two major antibody light chain isotypes.

[0175] By the term "synthetic antibody" as used herein, is meant an antibody molecule which is generated using recombinant DNA technology, such as, for example, an antibody molecule expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody molecule which has been generated by the synthesis of a DNA molecule encoding the antibody molecule and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

[0176] The term "apheresis" as used herein refers to the art-recognized extracorporeal process by which the blood of a donor or patient is removed from the donor or patient and passed through an apparatus that separates out selected particular constituent(s) and returns the remainder to the circulation of the donor or patient, e.g., by retransfusion. Thus, "an apheresis sample" refers to a sample obtained using apheresis.

[0177] "Anti-tumor effect" as the term is used herein, refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, decrease in tumor cell proliferation, decrease in tumor cell survival, or amelioration of various physiological symptoms associated with the cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the disclosure in prevention of the occurrence of tumor in the first place.

[0178] "Autologous" as the term is used herein refers to any material derived from the same individual to whom it is later to be re-introduced.

[0179] "Allogeneic" as the term is used herein refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.

[0180] "Cancer" as the term is used herein, refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like. In an embodiment, a cancer is characterized by expression of a PD-1 ligand, e.g., PD-L1 or PD-L2, on a cancer cell or in a tumor microenvironment. The term "cancer" includes all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term "cancer" or "tumor" includes premalignant, as well as malignant cancers and tumors.

[0181] The terms "cancer associated antigen" or "tumor antigen" or "proliferative disorder antigen" or "antigen associated with a proliferative disorder" interchangeably refers to a molecule (typically protein, carbohydrate or lipid) that is preferentially expressed on the surface of a cancer cell, either entirely or as a fragment (e.g., MHC / peptide), in comparison to a normal cell, and which is useful for the preferential targeting of a pharmacological agent to the cancer cell. In some embodiments, the tumor antigen is an antigen that is common to a specific proliferative disorder. In some embodiments, a cancer-associated antigen is a cell surface molecule that is overexpressed in a cancer cell in comparison to a normal cell, for instance, 1-fold over expression, 2-fold overexpression, 3-fold overexpression or more in comparison to a normal cell. In some enbodiments, a cancer-associated antigen is a cell surface molecule that is inappropriately synthesized in the cancer cell, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal cell. In some enbodiments, a cancer-associated antigen will be expressed exclusively on the cell surface of a cancer cell, entirely or as a fragment (e.g., MHC / peptide), and not synthesized or expressed on the surface of a normal cell. In some embodiments, the CARs of the present invention includes CARs comprising an antigen binding domain (e.g., antibody or antibody fragment) that binds to a MHC presented peptide. Normally, peptides derived from endogenous proteins fill the pockets of Major histocompatibility complex (MHC) class I molecules, and are recognized by T cell receptors (TCRs) on CD8 + T lymphocytes. The MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets for immunotherapy. TCR-like antibodies targeting peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, e.g., Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Bood, 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21) :1601-1608 ; Dao et al., Sci Transl Med 2013 5(176) :176ra33 ; Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example, TCR-like antibody can be identified from screening a library, such as a human scFv phage displayed library. Accordingly, the present invention provides CARs that comprising an antigen binding domain that binds to a MHC presented peptide of a molecule selected from the group of WT1, NY-ESO-1, LAGE-1a, MAGE-A1 and RAGE-1.

[0182] "Chimeric Antigen Receptor" or alternatively a "CAR" as the term is used herein, refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as "an intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and / or costimulatory molecule as defined below. In some embodiments, the domains in the CAR polypeptide construct are in the same polypeptide chain, e.g., comprise a chimeric fusion protein. In other embodiments, the domains in the CAR polypeptide construct are not contiguous with each other, e.g., are in different polypeptide chains, e.g., as provided in an RCAR as described herein.

[0183] In one aspect, the stimulatory molecule of the CAR is the zeta chain associated with the T cell receptor complex (e.g., CD3 zeta). In one aspect, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3-zeta). In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one aspect, the costimulatory molecule is chosen from 4-1BB (i.e., CD137), CD27, CD28 and / or ICOS. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more co-stimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one aspect the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane.

[0184] The portion of the CAR of use in the invention comprising an antibody or antibody fragment thereof may exist in a variety of forms, for example, where the antigen binding domain is expressed as part of a polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), or e.g., a humanized antibody, or bispecific antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one aspect, the antigen binding domain of a CAR composition of the invention comprises an antibody fragment. In a further aspect, the CAR comprises an antibody fragment that comprises a scFv.

[0185] A CAR that comprises an antigen binding domain (e.g., a scFv, a single domain antibody, or TCR (e.g., a TCR alpha binding domain or TCR beta binding domain)) that targets a specific cancer associated antigen (or tumor marker) X, such as those described herein, is also referred to as XCAR. For example, a CAR that comprises an antigen binding domain that targets CD19 is referred to as CD19CAR.

[0186] "CARX cell," as that term is used herein, refers to a cell comprising CAR. Any cell that is engineered to express a CAR can be used as a CARX cell. Typically the CARX cell is a T cell comprising a CAR, and is referred to as a CART cell. In the invention, a CART is a CD4 +< T cell comprising a CAR, and is referred to herein as a CD4 +< T cell comprising a CAR CD4+< . In the invention, a CART is a CD8+ T cell comprising a CAR, and is referred to herein as a CD8+ T cell comprising a CAR CD8+< . In an embodiment the CART cell is autologous to the patient. In an embodiment the CART is allogeneic to the patient. In the invention, a patient receives a CD4 +< T cell comprising a CAR CD4+< and a CD8+ T cell comprising a CAR CD8+< .

[0187] "CAR CD4+< " and "CAR CD8+< " as those terms are used herein, refer to a CAR associated with, respectively, a CD4 +< T cell and a CD8+ T cell. In an embodiment, the CAR CD4+< is provided in, designed to optimize the performance of, or optimizes the performance of, a CD4 +< T cell. In an embodiment, the CAR CD8+< is provided in, designed to optimize the performance of, or optimizes the performance of, a CD8+ T cell. Typically, a CAR CD4+< optimizes the performance of a CD4 +< T cell and, in the invention, comprises an ICOS domain. Typically, a CAR CD8+< optimizes the performance of a CD8+ T cell and, in embodiments, comprises a CD28 or 4-1BB domain. Thus, CARs can be optimized for a selected subset of T cells, e.g., CD4 +< T cells or CD8 +< T cells.

[0188] A CD4 +< T cell comprising a CAR CD4+< and a CD8+ T cell comprising a CAR CD8+< , e.g., when provided in a kit, composition, or administered to a patient, differ from one another in structure. The CAR CD4+< and the CAR CD8+< can differ from one another, e.g., wherein the intracellular signaling domain of the CAR CD4+< differs from the CAR CD8+< intracellular signaling domain, wherein the the CAR CD4+< comprises a first costimulatory domain not present on the the CAR CD8+< , wherein the the CAR CD4+< comprises a first costimulatory domain and the CAR CD8+< comprises a second costimulatory domain, or wherein the the CAR CD4+< comprises a first costimulatory domain, e.g., an ICOS domain, not present on the CAR CD8+< and the CAR CD8+< comprises a second costimulatory domain, e.g., a CD28 or 4-1BB domain, not present on the CAR CD4+< .

[0189] A CD4 +< T cell, as that term is used herein, refers to a T cell which expresses the surface protein CD4. In an embodiment, the T cell expresses the surface protein CD4 at a sufficient level to be detected, e.g., by flow cytometry.

[0190] A CD8 +< T cell, as that term is used herein, refers to a T cell which expresses the surface protein CD8. In an embodiment, the T cell expresses the surface protein CD8 at a sufficient level to be detected, e.g., by flow cytometry.

[0191] The terms "complementarity determining region" or "CDR," as used herein, refer to the sequences of amino acids within antibody variable regions which confer antigen specificity and binding affinity. For example, in general, there are three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme), or a combination thereof. Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments, the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In a combined Kabat and Chothia numbering scheme, in some embodiments, the CDRs correspond to the amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both. For instance, in some embodiments, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in a VH, e.g., a mammalian VH, e.g., a human VH; and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in a VL, e.g., a mammalian VL, e.g., a human VL.

[0192] "Costimulatory signaling domain," as that term is used herein, refers to an intracellular signaling domain of a molecule, e.g., an endogenous molecule, of the CART cell that, upon binding to its cognate counter ligand on a target cell, enhance, e.g., increases, an immune effector response. A costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. Costimulatory molecules include, but are not limited to an MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, and a ligand that specifically binds with CD83. "Costimulatory molecule" refers to a molecule comprising a "costimulatory signaling domain." A costimulatory intracellular signaling domain can be derived from the intracellular portion of a costimulatory molecule. The intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.

[0193] As used herein, the term "CD19" refers to the Cluster of Differentiation 19 protein, which is an antigenic determinant detectable on leukemia precursor cells. The human and murine amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found as UniProt / Swiss-Prot Accession No. P15391 and the nucleotide sequence encoding of the human CD19 can be found at Accession No. NM_001178098. As used herein, "CD19" includes proteins comprising mutations, e.g., point mutations, fragments, insertions, deletions and splice variants of full length wild-type CD19. CD19 is expressed on most B lineage cancers, including, e.g., acute lymphoblastic leukaemia, chronic lymphocyte leukaemia and non-Hodgkin lymphoma. Other cells with express CD19 are provided below in the definition of "disease associated with expression of CD19." It is also an early marker of B cell progenitors. See, e.g., Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one aspect the antigen-binding portion of the CART recognizes and binds an antigen within the extracellular domain of the CD19 protein. In one aspect, the CD19 protein is expressed on a cancer cell.

[0194] "Derived from" as that term is used herein, indicates a relationship between a first and a second molecule. It generally refers to structural similarity between the first molecule and a second molecule and does not conotate or include a process or source limitation on a first molecule that is derived from a second molecule. For example, in the case of an intracellular signaling domain that is derived from a CD3zeta molecule, the intracellular signaling domain retains sufficient CD3zeta structure such that is has the required function, namely, the ability to generate a signal under the appropriate conditions. It does not conotate or include a limitation to a particular process of producing the intracellular signaling domain, e.g., it does not mean that, to provide the intracellular signaling domain, one must start with a CD3zeta sequence and delete unwanted sequence, or impose mutations, to arrive at the intracellular signaling domain.

[0195] The phrase "disease associated with expression of a tumor marker as described herein" includes, but is not limited to, a disease associated with a cell that expresses a tumor marker as described herein or condition associated with a cell which expresses, or at any time expressed, a tumor marker as described herein including, e.g., proliferative diseases such as a cancer or malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia; or a noncancer related indication associated with a cell which expresses a tumor marker as described herein. In one aspect, a cancer associated with expression of a tumor marker as described herein is a hematological cancer. In one aspect, a cancer associated with expression of a tumor marker as described herein is a solid cancer. Further diseases associated with expression of a tumor marker as described herein include, but not limited to, e.g., atypical and / or non-classical cancers, malignancies, precancerous conditions or proliferative diseases associated with expression of a tumor marker as described herein. Non-cancer related indications associated with expression of a tumor marker as described herein include, but are not limited to, e.g., autoimmune disease, (e.g., lupus), inflammatory disorders (allergy and asthma) and transplantation.

[0196] The phrase "disease associated with expression of CD19" includes, but is not limited to, a disease associated with a cell that expresses CD19 or condition associated with a cell which expresses, or at any time expressed, CD19 including, e.g., proliferative diseases such as a cancer or malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia; or a noncancer related indication associated with a cell which expresses CD19. For the avoidance of doubt, a disease associated with expression of CD19 may include a condition associated with a cell which does not presently express CD19, e.g., because CD19 expression has been downregulated, e.g., due to treatment with a molecule targeting CD19, e.g., a CD19 CAR, but which at one time expressed CD19. In one aspect, a cancer associated with expression of CD19 is a hematological cancer. In one aspect, the hematolical cancer is a leukemia or a lymphoma. In one aspect, a cancer associated with expression of CD19 includes cancers and malignancies including, but not limited to, e.g., one or more acute leukemias including but not limited to, e.g., B-cell acute Lymphoid Leukemia (BALL), T-cell acute Lymphoid Leukemia (TALL), acute lymphoid leukemia (ALL); one or more chronic leukemias including but not limited to, e.g., chronic myelogenous leukemia (CML), Chronic Lymphoid Leukemia (CLL). Additional cancers or hematologic conditions associated with expression of CD19 comprise, but are not limited to, e.g., B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, Follicular lymphoma, Hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma (MCL), Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and "preleukemia" which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells, and the like. Further diseases associated with expression of CD19 expression include, but not limited to, e.g., atypical and / or non-classical cancers, malignancies, precancerous conditions or proliferative diseases associated with expression of CD19. Non-cancer related indications associated with expression of CD19 include, but are not limited to, e.g., autoimmune disease, (e.g., lupus), inflammatory disorders (allergy and asthma) and transplantation. In some embodiments, the tumor antigen-expressing cell expresses, or at any time expressed, mRNA encoding the tumor antigen. In an embodiment, the tumor antigen -expressing cell produces the tumor antigen protein (e.g., wild-type or mutant), and the tumor antigen protein may be present at normal levels or reduced levels. In an embodiment, the tumor antigen -expressing cell produced detectable levels of a tumor antigen protein at one point, and subsequently produced substantially no detectable tumor antigen protein.

[0197] "dsRNA," as that term is used herein, refers to a nucleic acid molecule, having at least a region of duplexed structure, that is capable of mediating sequence specific inhibition of the expression of a target gene. dsRNAs comprise short interfering RNA (siRNA) and short hairpin RNA (shRNA). In embodiments, shRNA is similar in structure to an siRNA but includes a moiety, typically one or more RNA monomers, that connect a duplex region of sense and an antisense sequence. In an embodiment the shRNA, after intracellular processing (e.g., by Dicer), results in a 19-23 nucleotide duplex siRNA with 2 nucleotide 3' overhangs.

[0198] "Endogenous" as that term is used herein, refers to any material, e.g., a polypeptide, from or produced inside an organism, cell, tissue or system.

[0199] "Exogenous" as that term is used herein, refers to any material, e.g., a polypeptide, or dimerization molecule, introduced from or produced outside an organism, cell, tissue or system.

[0200] "ICOS domain" as that term is used herein, refers to a functional (in terms of intracellular signaling) fragment, or analog, of an ICOS molecule. It can comprise the entire intracellular region, or a fragment of the intracellular region which is sufficient for generation of an intracellular signal, e.g., when an antigen binding domain to which it is fused binds cognate antigen. In embodiments the ICOS domain has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99 % sequence identity with, or differs by no more than 15, 10, 5, 4, 3, 2, or 1 amino acid residues from, the corresponding residues of the entire intracellular region, or a fragment of the intracellular region which is sufficient for generation of an intracellular signal, of a naturally occurring ICOS molecule, e.g., a human, or other mammalian, e.g., a nonhuman species, e.g., rodent, monkey, ape or murine intracellular costimulatory molecule. In embodiments the costimulatory domain has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99 % sequence identity with, or differs by no more than 15, 10, 5, 4, 3, 2, or 1 amino acid residues from, SEQ ID NO: 40.

[0201] "Immune effector cell," as that term is used herein, refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response. Examples of immune effector cells include T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloic-derived phagocytes.

[0202] "Immune effector function" or "immune effector response," as that term is used herein, refers to function or response, e.g., of an immune effector cell, that enhances or promotes an immune attack of a target cell. E.g., an immune effector function or response refers a property of a T or NK cell that promotes killing or the inhibition of growth or proliferation, of a target cell. In the case of a T cell, primary stimulation and costimulation are examples of immune effector function or response. An immune effector function or response can be promoted by the action of a CAR, and can, e.g., result in a CARX cell that is more effective at proliferation, cytokine production, cytotoxicity or upregulation of cell surface markers such as CD25, CD69, CD107a.

[0203] An "inhibitory extracellular domain," as that term is used herein, refers to polypeptide comprising an extracellular domain of an inhibitory molecule. Normally, binding to its counterligand has an inhibitory effect on the generation of an immune effector response. When linked, e.g., fused to an intracellular signaling domain, it redirects an interaction that normally inhibits the generation of an immune effector response into one that promotes an immune effector response.

[0204] "Inhibitory molecule," as that term is used herein, refers to a molecule, e.g., an endogenous molecule, of CARX cell, e.g., a CART cell that, upon binding to its cognate counter ligand on a target cell, minimizes, e.g., suppresses or inhibits, an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR beta.

[0205] The term "effector function" refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines

[0206] "Intracellular signaling domain," as the term is used herein, refers to an intracellular portion of a molecule. In embodiments, the intracellular signal domain transduces the effector function signal and directs the cell to perform a specialized function. While the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0207] In an embodiment, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In an embodiment, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation. For example, in the case of a CART, a primary intracellular signaling domain can comprise cytoplasmic sequences of the T cell receptor, and a costimulatory intracellular signaling domain can comprise cytoplasmic sequence from co-receptor or costimulatory molecule.

[0208] Primary intracellular signaling domains can comprise signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences include those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, and CD66d. Further examples of molecules containing a primary intracellular signaling domain that are of particular use in the invention include those of DAP10, DAP12, and CD32.

[0209] "Isolated" as that term is used herein refers to a nucleic acid or polypeptide means separated from at least one contaminating compound. With regard to a nucleic acid or polypeptide that exists in nature, it means free of a compound with which it occurs in nature, wherein in embodiments, the contaminating compound is a polynucleotide or polypeptide. With regard to a nucleic acid or polypeptide that is made synthetically, it means free of a sude reactant or compound used in its preparation, e.g., a solvent or starting reactant. For example, a nucleic acid or a polypeptide naturally present in a living animal is not "isolated," but the same nucleic acid or polypeptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0210] The term 'low, immune enhancing, dose" when used in conjuction with an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., RAD001 or rapamycin, or a catalytic mTOR inhibitor, refers to a dose of mTOR inhibitor that partially, but not fully, inhibits mTOR activity, e.g., as measured by the inhibition of P70 S6 kinase activity. Methods for evaluating mTOR activity, e.g., by inhibition of P70 S6 kinase, are discussed herein. The dose is insufficient to result in complete immune suppression but is sufficient to enhance the immune response. In an embodiment, the low, immune enhancing, dose of mTOR inhibitor results in a decrease in the number of PD-1 positive T cells and / or an increase in the number of PD-1 negative T cells; an increase in the ratio of PD-1 negative T cells / PD-1 positive T cells; an increase in the number of naive T cells; an increase in the number of memory T cell precursors (e.g., cells with any one or a combination of the following characteristics: increased CD62L high< , increased CD127 high< , increased CD27 +< , decreased KLRG1, and increased BCL2), or an increase in the expression of one or more of the memory T cell precursor markers CD62L high< , CD127 high< , CD27 +< , and BCL2; and / or a decrease in the expression of memory T cell precursor marker, KLRG1.

[0211] "Membrane anchor," as that term is used herein, refers to a polypeptide sufficient to anchor an extracellular domain to the plasma membrane.

[0212] "Membrane tethering domain", as that term is used herein, refers to a polypeptide or moiety, e.g., a myristoyl group, sufficient to anchor an extracellular or intracellular domain to the plasma membrane.

[0213] "Nucleic acid-based inhibitor," as that term is used herein, refers to a nucleic acid molecule that can inhibit expression of a target gene, e.g., an inhibitory molecule. It comprises double stranded RNA (dsRNA), including short hairpin RNA (shRNA) and short interfering RNA (siRNA), antisense RNA, and microRNA (miRNA). In an embodiment, the nucleic-acid based inhibitor binds to the target mRNA and inhibits the production of protein therefrom, e.g., by cleavage of the target mRNA.

[0214] "Refractory" as used herein refers to a disease, e.g., cancer, that does not respond to a treatment. In embodiments, a refractory cancer can be resistant to a treatment before or at the beginning of the treatment. In other embodiments, the refractory cancer can become resistant during a treatment. A refractory cancer is also called a resistant cancer.

[0215] "Relapsed" or a "relapse" as used herein refers to the reappearance of a disease (e.g., cancer) or the signs and symptoms of a disease such as cancer after a period of improvement or responsiveness, e.g., after prior treatment of a therapy, e.g., cancer therapy. For example, the period of responsiveness may involve the level of cancer cells falling below a certain threshold, e.g., below 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. The reappearance may involve the level of cancer cells rising above a certain threshold, e.g., above 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%.

[0216] "Subject", as that term is used herein, refers to living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic species thereof. In an embodiment the subject is a human.

[0217] "Transmembrane domain," as that term is used herein, refers to a polypeptide that spans the plasma membrane. In an embodiment, it links an extracellular sequence, e.g., a switch domain, an extracellular recognition element, e.g., an antigen binding domain, an inhibitory counter ligand binding domain, or costimulatory ECD domain, to an intracellular sequence, e.g., to a switch domain or an intracellular signaling domain. A transmembrane domain of particular use in this invention may include at least the transmembrane region(s) of e.g., the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp.

[0218] "Unit dosage form" as the term is used herein refers to a dosage for suitable one administration. By way of example a unit dosage form can be a tablet, a capsule, or an amount of therapeutic disposed in a delivery device, e.g., a syringe or intravenous drip bag. In an embodiment a unit dosage form is administered in a single administration. In an embodiment more than one unit dosage form, e.g., two tablets, can be administered simultaneously.

[0219] "Xenogeneic" as the term is used herein refers to a graft derived from an animal of a different species.

[0220] "Regulatable chimeric antigen receptor (RCAR),"as that term is used herein, refers to a set of polypeptides, typically two in the simplest embodiments, which when in a RCARX cell, provides the RCARX cell with specificity for a target cell, typically a cancer cell, and with regulatable intracellular signal generation or proliferation, which can optimize an immune effector property of the RCARX cell. An RCARX cell relies at least in part, on an antigen binding domain to provide specificity to a target cell that comprises the antigen bound by the antigen binding domain. In an embodiment, an RCAR includes a dimerization switch that, upon the presence of a dimerization molecule, can couple an intracellular signaling domain to the antigen binding domain.

[0221] "Switch domain," as that term is used herein, e.g., when referring to an RCAR, refers to an entity, typically a polypeptide-based entity, that, in the presence of a dimerization molecule, associates with another switch domain. The association results in a functional coupling of a first entity linked to, e.g., fused to, a first switch domain, and a second entity linked to, e.g., fused to, a second switch domain. A first and second switch domain are collectively referred to as a dimerization switch. In embodiments, the first and second switch domains are the same as one another, e.g., they are polypeptides having the same primary amino acid sequence, and are referred to collectively as a homodimerization switch. In embodiments, the first and second switch domains are different from one another, e.g., they are polypeptides having different primary amino acid sequences, and are referred to collectively as a heterodimerization switch. In embodiments, the switch is intracellular. In embodiments, the switch is extracellular. In embodiments, the switch domain is a polypeptide-based entity, e.g., FKBP or FRB-based, and the dimerization molecule is small molecule, e.g., a rapalogue. In embodiments, the switch domain is a polypeptide-based entity, e.g., an scFv that binds a myc peptide, and the dimerization molecule is a polypeptide, a fragment thereof, or a multimer of a polypeptide, e.g., a myc ligand or multimers of a myc ligand that bind to one or more myc scFvs. In embodiments, the switch domain is a polypeptide-based entity, e.g., myc receptor, and the dimerization molecule is an antibody or fragments thereof, e.g., myc antibody.

[0222] "Dimerization molecule," as that term is used herein, e.g., when referring to an RCAR, refers to a molecule that promotes the association of a first switch domain with a second switch domain. In embodiments, the dimerization molecule does not naturally occur in the subject, or does not occur in concentrations that would result in significant dimerization. In embodiments, the dimerization molecule is a small molecule, e.g., rapamycin or a rapalogue, e.g, RAD001.

[0223] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.DESCRIPTION Chimeric Antigen Receptor (CAR)

[0224] The present invention may utilise a recombinant DNA construct comprising sequences encoding a CAR, wherein the CAR comprises an antigen binding domain (e.g., antibody, antibody molecule, or antibody fragment, TCR or TCR fragment) that binds specifically to a tumor antigen, e.g., a tumor antigen described herein, wherein the sequence of the antigen binding domain is contiguous with and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. The intracellular signaling domain can comprise a costimulatory signaling domain and / or a primary signaling domain, e.g., a zeta chain. The costimulatory signaling domain refers to a portion of the CAR comprising at least a portion of the intracellular domain of a costimulatory molecule.

[0225] In specific aspects, a CAR construct of use in the invention comprises a scFv domain, wherein the scFv may be preceded by an optional leader sequence such as provided in SEQ ID NO: 2, and followed by an optional hinge sequence such as provided in SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10, a transmembrane region such as provided in SEQ ID NO:12 or SEQ ID NO: 42, an intracellular signaling domain, e.g., a costimulatory signaling domain, that includes SEQ ID NO:14,SEQ ID NO:16, SEQ ID NO: 40 or SEQ ID NO: 44 and a CD3 zeta sequence that includes SEQ ID NO:18 or SEQ ID NO:20, e.g., wherein the domains are contiguous with and in the same reading frame to form a single fusion protein.

[0226] In one aspect, an exemplary CAR constructs comprise an optional leader sequence (e.g., a leader sequence described herein), an extracellular antigen binding domain (e.g., an antigen binding domain described herein), a hinge (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular stimulatory domain (e.g., an intracellular stimulatory domain described herein). In one aspect, an exemplary CAR construct comprises an optional leader sequence (e.g., a leader sequence described herein), an extracellular antigen binding domain (e.g., an antigen binding domain described herein), a hinge (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein), an intracellular costimulatory signaling domain (e.g., a costimulatory signaling domain described herein) and / or an intracellular primary signaling domain (e.g., a primary signaling domain described herein).

[0227] An exemplary leader sequence is provided as SEQ ID NO: 2. An exemplary hinge / spacer sequence is provided as SEQ ID NO: 4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10. An exemplary transmembrane domain sequence is provided as SEQ ID NO:12 or SEQ ID NO: 42. An exemplary sequence of the intracellular signaling domain of the 4-1BB protein is provided as SEQ ID NO: 14. An exemplary sequence of the intracellular signaling domain of CD27 is provided as SEQ ID NO:16. An exemplary CD3zeta domain sequence is provided as SEQ ID NO: 18 or SEQ ID NO:20. An exemplary intracellular signaling domain of CD28 is provided as SEQ ID NO: 44. An exemplary intracellular signaling domain of ICOS is provided as SEQ ID NO: 40.

[0228] Sequences of some examples of various components of CARs of use in the invention, and nucleic acids that encode them are listed in Table 1, where aa stands for amino acids, and na stands for nucleic acids that encode the corresponding peptide. Table 1. Sequences of various components of CAR (aa - amino acids, na - nucleic acids that encodes the corresponding protein)SEQ ID NODescriptionSequence1EF-1 promoter (na)2Leader (aa)MALPVTALLLPLALLLHAARP3Leader (na)4CD 8 hinge (aa)TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD5CD8 hinge (na)6Ig4 hinge (aa)7Ig4 hinge (na)8IgD hinge (aa)9IgD hinge (na)10GS hinge / linker (aa)GGGGSGGGGS11GS hinge / linker (na)GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC12CD8TM (aa)IYIWAPLAGTCGVLLLSLVITLYC13CD8 TM (na)144-1BB intracellular domain (aa)KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL154-1BB intracellular domain (na)16CD27 (aa)QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP17CD27 (na)18CD3-zeta (aa)19CD3-zeta (na)20CD3-zeta (aa)21CD3-zeta (na)22linkerGGGGS23linkerGGTGGCGGAGGTTCTGGAGGTGGAGGTTCC24PD-1 extracellular domain (aa)25PD-1 extracellular domain (na) 26PD-1 CAR (aa) with signal27PD-1 CAR (na)28linker(Gly-Gly-Gly-Ser) n , where n = 1-1029linker(Gly4 Ser)430linker(Gly4 Ser)331linker(Gly3Ser)32polyA (2000 A's)a[a] 1999 33polyA (150 A's)a[a] 149 34polyA (5000 A's)a[a] 4999 35polyA (100 T's)t[t] 99 36polyA (500 T's)t[t] 499 37polyA (64 A's)a[a]6338polyA (400 A's)a[a] 399 39PD1 CAR (aa)40ICOS ICD domain (aa)41ICOS ICD domain (na)42ICOS TM domain (aa)43ICOS TM domain (na)44CD28 domain (aa)RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS45CD28 domain (na)46ICOS (FMFM)TKKKYSSSVHDPNGEFMFMRAVNTAKKSRLTDVTLICD domain (aa) ("FMFM" disclosed as SEQ ID NO: 47)

[0229] In embodiments, CAR scFv fragments are cloned into lentiviral vectors to create a full length CAR construct in a single coding frame, and using a promoter, e.g., EF1 alpha promoter, for expression (SEQ ID NO: 1).

[0230] In one aspect, the present invention may utilise a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises the nucleic acid sequence encoding an antigen binding domain, e.g., described herein, that is contiguous with and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain.

[0231] In one aspect, the present invention may utilise a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an antigen binding domain, wherein the sequence is contiguous with and in the same reading frame as the nucleic acid sequence encoding an intracellular signaling domain. An exemplary intracellular signaling domain that can be used in the CAR includes, but is not limited to, one or more intracellular signaling domains of, e.g., CD3-zeta, CD28, CD27, 4-1BB, ICOS, and the like. In some instances, the CAR can comprise any combination of CD3-zeta, CD28, 4-1BB, ICOS and the like.

[0232] In an the invention, the CAR comprises an ICOS domain and is provided in a CD4 + T cell. In an embodiment, the CAR comprises a CD28 or 4-1BB domain, and is provided in a CD8 + T cell.

[0233] The nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the nucleic acid molecule, by deriving the nucleic acid molecule from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the nucleic acid of interest can be produced synthetically, rather than cloned.

[0234] The present invention may utilise retroviral and lentiviral vector constructs expressing a CAR that can be directly transduced into a cell.

[0235] The present invention may utilise an RNA construct that can be directly transfected into a cell. A method for generating mRNA for use in transfection involves in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3' and 5' untranslated sequence ("UTR") (e.g., a 3' and / or 5' UTR described herein), a 5' cap (e.g., a 5' cap described herein) and / or Internal Ribosome Entry Site (IRES) (e.g., an IRES described herein), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length (SEQ ID NO:32). RNA so produced can efficiently transfect different kinds of cells. In one embodiment, the template includes sequences for the CAR. In an embodiment, an RNA CAR vector is transduced into a cell, e.g., a T cell by electroporation.ANTIGEN BINDING DOMAIN

[0236] The CARs described herein can include an antigen binding domain in the extracellular region.

[0237] In one embodiment, the antigen binding domain is a murine antibody or antibody fragment described herein. In one embodiment, the antigen binding domain is a humanized antibody or antibody fragment.

[0238] The choice of an antigen binding domain can depend upon the type and number of ligands or receptors that define the surface of a target cell. For example, the antigen binding domain may be chosen to recognize an antigen that acts as a cell surface marker on target cells associated with a particular disease state. Examples of cell surface markers that may act as ligands or receptors include a cell surface marker associated with a particular disease state, e.g., cell surface makers for viral diseases, bacterial diseases parasitic infections, autoimmune diseases and disorders associated with unwanted cell proliferation, e.g., a cancer, e.g., a cancer described herein.

[0239] In certain aspects, the proliferative disorder antigens of the present disclosure are derived from, cancers including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer (e.g., NSCLC or SCLC), liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemias, multiple myeloma, glioblastoma, neuroblastoma, uterine cancer, cervical cancer, renal cancer, thyroid cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer and the like. In some embodiments, the cancer is B-cell acute lymphoid leukemia ("BALL"), T-cell acute lymphoid leukemia ("TALL"), acute lymphoid leukemia (ALL), acute myelogenous leukemia (AML); one or more chronic leukemias including but not limited to chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); additional hematologic cancers or hematologic conditions including, but not limited to B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia.

[0240] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes immunologically recognized by tumor infiltrating lymphocytes (TIL) derived from a cancer tumor of a mammal.

[0241] Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T-cell mediated immune responses. The selection of the antigen binding domain of use in the invention will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, IL-11Ra, IL-13Ra, EGFR, FAP, B7H3, Kit, CA-IX, CS-1, MUC1, BCMA, bcr-abl, HER2, β-human chorionic gonadotropin, alphafetoprotein (AFP), ALK, CD19, CD123, cyclin B1, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RU1, RU2, SSX2, AKAP-4, LCK, OY-TES1, PAX5, SART3, CLL-1, fucosyl GM1, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, plysialic acid, PLAC1, RU1, RU2 (AS), intestinal carboxyl esterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYP1B1, BORIS, prostase, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-1a, LMP2, NCAM, p53, p53 mutant, Ras mutant, gp100, prostein, OR51E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-beta, survivin and telomerase, legumain, HPV E6,E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate-carcinoma tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1,MAD-CT-1, MAD-CT-2, MelanA / MART1, XAGE1, ELF2M, ERG (TMPRSS2 ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoints, NY-BR-1, ephrinB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1, Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin. In a preferred embodiment, the tumor antigen is selected from the group consisting of folate receptor (FRa), mesothelin, EGFRvIII, IL-13Ra, CD123, CD19, CD33, BCMA, GD2, CLL-1, CA-IX, MUC1, HER2, and any combination thereof.

[0242] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include but are not limited to tissue-specific antigens such as MART-1, tyrosinase and GP 100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target antigens include transformation-related molecules such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are onco-fetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma the tumor-specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor. B-cell differentiation antigens such as CD19, CD20 and CD37 are other candidates for target antigens in B-cell lymphoma.

[0243] Non-limiting examples of tumor antigens include the following: Differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

[0244] In some embodiments, the tumor antigen is a tumor antigen described in International Application PCT / US2015 / 020606 (publication number WO2015 / 142675). In some embodiments, the tumor antigen is chosen from one or more of: CD19; CD123; CD22; CD30; CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-Like Tyrosine Kinase 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); Protease Serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2 (Her2 / neu); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2 (EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer / testis antigen 1 (NY-ESO-1); Cancer / testis antigen 2 (LAGE-1a); Melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase; prostate carcinoma tumor antigen-1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MART1); Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1 (CYP1B1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced Glycation Endproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1). In some embodiments, the tumor antigen is GFRa4 (see Spinasanta, "The Endocrine Society's 97th Annual Meeting & Expo: Targeted Therapies in Medullary Thyroid Cancer" March 13, 2015).

[0245] In some embodiments, tumor antigen bound by the CAR molecule is chosen from one or more of: TSHR, CD171, CS-1, CLL-1, GD3, Tn Ag, FLT3, CD38, CD44v6, B7H3, KIT, IL-13Ra2, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, MUC1, EGFR, NCAM, CAIX, LMP2, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53 mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1.

[0246] In certain embodiments, the tumor antigen bound by the CAR molecule is chosen from one or more of: TSHR, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, and OR51E2.

[0247] Depending on the desired antigen to be targeted, the CAR of use in the invention can be engineered to include the appropriate antigen bind domain that is specific to the desired antigen target.

[0248] A CAR as described herein, can include an antigen binding domain (e.g., antibody or antibody fragment) that binds to a MHC presented-peptide. Normally, peptides derived from endogenous proteins fill the pockets of Major histocompatibility complex (MHC) class I molecules, and are recognized by T cell receptors (TCRs) on CD8 + T lymphocytes. The MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets for immunotherapy. TCR-like antibodies targeting peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, e.g., Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Bood, 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21) :1601-1608 ; Dao et al., Sci Transl Med 2013 5(176) :176ra33 ; Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example, TCR-like antibody can be identified from screening a library, such as a human scFv phage displayed library. Accordingly, the present invention may utilise a CAR that comprises an antigen binding domain that binds to a MHC presented peptide of a molecule selected from any tumor antigen described above that is expressed intracellularly, e.g., p53, BCR-Abl, Ras, K-ras, and c-met.

[0249] In one aspect, the CARX cell described herein can further comprise a second CAR, e.g., a second CAR that includes a different antigen binding domain, e.g., to the same target (a cancer associated antigen as described herein) or a different target (e.g., CD19, CD123, CD22, CD30, CD34, CD171, CS-1, CLL-1, CD33, EGFRvIII , GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, Plysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, legumain, HPV E6,E7, MAGE-A1, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, or IGLL1). In one embodiment, when the CARX cell comprises two or more different CARs, the antigen binding domains of the different CARs can be such that the antigen binding domains do not interact with one another. For example, a cell expressing a first and second CAR can have an antigen binding domain of the first CAR, e.g., as a fragment, e.g., an scFv, that does not form an association with the antigen binding domain of the second CAR, e.g., the antigen binding domain of the second CAR is a VHH.Antigen binding domains derived from an Antibody Molecule

[0250] The antigen binding domain can be derived from an antibody molecule, e.g., one or more of monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-domain antibodies e.g., a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) from, e.g., human or camelid origin. In some instances, it is beneficial for the antigen binding domain to be derived from the same species in which the CAR will ultimately be used in, e.g., for use in humans, it may be beneficial for the antigen binding domain of the CAR, described herein, to comprise a human or a humanized antigen binding domain. Antibodies can be obtained using known techniques known in the art.

[0251] In embodiments, the antigen binding domain comprises a fragment of an antibody that is sufficient to confer recognition and specific binding to the target antigen. Examples of an antibody fragment include, but are not limited to, an Fab, Fab', F(ab') 2 , or Fv fragment, an scFv antibody fragment, a linear antibody, single domain antibody such as an sdAb (either VL or VH), a camelid VHH domain, and multi-specific antibodies formed from antibody fragments.

[0252] In an embodiment, the antigen binding domain is a "scFv," which can comprise a fusion protein comprising a VL chain and a VH chain of an antibody, where the VH and VL are, e.g., linked via a short flexible polypeptide linker, e.g., a linker described herein. The scFv is capable of being expressed as a single chain polypeptide and retains the specificity of the intact antibody from which it is derived. Moreover, the VL and VH variable chains can be linked in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL. An scFv that can be prepared according to method known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).

[0253] As described above and elsewhere, scFv molecules can be produced by linking VH and VL chians together using flexible polypeptide linkers. In some embodiments, the scFv molecules comprise flexible polypeptide linker with an optimized length and / or amino acid composition. The flexible polypeptide linker length can greatly affect how the variable regions of a scFv fold and interact. In fact, if a short polypeptide linker is employed (e.g., between 5-10 amino acids, intrachain folding is prevented. For examples of linker orientation and size see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent Application Publication Nos. 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and PCT publication Nos. WO2006 / 020258 and WO2007 / 024715 . In one embodiment, the peptide linker of the scFv consists of amino acids such as glycine and / or serine residues used alone or in combination, to link variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker and, e.g., comprises the amino acid sequence (Gly-Gly-Gly-Ser)n (SEQ ID NO: 28), where n is a positive integer equal to or greater than 1. For example, n=1, n=2, n=3. n=4, n=5 and n=6, n=7, n=8, n=9 and n=10. In one embodiment, the flexible polypeptide linkers include, but are not limited to, (Gly4 Ser)4 (SEQ ID NO: 29) or (Gly4 Ser)3 (SEQ ID NO: 30). In another embodiment, the linkers include multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser) (SEQ ID NO: 31).

[0254] In some embodiments, the antigen binding domain is a single domain antigen binding (SDAB) molecules. A SDAB molecule includes molecules whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain variable domains, binding molecules naturally devoid of light chains, single domains derived from conventional 4-chain antibodies, engineered domains and single domain scaffolds other than those derived from antibodies (e.g., described in more detail below). SDAB molecules may be any of the art, or any future single domain molecules. SDAB molecules may be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine. This term also includes naturally occurring single domain antibody molecules from species other than Camelidae and sharks.

[0255] In one aspect, an SDAB molecule can be derived from a variable region of the immunoglobulin found in fish, such as, for example, that which is derived from the immunoglobulin isotype known as Novel Antigen Receptor (NAR) found in the serum of shark. Methods of producing single domain molecules derived from a variable region of NAR ("IgNARs") are described in WO 03 / 014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

[0256] According to another aspect, an SDAB molecule is a naturally occurring single domain antigen binding molecule known as a heavy chain devoid of light chains. Such single domain molecules are disclosed in WO 9404678 and Hamers-Casterman, C. et al. (1993) Nature 363:446-448, for example. For clarity reasons, this variable domain derived from a heavy chain molecule naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain molecules naturally devoid of light chain; such VHHs are within the scope of the invention.

[0257] Antibody proteins obtained from members of the camel and dromedary (Camelus bactrianus and Calelus dromaderius) family including new world members such as llama species (Lama paccos, Lama glama and Lama vicugna) have been characterized with respect to size, structural complexity and antigenicity for human subjects. Certain IgG antibodies from this family of mammals as found in nature lack light chains, and are thus structurally distinct from the typical four chain quaternary structure having two heavy and two light chains, for antibodies from other animals. See PCT / EP93 / 02214 (WO 94 / 04678 published 3 March 1994).

[0258] A region of the camelid antibody which is the small single variable domain identified as VHH can be obtained by genetic engineering to yield a small protein having high affinity for a target, resulting in a low molecular weight antibody-derived protein known as a "camelid nanobody". See U.S. patent number 5,759,808 issued June 2, 1998; see also Stijlemans et al., (2004) J Biol Chem 279:1256-1261; Dumoulin et al., (2003) Nature 424:783-788; Pleschberger et al., (2003) Bioconjugate Chem 14:440-448; Cortez-Retamozo et al., (2002) Int J Cancer 89:456-62; and Lauwereys et al., (1998) EMBO J 17:3512-3520. Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium (e.g., US20060115470; Domantis (US20070065440, US20090148434). As with other antibodies of non-human origin, an amino acid sequence of a camelid antibody can be altered recombinantly to obtain a sequence that more closely resembles a human sequence, i.e., the nanobody can be "humanized". Thus the natural low antigenicity of camelid antibodies to humans can be further reduced.

[0259] The camelid nanobody has a molecular weight approximately one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers. One consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins, i.e., camelid nanobodies are useful as reagents detect antigens that are otherwise cryptic using classical immunological techniques, and as possible therapeutic agents. Thus yet another consequence of small size is that a camelid nanobody can inhibit as a result of binding to a specific site in a groove or narrow cleft of a target protein, and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug than that of a classical antibody.

[0260] The low molecular weight and compact size further result in camelid nanobodies being extremely thermostable, stable to extreme pH and to proteolytic digestion, and poorly antigenic. Another consequence is that camelid nanobodies readily move from the circulatory system into tissues, and even cross the blood-brain barrier and can treat disorders that affect nervous tissue. Nanobodies can further facilitated drug transport across the blood brain barrier. See U.S. patent application 20040161738 published August 19, 2004. These features combined with the low antigenicity to humans indicate great therapeutic potential. Further, these molecules can be fully expressed in prokaryotic cells such as E. coli and are expressed as fusion proteins with bacteriophage and are functional.

[0261] An antigen binding domain can comprise a camelid antibody or nanobody, or an antigen binding fragment thereof. Such antibodies can have high affinity for its cognate antigen. In certain embodiments herein, the camelid antibody or nanobody is naturally produced in the camelid animal, i.e., is produced by the camelid following immunization with antigen or a peptide fragment thereof. Alternatively, the camelid nanobody is engineered, i.e., produced by selection for example from a library of phage displaying appropriately mutagenized camelid nanobody proteins using panning procedures with the target antigen. Engineered nanobodies can further be customized by genetic engineering to have a half life in a recipient subject of from 45 minutes to two weeks. In a specific embodiment, the camelid antibody or nanobody is obtained by grafting the CDRs sequences of the heavy or light chain of the human antibodies of the disclosure into nanobody or single domain antibody framework sequences, as described for example in PCT / EP93 / 02214 (published as WO94 / 04678).

[0262] An antigen binding domain can comprise a single domain antibody, e.g., which relies only on a heavy chain variable region for binding, e.g., a nanobody. Nanobodies suitable for use herein can be made by the methods described in US2010 / 0028341, WO2009 / 030285, and WO2010 / 007376.

[0263] In certain embodiments, the SDAB molecule is a single chain fusion polypeptide comprising one or more single domain molecules (e.g., nanobodies), devoid of a complementary variable domain or an immunoglobulin constant, e.g., Fc, region, that binds to one or more target antigens.

[0264] The SDAB molecules can be recombinant, CDR-grafted, humanized, camelized, de-immunized and / or in vitro generated (e.g., selected by phage display).

[0265] In one embodiment, the antigen biding domain portion comprises a human antibody or a fragment thereof.

[0266] In some embodiments, a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human. In an embodiment, the antigen binding domain is humanized.

[0267] Non human antibodies can be humanized using a variety of techniques known in the art, e.g., CDR-grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089 ), veneering or resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91:969-973), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332), and techniques disclosed in, e.g., U.S. Patent Application Publication No. US2005 / 0042664, U.S. Patent Application Publication No. US2005 / 0048617, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO 9317105, Tan et al., 2002, J. Immunol., 169:1119-25; Caldas et al., 2000, Protein Eng., 13(5):353-60; Morea et al., 2000, Methods, 20:267-79; Baca et al., 1997, J. Biol. Chem., 272:10678-84; Roguska et al., 1996, Protein Eng., 9(10):895-904; Couto et al., 1995, Cancer Res., 55 :5973s-5977; Couto et al., 1995, Cancer Res., 55(8):1717-22; Sandhu 1994 Gene, 150(2):409-10; and Pedersen et al., 1994, J. Mol. Biol., 235(3):959-73 .

[0268] Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, for example improve, antigen binding. These framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323).

[0269] In preferred embodiments, the humanized antibody molecule comprises a sequence described herein, e.g., a variable light chain and / or a variable heavy chain described herein, e.g., a humanized variable light chain and / or variable heavy chain described in Table X.

[0270] A humanized antibody has one or more amino acid residues introduced into it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Thus, humanized antibodies comprise one or more CDRs from nonhuman immunoglobulin molecules and framework regions from human. Humanization of antibodies is well-known in the art and can essentially be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, i.e., CDR-grafting (EP 239,400; PCT Publication No. WO 91 / 09967; and U.S. Pat. Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640).

[0271] In such humanized chimeric antibodies, substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework (FR) residues are substituted by residues from analogous sites in rodent antibodies. Humanization of antibodies can also be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332).

[0272] In some embodiments, the antibody of the disclosure is further prepared using an antibody having one or more of the VH and / or VL sequences disclosed herein can be used as starting material to engineer a modified antibody, which modified antibody may have altered properties as compared to the starting antibody. In various embodiments, the antibody is engineered by modifying one or more amino acids within one or both variable regions (i.e., VH and / or VL), for example within one or more CDR regions and / or within one or more framework regions.

[0273] In another aspect, the antigen binding domain is a T cell receptor ("TCR"), or a fragment thereof, for example, a single chain TCR (scTCR). Methods to make such TCRs are known in the art. See, e.g., Willemsen RA et al, Gene Therapy 7: 1369-1377 (2000); Zhang T et al, Cancer Gene Ther 11: 487-496 (2004); Aggen et al, Gene Ther. 19(4):365-74 (2012) .

[0274] For example, scTCR can be engineered that contains the Vα and Vβ genes from a T cell clone linked by a linker (e.g., a flexible peptide). This approach is very useful to cancer associated target that itself is intracellular, however, a fragment of such antigen (peptide) is presented on the surface of the cancer cells by MHC.

[0275] An antigen binding domain can comprise a sequence from Table 2. Table 2: Exemplary Sequences for Antigen Binding DomainsTarget AntigenNameAmino Acid SequenceSEQ ID NO:CD19huscFv148CD19huscFv249CD19huscFv350CD19huscFv451CD19huscFv5 52CD19huscFv653CD19huscFv754CD19huscFv855CD19huscFv956CD19Hu scFv1057CD19Hu scFv1158CD19Hu scFv1259CD19muCTL 01960CD123Mu117261CD123Mu1176 62CD123huscFv163CD123huscFv264CD123huscFv365CD123huscFv466CD123huscFv567CD123huscFv668CD123huscFv769CD123huscFv870EGFR vIIIhuscFv171EGFR vIIIhuscFv272EGFR vIIIhuscFv373EGFR vIIIhuscFv474EGFR vIIIhuscFv575EGFRhuscFv6 76EGFR vIIIhuscFv777EGFR vIIIhuscFv878EGFR vIIIMu310 C79mesotheli nssl80Folate Receptor αMOv1981

[0276] In an embodiment, the antigen binding domain comprises any antibody, or a fragment thereof, e.g., an scFv, known in the art that targets or specifically binds to any one of the following: BCMA (also known as TNFRSF17, Tumor Necrosis Factor Receptor Superfamily, Member 17, or B Cell Maturation Antigen), CD33, CLL-1 (also known as C-type Lectin-Like domain family 1, or CLECL1), claudin-6 (CLDN6) or WT-1 (Wilms tumor 1). The antibody, or fragment thereof, can be a murine, humanized, or fully human antibody or fragment thereof.

[0277] In some embodiments, the antigen binding domain comprises a HC CDR1, a HC CDR2, and a HC CDR3 of any heavy chain binding domain amino acid sequences listed in Table 2. In embodiments, the antigen binding domain further comprises a LC CDR1, a LC CDR2, and a LC CDR3. In embodiments, the antigen binding domain comprises a LC CDR1, a LC CDR2, and a LC CDR3 of any light chain binding domain amino acid sequences listed in Table 2.

[0278] In some embodiments, the antigen binding domain comprises one, two or all of LC CDR1, LC CDR2, and LC CDR3 of any light chain binding domain amino acid sequences listed in Table 2, and one, two or all of HC CDR1, HC CDR2, and HC CDR3 of any heavy chain binding domain amino acid sequences listed in Table 2.

[0279] In some embodiments, the CDRs are defined according to the Kabat numbering scheme, the Chothia numbering scheme, or a combination thereof.

[0280] In embodiments, the order in which the VL and VH domains appear in the scFv is varied (i.e., VL-VH, or VH-VL orientation), and where either three or four copies of the "G4S" (SEQ ID NO:31) subunit, in which each subunit comprises the sequence GGGGS (SEQ ID NO:31) (e.g., (G4S)3 (SEQ ID NO:30) or (G4S)4(SEQ ID NO:29)), connect the variable domains to create the entirety of the scFv domain. Alternatively, the CAR construct can include, for example, a linker including the sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 86)

[0281] Exemplary sequences of various scFv fragments and other CAR components are provided herein. It is noted that these CAR components (e.g., of SEQ ID NOs. 26, 89) without a leader sequence (e.g., without the amino acid sequence of SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO:3), are also provided herein.

[0282] In embodiments, the CAR sequences described herein contain a Q / K residue change in the signal domain of the co-stimulatory domain derived from CD3zeta chain.

[0283] In one embodiment, the portion of the CAR comprising the antigen binding domain comprises an antigen binding domain that binds specifically to CD19. In one aspect, the antigen binding domain targets human CD19. In one aspect, the antigen binding domain of the CAR has the same or a similar binding specificity as the FMC63 scFv fragment described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one embodiment, the antigen binding domain of the CAR includes the scFv fragment described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). A CD19 antibody molecule can be, e.g., an antibody molecule (e.g., a humanized anti-CD19 antibody molecule) described in WO2014 / 153270.

[0284] WO2014 / 153270 also describes methods of assaying the binding and efficacy of various CART constructs.

[0285] In one aspect, the parental murine scFv sequence is the CAR19 construct provided in PCT publication WO2012 / 079000 and provided herein as SEQ ID NO:60. In one embodiment, the anti-CD19 binding domain is a scFv described in WO2012 / 079000 and provided in SEQ ID NO:60.

[0286] In some aspects, the antibodies of the disclosure are incorporated into a chimeric antigen receptor (CAR). In one aspect, the CAR comprises the polypeptide sequence provided as SEQ ID NO: 12 in PCT publication WO2012 / 079000, and provided herein as SEQ ID NO: 89, wherein the scFv domain is substituted by one or more sequences selected from SEQ ID NOS: 48-59. In one aspect, the scFv domains of SEQ ID NOS:48-59 are humanized variants of the scFv domain of SEQ ID NO:60 which is an scFv fragment of murine origin that specifically binds to human CD19. Humanization of this mouse scFv may be desired for the clinical setting, where the mouse-specific residues may induce a human-anti-mouse antigen (HAMA) response in patients who receive CART19 treatment, e.g., treatment with T cells transduced with the CAR19 construct.

[0287] The CD19 CAR provided as SEQ ID NO: 12 in PCT publication WO2012 / 079000 is:

[0288] In an embodiment, the antigen binding domain comprises an anti-CD19 antibody, or fragment thereof, e.g., an scFv. For example, the antigen binding domain comprises a variable heavy chain and a variable light chain listed in Table 3. The linker sequence joining the variable heavy and variable light chains can be any of the linker sequences described herein, or alternatively, can be GSTSGSGKPGSGEGSTKG (SEQ ID NO:86). Table 3: Anti-CD 19 antibody binding domainsAntibodyVH SequenceVL SequenceSJ25-C1

[0289] Any known CD19 CAR, e.g., the CD19 antigen binding domain of any known CD19 CAR, in the art can be used in accordance with the instant invention. For example, LG-740; CD19 CAR described in the US Pat. No. 8,399,645; US Pat. No. 7,446,190; Xu et al., Leuk Lymphoma. 2013 54(2):255-260(2012); Cruz et al., Blood 122(17):2965-2973 (2013); Brentjens et al., Blood, 118(18):4817-4828 (2011); Kochenderfer et al., Blood 116(20):4099-102 (2010); Kochenderfer et al., Blood 122 (25):4129-39(2013); and 16th Annu Meet Am Soc Gen Cell Ther (ASGCT) (May 15-18, Salt Lake City) 2013, Abst 10.

[0290] Exemplary target antigens that can be targeted using the CAR-expressing cells, include, but are not limited to, CD19, CD123, EGFRvIII, mesothelin, among others, as described in, for example, WO 2014 / 130635, WO 2014 / 130657, and WO 2015 / 090230.

[0291] In one embodiment, the CAR T cell that specifically binds to CD19 has the USAN designation TISAGENLECLEUCEL-T. CTL019 is made by a gene modification of T cells is mediated by stable insertion via transduction with a self-inactivating, replication deficient Lentiviral (LV) vector containing the CTL019 transgene under the control of the EF-1 alpha promoter. CTL019 can be a mixture of transgene positive and negative T cells that are delivered to the subject on the basis of percent transgene positive T cells.

[0292] In other embodiments, the CAR-expressing cells can specifically bind to human CD19, e.g., can include a CAR molecule, or an antigen binding domain (e.g., a humanized antigen binding domain) according to Table 3 of WO2014 / 153270 .

[0293] In other embodiments, the CAR-expressing cells can specifically bind to CD123, e.g., can include a CAR molecule (e.g., any of the CAR1-CAR8), or an antigen binding domain according to Tables 1-2 of WO 2014 / 130635 .

[0294] In other embodiments, the CAR-expressing cells can specifically bind to EGFRvIII, e.g., can include a CAR molecule, or an antigen binding domain according to Table 2 or SEQ ID NO:11 of WO 2014 / 130657.

[0295] In other embodiments, the CAR-expressing cells can specifically bind to mesothelin, e.g., can include a CAR molecule, or an antigen binding domain according to Tables 2-3 of WO 2015 / 090230 .

[0296] In one embodiment, the antigen binding domain comprises one, two three (e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2 and HC CDR3, from an antibody listed above, and / or one, two, three (e.g., all three) light chain CDRs, LC CDR1, LC CDR2 and LC CDR3, from an antibody listed above. In one embodiment, the antigen binding domain comprises a heavy chain variable region and / or a variable light chain region of an antibody listed or described above.Bispecific CARs

[0297] In an embodiment a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In an embodiment the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In an embodiment the first and second epitopes overlap. In an embodiment the first and second epitopes do not overlap. In an embodiment the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In an embodiment a bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a second epitope. In an embodiment a bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In an embodiment a bispecific antibody molecule comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In an embodiment a bispecific antibody molecule comprises a scFv, or fragment thereof, have binding specificity for a first epitope and a scFv, or fragment thereof, have binding specificity for a second epitope.

[0298] In certain embodiments, the antibody molecule is a multi-specific (e.g., a bispecific or a trispecific) antibody molecule. Protocols for generating bispecific or heterodimeric antibody molecules are known in the art; including but not limited to, for example, the "knob in a hole" approach described in, e.g., US 5731168; the electrostatic steering Fc pairing as described in, e.g., WO 09 / 089004, WO 06 / 106905 and WO 2010 / 129304; Strand Exchange Engineered Domains (SEED) heterodimer formation as described in, e.g., WO 07 / 110205; Fab arm exchange as described in, e.g., WO 08 / 119353, WO 2011 / 131746, and WO 2013 / 060867; double antibody conjugate, e.g., by antibody cross-linking to generate a bi-specific structure using a heterobifunctional reagent having an amine-reactive group and a sulfhydryl reactive group as described in, e.g., US 4433059; bispecific antibody determinants generated by recombining half antibodies (heavy-light chain pairs or Fabs) from different antibodies through cycle of reduction and oxidation of disulfide bonds between the two heavy chains, as described in, e.g., US 4444878; trifunctional antibodies, e.g., three Fab' fragments cross-linked through sulfhdryl reactive groups, as described in, e.g., US5273743; biosynthetic binding proteins, e.g., pair of scFvs cross-linked through C-terminal tails preferably through disulfide or amine-reactive chemical cross-linking, as described in, e.g., US5534254; bifunctional antibodies, e.g., Fab fragments with different binding specificities dimerized through leucine zippers (e.g., c-fos and c-jun) that have replaced the constant domain, as described in, e.g., US5582996; bispecific and oligospecific mono-and oligovalent receptors, e.g., VH-CH1 regions of two antibodies (two Fab fragments) linked through a polypeptide spacer between the CH1 region of one antibody and the VH region of the other antibody typically with associated light chains, as described in, e.g., US5591828; bispecific DNA-antibody conjugates, e.g., crosslinking of antibodies or Fab fragments through a double stranded piece of DNA, as described in, e.g., US5635602; bispecific fusion proteins, e.g., an expression construct containing two scFvs with a hydrophilic helical peptide linker between them and a full constant region, as described in, e.g., US5637481; multivalent and multispecific binding proteins, e.g., dimer of polypeptides having first domain with binding region of Ig heavy chain variable region, and second domain with binding region of Ig light chain variable region, generally termed diabodies (higher order structures are also encompassed creating for bispecifc, trispecific, or tetraspecific molecules, as described in, e.g., US5837242; minibody constructs with linked VL and VH chains further connected with peptide spacers to an antibody hinge region and CH3 region, which can be dimerized to form bispecific / multivalent molecules, as described in, e.g., US5837821; VH and VL domains linked with a short peptide linker (e.g., 5 or 10 amino acids) or no linker at all in either orientation, which can form dimers to form bispecific diabodies; trimers and tetramers, as described in, e.g., US5844094; String of VH domains (or VL domains in family members) connected by peptide linkages with crosslinkable groups at the C-terminus futher associated with VL domains to form a series of FVs (or scFvs), as described in, e.g., US5864019; and single chain binding polypeptides with both a VH and a VL domain linked through a peptide linker are combined into multivalent structures through non-covalent or chemical crosslinking to form, e.g., homobivalent, heterobivalent, trivalent, and tetravalent structures using both scFV or diabody type format, as described in, e.g., US5869620. Additional exemplary multispecific and bispecific molecules and methods of making the same are found, for example, in US5910573, US5932448, US5959083, US5989830, US6005079, US6239259, US6294353, US6333396, US6476198, US6511663, US6670453, US6743896, US6809185, US6833441, US7129330, US7183076, US7521056, US7527787, US7534866, US7612181, US2002004587A1, US2002076406A1, US2002103345A1, US2003207346A1, US2003211078A1, US2004219643A1, US2004220388A1, US2004242847A1, US2005003403A1, US2005004352A1, US2005069552A1, US2005079170A1, US2005100543A1, US2005136049A1, US2005136051A1, US2005163782A1, US2005266425A1, US2006083747A1, US2006120960A1, US2006204493A1, US2006263367A1, US2007004909A1, US2007087381A1, US2007128150A1, US2007141049A1, US2007154901A1, US2007274985A1, US2008050370A1, US2008069820A1, US2008152645A1, US2008171855A1, US2008241884A1, US2008254512A1, US2008260738A1, US2009130106A1, US2009148905A1, US2009155275A1, US2009162359A1, US2009162360A1, US2009175851A1, US2009175867A1, US2009232811A1, US2009234105A1, US2009263392A1, US2009274649A1, EP346087A2, WO0006605A2, WO02072635A2, WO04081051A1, WO06020258A2, WO2007044887A2, WO2007095338A2, WO2007137760A2, WO2008119353A1, WO2009021754A2, WO2009068630A1, WO9103493A1, WO9323537A1, WO9409131A1, WO9412625A2, WO9509917A1, WO9637621A2, WO9964460A1.

[0299] Within each antibody or antibody fragment (e.g., scFv) of a bispecific antibody molecule, the VH can be upstream or downstream of the VL. In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VH1) upstream of its VL (VL1) and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VL2) upstream of its VH (VH2), such that the overall bispecific antibody molecule has the arrangement VH1-VL1-VL2-VH2. In other embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VL1) upstream of its VH (VH1) and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VH2) upstream of its VL (VL2), such that the overall bispecific antibody molecule has the arrangement VL1-VH1-VH2-VL2. Optionally, a linker is disposed between the two antibodies or antibody fragments (e.g., scFvs), e.g., between VL1 and VL2 if the construct is arranged as VH1-VL1-VL2-VH2, or between VH1 and VH2 if the construct is arranged as VL1-VH1-VH2-VL2. The linker may be a linker as described herein, e.g., a (Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 4 (SEQ ID NO: 29). In general, the linker between the two scFvs should be long enough to avoid mispairing between the domains of the two scFvs. Optionally, a linker is disposed between the VL and VH of the first scFv. Optionally, a linker is disposed between the VL and VH of the second scFv. In constructs that have multiple linkers, any two or more of the linkers can be the same or different. Accordingly, in some embodiments, a bispecific CAR comprises VLs, VHs, and optionally one or more linkers in an arrangement as described herein.

[0300] In one aspect, the bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence, e.g., a scFv, which has binding specificity for a first cancer-associated antigen, e.g., comprises a scFv as described herein, e.g., as described in Table 2, or comprises the light chain CDRs and / or heavy chain CDRs from a scFv described herein, and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope on a different antigen. In some aspects the second immunoglobulin variable domain sequence has binding specificity for an antigen expressed on AML cells. For example, the second immunoglobulin variable domain sequence has binding specificity for CD123. As another example, the second immunoglobulin variable domain sequence has binding specificity for CD33. As another example, the second immunoglobulin variable domain sequence has binding specificity for CLL-1. As another example, the second immunoglobulin variable domain sequence has binding specificity for CD34. As another example, the second immunoglobulin variable domain sequence has binding specificity for FLT3. For example, the second immunoglobulin variable domain sequence has binding specificity for folate receptor beta. In some aspects, the second immunoglobulin variable domain sequence has binding specificity for an antigen expressed on B-cells, for example, CD19, CD20, CD22 or ROR1.Chimeric TCR

[0301] In one aspect, the antibodies and antibody fragments disclosed herein (for example, those disclosed in Table 2) can be grafted to one or more constant domain of a T cell receptor ("TCR") chain, for example, a TCR alpha or TCR beta chain, to create an chimeric TCR that binds specifically to a cancer associated antigen. Without being bound by theory, it is believed that chimeric TCRs will signal through the TCR complex upon antigen binding. For example, an scFv as disclosed herein, can be grafted to the constant domain, e.g., at least a portion of the extracellular constant domain, the transmembrane domain and the cytoplasmic domain, of a TCR chain, for example, the TCR alpha chain and / or the TCR beta chain. As another example, an antibody fragment, for example a VL domain as described herein, can be grafted to the constant domain of a TCR alpha chain, and an antibody fragment, for example a VH domain as described herein, can be grafted to the constant domain of a TCR beta chain (or alternatively, a VL domain may be grafted to the constant domain of the TCR beta chain and a VH domain may be grafted to a TCR alpha chain). As another example, the CDRs of an antibody or antibody fragment, e.g., the CDRs of an antibody or antibody fragment as described in Table 3 may be grafted into a TCR alpha and / or beta chain to create a chimeric TCR that binds specifically to a cancer associated antigen. For example, the LC CDRs disclosed herein may be grafted into the variable domain of a TCR alpha chain and the HC CDRs disclosed herein may be grafted to the variable domain of a TCR beta chain, or vice versa. Such chimeric TCRs may be produced by any appropriate method (For example, Willemsen RA et al, Gene Therapy 2000; 7: 1369-1377; Zhang T et al, Cancer Gene Ther 2004; 11: 487-496; Aggen et al, Gene Ther. 2012 Apr;19(4):365-74).Non-Antibody Scaffolds

[0302] In embodiments, the antigen binding domain comprises a non antibody scaffold, e.g., a fibronectin, ankyrin, domain antibody, lipocalin, small modular immuno-pharmaceutical, maxybody, Protein A, or affilin. The non antibody scaffold has the ability to bind to target antigen on a cell. In embodiments, the antigen binding domain is a polypeptide or fragment thereof of a naturally occurring protein expressed on a cell. In some embodiments, the antigen binding domain comprises a non-antibody scaffold. A wide variety of non-antibody scaffolds can be employed so long as the resulting polypeptide includes at least one binding region which specifically binds to the target antigen on a target cell.

[0303] Non-antibody scaffolds include: fibronectin (Novartis, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge, MA, and Ablynx nv, Zwijnaarde, Belgium), lipocalin (Pieris Proteolab AG, Freising, Germany), small modular immuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA), maxybodies (Avidia, Inc., Mountain View, CA), Protein A (Affibody AG, Sweden), and affilin (gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).

[0304] Fibronectin scaffolds can be based on fibronectin type III domain (e.g., the tenth module of the fibronectin type III ( 10< Fn3 domain)). The fibronectin type III domain has 7 or 8 beta strands which are distributed between two beta sheets, which themselves pack against each other to form the core of the protein, and further containing loops (analogous to CDRs) which connect the beta strands to each other and are solvent exposed. There are at least three such loops at each edge of the beta sheet sandwich, where the edge is the boundary of the protein perpendicular to the direction of the beta strands (see US 6,818,418). Because of this structure, this non-antibody scaffold mimics antigen binding properties that are similar in nature and affinity to those of antibodies. These scaffolds can be used in a loop randomization and shuffling strategy in vitro that is similar to the process of affinity maturation of antibodies in vivo.

[0305] The ankyrin technology is based on using proteins with ankyrin derived repeat modules as scaffolds for bearing variable regions which can be used for binding to different targets. The ankyrin repeat module is a 33 amino acid polypeptide consisting of two anti-parallel α-helices and a β-turn. Binding of the variable regions is mostly optimized by using ribosome display.

[0306] Avimers are derived from natural A-domain containing protein such as HER3. These domains are used by nature for protein-protein interactions and in human over 250 proteins are structurally based on A-domains. Avimers consist of a number of different "A-domain" monomers (2-10) linked via amino acid linkers. Avimers can be created that can bind to the target antigen using the methodology described in, for example, U.S. Patent Application Publication Nos. 20040175756; 20050053973; 20050048512; and 20060008844.

[0307] Affibody affinity ligands are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of Protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. This scaffold domain consists of 58 amino acids, 13 of which are randomized to generate affibody libraries with a large number of ligand variants (See e.g., US 5,831,012). Affibody molecules mimic antibodies, they have a molecular weight of 6 kDa, compared to the molecular weight of antibodies, which is 150 kDa. In spite of its small size, the binding site of affibody molecules is similar to that of an antibody.

[0308] Protein epitope mimetics (PEM) are medium-sized, cyclic, peptide-like molecules (MW 1-2kDa) mimicking beta-hairpin secondary structures of proteins, the major secondary structure involved in protein-protein interactions. Antigen binding domains, e.g., those comprising scFv, single domain antibodies, or camelid antibodies, can be directed to any target receptor / ligand described herein, e.g., the the PD1 receptors, PD-L1 or PD-L2.

[0309] In an embodiment the antigen binding domain comprises the extracellular domain, or a counter-ligand binding fragment thereof, of molecule that binds a counterligand on the surface of a target cell.

[0310] An antigen binding domain can comprise the extracellular domain of an inhibitory receptors. Engagement with a counterligand of the coinhibitory molecule is redirected into an optimization of immune effector response.

[0311] An antigen binding domain can comprise the extracellular domain of a costimulatory molecule, referred to as a Costimulatory ECD domain, Engagement with a counter ligand of the costimulatory molecule results in optimization of immune effector response.TRANSMEMBRANE DOMAIN

[0312] In embodiments, a CAR of use in the invention comprises a transmembrane domain that is fused to an extracellular sequence, e.g., an extracellular recognition element, which can comprise an antigen binding domain, an inhibitory counter ligand binding domain, or a costimulatory ECD domain. In an embodiment, the transmembrane domain is one that naturally is associated with one of the domains in the CAR. In an embodiment, the transmembrane domain is one that is not naturally associated with one of the domains in the CAR.

[0313] A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region). In one aspect, the transmembrane domain is one that is associated with one of the other domains of the CAR e.g., in one embodiment, the transmembrane domain may be from the same protein that the signaling domain, costimulatory domain or the hinge domain is derived from. In another aspect, the transmembrane domain is not derived from the same protein that any other domain of the CAR is derived from.

[0314] In embodiments, the transmembrane domain is one which minimizes interactions with other elements, e.g., other transmembrane domains. In some instances, the transmembrane domain minimizes binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex. Suitable examples can be derived by selection or modification of amino acid substitution of a known transmembrane domain. In an embodiment, the transmembrane domain is capable of promoting homodimerization with another CAR on the cell surface. In a different aspect the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same CAR-expressing cell.

[0315] The transmembrane domain may comprise a naturally occurring, or a non-naturally occurring synthetic sequence. Where naturally occurring, the transmembrane domain may be derived from any membrane-bound or transmembrane protein.

[0316] Transmembrane regions suitable for use in molecules described herein may be derived from any one or more of e.g., the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp , NKG2D, and NKG2C. In an embodiment the transmembrane domain is derived from CD8. In an embodiment the transmembrane domain is derived from CD28. In one aspect, the transmembrane domain is a transmembrane domain from the sequence provided as SEQ ID NO: 12 or SEQ ID NO: 42.

[0317] In an embodiment, a sequence, e.g., a hinge or spacer sequence, can be disposed between a transmembrane domain and another sequence or domain to which it is fused. In embodiments, a variety of human hinges (aka "spacers") can be employed as well, e.g., including but not limited to the human Ig (immunoglobulin) hinge. In one embodiment, the hinge can be a human Ig (immunoglobulin) hinge (e.g., an IgG4 hinge an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge or a CD8a hinge. Optionally, a short oligo- or polypeptide linker, between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and another domain, e.g., an intracellular signaling domain or costimulatory domain, of a CAR. A glycine-serine doublet provides a particularly suitable linker. In one aspect, the hinge or spacer is the amino acid sequence provided as SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. In one aspect, the hinge or spacer comprises a KIR2DS2 hinge.

[0318] In an embodiment, the transmembrane domain may be a non-naturally occurring sequence, in which case can comprise predominantly hydrophobic residues such as leucine and valine. In an embodiment, a triplet of phenylalanine, tryptophan and valine will be found at each end of a transmembrane domain.

[0319] Optionally, a short oligo- or polypeptide linker, between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic region of the CAR. A glycine-serine doublet provides a particularly suitable linker. For example, in one aspect, the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO:10). In some embodiments, the linker is encoded by a nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO:11).INTRACELLULAR SIGNALING DOMAIN

[0320] In embodiments, an intracellular signaling domain produces an intracellular signal when an extracellular domain, e.g., an antigen binding domain, to which it is fused, binds a counter ligand. Intracellular signaling domains can include primary intracellular signaling domains and costimulatory signaling domains. In an embodiment, a CAR molecule can be constructed for expression in an immune cell, e.g., a T cell, such that the CAR molecule comprises a domain, e.g., a primary intracellular signaling domains, costimulatory signaling domain, inhibitory domains, etc., that is derived from a polypeptide that is typically associated with the immune cell. For example, a CAR for expression in a T cell can comprise a 41BB domain and a CD3 zeta domain. In this instance, both the 41BB and CD3 zeta domains are derived from polypeptides associated with the T cell. In another embodiment, a CAR for expression in a T cell can comprise a CD28 domain and a CD3 zeta domain. In another embodiment, a CAR for expression in a T cell can comprise an ICOS domain and a CD3 zeta domain. In another embodiment, a CAR for expression in a T cell can comprise a CD27 deomain and a CD3 zeta domain. In another embodiment, a CAR molecule can be constructed for expression in an immune cell e.g., a T cell, such that the CAR molecule comprises a domain that is derived from a polypeptide that is not typically associated with the immune cell.

[0321] The CARs of use in the invention comprise intracellular signalling domains in accordance with the claims.PRIMARY INTRACELLULAR SIGNALING DOMAIN

[0322] In an embodiment, a primary intracellular signaling domain produces an intracellular signal when an extracellular domain, e.g., an antigen binding domain, to which it is fused binds cognate antigen. The primary intracellular signaling domain is derived from a primary stimulatory molecule, e.g., it comprises intracellular sequence of a primary stimulatory molecule. The primary intracellular signaling domain comprises sufficient primary stimulatory molecule sequence to produce an intracellular signal, e.g., when an antigen binding domain to which it is fused binds cognate antigen.

[0323] A primary stimulatory molecule, is a molecule, that upon binding cognate ligand, mediates an immune effector response, e.g., in the cell in which it is expressed. Typically, it generates an intracellular signal that is dependent on binding to a cognate ligand that comprises antigen. The TCR / CD3 complex is an exemplary primary stimulatory molecule; it generates an intracellular signal upon binding to cognate ligand, e.g., an MHC molecule loaded with a peptide. Typically, e.g., in the case of the TCR / CD3 primary stimulatory molecule, the generation of an intracellular signal by a primary intracellular signaling domain is dependent on binding of the primary stimulatory molecule to antigen.

[0324] Primary stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-β, and / or reorganization of cytoskeletal structures, and the like. Stimulation, can, e.g., in the presence of costimulation, result in an optimization, e.g., an increase, in an immune effector function of the CARX cell, e.g., CART cell. Stimulation, e.g., in the context of a CART cell, can mediate a T cell response, e.g., proliferation, activation, differentiation, and the like.

[0325] In an embodiment, the primary intracellular signaling domain comprises a signaling motif, e.g., an immunoreceptor tyrosine-based activation motif or ITAMs. A primary intracellular signaling domain can comprise ITAM containing cytoplasmic signaling sequences from (for example) TCR zeta (CD3 zeta), common FcR gamma, (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, DAP10, DAP12, and CD66d.

[0326] Exemplary primary intracellular signaling domains are provided in Table 4. Table 4. Primary Intracellular Signaling Domains In embodiments the domain comprises an ITAMTCR zetaFcR gammaFcR betaCD3 gammaCD3 deltaCD3 epsilonCD3 zetaCD5CD22CD79aCD79bCD66dDAP10DAP12CD32

[0327] The CARs of use in the invention comprise a CD3 zeta intracellular signalling domain in accordance with the claims.

[0328] A primary intracellular signaling domain comprises a functional fragment, or analog, of a primary stimulatory molecule (e.g., CD3 zeta - GenBank Acc. No. BAG36664.1). The primary intracellular signalin domain can comprise the entire intracellular region or a fragment of the intracellular region which is sufficient for generation of an intracellular signal when an antigen binding domain to which it is fused binds cognate antigen. In embodiments the primary intracellular signaling domain has at least 70, 75, 80, 85, 90, 95, 98, or 99 % sequence identity with the entire intracellular region, or a fragment of the intracellular region which is sufficient for generation of an intracellular signal, of a naturally occurring primary stimulatory molecule, e.g., a human (GenBank Acc No. BAG36664.1), or other mammalian, e.g., a nonhuman species, e.g., rodent, monkey, ape or murine intracellular primary stimulatory molecule. In embodiments the primary intracellular signaling domain has at least 70, 75, 80, 85, 90, 95, 98, or 99 % sequence identity with SEQ ID NO: 18 or SEQ ID NO: 20.

[0329] In embodiments the primary intracellular signaling domain, has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with, or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues from the corresponding residues of the entire intracellular region, or a fragment of the intracellular region which is sufficient for generation of an intracellular signal, of a naturally occurring human primary stimulatory molecule, e.g., a naturally occurring human primary stimulatory molecule disclosed herein.COSTIMULATORY SIGNALING DOMAIN

[0330] In an embodiment, a costimulatory signaling domain produces an intracellular signal when an extracellular domain, e.g., an antigen binding domain to which it is fused binds cognate ligand. The costimulatory signaling domain is derived from a costimulatory molecule. The costimulatory signaling domain comprises sufficient primary costimulatory molecule sequence to produce an intracellular signal, e.g., when an extracellular domain, e.g., an antigen binding domain, to which it is fused binds cognate ligand.

[0331] The costimulatory domain can be one which optimizes the performance, e.g., the persistence, or immune effector function, of a T cell that comprises a CAR which comprises the costimulatory domain. E.g., a CAR (a CAR CD4+< ) for use in CD4 +< T cells can comprise an ICOS domain. E.g., a CAR (a CAR CD8+< ) for use in CD8 +< T cells can comprise a CD28 or a 4-1BB domain.

[0332] Costimulatory molecules are cell surface molecules, other than antigen receptors or their counter ligands that promote an immune effector response. In some cases they are required for an efficient or enhanced immune response. Typically, a costimulatory molecule generates an intracellular signal that is dependent on binding to a cognate ligand that is, in embodiments, other than an antigen, e.g., the antigen recognized by an antigen binding domain of a CARX cell, e.g., CART cell. Typically, signaling from a primary stimulatory molecule and a costimulatory molecule contribute to an immune effector response, and in some cases both are required for efficient or enhanced generation of an immune effector response.

[0333] A costimulatory domain comprises a functional fragment, or analog, of a costimulatory molecule (e.g., ICOS, CD28, or 4-1BB). It can comprise the entire intracellular region or a fragment of the intracellular region which is sufficient for generation of an intracellular signal, e.g., when an antigen binding domain to which it is fused binds cognate antigen. In embodiments the costimulatory domain has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99 % sequence identity with the entire intracellular region, or a fragment of the intracellular region which is sufficient for generation of an intracellular signal, of a naturally occurring costimulatory molecule, e.g., a human, or other mammalian, e.g., a nonhuman species, e.g., rodent, monkey, ape or murine intracellular costimulatory molecule. In embodiments the costimulatory domain has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99 % sequence identity with SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 40, or SEQ ID NO: 44.

[0334] Exemplary costimulatory signaling domains (intracellular signaling domains) are provided in Table 5. Table 5: Costimulatory Signaling Domains for CARX (identified by the Costimulatory Molecules from which they are derived)CD27CD284-1BB (CD137)OX40CD30CD40ICOS (CD278)ICAM-1LFA-1 (CD11a / CD18)CD2CD7LIGHTNKG2CB7-H3a ligand that specifically binds with CD83CDSGITRBAFFRHVEM (LIGHTR)SLAMf7NKP80 (KLRF1)CD160 (BY55)CD19CD4CD8 alphaCD8 betaIL2R betaIL2R gammaIL7R alphaITGA4VLA1CD49aITGA4IA4CD49DITGA6VLA-6C49fITGADCD11dITGAECD103ITGALCD11aLFA-1ITGAMCD11bITGAXCD11cITGB1CD29ITGB2CD18ITGB7TNFR2TRANCE / RANKLDNAM1 (CD226)SLAMF4 (C244, 2B4)CD84CD96 (Tactile)CEACAM1CRTAMLy9 (CD229)PSGL1C100 (SEMA4D)CD69SLAMF6 (NTB-A, Ly108)SLAM (SLAMF1, CD150, IPO-3)BLAME (SLAMF8)SELPLG (CD162)LTBRLATGADSPAG / Cbp

[0335] The CARs of use in the invention comprise costimulatory domains as defined in the claims.

[0336] In embodiments the costimulatory signaling domain, has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with, or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues from the corresponding residues of the entire intracellular region, or a fragment of the intracellular region which is sufficient for generation of an intracellular signal, of, a naturally occurring human costimulatory molecule, e.g., a naturally occurring human costimulatory molecule disclosed herein.INHIBITORY MOLECULES: INHIBITION

[0337] Inhibitory molecules, e.g., PD1, can, in some embodiments, decrease the ability of a CARX cell to mount an immune effector response. Inhibition of an inhibitory molecule, e.g., by inhibition at the DNA, RNA or protein level, can optimize CARX cell performance. In embodiments an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, can be used to inhibit expression of an inhibitory molecule in the CARX cell. In an embodiment the inhibitor is a shRNA. In an embodiment, the inhibitory molecule is inhibited within a CARX cell. In these embodiments, a dsRNA molecule that inhibits expression of the inhibitory molecule is linked to the nucleic acid that encodes a component, e.g., all of the components, of the CAR.

[0338] Exemplary inhibitory molecules, useful e.g., as shRNA targets, are provided in Table 6. Table 6: Inhibitory moleculesCD1602B4PD1TIM3LAG3TIGITCTLA-4BTLALAIR1PD-L1VISTA

[0339] In another aspect, the CAR-expressing cell described herein can further express another agent, e.g., an agent which enhances the activity of a CAR-expressing cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Inhibitory molecules, e.g., PD1, can, in some embodiments, decrease the ability of a CAR-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR beta. In one embodiment, the agent which inhibits an inhibitory molecule, e.g., is a molecule described herein, e.g., an agent that comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR beta, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 4-1BB, ICOS, CD27 or CD28, e.g., as described herein) and / or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein). In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of an extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and / or a CD3 zeta signaling domain described herein). In embodiments, the CAR-expressing cell comprises a switch costimulatory receptor, e.g., as described in WO 2013 / 019615 .

[0340] PD1 is an inhibitory member of the CD28 family of receptors that also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2 have been shown to downregulate T cell activation upon binding to PD1 (Freeman et a. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094). Immune suppression can be reversed by inhibiting the local interaction of PD1 with PD-L1.REDIRECTED SWITCHABLE INHIBITORY RECEPTORS: INHIBITORY EXTRACELLULAR DOMAINS

[0341] Extracellular domains of inhibitory receptors can be coupled to intracellular signaling domains that promote an immune effector response. Thus, engagement with a counterligand of the coinhibitory molecule is redirected into an optimization of immune effector response.

[0342] In an embodiment the CAR comprising an extracellular domain (ECD) of an inhibitory molecule is a CAR CD4+< , e.g., comprising an ICOS doamain, and is disposed in a CD4 +< T cell. In an embodiment the CAR comprising an extracellular domain (ECD) of an inhibitory molecule is a CAR CD8+< , e.g., comprising a CD28 or 4-1BB domain, and is disposed in a CD8+ T cell.

[0343] In one embodiment, the extracellular domain (ECD) of an inhibitory molecule, e.g., an inhibitory molecule described herein such as, e.g., Programmed Death 1 (PD1), can be fused to a transmembrane domain and intracellular signaling domain described herein, e.g., an intracellular signaling domain comprising a costimulatory signaling domain such as, e.g., 41BB OX40, Cd28, CD27, ICOS, and / or a primary signaling domain, e.g., of CD3 zeta. In one embodiment, the inhibitory molecule CAR, e.g., PD1 CAR, can be used alone. In one embodiment, the inhibitory molecule CAR, e.g., inhibitory molecule CAR, e.g., PD1 CAR, can be used in combination with another CAR, e.g., CD19CAR (e.g., a CD19CAR). In one embodiment, the PD1 CAR improves the persistence of the T cell. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR beta. In one embodiment, the inhibitory molecule CAR comprises a first polypeptide, e.g., of an inhibitory molecule such as PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR beta, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 41BB, ICOS, CD27 or CD28, e.g., as described herein) and / or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein).

[0344] In one embodiment, the inhibitory molecule CAR comprises the extracellular domain (ECD) of PD1 fused to a transmembrane domain and intracellular signaling domains such as 41BB and CD3 zeta (also referred to herein as a PD1 CAR). In one embodiment, the PD1 CAR improves the persistence of the cell CAR-expressing cell. In one embodiment, the PD1 CAR comprises the extracellular domain of PD1; the amino acid sequence of the extracellular domain of PD1 is provided as SEQ ID NO: 24.

[0345] In one embodiment, the PD1 CAR comprises the amino acid sequence of SEQ ID NO: 26 or 39.

[0346] In one embodiment, the PD1 CAR, e.g., the PD1 CAR described herein, is encoded by a nucleic acid sequence of SEQ ID NO: 27, or at least comprises the nucleic acid sequence of SEQ ID NO: 25 encoding the extracellular domain of PD-1.

[0347] Exemplary inhibitory extracellular domains are provided in Table 7. Table 7: Extracellular counter ligand binding domains from coinhibitory molecules (identified by the Coinibitory Molecules from which they are derived)B7-H1B7-1CD160P1H2B4PD1TIM3LAG3TIGITCTLA-4BTLALAIR1TGF-beta receptor

[0348] In embodiments the inhibitory extracellular domain, has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with, or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues from the corresponding residues of the entire extracellular region, or a fragment of the extracellular region which is sufficient for engagement with the counter ligand, of a naturally occurring human inhibitory molecule, e.g., a naturally occurring human primary stimulatory molecule disclosed herein.COSTIMULATORY MOLECULE LIGAND BINDING DOMAINS

[0349] Extracellular ligand binding domains of costimulatory molecules, referred to as a costimulatory ECD domain, can be coupled to intracellular signaling domains that promote an immune effector response. Thus, engagement with a counter ligand of the costimulatory molecule results in optimization of immune effector response.

[0350] In an embodiment the CAR comprising a costimulatory ECD domain is a CAR CD4+< , comprising, e.g., an ICOS domain, and is disposed in a CD4 +< T cell. In an embodiment the CAR comprising a costimulatory ECD domain is a CAR CD8+< , comprising, e.g., a CD28 or 4-1BB domain, and is disposed in a CD8+ T cell.

[0351] Exemplary Costimulatory ECD domains are provided in the Table 8 Table 8: Costimulatory ECD domains from costimulatory molecules (identified by the Costimulatory Molecules from which they are derived)ICOSCD28CD27HVEMLIGHTCD40L4-1BBOX40DR3GITRCD30TIM1SLAMCD2CD226

[0352] In embodiments the Costimulatory ECD domain, has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with, or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues from the corresponding residues of the entire extracellular region, or a fragment of the extracellular region which is sufficient for engagement with the counter ligand, of a naturally occurring human inhibitory molecule, e.g., a naturally occurring human costimulatory molecule disclosed herein.ICARs

[0353] CARs disclosed herein can include an inhibitory CAR (iCAR) member. An iCAR member comprises: an antigen binding domain (or other extracelluar domain) that recognizes an antigen on a non-target, e.g., a noncancer, cell; a transmembrane domain; and, a domain from an inhibitory molecule, e.g., an intracellular domain from an inhibitory molecule, e.g., from PD-1, CTLA4, or from a protein listed in Table 9. In an embodiment, the iCAR member comprises a second inhibitory intracellular signaling domain, e.g., from PD-1, CTLA4, or from a protein listed in Table 9.

[0354] Upon engagement of the antigen binding domain (or other extracelluar domain) of the iCAR member with its target antigen (or counter-ligand), the iCAR contributes to inhibiting, e.g., reversibly inhibiting, or minimizing, activation of the cell comprising the iCAR. As such, inclusion of an iCAR member in a CAR, e.g., and CART, cell, can limit damage to non-target, e.g., bystander, cells. While not wishing to be bound by theory, it is believed that an iCAR member, upon engagement with its antigen (or counter-ligand), limits one or more of cytokine secretion, cytotoxicity, and proliferation. In embodiments the effect is temporary, and upon subsequent engagement with a target cell the CAR, e.g., CART, cell is activated and attacks the target cell.

[0355] A target antigen for an iCAR member can be an antigen that has an expression profile on target cells and non-target cells such that an acceptably high level of attack on target cells and an acceptably low level of attack on non-target cells is achieved. Not only choice of antigen, but iCAR affinity for its antigen (or counter-ligand), CAR affinity for its antigen, level of expression of the iCAR, or levels of expression of the CAR can be used to optimize the ratio of on-target / off-target response.

[0356] In an embodiment, the antigen is absent, or down-regulated on tumor cells. In an embodiment the antigen comprises an HLA molecule. In an embodiment the antigen comprises a cell suface tumor suppressor antigen. In an embodiment the antigen comprises PCML (or another antigen that is down-regulated in lymphomas, breast or prostate cancer), HYAL2, DCC, or SMAR1.

[0357] In an embodiment, the antigen comprises a protein, carbohydrate, lipid, or a post-translational modification of a cell surface moiety, e.g., a mucin-type O-glycan (a core 3 O-glycan).

[0358] In an embodiment, the antigen comprises a moiety that is down-regulated by tumor cells undergoing an epithelial to mesenchymal transition.

[0359] In an embodiment, the antigen comprises E-cadherin.

[0360] In an embodiment a domain from an inhibitory molecule,, e.g., an intracellular signaling domain from PD-1 or CTLA4, produces an intracellular signal when an extracellular domain, e.g., an antigen binding domain, to which it is fused binds cognate antigen (or counter ligand). The inhibitory intracellular signaling domain is derived from an inhibitory molecule, e.g., it comprises intracellular sequence of an inhibitory molecule. It comprises sufficient inhibitory molecule sequence to produce an intracellular signal, e.g., when an antigen binding domain to which it is fused binds its cognate antigen.

[0361] In an embodiment, the primary intracellular signaling domain comprises a signaling motif, e.g., an immunoreceptor tyrosine-based activation motif or ITIM.

[0362] A domain from an inhibitory molecule, comprises a functional fragment, or analog, of an inhibitory molecule intracellular domain. It can comprise the entire intracellular region or a fragment of the intracellular region which is sufficient for generation of an intracellular signal when an antigen binding domain to which it is fused, binds cognate antigen. In embodiments the inhibitory intracellular signaling domain has at least 70, 75, 80, 85, 90, 95, 98, or 99 % sequence identity with, or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues from, the corresponding residues oa naturally occurring inhibitory molecule, e.g., a a molecule from Table 9.

[0363] Exemplary inhibitory molecules which can provide intracellular signaling domains are provided in Table 9. Table 9: Inhibitory moleculesB7-H1B7-1CD160P1H2B4PD1TIM3LAG3TIGITCTLA-4BTLALAIR1TGF-beta receptor

[0364] Thus, in one, aspect, disclosed herein is, an CAR comprising an iCAR member. The iCAR member comprises: an antigen binding domain (or other extracelluar domain) that recognizes an antigen on a non-target, e.g., a noncancer cell; a transmembrane domain; and a domain from an inhibitory molecule,, e.g., from PD-1, CTLA4, or from a protein listed in Table 4.

[0365] In an embodiment, the iCAR member comprises a second inhibitory intracellular signaling domain, e.g., from PD-1, CTLA4, or from a protein listed in Table 9.NATURAL KILLER CELL RECEPTOR (NKR) CARs

[0366] In an embodiment, a CAR molecule comprises one or more components of a natural killer cell receptor (NKR), thereby forming an NKR-CAR. The NKR component can be a transmembrane domain, a hinge domain, or a cytoplasmic domain from any of the following natural killer cell receptors: killer cell immunoglobulin-like receptor (KIR), e.g., KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3DP1; natural cyotoxicity receptor (NCR), e.g., NKp30, NKp44, NKp46; signaling lymphocyte activation molecule (SLAM) family of immune cell receptors, e.g., CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10; Fc receptor (FcR), e.g., CD16, and CD64; and Ly49 receptors, e.g., LY49A, LY49C. The NKR-CAR molecules described herein may interact with an adaptor molecule or intracellular signaling domain, e.g., DAP12. Exemplary configurations and sequences of CAR molecules comprising NKR components are described in International Publication No. WO2014 / 145252 .STRATEGIES FOR REGULATING CHIMERIC ANTIGEN RECEPTORS

[0367] There are many ways CAR activities can be regulated. In some embodiments, a regulatable CAR (RCAR) where the CAR activity can be controlled is desirable to optimize the safety and efficacy of a CAR therapy. For example, inducing apoptosis using, e.g., a caspase fused to a dimerization domain (see, e.g., Di et al., N Engl. J. Med. 2011 Nov. 3; 365(18):1673-1683), can be used as a safety switch in the CAR therapy of the instant invention. In another example, CAR-expressing cells can also express an inducible Caspase-9 (iCaspase-9) molecule that, upon administration of a dimerizer drug (e.g., rimiducid (also called AP1903 (Bellicum Pharmaceuticals) or AP20187 (Ariad)) leads to activation of the Caspase-9 and apoptosis of the cells. The iCaspase-9 molecule contains a chemical inducer of dimerization (CID) binding domain that mediates dimerization in the presence of a CID. This results in inducible and selective depletion of CAR-expressing cells. In some cases, the iCaspase-9 molecule is encoded by a nucleic acid molecule separate from the CAR-encoding vector(s). In some cases, the iCaspase-9 molecule is encoded by the same nucleic acid molecule as the CAR-encoding vector. The iCaspase-9 can provide a safety switch to avoid any toxicity of CAR-expressing cells. See, e.g., Song et al. Cancer Gene Ther. 2008; 15(10):667-75; Clinical Trial Id. No. NCT02107963; and Di Stasi et al. N. Engl. J. Med. 2011; 365:1673-83.

[0368] Alternative strategies for regulating the CAR therapy of the instant invention include utilizing small molecules or antibodies that deactivate or turn off CAR activity, e.g., by deleting CAR-expressing cells, e.g., by inducing antibody dependent cell-mediated cytotoxicity (ADCC). For example, CAR-expressing cells described herein may also express an antigen that is recognized by molecules capable of inducing cell death, e.g., ADCC or compliment-induced cell death. For example, CAR expressing cells described herein may also express a receptor capable of being targeted by an antibody or antibody fragment. Examples of such receptors include EpCAM, VEGFR, integrins (e.g., integrins αvβ3, α4, αI3 / 4β3, α4β7, α5β1, αvβ3, αv), members of the TNF receptor superfamily (e.g., TRAIL-R1 , TRAIL-R2), PDGF Receptor, interferon receptor, folate receptor, GPNMB, ICAM-1 , HLA-DR, CEA, CA-125, MUC1 , TAG-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD1 1 , CD1 1 a / LFA-1 , CD15, CD18 / ITGB2, CD19, CD20, CD22, CD23 / lgE Receptor, CD25, CD28, CD30, CD33, CD38, CD40, CD41 , CD44, CD51 , CD52, CD62L, CD74, CD80, CD125, CD147 / basigin, CD152 / CTLA-4, CD154 / CD40L, CD195 / CCR5, CD319 / SLAMF7, and EGFR, and truncated versions thereof (e.g., versions preserving one or more extracellular epitopes but lacking one or more regions within the cytoplasmic domain). For example, CAR-expressing cells described herein may also express a truncated epidermal growth factor receptor (EGFR) which lacks signaling capacity but retains the epitope that is recognized by molecules capable of inducing ADCC, e.g., cetuximab (ERBITUX ®< ), such that administration of cetuximab induces ADCC and subsequent depletion of the CAR-expressing cells (see, e.g., WO2011 / 056894, and Jonnalagadda et al., Gene Ther. 2013; 20(8)853-860). Another strategy includes expressing a highly compact marker / suicide gene that combines target epitopes from both CD32 and CD20 antigens in the CAR-expressing cells described herein, which binds rituximab, resulting in selective depletion of the CAR-expressing cells, e.g., by ADCC (see, e.g., Philip et al., Blood. 2014; 124(8)1277-1287). Other methods for depleting CAR-expressing cells described herein include administration of CAMPATH ®< , a monoclonal anti-CD52 antibody that selectively binds and targets mature lymphocytes, e.g., CAR-expressing cells, for destruction, e.g., by inducing ADCC. In other embodiments, the CAR-expressing cell can be selectively targeted using a CAR ligand, e.g., an anti-idiotypic antibody. In some embodiments, the anti-idiotypic antibody can cause effector cell activity, e.g, ADCC or ADC activities, thereby reducing the number of CAR-expressing cells. In other embodiments, the CAR ligand, e.g., the anti-idiotypic antibody can be coupled to an agent that induces cell killing, e.g., a toxin, thereby reducing the number of CAR-expressing cells. In other embodiments, CAR-expressing cells can be selectively targeted using a CAR ligand, e.g., an anti-idiotypic antibody. In some embodiments, the anti-idiotypic antibody can cause effector cell activity, e.g, ADCC or ADC activities, thereby reducing the number of CAR-expressing cells. In other embodiments, the CAR ligand, e.g., the anti-idiotypic antibody, can be coupled to an agent that induces cell killing, e.g., a toxin, thereby reducing the number of CAR-expressing cells. Alternatively, the CAR molecules themselves can be configured such that the activity can be regulated, e.g., turned on and off, as described below.

[0369] In an aspect, a RCAR comprises a set of polypeptides, typically two in the simplest embodiments, in which the components of a standard CAR described herein, e.g., an antigen binding domain and an intracellular signaling domain, are partitioned on separate polypeptides or members. In some embodiments, the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen binding domain to an intracellular signaling domain. In one embodiment, the CARs of the present invention utilizes a dimerization switch as those described in, e.g., WO2014127261 . Additional description and exemplary configurations of such regulatable CARs are provided herein and in International Publiciation No. WO 2015 / 090229 .

[0370] In an aspect, an RCAR comprises two polypeptides or members: 1) an intracellular signaling member comprising an intracellular signaling domain, e.g., a primary intracellular signaling domain described herein, and a first switch domain; 2) an antigen binding member comprising an antigen binding domain, e.g., that binds specifically to CD19, as described herein and a second switch domain. Optionally, the RCAR comprises a transmembrane domain described herein. In an embodiment, a transmembrane domain can be disposed on the intracellular signaling member, on the antigen binding member, or on both. (Unless otherwise indicated, when members or elements of an RCAR are described herein, the order can be as provided, but other orders are included as well. In other words, in an embodiment, the order is as set out in the text, but in other embodiments, the order can be different. E.g., the order of elements on one side of a transmembrane region can be different from the example, e.g., the placement of a switch domain relative to a intracellular signaling domain can be different, e.g., reversed).

[0371] In an embodiment, the first and second switch domains can form an intracellular or an extracellular dimerization switch. In an embodiment, the dimerization switch can be a homodimerization switch, e.g., where the first and second switch domain are the same, or a heterodimerization switch, e.g., where the first and second switch domain are different from one another.

[0372] In embodiments, an RCAR can comprise a "multi switch." A multi switch can comprise heterodimerization switch domains or homodimerization switch domains. A multi switch comprises a plurality of, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, switch domains, independently, on a first member, e.g., an antigen binding member, and a second member, e.g., an intracellular signaling member. In an embodiment, the first member can comprise a plurality of first switch domains, e.g., FKBP-based switch domains, and the second member can comprise a plurality of second switch domains, e.g., FRB-based switch domains. In an embodiment, the first member can comprise a first and a second switch domain, e.g., a FKBP-based switch domain and a FRB-based switch domain, and the second member can comprise a first and a second switch domain, e.g., a FKBP-based switch domain and a FRB-based switch domain.

[0373] In an embodiment, the intracellular signaling member comprises one or more intracellular signaling domains, e.g., a primary intracellular signaling domain and one or more costimulatory signaling domains.

[0374] In an embodiment, the antigen binding member may comprise one or more intracellular signaling domains, e.g., one or more costimulatory signaling domains. In an embodiment, the antigen binding member comprises a plurality, e.g., 2 or 3 costimulatory signaling domains described herein, e.g., selected from 4-1BB, CD28, CD27, ICOS, and OX40, and in embodiments, no primary intracellular signaling domain. In an embodiment, the antigen binding member comprises the following costimulatory signaling domains, from the extracellular to intracellular direction: 4-1BB - CD27; 4-1BB - CD27; CD27 - 4-1BB; 4-1BB - CD28; CD28 - 4-1BB; OX40 - CD28; CD28 - OX40; CD28 - 4-1BB; or 4-1BB - CD28. In the invention, the intracellular binding member comprises a CD3zeta domain. In one such embodiment the RCAR comprises (1) an antigen binding member comprising, an antigen binding domain, a transmembrane domain, and two costimulatory domains and a first switch domain; and (2) an intracellular signaling domain comprising a transmembrane domain or membrane tethering domain and at least one primary intracellular signaling domain, and a second switch domain. The CARs utilised in the invention include signaling domains defined in the claims.

[0375] An embodiment provides RCARs wherein the antigen binding member is not tethered to the surface of the CAR cell. This allows a cell having an intracellular signaling member to be conveniently paired with one or more antigen binding domains, without transforming the cell with a sequence that encodes the antigen binding member. In such embodiments, the RCAR comprises: 1) an intracellular signaling member comprising: a first switch domain, a transmembrane domain, an intracellular signaling domain, e.g., a primary intracellular signaling domain, and a first switch domain; and 2) an antigen binding member comprising: an antigen binding domain, and a second switch domain, wherein the antigen binding member does not comprise a transmembrane domain or membrane tethering domain, and, optionally, does not comprise an intracellular signaling domain. In some embodiments, the RCAR may further comprise 3) a second antigen binding member comprising: a second antigen binding domain, e.g., a second antigen binding domain that binds a different antigen than is bound by the antigen binding domain; and a second switch domain.

[0376] Also provided herein are RCARs wherein the antigen binding member comprises bispecific activation and targeting capacity. In this embodiment, the antigen binding member can comprise a plurality, e.g., 2, 3, 4, or 5 antigen binding domains, e.g., scFvs, wherein each antigen binding domain binds to a target antigen, e.g. different antigens or the same antigen, e.g., the same or different epitopes on the same antigen. In an embodiment, the plurality of antigen binding domains are in tandem, and optionally, a linker or hinge region is disposed between each of the antigen binding domains. Suitable linkers and hinge regions are described herein.

[0377] An embodiment provides RCARs having a configuration that allows switching of proliferation. In this embodiment, the RCAR comprises: 1) an intracellular signaling member comprising: optionally, a transmembrane domain or membrane tethering domain; one or more co-stimulatory signaling domain, e.g., selected from 4-1BB, CD28, CD27, ICOS, and OX40, and a switch domain; and 2) an antigen binding member comprising: an antigen binding domain, a transmembrane domain, and a primary intracellular signaling domain, e.g., a CD3zeta domain, wherein the antigen binding member does not comprise a switch domain, or does not comprise a switch domain that dimerizes with a switch domain on the intracellular signaling member. In an embodiment, the antigen binding member does not comprise a co-stimulatory signaling domain. In an embodiment, the intracellular signaling member comprises a switch domain from a homodimerization switch. In an embodiment, the intracellular signaling member comprises a first switch domain of a heterodimerization switch and the RCAR comprises a second intracellular signaling member which comprises a second switch domain of the heterodimerization switch. In such embodiments, the second intracellular signaling member comprises the same intracellular signaling domains as the intracellular signaling member. In an embodiment, the dimerization switch is intracellular. In an embodiment, the dimerization switch is extracellular.

[0378] In any of the RCAR configurations described here, the first and second switch domains comprise a FKBP-FRB based switch as described herein.

[0379] Also provided herein are cells comprising an RCAR described herein. Any cell that is engineered to express a RCAR can be used as a RCARX cell. In an embodiment the RCARX cell is a T cell, and is referred to as a RCART cell. In an instance the RCARX cell is an NK cell, and is referred to as a RCARN cell.

[0380] Also provided herein are nucleic acids and vectors comprising RCAR encoding sequences. Sequence encoding various elements of an RCAR can be disposed on the same nucleic acid molecule, e.g., the same plasmid or vector, e.g., viral vector, e.g., lentiviral vector. In an embodiment, (i) sequence encoding an antigen binding member and (ii) sequence encoding an intracellular signaling member, can be present on the same nucleic acid, e.g., vector. Production of the corresponding proteins can be achieved, e.g., by the use of separate promoters, or by the use of a bicistronic transcription product (which can result in the production of two proteins by cleavage of a single translation product or by the translation of two separate protein products). In an embodiment, a sequence encoding a cleavable peptide, e.g., a P2A or F2A sequence, is disposed between (i) and (ii). In an embodiment, a sequence encoding an IRES, e.g., an EMCV or EV71 IRES, is disposed between (i) and (ii). In these embodiments, (i) and (ii) are transcribed as a single RNA. In an embodiment, a first promoter is operably linked to (i) and a second promoter is operably linked to (ii), such that (i) and (ii) are transcribed as separate mRNAs.

[0381] Alternatively, the sequence encoding various elements of an RCAR can be disposed on the different nucleic acid molecules, e.g., different plasmids or vectors, e.g., viral vector, e.g., lentiviral vector. E.g., the (i) sequence encoding an antigen binding member can be present on a first nucleic acid, e.g., a first vector, and the (ii) sequence encoding an intracellular signaling member can be present on the second nucleic acid, e.g., the second vector.Dimerization switches

[0382] Dimerization switches can be non-covalent or covalent. In a non-covalent dimerization switch, the dimerization molecule promotes a non-covalent interaction between the switch domains. In a covalent dimerization switch, the dimerization molecule promotes a covalent interaction between the switch domains.

[0383] In an embodiment, the RCAR comprises a FKBP / FRAP, or FKBP / FRB,-based dimerization switch. FKBP12 (FKBP, or FK506 binding protein) is an abundant cytoplasmic protein that serves as the initial intracellular target for the natural product immunosuppressive drug, rapamycin. Rapamycin binds to FKBP and to the large PI3K homolog FRAP (RAFT, mTOR). FRB is a 93 amino acid portion of FRAP, that is sufficient for binding the FKBP-rapamycin complex (Chen, J., Zheng, X. F., Brown, E. J. & Schreiber, S. L. (1995) Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proc Natl Acad Sci U S A 92: 4947-51.)

[0384] In embodiments, an FKBP / FRAP, e.g., an FKBP / FRB, based switch can use a dimerization molecule, e.g., rapamycin or a rapamycin analog.

[0385] The amino acid sequence of FKBP is as follows:

[0386] In embodiments, an FKBP switch domain can comprise a fragment of FKBP having the ability to bind with FRB, or a fragment or analog thereof, in the presence of rapamycin or a rapalog, e.g., the underlined portion of SEQ ID NO: 90, which is:

[0387] The amino acid sequence of FRB is as follows:

[0388] "FKBP / FRAP, e.g., an FKBP / FRB, based switch" as that term is used herein, refers to a dimerization switch comprising: a first switch domain, which comprises an FKBP fragment or analog thereof having the ability to bind with FRB, or a fragment or analog thereof, in the presence of rapamycin or a rapalog, e.g., RAD001, and has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with, or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues from, the FKBP sequence of SEQ ID NO: 90 or 91; and a second switch domain, which comprises an FRB fragment or analog thereof having the ability to bind with FRB, or a fragment or analog thereof, in the presence of rapamycin or a rapalog, and has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with, or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues from, the FRB sequence of SEQ ID NO: 92. In an embodiment, a RCAR comprises one switch domain comprises amino acid residues disclosed in SEQ ID NO: 90 (or SEQ ID NO: 91), and one switch domain comprises amino acid residues disclosed in SEQ ID NO: 92.

[0389] In embodiments, the FKBP / FRB dimerization switch comprises a modified FRB switch domain that exhibits altered, e.g., enhanced, complex formation between an FRB-based switch domain, e.g., the modified FRB switch domain, a FKBP-based switch domain, and the dimerization molecule, e.g., rapamycin or a rapalogue, e.g., RAD001. In an embodiment, the modified FRB switch domain comprises one or more mutations, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, selected from mutations at amino acid position(s) L2031, E2032, S2035, R2036, F2039, G2040, T2098, W2101, D2102, Y2105, and F2108, where the wild-type amino acid is mutated to any other naturally-occurring amino acid. In an embodiment, a mutant FRB comprises a mutation at E2032, where E2032 is mutated to phenylalanine (E2032F), methionine (E2032M), arginine (E2032R), valine (E2032V), tyrosine (E2032Y), isoleucine (E2032I), e.g., SEQ ID NO: 93, or leucine (E2032L), e.g., SEQ ID NO: 94. In an embodiment, a mutant FRB comprises a mutation at T2098, where T2098 is mutated to phenylalanine (T2098F) or leucine (T2098L), e.g., SEQ ID NO: 95. In an embodiment, a mutant FRB comprises a mutation at E2032 and at T2098, where E2032 is mutated to any amino acid, and where T2098 is mutated to any amino acid, e.g., SEQ ID NO: 96. In an embodiment, a mutant FRB comprises an E2032I and a T2098L mutation, e.g., SEQ ID NO: 97. In an embodiment, a mutant FRB comprises an E2032L and a T2098L mutation, e.g., SEQ ID NO: 98. Table 10. Exemplary mutant FRB having increased affinity for a dimerization molecule.FRB mutant Amino Acid Sequence SEQ ID NO: E2032I mutant93E2032L mutant94T2098L mutant95E2032, T2098 mutant96E2032I, T2098L mutant97E2032L, T2098L mutant98

[0390] Other suitable dimerization switches include a GyrB-GyrB based dimerization switch, a Gibberellin-based dimerization switch, a tag / binder dimerization switch, and a halo-tag / snap-tag dimerization switch. Following the guidance provided herein, such switches and relevant dimerization molecules will be apparent to one of ordinary skill.Dimerization molecule

[0391] Association between the switch domains is promoted by the dimerization molecule. In the presence of dimerization molecule interaction or association between switch domains allows for signal transduction between a polypeptide associated with, e.g., fused to, a first switch domain, and a polypeptide associated with, e.g., fused to, a second switch domain. In the presence of non-limiting levels of dimerization molecule signal transduction is increased by 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 5, 10, 50, 100 fold, e.g., as measured in a system described herein.

[0392] Rapamycin and rapamycin analogs (sometimes referred to as rapalogues), e.g., RAD001, can be used as dimerization molecules in a FKBP / FRB-based dimerization switch described herein. In an embodiment the dimerization molecule can be selected from rapamycin (sirolimus), RAD001 (everolimus), zotarolimus, temsirolimus, AP-23573 (ridaforolimus), biolimus and AP21967. Additional rapamycin analogs suitable for use with FKBP / FRB-based dimerization switches are further described in the section entitled "Combination Therapies", or in the subsection entitled "mTOR inhibitors".SPLIT CAR

[0393] In some embodiments, the CAR-expressing cell uses a split CAR. The split CAR approach is described in more detail in publications WO2014 / 055442 and WO2014 / 055657. Briefly, a split CAR system comprises a cell expressing a first CAR having a first antigen binding domain and a costimulatory domain (e.g., 4-1BB), and the cell also expresses a second CAR having a second antigen binding domain and an intracellular signaling domain ( CD3 zeta). When the cell encounters the first antigen, the costimulatory domain is activated, and the cell proliferates. When the cell encounters the second antigen, the intracellular signaling domain is activated and cell-killing activity begins. Thus, the CAR-expressing cell is only fully activated in the presence of both antigens. In embodiments, the first antigen binding domain recognizes a cancer associated antigen described herein (e.g., CD19, CD123, CD22, CD30, CD34, CD171, CS-1, CLL-1, CD33, EGFRvIII , GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, Plysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, legumain, HPV E6,E7, MAGE-A1, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, or IGLL1).NUCLEIC ACID CONSTRUCTS ENCODING A CAR

[0394] The present invention may utilise nucleic acid molecules encoding one or more CAR constructs described herein. In one aspect, the nucleic acid molecule is provided as a messenger RNA transcript. In one aspect, the nucleic acid molecule is provided as a DNA construct. The CARs utilised in the present invention are defined in the claims.

[0395] Accordingly, in one aspect, the invention may utilise a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain that binds to a tumor antigen described herein, a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular signaling domain comprising a costimulatory signaling domain and a primary signaling domain in accordance with the claims.

[0396] In one embodiment, the transmembrane domain is transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In some embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp.

[0397] In one embodiment, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In some embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp. In one embodiment, the transmembrane domain comprises a sequence that encodes SEQ ID NO: 12 or SEQ ID NO: 42, or a sequence with 95-99% identity thereof.

[0398] In one embodiment, the antigen binding domain is connected to the transmembrane domain by a hinge region, e.g., a hinge described herein. In one embodiment, the hinge region comprises a sequence that encodes SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10, or a sequence with 95-99% identity thereof.

[0399] The isolated nucleic acid molecule further comprises a sequence encoding a costimulatory domain. The costimulatory domain may be a functional signaling domain of a protein selected from the group consisting of MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, and a ligand that specifically binds with CD83. The costimulatory domain may comprise 4-1BB, CD27, CD28 or ICOS. In the invention, the CAR molecules comprise a costimulatory domain and an intracellular signaling domain as defined in the claims.

[0400] In one embodiment, the costimulatory domain comprises a sequence that encodes SEQ ID NO:16, or a sequence with 95-99% identity thereof. In one embodiment, the intracellular signaling domain comprises a functional signaling domain of 4-1BB and a functional signaling domain of CD3 zeta. In one embodiment, the intracellular signaling domain comprises a sequence that encodes SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 40 or SEQ ID NO: 44, or a sequence with 95-99% identity thereof, and a sequence that encodes SEQ ID NO: 18 or SEQ ID NO:20, or a sequence with 95-99% identity thereof, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.

[0401] In another aspect, the invention may utilise an isolated nucleic acid molecule encoding a CAR construct comprising a leader sequence of SEQ ID NO: 3, a scFv domain as described herein, a hinge region of SEQ ID NO:5 or SEQ ID NO:7 or SEQ ID NO:9 or SEQ ID NO:11 (or a sequence with 95-99% identity thereof), a transmembrane domain having a sequence of SEQ ID NO: 13 or SEQ ID NO: 43 (or a sequence with 95-99% identity thereof), a 4-1BB costimulatory domain having a sequence of SEQ ID NO:15 or a CD27 costimulatory domain having a sequence of SEQ ID NO:17 or a ICOS costimulatory domain having a sequence of SEQ ID NO: 41 or 45 (or a sequence with 95-99% identity thereof), and a CD3 zeta stimulatory domain having a sequence of SEQ ID NO:19 (mutant CD3 zeta) or SEQ ID NO: 21 (wild-type human CD3 zeta) or a sequence with 95-99% identity thereof.

[0402] In another aspect, the invention may utilise a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule that comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulatory domain, and wherein said antigen binding domain binds to a tumor antigen selected from a group consisting of: CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII , GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PRSS21, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, Plysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6,E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1.

[0403] The nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.VECTORS ENCODING A CAR

[0404] The present invention may utilise vectors in which a DNA of the present disclosure is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. A retroviral vector may also be, e.g., a gammaretroviral vector. A gammaretroviral vector may include, e.g., a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTR), and a transgene of interest, e.g., a gene encoding a CAR. A gammaretroviral vector may lack viral structural gens such as gag, pol, and env. Exemplary gammaretroviral vectors include Murine Leukemia Virus (MLV), Spleen-Focus Forming Virus (SFFV), and Myeloproliferative Sarcoma Virus (MPSV), and vectors derived therefrom. Other gammaretroviral vectors are described, e.g., in Tobias Maetzig et al., "Gammaretroviral Vectors: Biology, Technology and Application" Viruses. 2011 Jun; 3(6): 677-713.

[0405] In another embodiment, the vector comprising the nucleic acid encoding the desired CAR of use in the invention is an adenoviral vector (A5 / 35). In another embodiment, the expression of nucleic acids encoding CARs can be accomplished using of transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See below June et al. 2009 Nature Reviews Immunology 9.10: 704-716.

[0406] In brief summary, the expression of natural or synthetic nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to a promoter, and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0407] The expression constructs of the present disclosure may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466.

[0408] In another embodiment, the invention may utilise a gene therapy vector.

[0409] The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0410] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1 -4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0411] A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used. Exemplary promoters include the CMV IE gene, EF-1α, ubiquitin C, or phosphoglycerokinase (PGK) promoters. In an embodiment, the promoter is a PGK promoter, e.g., a truncated PGK promoter as described herein.

[0412] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[0413] An example of a promoter that is capable of expressing a CAR encoding nucleic acid molecule in a mammalian T cell is the EF1a promoter. The native EF1a promoter drives expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome. The EF1a promoter has been extensively used in mammalian expression plasmids and has been shown to be effective in driving CAR expression from nucleic acid molecules cloned into a lentiviral vector. See, e.g., Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). In one aspect, the EF1a promoter comprises the sequence provided as SEQ ID NO:1.

[0414] Another example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor-1α promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0415] Another example of a promoter is the phosphoglycerate kinase (PGK) promoter. In embodiments, a truncated PGK promoter (e.g., a PGK promoter with one or more, e.g., 1, 2, 5, 10, 100, 200, 300, or 400, nucleotide deletions when compared to the wild-type PGK promoter sequence) may be desired. The nucleotide sequences of exemplary PGK promoters are provided below.

[0416] WT PGK Promoter:

[0417] Exemplary truncated PGK Promoters: PGK100: PGK200: PGK300: PGK400:

[0418] A vector may also include, e.g., a signal sequence to facilitate secretion, a polyadenylation signal and transcription terminator (e.g., from Bovine Growth Hormone (BGH) gene), an element allowing episomal replication and replication in prokaryotes (e.g. SV40 origin and ColE1 or others known in the art) and / or elements to allow selection (e.g., ampicillin resistance gene and / or zeocin marker).

[0419] In order to assess the expression of a CAR polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co- transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

[0420] Reporter genes are used for identifying potentially transfected or transduced cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0421] In embodiments, the vector may comprise two or more nucleic acid sequences encoding a CAR, e.g., a first CAR and a second CAR in accordance with the claims that specifically binds to a second antigen, e.g., another cancer associated antigen described herein (e.g., CD19, CD123, CD22, CD30, CD34, CD171, CS-1, CLL-1, CD33, EGFRvIII , GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, Plysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, legumain, HPV E6,E7, MAGE-A1, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, or IGLL1). In such embodiments, the two or more nucleic acid sequences encoding the CAR are encoded by a single nucleic molecule in the same frame and as a single polypeptide chain. In this aspect, the two or more CARs, can, e.g., be separated by one or more peptide cleavage sites. (e.g., an auto-cleavage site or a substrate for an intracellular protease). Examples of peptide cleavage sites include the following, wherein the GSG residues are optional: T2A:(GSG)EGRGSLLTCGDVEENPGP (SEQ ID NO: 104)P2A:(GSG)ATNFSLLKQAGDVEENPGP (SEQ ID NO: 105)E2A:(GSG)QCTNYALLKLAGDVESNPGP (SEQ ID NO: 106)F2A:(GSG)VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 107)

[0422] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0423] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1 -4, Cold Spring Harbor Press, NY). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection

[0424] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0425] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g. , an artificial membrane vesicle). Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system.

[0426] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a "collapsed" structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0427] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. "Liposome" is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[0428] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present disclosure, in order to confirm the presence of the recombinant DNA or RNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.

[0429] The present invention further may further utilise a vector comprising a CAR encoding nucleic acid molecule. In one aspect, a CAR vector can be directly transduced into a cell, e.g., a T cell. In one aspect, the vector is a cloning or expression vector, e.g., a vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double minute chromosomes), retroviral and lentiviral vector constructs. In one aspect, the vector is capable of expressing the CAR construct in T cells, e.g., CD4 +< T cells or CD8 +< T cells. In one aspect, the mammalian T cell is a human T cell.RNAs ENCODING CARS; RNA TRANSFECTION

[0430] Also disclosed herein are methods for producing an in vitro transcribed RNA CAR. The present disclosure includes a CAR encoding RNA construct that can be directly transfected into a cell. A method for generating mRNA for use in transfection can involve in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3' and 5' untranslated sequence ("UTR"), a 5' cap and / or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length (SEQ ID NO:32). RNA so produced can efficiently transfect different kinds of cells. In one aspect, the template includes sequences for the CAR.

[0431] In one aspect, a CAR of use in the invention is encoded by a messenger RNA (mRNA). In one aspect, the mRNA encoding a CAR described herein is introduced into a T cell for production of a CART cell.

[0432] In one embodiment, the in vitro transcribed RNA CAR can be introduced to a cell as a form of transient transfection. The RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA. The desired temple for in vitro transcription is a CAR described herein. For example, the template for the RNA CAR comprises an extracellular region comprising a single chain variable domain of an antibody to a tumor associated antigen described herein; a hinge region (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein such as a transmembrane domain of CD8a); and a cytoplasmic region that includes an intracellular signaling domain, e.g., an intracellular signaling domain described herein, e.g., comprising the signaling domain of CD3-zeta and the signaling domain of 4-1BB.

[0433] In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the nucleic acid can include some or all of the 5' and / or 3' untranslated regions (UTRs). The nucleic acid can include exons and introns. In one embodiment, the DNA to be used for PCR is a human nucleic acid sequence. In another embodiment, the DNA to be used for PCR is a human nucleic acid sequence including the 5' and 3' UTRs. The DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.

[0434] PCR is used to generate a template for in vitro transcription of mRNA which is used for transfection. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR. "Substantially complementary," as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary, or mismatched. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR. The primers can be designed to be substantially complementary to any portion of the DNA template. For example, the primers can be designed to amplify the portion of a nucleic acid that is normally transcribed in cells (the open reading frame), including 5' and 3' UTRs. The primers can also be designed to amplify a portion of a nucleic acid that encodes a particular domain of interest. In one embodiment, the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5' and 3' UTRs. Primers useful for PCR can be generated by synthetic methods that are well known in the art. "Forward primers" are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified. "Upstream" is used herein to refer to a location 5, to the DNA sequence to be amplified relative to the coding strand. "Reverse primers" are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified. "Downstream" is used herein to refer to a location 3' to the DNA sequence to be amplified relative to the coding strand.

[0435] Any DNA polymerase useful for PCR can be used in the methods disclosed herein. The reagents and polymerase are commercially available from a number of sources.

[0436] Chemical structures with the ability to promote stability and / or translation efficiency may also be used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is between one and 3000 nucleotides in length. The length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.

[0437] The 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the nucleic acid of interest. Alternatively, UTR sequences that are not endogenous to the nucleic acid of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the nucleic acid of interest can be useful for modifying the stability and / or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3' UTR sequences can decrease the stability of mRNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.

[0438] In one embodiment, the 5' UTR can contain the Kozak sequence of the endogenous nucleic acid. Alternatively, when a 5' UTR that is not endogenous to the nucleic acid of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art. In other embodiments the 5' UTR can be 5'UTR of an RNA virus whose RNA genome is stable in cells. In other embodiments various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the mRNA.

[0439] To enable synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In one preferred embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

[0440] In a preferred embodiment, the mRNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell. On a circular DNA template, for instance, plasmid DNA, RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of the 3' UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is polyadenylated after transcription.

[0441] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

[0442] The conventional method of integration of polyA / T stretches into a DNA template is molecular cloning. However polyA / T sequence integrated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes cloning procedures not only laborious and time consuming but often not reliable. That is why a method which allows construction of DNA templates with polyA / T 3' stretch without cloning highly desirable.

[0443] The polyA / T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100T tail (SEQ ID NO: 35) or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. In one embodiment, the poly(T) tail is from 50 to 5000 nucleotides in length (SEQ ID NO: 87). Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 nucleotides in length (SEQ ID NO: 88).

[0444] Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP). In one embodiment, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides (SEQ ID NO: 38) results in about a two-fold increase in the translation efficiency of the RNA. Additionally, the attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachment can contain modified / artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.

[0445] 5' caps on also provide stability to RNA molecules. In a preferred embodiment, RNAs produced by the methods disclosed herein include a 5' cap. The 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0446] The RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.

[0447] There are several advantages of RNA transfection methods of the disclosure. For example, RNA transfection is essentially transient and a vector-free: An RNA transgene can be delivered to a lymphocyte and expressed therein following a brief in vitro cell activation, as a minimal expressing cassette without the need for any additional viral sequences. Under these conditions, integration of the transgene into the host cell genome is unlikely. Cloning of cells is not necessary because of the efficiency of transfection of the RNA and its ability to uniformly modify the entire lymphocyte population. Furthermore, gene expression from an RNA source does not require transcription and the protein product is produced rapidly after the transfection. Since the RNA has to only gain access to the cytoplasm, rather than the nucleus, typical transfection methods result in an extremely high rate of transfection.

[0448] RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as "gene guns" (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).NON-VIRAL DELIVERY METHODS

[0449] In some aspects, non-viral methods can be used to deliver a nucleic acid encoding a CAR described herein into a cell or tissue or a subject.

[0450] In some embodiments, the non-viral method includes the use of a transposon (also called a transposable element). In some embodiments, a transposon is a piece of DNA that can insert itself at a location in a genome, for example, a piece of DNA that is capable of self-replicating and inserting its copy into a genome, or a piece of DNA that can be spliced out of a longer nucleic acid and inserted into another place in a genome. For example, a transposon comprises a DNA sequence made up of inverted repeats flanking genes for transposition.

[0451] Exemplary methods of nucleic acid delivery using a transposon include a Sleeping Beauty transposon system (SBTS) and a piggyBac (PB) transposon system. See, e.g., Aronovich et al. Hum. Mol. Genet. 20.R1(2011):R14-20; Singh et al. Cancer Res. 15(2008):2961-2971; Huang et al. Mol. Ther. 16(2008):580-589; Grabundzija et al. Mol. Ther. 18(2010):1200-1209; Kebriaei et al. Blood. 122.21(2013):166; Williams. Molecular Therapy 16.9(2008):1515-16; Bell et al. Nat. Protoc. 2.12(2007):3153-65; and Ding et al. Cell. 122.3(2005):473-83 .

[0452] The SBTS includes two components: 1) a transposon containing a transgene and 2) a source of transposase enzyme. The transposase can transpose the transposon from a carrier plasmid (or other donor DNA) to a target DNA, such as a host cell chromosome / genome. For example, the transposase binds to the carrier plasmid / donor DNA, cuts the transposon (including transgene(s)) out of the plasmid, and inserts it into the genome of the host cell. See, e.g., Aronovich et al. supra.

[0453] Exemplary transposons include a pT2-based transposon. See, e.g., Grabundzija et al. Nucleic Acids Res. 41.3(2013):1829-47; and Singh et al. Cancer Res. 68.8(2008): 2961-2971.

[0454] Exemplary transposases include a Tc1 / mariner-type transposase, e.g., the SB10 transposase or the SB11 transposase (a hyperactive transposase which can be expressed, e.g., from a cytomegalovirus promoter). See, e.g., Aronovich et al.; Kebriaei et al.; and Grabundzija et al..

[0455] Use of the SBTS permits efficient integration and expression of a transgene, e.g., a nucleic acid encoding a CAR described herein. Provided herein are methods of generating a cell, e.g., T cell or NK cell, that stably expresses a CAR described herein, e.g., using a transposon system such as SBTS.

[0456] In accordance with methods described herein, in some embodiments, one or more nucleic acids, e.g., plasmids, containing the SBTS components are delivered to a cell .

[0457] For example, the nucleic acid(s) are delivered by standard methods of nucleic acid (e.g., plasmid DNA) delivery, e.g., methods described herein, e.g., electroporation, transfection, or lipofection. In some embodiments, the nucleic acid contains a transposon comprising a transgene, e.g., a nucleic acid encoding a CAR described herein. In some embodiments, the nucleic acid contains a transposon comprising a transgene (e.g., a nucleic acid encoding a CAR described herein) as well as a nucleic acid sequence encoding a transposase enzyme. In other embodiments, a system with two nucleic acids is provided, e.g., a dual-plasmid system, e.g., where a first plasmid contains a transposon comprising a transgene, and a second plasmid contains a nucleic acid sequence encoding a transposase enzyme. For example, the first and the second nucleic acids are co-delivered into a host cell.

[0458] In some embodiments, cells are generated that express a CAR in accordance with the claims by using a combination of gene insertion using the SBTS and genetic editing using a nuclease (e.g., Zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR / Cas system, or engineered meganuclease re-engineered homing endonucleases).

[0459] In some embodiments, use of a non-viral method of delivery permits reprogramming of cells and direct infusion of the cells into a subject. Advantages of non-viral vectors include but are not limited to the ease and relatively low cost of producing sufficient amounts required to meet a patient population, stability during storage, and lack of immunogenicity.IMMUNE EFFECTOR CELLS, E.G., T CELLS

[0460] Methods described herein provide CD4 +< cells having a CAR CD4+< and CD8 +< T cells having a CAR CD8+< , wherein the CAR CD4+< and CAR CD8+< differ from one another. T cells can be obtained by a variety of methods from a variety of sources, e.g., as described in the section herein entitled "SOURCES OF CELLS ." It can be desirable to have separate populations of CD4 +< and CD8 +< T cells for methods described herein. Preparations of CD4 +< and preparations of CD8 +< T cells can be obtained by a variety of methods e.g., as described in the section herein entitled "SEPARATION OF T CELLS." The T cells are transformed with CARs and expanded. CD4 +< cells having a CAR CD4+< and CD8 +< cells having a CAR CD8+< can be expanded, by a variety of methods, e.g., methods described in the section herein entitled "ACTIVATION AND EXPANSION OF T CELLS. " CD4 +< T cells can be Th17 polarized. Th17 polarized CD4 +< cells having a CAR CD4+< can be polarized, and expanded, by a variety of methods, e.g., methods described in the section herein entitled "ACTIVATION AND EXPANSION OF TH17 CELLS ." CD4 +< T cells, CD8 +< T cells, and Th17 cells can be transformed by a variety of methods, e.g., methods described in the section herein entitled "NUCLEIC ACID CONSTRUCTS ENCODING A CAR ."SOURCES OF CELLS

[0461] In embodiments, prior to expansion and genetic modification or other modification, a source of T cells can be obtained from a subject. Examples of subjects include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.

[0462] In embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll ™< separation. In an embodiment, cells from the circulating blood of an individual are obtained by 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 an embodiment, the cells collected by apheresis may be washed to remove the plasma fraction and optionally to place the cells in an appropriate buffer or media for subsequent processing steps. In an embodiment, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Surprisingly, the initial activation steps in the absence of calcium lead to magnified signal activation. A washing step may be accomplished by methods known to those in the art, such as by using a semi-automated "flow-through" centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

[0463] It is recognized that the methods of the application can utilize culture media conditions comprising 5% or less, for example 2%, human AB serum, and employ known culture media conditions and compositions, for example those described in Smith et al., "Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement" Clinical & Translational Immunology (2015) 4, e31; doi:10.1038 / cti.2014.31.

[0464] In an embodiment, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL ™< gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3+, CD28+, CD4 +< , CD8+, CD45RA+, and CD45RO+T cells, can be further isolated by positive or negative selection techniques.

[0465] T cell lines available in the art, may be used.SEPARATION OF T CELLS

[0466] Methods described herein provide CD4 +< cells having a CAR CD4+< and CD8 +< cells having a CAR CD8+< , wherein the a CAR CD4+< and a CAR CD8+< differ from one another. It can be desirable to have separate populations of CD4 +< and CD8 +< T cells for these methods.

[0467] In an embodiment, a CD4 +< T cell or a population of CD4 +< T cells is isolated by positive selection. For example, the T cells isolated from the blood of a subject can be incubated with an antibody that specifically recognizes CD4 under condition suitable for antibody labelling of the CD4 +< T cells. Antibodies that specifically recognize CD4 are known in the art, e.g., anti-CD4 antibody clone M-T466 (Miltenyi Biotech), anti-CD4 antibody clone OKT4 (Affymetrix). In an embodiment, the anti-CD4 antibody is conjugated to a fluorescent molecule, e.g., FITC, and the T cells are sorted using flow cytometry to separate the CD4-expressing T cells from T cells that do not express CD4, e.g., CD8 +< T cells. In some embodiments, the anti-CD4 antibody is conjugated to a surface or a solid support, e.g., a magnetic bead, and the CD4 +< cells can be isolated using chromatography methods known in the art.

[0468] A CD4 +< T cell or population of CD4 +< T cells can also be enriched for by negative selection. In an embodiment, an antibody cocktail is utilized that includes antibodies against markers that are present on cells that do not express CD4, e.g., CD8, CD16, CD19, CD36, CD56, CD66b, TCRγ / δ, and glycophorin A. The antibody cocktail is added to the blood cell sample, mixed, and incubated, e.g., for 20 minutes at room temperature. A density medium is layered over the sample, and the mixture is centrifuged. The enriched cells expressing CD4 +< will be present at the interface of the plasma and the density medium, and can be isolated. The unwanted cells, e.g., cells that do not express CD4, are found at the bottom of the tube, e.g., below the density medium. In another embodiment, the antibody cocktail can be conjugated to a surface or a bead, and the CD4 +< cells can be collected from flow through over the surface or bead, while unwanted cells that do not express CD4 are immobilized on the surface or bead. In another embodiment, the monoclonal antibody cocktail includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0469] In an embodiment, a CD8 +< T cell or a population of CD8 +< T cells is isolated by positive selection. For example, the T cells isolated from the blood of a subject can be incubated with an antibody that specifically recognizes CD8 under condition suitable for antibody labelling of the CD8 +< T cells. Antibodies that specifically recognize CD8 are known in the art, e.g., anti-CD8 antibody clone OKT8 (Affymetrix), anti-CD8 antibody clone C8 / 144B (Dako). In an embodiment, the anti-CD8 antibody is conjugated to a fluorescent molecule, e.g., FITC, and the T cells are sorted using flow cytometry to separate the CD8-expressing T cells from T cells that do not express CD8, e.g., CD4 +< T cells. In some embodiments, the anti-CD8 antibody is conjugated to a surface or a solid support, e.g., a magnetic bead, and the CD8 +< cells can be isolated using chromatography methods known in the art.

[0470] A CD8 +< T cell or population of CD8 +< T cells can also be enriched for by negative selection. In an embodiment, an antibody cocktail is utilized that includes antibodies against markers present on cells that do not express CD8, e.g., antibodies against CD4, CD16, CD19, CD36, CD56, CD66b, CD123, and TCRγ / δ. The antibody cocktail is added to the blood cell sample, mixed, and incubated, e.g., for 20 minutes at room temperature. A density medium is layered over the sample, and the mixture is centrifuged. The enriched cells expressing CD8 +< will be present at the interface of the plasma and the density medium, and can be isolated. The unwanted cells, e.g., cells that do not express CD8, are found at the bottom of the tube, e.g., below the density medium. In another embodiment, the antibody cocktail can be conjugated to a surface or a bead, and the CD8 +< cells can be collected from flow through over the surface or bead, while unwanted cells that do not express CD8 are immobilized on the surface or bead.

[0471] The methods described herein can include, e.g., selection of a specific subpopulation of immune effector cells, e.g., T cells, e.g., CD4 +< and / or CD8 +< T cells, that are a T regulatory cell-depleted population, CD25+ depleted cells, using, e.g., a negative selection technique, e.g., described herein. Preferably, the population of T regulatory depleted cells contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells.

[0472] In one embodiment, T regulatory cells, e.g., CD25+ T cells, are removed from the population using an anti-CD25 antibody, or fragment thereof, or a CD25-binding ligand, IL-2. In one embodiment, the anti-CD25 antibody, or fragment thereof, or CD25-binding ligand is conjugated to a substrate, e.g., a bead, or is otherwise coated on a substrate, e.g., a bead. In one embodiment, the anti-CD25 antibody, or fragment thereof, is conjugated to a substrate as described herein.

[0473] In one embodiment, the T regulatory cells, e.g., CD25+ T cells, are removed from the population using CD25 depletion reagent from Miltenyi ™< . In one embodiment, the ratio of cells to CD25 depletion reagent is 1e7 cells to 20 uL, or 1e7 cells to15 uL, or 1e7 cells to 10 uL, or 1e7 cells to 5 uL, or 1e7 cells to 2.5 uL, or 1e7 cells to 1.25 uL. In one embodiment, e.g., for T regulatory cells, e.g., CD25+ depletion, greater than 500 million cells / ml is used. In a further aspect, a concentration of cells of 600, 700, 800, or 900 million cells / ml is used.

[0474] In one embodiment, the population of immune effector cells to be depleted includes about 6 x 10 9< CD25+ T cells. In other aspects, the population of immune effector cells to be depleted include about 1 x 10 9< to 1x 10 10< CD25+ T cell, and any integer value in between. In one embodiment, the resulting population T regulatory depleted cells has 2 x 10 9< T regulatory cells, e.g., CD25+ cells, or less (e.g., 1 x 10 9< , 5 x 10 8< , 1 x 10 8< , 5 x 10 7< , 1 x 10 7< , or less CD25+ cells).

[0475] In one embodiment, the T regulatory cells, e.g., CD25+ cells, are removed from the population using the CliniMAC system with a depletion tubing set, such as, e.g., tubing 162-01. In one embodiment, the CliniMAC system is run on a depletion setting such as, e.g., DEPLETION2.1.

[0476] Without wishing to be bound by a particular theory, decreasing the level of negative regulators of immune cells (e.g., decreasing the number of unwanted immune cells, e.g., T REG cells), in a subject prior to apheresis or during manufacturing of a CAR-expressing cell product can reduce the risk of subject relapse. For example, methods of depleting T REG cells are known in the art. Methods of decreasing T REG cells include, but are not limited to, cyclophosphamide, anti-GITR antibody (an anti-GITR antibody described herein), CD25-depletion, and combinations thereof.

[0477] In some instances, the manufacturing methods comprise reducing the number of (e.g., depleting) T REG cells prior to manufacturing of the CAR-expressing cell. For example, manufacturing methods comprise contacting the sample, e.g., the apheresis sample, with an anti-GITR antibody and / or an anti-CD25 antibody (or fragment thereof, or a CD25-binding ligand), e.g., to deplete T REG cells prior to manufacturing of the CAR-expressing cell (e.g., T cell, NK cell) product.

[0478] In some instances, the manufacturing methods comprise reducing the number of (e.g., depleting) T REG cells prior to manufacturing of the CAR-expressing cell. For example, manufacturing methods comprise contacting the sample, e.g., the apheresis sample, with an anti-GITR antibody and / or an anti-CD25 antibody (or fragment thereof, or a CD25-binding ligand), e.g., to deplete T REG cells prior to manufacturing of the CAR-expressing cell (e.g., T cell, NK cell) product.

[0479] In an embodiment, a subject is pre-treated with one or more therapies that reduce T REG cells prior to collection of cells for CAR-expressing cell product manufacturing, thereby reducing the risk of subject relapse to CAR-expressing cell treatment. In an embodiment, methods of decreasing T REG cells include, but are not limited to, administration to the subject of one or more of cyclophosphamide, anti-GITR antibody, CD25-depletion, or a combination thereof. Administration of one or more of cyclophosphamide, anti-GITR antibody, CD25-depletion, or a combination thereof, can occur before, during or after an infusion of the CAR-expressing cell product.

[0480] In an embodiment, a subject is pre-treated with cyclophosphamide prior to collection of cells for CAR-expressing cell product manufacturing, thereby reducing the risk of subject relapse to CAR-expressing cell treatment. In an embodiment, a subject is pre-treated with an anti-GITR antibody prior to collection of cells for CAR-expressing cell product manufacturing, thereby reducing the risk of subject relapse to CAR-expressing cell treatment.

[0481] In one embodiment, the population of cells to be removed are neither the regulatory T cells or tumor cells, but cells that otherwise negatively affect the expansion and / or function of CART cells, e.g. cells expressing CD14, CD11b, CD33, CD15, or other markers expressed by potentially immune suppressive cells. In one embodiment, such cells are envisioned to be removed concurrently with regulatory T cells and / or tumor cells, or following said depletion, or in another order.

[0482] The methods described herein can include more than one selection step, e.g., more than one depletion step. Enrichment of a T cell population by negative selection can be accomplished, e.g., with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail can include antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0483] The methods described herein can further include removing cells from the population which express a tumor antigen, e.g., a tumor antigen that does not comprise CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14 or CD11b, to thereby provide a population of T regulatory depleted, e.g., CD25+ depleted, and tumor antigen depleted cells that are suitable for expression of a CAR, e.g., a CAR described herein. In one embodiment, tumor antigen expressing cells are removed simultaneously with the T regulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody, or fragment thereof, and an anti-tumor antigen antibody, or fragment thereof, can be attached to the same substrate, e.g., bead, which can be used to remove the cells or an anti-CD25 antibody, or fragment thereof, or the anti-tumor antigen antibody, or fragment thereof, can be attached to separate beads, a mixture of which can be used to remove the cells. In other embodiments, the removal of T regulatory cells, e.g., CD25+ cells, and the removal of the tumor antigen expressing cells is sequential, and can occur, e.g., in either order.

[0484] Also provided are methods that include removing cells from the population which express a checkpoint inhibitor, e.g., a checkpoint inhibitor described herein, e.g., one or more of PD1+ cells, LAG3+ cells, and TIM3+ cells, to thereby provide a population of T regulatory depleted, e.g., CD25+ depleted cells, and check point inhibitor depleted cells, e.g., PD1+, LAG3+ and / or TIM3+ depleted cells. Exemplary check point inhibitors include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR beta. In one embodiment, check point inhibitor expressing cells are removed simultaneously with the T regulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody, or fragment thereof, and an anti-check point inhibitor antibody, or fragment thereof, can be attached to the same bead which can be used to remove the cells, or an anti-CD25 antibody, or fragment thereof, and the anti-check point inhibitor antibody, or fragment there, can be attached to separate beads, a mixture of which can be used to remove the cells. In other embodiments, the removal of T regulatory cells, e.g., CD25+ cells, and the removal of the checkpoint inhibitor expressing cells is sequential, and can occur, e.g., in either order.

[0485] Methods described herein can include a positive selection step. For example, T cells (e.g CD4+ T cells or CD8 +< T cells) can be isolated by incubation with anti-CD3 / anti-CD28 (e.g., 3x28)-conjugated beads, such as DYNABEADS ®< M-450 CD3 / CD28 T, for a time period sufficient for positive selection of the desired T cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment, the time period is 10 to 24 hours, e.g., 24 hours. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3 / CD28 beads and / or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points.

[0486] In one embodiment, a T cell population (e.g., T cells such as CD4 +< T cells or CD8+ T cells) can be selected that expresses one or more of IFN- γ< , TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other appropriate molecules, e.g., other cytokines. Methods for screening for cell expression can be determined, e.g., by the methods described in PCT Publication No.: WO 2013 / 126712.

[0487] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. In an embodiment, a concentration of 10 billion cells / ml, 9 billion / ml, 8 billion / ml, 7 billion / ml, 6 billion / ml, or 5 billion / ml is used. In an embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8 +< T cells that normally have weaker CD28 expression.

[0488] In a related embodiment it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells is minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4 +< T cells express higher levels of CD28 and are more efficiently captured than CD8 +< T cells in dilute concentrations. In an embodiment, the concentration of cells used is 5 X 10 6< / ml. In other embodiments, the concentration used can be from about 1 X 10 5< / ml to 1 X 10 6< / ml, and any integer value in between. In other embodiments, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10°C or at room temperature.

[0489] T cells for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20° C or in liquid nitrogen.

[0490] In certain embodiments, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods described herein.

[0491] In an embodiment the collection of blood samples or apheresis product from a subject is made at a time period prior to when the expanded cells might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in, e.g., T cell therapy for any number of diseases or conditions that would benefit from such T cell therapy. In an embodiment a blood sample or an apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, the T cells may be expanded, frozen, and used at a later time. In certain embodiments, samples are collected from a patient shortly after diagnosis of a particular disease but prior to any treatments. In a further embodiment, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). In a further embodiment, the cells are isolated for a patient and frozen for later use in conjunction with (e.g., before, simultaneously or following) bone marrow or stem cell transplantation, T cell ablative therapy using ...

Claims

1. A subset-optimized CART cell combination for use in a method of treating a subject having cancer, the method comprising administering to the subject an effective amount of: (a) a CD4+ T cell comprising a CAR (the CARCD4+) comprising: an antigen binding domain; a transmembrane domain; an ICOS costimulatory domain; and an intracellular signaling domain comprising a CD3 zeta (CD3Z) domain; (b) a CD8+ T cell comprising a CAR (the CARCD8+) comprising: an antigen binding domain; a transmembrane domain; a costimulatory domain selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICAM-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, GITR, BAFFR, HVEM (LIGHTR), SLAMf7, NKP80 (KLRF1), CD160 (BY55), CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, C49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (C244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, C100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, and PAG / Cbp; and an intracellular signaling domain comprising a CD3 zeta domain (CD3Z); and (c) a second CD8+ T cell comprising a CAR (the second CARCD8+), wherein the CARCD4+, the CARCD8+, and the second CARCD8+ differ from one another, comprising: an antigen binding domain; a transmembrane domain; a costimulatory domain selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICAM-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, GITR, BAFFR, HVEM (LIGHTR), SLAMf7, NKP80 (KLRF1), CD160 (BY55), CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, C49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (C244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, C100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, and PAG / Cbp; and an intracellular signaling domain comprising a CD3 zeta domain (CD3Z); wherein the subset-optimized CART cell combination enhances CAR T cells in vivo persistence.

2. The subset-optimized CART cell combination for use according to claim 1, wherein the CD4+ T cell comprising the CARCD4+, the CD8+ T cell comprising the CARCD8+, and / or the second CD8+ T cell comprising the second CARCD8+ are administered simultaneously, in the same or in separate compositions, or sequentially.

3. The subset-optimized CART cell combination for use of claim 1 or 2, wherein: (a) the CD4+ T cell comprising a CARCD4+ persists in the subject for at least 10, 15, 30, 45, 60, or 90 days after administration; and / or (b) the CD8+ T cell comprising a CARCD8+ persists in the subject for at least 10, 15, 30, 45, 60, or 90 days after administration; and / or (c) the second CD8+ T cell persists in the subject for at least 10, 15, 30, 45, 60, or 90 days after administration.

4. The subset-optimized CART cell combination for use of any one of claims 1-3, wherein: (a) at least 10, 20, 30, 40, 50, 60, 70, or 80% of the subset-optimized CAR T cells are CD4+ T cells comprising a CARCD4+; and / or (b) at least 10, 20, 30, 40, 50, 60, 70, or 80% of the subset-optimized CAR T cells are CD8+ T cells comprising a CARCD8+; and / or (c) at least 10, 20, 30, 40, 50, 60, 70, or 80% of the subset-optimized CAR T cells are CD8+ T cells comprising a second CARCD8+ further wherein the total of CD4+ T cells comprising a CARCD4+ and CD8+ T cells comprising a CARCD8+ does not exceed 100%.

5. The subset-optimized CART cell combination for use according to any one of claims 1-4, wherein: (a) the CD4+ T cell comprising the CARCD4+, the CD8+ T cell comprising the CARCD8+, and / or the CD8+ T cell comprising the second CARCD8+ is an autologous T cell; (b) the CD4+ T cell comprising the CARCD4+, the CD8+ T cell comprising the CARCD8+, and / or the CD8+ T cell comprising the second CARCD8+ is an allogeneic T cell; (c) the CD4+ T cell comprising the CARCD4+ is an autologous T cell and the CD8+ T cell comprising the CAR is an allogeneic T cell; or (d) the CD4+ T cell comprising the CARCD4+ is an allogeneic T cell and the CD8+ T cell comprising the CAR is an autologous T cell.

6. The subset-optimized CART cell combination for use according to any one of claims 1-5, wherein: (a) the intracellular signaling domain of the CARCD4+, CARCD8+, or the second CARCD8+ comprises the amino acid sequence set forth in SEQ ID NO: 18 or 20; (b) the costimulatory domain of the CARCD4+ comprises the amino acid sequence set forth in SEQ ID NO: 40, or 46; or (c) the costimulatory domain of the CARCD8+ or the second CARCD8+ comprises the amino acid sequence set forth in SEQ ID NO: 14, 16, or 44.

7. The subset-optimized CART cell combination for use according to any one of claims 1-6, wherein: (a) the CARCD8+ comprises a 4-1BB costimulatory domain and the second CARCD8+ comprises a CD28 costimulatory domain; (b) the CARCD8+ comprises a 4-1BB costimulatory domain and the second CARCD8+ comprises a CD27 costimulatory domain; (c) the CARCD8+ comprises a CD28 costimulatory domain and the second CARCD8+ comprises a 4-1BB costimulatory domain; or (d) the CARCD8+ comprises a 4-1BB costimulatory domain and the second CARCD8+ comprises a 4-1BB costimulatory domain.

8. The subset-optimized CART cell combination for use according to any one of claims 1-7, wherein: (a) CD4+ T cells with an ICOS-based CAR enhance the persistence of CD4+ T cells and the accompanying CD8+ T cells; (b) CD8+ T cells enhance the persistence of CD8+ T cells and the accompanying CD4+ T cells when redirected with CARs containing a 4-1BB costimulatory domain; and / or (c) CD8+ T cells enhance cytokine release and antitumor effect of the CD8+ T cells and the accompanying CD4+ T cells when redirected with CARs containing a CD28 costimulatory domain.

9. The subset-optimized CART cell combination for use according to any one of claims 1-8, wherein when the CARCD8+ comprises a 4-1BB costimulatory domain, the subset-optimized CART cell combination enhances in vivo persistence of CD4+ and CD8+ T cells by at least about: (a) 5-fold when compared to a CART cell combination comprising a CD4+ T cell comprising a CARCD4+ with a ICOS costimulatory domain and a CD8+ T cell comprising a CARCD8+ with a ICOS costimulatory domain; (b) 7-fold when compared to a CART cell combination comprising a CD4+ T cell comprising a CARCD4+ lacking a costimulatory domain and a CD8+ T cell comprising a CARCD8+ lacking a costimulatory domain; (c) 17-fold when compared to a CART cell combination comprising a CD4+ T cell comprising a CARCD4+ with a 4-1BB costimulatory domain and a CD3Z signaling domain and a CD8+ T cell comprising a CARCD8+ with a 4-1BB costimulatory domain and a CD3Z signaling domain; or (d) 2800-fold when compared to a CART cell combination comprising a CD4+ T cell comprising a CARCD4+ with a CD28 costimulatory domain and a CD3Z signaling domain and a CD8+ T cell comprising a CARCD8+ with a CD28 costimulatory domain and a CD3Z signaling domain.

10. The subset-optimized CART cell combination for use according to any one of claims 1-9, wherein the CD4+ T cell: (a) is a Th17 polarized cell; (b) produces IL-17A; and / or (c) expresses IL-23R and / or CD161 on the cell surface.

11. The subset-optimized CART cell combination for use according to any one of claims 1-10, wherein: (a) the antigen binding domain of the CARCD4+, the CARCD8+, and the second CARCD8+ are the same; or (b) the antigen binding domain of the CARCD4+, the CARCD8+, and the second CARCD8+ target different antigens; or (c) the antigen binding domain of the CARCD4+, the CARCD8+, or the second CARCD8+ each comprises an antibody variable domain, an scFv, or a nanobody, or an antigen binding fragment thereof.

12. The subset-optimized CART cell combination for use according to any one of claims 1-11, wherein the CARCD4+ and the CARCD8+ each comprises an antigen binding domain that targets a tumor antigen selected from the group consisting of CD19, CD20, CD22, ROR1, mesothelin, IL-13Ra, PSCA, EGFR, MUC1, TN Ag, BCMA, CLL-1, HER2, CD33 / IL3Ra, c-Met, PSMA, PSA, Glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, Folate receptor-α; , MAGE A3 TCR, and any combination thereof.

13. The subset-optimized CART cell combination for use according to any one of claims 1-10, wherein the CARCD4+ and the CARCD8+ each comprises an antigen binding domain selected from SEQ ID NO: 48-81, or an antigen binding domain comprising a variable heavy chain comprising SEQ ID NO: 82 and a variable light chain comprising SEQ ID NO: 83.

14. The subset-optimized CART cell combination for use according to any one of claims 1-10, wherein: (a) the CD4+ T cell comprises SEQ ID NO: 85; or (b) the CD8+ T cell comprises SEQ ID NO: 89.

15. A kit comprising: (a) a CD4+ T cell comprising a CARCD4+ as defined in any one of claims 1-14; (b) a CD8+ T cell comprising a CARCD8+ as defined in any one of claims 1-14; and (c) a CD8+ T cell comprising a second CARCD8+ as defined in any one of claims 1-14.