CHIMERATED ANTIGEN RECEPTORS

Description

FIELD OF INVENTION

[0001] Provided herein are chimeric antigen receptors for treating cancers.BACKGROUND

[0002] The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Adoptive T-cell immunotherapy has risen to the forefront of treatment approaches for cancer. T cells can be engineered to express the genes of chimeric antigen receptors (CARs) that recognize tumor associated antigens. CARs are synthetic immune-receptors, which can redirect T cells to selectively kill tumor cells. The general premise for their use in cancer immunotherapy is to rapidly generate tumor-targeted T cells, bypassing the barriers and incremental kinetics of active immunization and thereby act as 'living drugs'. Unlike the physiologic T-cell receptor (TCR), which engages HLA-peptide complexes, CARs engage molecules that do not require peptide processing or HLA expression to be recognized. CARs therefore recognize antigen on any HLA background, in contrast to TCRs, which need to be matched to the haplotype of the patient. Furthermore, CARs can target tumor cells that have down-regulated HLA expression or proteasomal antigen processing, two mechanisms that contribute to tumor escape from TCR-mediated immunity. Another feature of the broad applicability of CARs is their ability to bind not only to proteins but also to carbohydrate and glycolipid structures, again expanding the range of potential targets.

[0004] Initial first-generation CARs were constructed through the fusion of a scFv (single chain fragment variable)-based antigen binding domain to an inert CD8 transmembrane domain, linked to a cytoplasmic signaling domain derived from the CD3-ζ or Fc receptor γ chains ( Fig. 1).

[0005] Although CD3-ζ chain aggregation is sufficient to enable lytic activity of T-cells, they failed to elicit a robust cytokine response, including interleukin-2 (IL-2), and support T-cell expansion upon repeated exposure to antigen. For optimal activation and proliferation, T cells require both T-cell receptor engagement and signaling, as well as costimulatory signaling through costimulatory receptors (i.e., CD28, 4-1BB, OX-40) on T cells binding to cognate ligands (i.e., CD80 / 86, 4-1BBL, OX-40L) expressed either by the targeted tumor cell or the antigen-presenting cells. To overcome the lack of T-cell co-stimulation, first generation CARs were further modified by incorporating the cytoplasmic signaling domains of T-cell costimulatory receptors. These second-generation CARs ( Fig. 1) enhanced signaling strength and persistence of the modified T cells, leading to superior antitumor activity. But, which second-generation CAR is the best is not known and each one has their own strengths and weakness. For example, in clinics the 4-1BB design appears to favor persistence, with CD19-CAR T cells detectable out to at least 6 months in a majority of patients, whereas the CD28 costimulatory domain containing CD19 CAR T cells were typically undetectable beyond 3 months, but less severe cytokine release syndrome (CRS) and a lower CD19-negative relapse rate were observed using the CD28-based CAR T cells.

[0006] Despite the success with CAR-T cells, there are several limitation to this approach. In majority of patients who respond to engineered CAR T cells, excessive release of proinflammatory cytokines causes symptoms that include fevers, hypotension, hypoxemia, cardiac dysfunction, kidney failure and electrolyte abnormalities, collectively termed as "Cytokine release syndrome' (CRS). In some cases, CAR therapy can lead to neurologic symptoms including tremor, seizures and can be fatal. Strategies to counteract CRS and neurological complications include immunosuppressive agents (e.g., steroids) and Tocilizumab, a monoclonal antibody against IL-6 receptor. However, these strategies are not uniformly successful. The instant invention provides novel approaches to control the activity of CAR-T cells so as to prevent and / or treat the above complications.

[0007] The CAR constructs in current clinical use are artificial in design as they represent fusion of several different proteins. In particular, inclusion of costimulatory domain in the CAR construct results in non-physiological signaling through the receptor, which in turn could contribute to their toxicity. Some CARs show tonic antigen-independent signaling, which leads to unrestrained cellular activation, eventually resulting in apoptosis, excessive cytokine release independent of cognate antigens, and immunologic exhaustion. Frigault et al demonstrated that expression of some CARs containing CD28 and CD3z tandem signaling domains leads to constitutive activation and proliferation of the transduced primary human T cells which was related to inferior in vivo efficacy (Frigault et al., Cancer immunology research 3:356-67, 2015). One mechanism that was found to result in the phenotype of CARs with continuous T-cell proliferation was high density of CARs at the cell surface (Frigault et al., Cancer immunology research 3:356-67, 2015). Long et al demonstrated that early T cell exhaustion is a primary factor limiting the antitumor efficacy of CAR-expressing T cells and that CAR structure has a central role in predisposing CAR T cells to chronic activation and exhaustion (Long et al., Nat Med 21:581-90, 2015). They showed that tonic CAR CD3-z phosphorylation, triggered by antigen-independent clustering of CAR single-chain variable fragments, can induce early exhaustion of CAR T cells that limits antitumor efficacy (Long et al., Nat Med 21:581-90, 2015). Thus, there is a need for improving the CAR design to achieve long term persistence of CAR modified T cells without the risk of excessive toxicity, such as CRS.

[0008] Most CAR constructs in current clinical trials are based on murine monoclonal antibodies. Immunogenicity of these murine monoclonal antibodies based construct has been also postulated to limit their in vivo persistence and efficacy. This problem can be overcome with the use of CAR constructs described herein that are based on human or humanized antibodies.

[0009] The polyclonal nature of the immune response is key to its success in controlling various infections. In contrast, the current CAR therapies generally rely on targeting of a single antigen and / or single epitope of a single antigen. Loss of the targeted antigen or the targeted epitope is a frequent cause of failure of the current CAR therapies. To overcome this limitation, the instant invention provides CAR against multiple antigens and against multiple epitopes of a single antigen. These CARs can be used in suitable combinations to provide a polyclonal adaptive immune response for the prevention or treatment of diseases, such as cancer, infectious diseases, autoimmune diseases, allergic diseases and degenerative diseases.

[0010] CD19 is an attractive target for immunotherapy because it is uniformly expressed by the vast majority of B-cell malignancies, it is not expressed by normal non-hematopoietic tissues, and among hematopoietic cells, it is only expressed by B-lineage lymphoid cells. Early experiments demonstrated that anti-CD19 CARs could activate T cells in a CD19-specific manner. T cells genetically modified to express these CARs could kill CD19 +< primary leukemia cells in vitro and eliminate CD19 +< target cells in murine xenograft models. Even though, initial clinical results with first-generation CD19-targeted CAR-modified T cells were disappointing, T cells engineered to express second-generation CARs have demonstrated impressive clinical efficacy with significant improvements in patient outcomes for a number of B-cell malignancies, the notable being the dramatic clinical responses observed in patients with relapsed B-cell ALL.

[0011] Anti-CD19 CART therapy as proof-of-concept has been successful in part due to the tissue restriction of CD19 to B cells and by the clinical tolerability of prolonged B-cell depletion. However, in other settings, CART-based targeting of antigens expressed at low levels by normal tissues has led to significant toxicities. The paucity of well-characterized, truly tumor-specific surface antigens in myeloid malignancies, such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and myelodysplastic syndrome MDS, has necessitated consideration of CAR-T tumor-targeting strategies that may also affect normal tissues, such as bone marrow. WO2014 / 152177A1 discloses T lymphocytes expressing artificial cell death polypeptides. In some disclosed examples the T lymphocytes comprise a chimeric antigen receptor comprising an extracellular domain that binds to an antigen, a transmembrane domain, and an intracellular signalling domain. In certain examples, the intracellular domain of the CAR comprises an ITAM.SUMMARY

[0012] The present invention provides a cell comprising nucleic acids encoding a chimeric antigen receptor (CAR) and Kaposi's sarcoma-associated herpesvirus-encoded viral FLICE Inhibitory Protein (vFLIP) K13 (K13-vFLIP) signaling protein, wherein the CAR comprises an a) extracellular antigen specific domain, b) a transmembrane domain and c) an intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM); wherein c) is located at the C-terminus of the chimeric receptor.

[0013] The present invention further provides nucleic acids comprising a first polynucleotide encoding the CAR and a second polynucleotide encoding the intracellular signaling protein, and optionally further comprising a third polynucleotide encoding the MC159-vFLIP signaling protein.

[0014] The following arrangements, embodiments and aspects thereof are described and illustrated in conjunction with systems, compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0015] In certain arrangements there is provided compositions comprising genetically engineered effector cells (such as NK cells and T cells) that include polynucleotides that encode chimeric antigen receptors that can be used on adoptive cell therapy for treatment of cancer, infectious, autoimmune and degenerative diseases.

[0016] In some arrangements, provided herein are polynucleotides encoding chimeric antigen receptors (CARs) comprising an antigen specific domain (ASD), an extracellular hinge or spacer region (HR), a transmembrane domain (TMD), a costimulatory domain (CSD) and intracellular signaling domain (ISD), wherein the antigen binding domain targets any one or more of the targets set forth in Table 21. In one embodiment, the CAR targets CD19 and comprises the V L and V H of Bu12 described in Table 21 and set forth in SEQ ID NO: 1470 and SEQ ID NO: 3215. In one embodiment, the CAR targets CD19 and comprises the V L and V H of MM described in Table 21 and set forth in SEQ ID NO: 1471 and SEQ ID NO: 3216.

[0017] In some arrangements, provided herein are polynucleotides encoding any of conventional CARs (Table 1) and backbones 1-62 (Table 2), wherein each backbone comprises a conventional CAR component (Table 1) and one or more accessory modules as shown in Table 2. In some arrangements, the conventional CAR component and the one or more accessory components of each backbone are encoded by a single polynucleotide molecule. In some arrangements, the conventional CAR component is encoded by a first polynucleotide molecule and the one or more accessory modules are encoded by a second polynucleotide molecule. In some arrangements, backbones comprising a conventional CAR component and more than one accessory module, the first polynucleotide molecule encodes the conventional CAR component, a second polynucleotide molecule encodes the first accessory molecule and a third polynucleotide molecule encodes the second accessory molecule. For example, backbone 1 comprising conventional CAR I and K13-vFLIP may be encoded by a single polynucleotide molecule or by two polynucleotide molecules, wherein the first polynucleotide molecule encodes the conventional CAR I and the second polynucleotide molecule encodes K13-vFLIP. In various arrangements, the polynucleotide molecules encoding the CAR component of the conventional CARs I to III (Table 1) or the backbones (Table 2) described herein encodes one or more antigen specific domains. In some arrangements, the antigen specific domain comprises one or more V L (or vL) fragments. In some arrangements, the antigen specific domain comprises one or more V H (or vH) fragments. In some arrangements, the antigen specific domain comprises one or more scFVs (or scFvs) specific to the antigens on target cells such as cancer cells. In some arrangements, the antigen specific domain comprises one or more Fv fragments. In some arrangements, the antigen specific domain comprises one or more Fab fragments. In some arrangements, the antigen specific domain comprises one or more (Fab')2 fragments. In some arrangements, the antigen specific domain comprises one or more single domain antibodies (SDAB). In some arrangements, the antigen specific domain comprises one or more camelid V HH (or vHH) domains. In some arrangements, the antigen specific domain comprises one or more non-immunoglobulin scaffold such as a DARPIN, an affibody, an affilin, an adnectin, an affitin, an obodies, a repebody, a fynomer, an alphabody, an avimer, an atrimer, a centyrin, a pronectin, an anticalin, a kunitz domain, an Armadillo repeat protein, an autoantigen, a receptor or a ligand. In some arrangements, the antigen specific domain comprises one or more ligands.

[0018] Also provided herein are polypeptides encoded by the polynucleotides described herein. In various arrangements, polypeptides encoded by the polynucleotide molecule encoding the CAR component of the conventional CARs I to III (Table 1) or the backbones (Table 2) described herein encodes one or more antigen specific domains. In some arrangements, the antigen specific domain comprises one or more V L (or vL) fragments. In some arrangements, the antigen specific domain comprises one or more V H (or vH) fragments. In some arrangements, the antigen specific domain comprises one or more scFVs specific to the antigens on target cells such as cancer cells. In some arrangements, the antigen specific domain comprises one or more Fv fragments. In some arrangements, the antigen specific domain comprises one or more Fab fragments. In some arrangements, the antigen specific domain comprises one or more (Fab')2 fragments. In some arrangements, the antigen specific domain comprises one or more single domain antibodies (SDAB). In some arrangements, the antigen specific domain comprises one or more camelid V HH (vHH) domains. In some arrangements, the antigen specific domain comprises one or more Fv fragments. In some arrangements, the antigen specific domain comprises one or more non-immunoglobulin scaffold such as a DARPIN, an affibody, an affilin, an adnectin, an affitin, an obodies, a repebody, a fynomer, an alphabody, an avimer, an atrimer, a centyrin, a pronectin, an anticalin, a kunitz domain, an Armadillo repeat protein, an autoantigen, a receptor or a ligand. In some arrangements, the antigen specific domain comprises one or more ligands.

[0019] Further provided herein are one or more vectors comprising nucleic acids encoding any of conventional CARS I to III (Table 1) or backbones 1-62 (Table 2). In some arrangements, all the components of each backbone are encoded by a single vector. In some arrangements, each component of the backbone is encoded by a different vector. For examples, a backbone comprising a conventional CAR component and a single accessory module, a first vector comprises polynucleotides encoding the conventional CAR component and a second vector comprises polynucleotides encoding the accessory module. For example, a backbone comprising a conventional CAR component and a more than one accessory module, a first vector comprises polynucleotides encoding the conventional CAR component and a second vector comprises polynucleotides encoding the accessory modules. Alternately, a first vector comprises polynucleotides encoding the conventional CAR component, a second vector comprises polynucleotides encoding the first accessory module and a third vector comprises polynucleotides encoding the second accessory module. In various arrangements, the vectors encoding the polynucleotide molecule encoding the CAR component of the conventional CARs I to III or the backbones described herein encodes one or more antigen specific domains. In some arrangements, the antigen specific domain comprises one or more V L fragments. In some arrangements, the antigen specific domain comprises one or more V H fragments. In some arrangements, the antigen specific domain comprises one or more scFVs specific to the antigens on target cells such as cancer cells. In some arrangements, the antigen specific domain comprises one or more Fv fragments. In some arrangements, the antigen specific domain comprises one or more Fab fragments. In some arrangements, the antigen specific domain comprises one or more (Fab')2 fragments. In some arrangements, the antigen specific domain comprises one or more single domain antibodies (SDAB). In some arrangements, the antigen specific domain comprises one or more camelid V HH domains. In some arrangements, the antigen specific domain comprises one or more Fv fragments. In some arrangements, the antigen specific domain comprises one or more non-immunoglobulin scaffold such as a DARPIN, an affibody, an affilin, an adnectin, an affitin, an obodies, a repebody, a fynomer, an alphabody, an avimer, an atrimer, a centyrin, a pronectin, an anticalin, a kunitz domain, an Armadillo repeat protein, an autoantigen, a receptor or a ligand. In some arrangements, the antigen specific domain comprises one or more ligand.

[0020] Further provided herein are genetically modified cells comprising vectors encoding polynucleotides encoding the conventional CARs I to III or backbones 1-62 described herein, wherein the CARs of the conventional CARs I to III or the backbones comprise antigen specific domains as described herein. In various arrangements, the genetically modified cells target antigen on target cells such as cancer cells via the V L fragments, V H fragments, V HH domains, scFvs, Fv fragments, Fab fragments, (Fab')2 fragments, single domain antibody (SDAB) fragments, and / or non-immunoglobulin scaffold each of which are encoded by the antigen specific domains.

[0021] In some arrangements, the antigen specific domains of the CARs comprise one or more V L fragments. In exemplary arrangements, the one or more V L fragments are described in Table 4. In some arrangements, the polynucleotides encoding the one more V L fragments comprise, consist of or consist essentially of sequences set forth in any one or more of SEQ ID NOs: 11-249. In some arrangements, the polypeptides encoding the one more V L fragments comprise, consist of or consist essentially of sequences set forth in any one or more of SEQ ID NOS: 1785-2023 or sequences with 80-99% identity to sequences set forth in any one or more of SEQ ID NOS: 1785-2023 or sequences with 80-99% identity in the three complementarity determining regions (CDRs) to the sequences set forth in any one or more of SEQ ID NOS: 1785-2023 or sequences that bind to the same target antigens or the same epitopes on the target antigens as the sequences set forth in any one or more of SEQ ID NOS: 1785-2023. In some arrangements, the antigen specific domains of the CARs comprise one or more V H fragments. In exemplary arrangements, the one or more V H fragments are described in Table 6. In some arrangements, the polynucleotides encoding the one more V H fragments comprise, consist of or consist essentially of sequences set forth in any one or more of SEQ ID NOS: 259-498. In some arrangements, the polypeptides encoding the one more V H fragments comprise, consist of or consist essentially of sequences set forth in any one or more of SEQ ID NOS: 2028-2268 or sequences with 80-99% identity to sequences set forth in any one or more of SEQ ID NOS: 2028-2268 or sequences with 80-99% identity in the three complementarity determining regions (CDRs) to the sequences set forth in any one or more of SEQ ID NOS: 2028-2268 or sequences that bind to the same target antigens or the same epitopes on the target antigens as the sequences set forth in any one or more of SEQ ID NOS:2028-2268.

[0022] In some arrangements, the antigen specific domains of the CARs comprise one or more nanobodies (V HH domains). In exemplary arrangements, the one or more V HH domains are described in Table 7. In some arrangements, the polynucleotides encoding the one more V HH fragments comprise, consist of or consist essentially of sequences set forth in any one or more of SEQ ID NOS: 499-523. In some arrangements, the polypeptides encoding the one more V HH fragments comprise, consist of or consist essentially of sequences set forth in any one or more of SEQ ID NOS: 2269-2293 or sequences with 80-99% identity to sequences set forth in any one or more of SEQ ID NOS: 2269-2293 or sequences with 80-99% identity in the three complementarity determining regions (CDRs) to sequences set forth in any one or more of SEQ ID NOS: 2269-2293 or sequences that bind to the same target antigens or the same epitopes on the target antigens as the sequences set forth in any one or more of SEQ ID NOS: 2269-2293.

[0023] In some arrangements, the antigen specific domains of the CARs comprise one or more non-immunoglobulin antigen binding scaffolds. In exemplary arrangements, the one or more non-immunoglobulin antigen binding scaffolds are described in Table 8. In some arrangements, the polynucleotides encoding the one more non-immunoglobulin antigen binding scaffolds comprise, consist of or consist essentially of sequences set forth in any one or more of SEQ ID NOS: 524-528. In some arrangements, the polypeptides encoding the one more non-immunoglobulin antigen binding scaffolds comprise, consist of or consist essentially of sequences set forth in any one or more of SEQ ID NOS: 2294-2298 or sequences with 80-99% identity to sequences set forth in any one or more of SEQ ID NOS:2294-2298 or sequences that bind to the same target antigens or the same epitopes on the target antigens as the sequences set forth in any one or more of SEQ ID NOS: 2269-2293.

[0024] In some arrangements, the antigen specific domains of the CARs comprise one or more ligands. In exemplary arrangements, the ligands are described in Table 10. In some arrangements, the polynucleotides encoding the one more ligands comprise, consist of or consist essentially of sequences set forth in any one or more of SEQ ID NOS: 553-563. In some arrangements, the polypeptides encoding the one more ligand binding domains comprise, consist of or consist essentially of sequences set forth in any one or more of SEQ ID NOS: 2323-2333 or sequences with 80-99% identity to sequences set forth in any one or more of SEQ ID NOS: 2323-2333 or sequences that bind to the same target antigens or the same epitopes on the target antigens as the sequences set forth in any one or more of SEQ ID NOS: 2323-2333.

[0025] In some arrangements, the antigen specific domains of the CARs comprise one or more scFvs. In exemplary arrangements, the scFv fragments are described in Table 11. In some arrangements, the polynucleotides encoding the one more scFv fragments comprise, consist of or consist essentially of sequences set forth in any one or more of SEQ ID NOS: 564-798. In some arrangements, the polynucleotides encoding the one more scFv fragments comprise, consist of or consist essentially of sequences that encode for polypeptide sequences set forth in any one or more of SEQ ID NOS: 2334-2568 or that encode for polypeptide sequences with 80-99% identity in the six complementarity determining regions (CDRs) to sequences set forth in any one or more of SEQ ID NOS: 2334-2568 or that encode for polypeptide sequences that bind to the same target antigens or the same epitopes on the target antigens as the sequences set forth in any one or more of SEQ ID NOS: 2334-2568. In some arrangements, the polypeptides encoding the one more scFv fragments comprise, consist of or consist essentially of sequences set forth in any one or more of SEQ ID NOS: 2334-2568 or sequences with 80-99% identity to sequences set forth in any one or more of SEQ ID NOS: 2334-2568 or sequences with 80-99% identity in the six complementarity determining regions (CDRs) to sequences set forth in any one or more of SEQ ID NOS: 2334-2568 or sequences that bind to the same target antigens or the same epitopes on the target antigens as the sequences set forth in any one or more of SEQ ID NOS: 2334-2568.

[0026] In some arrangements, provided herein are polynucleotides encoding conventional CAR (Table 1) and any of backbones 1-62 as described herein in Table 2, wherein each backbone comprises a conventional CAR component (Table 1) and one or more accessory modules as shown in Table 2. In some arrangements, the conventional CAR component and the one or more accessory components of each backbone are encoded by a single polynucleotide molecule. In some arrangements, the conventional CAR component is encoded by a first polynucleotide molecule and the one or more accessory modules are encoded by a second polynucleotide molecule. In some arrangements, backbones comprising a conventional CAR component and more than one accessory module, the first polynucleotide molecule encodes the conventional CAR component, a second polynucleotide molecule encodes the first accessory molecule and a third polynucleotide molecule encodes the second accessory molecule.

[0027] In some arrangements, provided herein are polypeptides encoding conventional CAR (Table 1) and any of backbones 1-62 as described herein in Table 2, wherein each backbone comprises a conventional CAR component (Table 1) and one or more accessory modules as shown in Table 2. In some arrangements, the polypeptide encoding the conventional CAR component and the one or more accessory module components of each backbone are encoded by a single polynucleotide molecule. In some arrangements, the polypeptide encoding the conventional CAR component is encoded by a first polynucleotide molecule and the one or more accessory modules are encoded by a second polynucleotide molecule. In some arrangements, the polypeptide encoding the conventional CAR component and the one or more accessory module components of each backbone are encoded by a single polypeptide molecule. In some arrangements, the polypeptide encoding the conventional CAR component is encoded by a first polypeptide molecule and the one or more accessory modules are encoded by a second or more polypeptide molecules. In some arrangements, backbones comprising a conventional CAR component and more than one accessory module, the first polynucleotide molecule encodes the conventional CAR component, a second polynucleotide molecule encodes the first accessory molecule and a third polynucleotide molecule encodes the second accessory molecule. In some arrangements, backbones comprising a conventional CAR component and more than one accessory module, the first polypeptide molecule encodes the conventional CAR component, a second polypeptide molecule encodes the first accessory molecule and a third polypeptide molecule encodes the second accessory molecule.

[0028] In some arrangements, the polynucleotides encoding the one more accessory module comprise, consist of or consist essentially of sequences set forth in any one or more of SEQ ID NOS: 850-904. In some arrangements, the polynucleotides encoding the one more accessory module comprise, consist of or consist essentially of sequences that encode for polypeptide sequences as set forth in any one or more of SEQ ID NOS: 2613-2667 or encode for polypeptide sequences with 70-99% identity to sequences set forth in any one or more of SEQ ID NOS: 2613-2667. In some arrangements, the polypeptides encoding the one more accessory modules comprise, consist of or consist essentially of sequences set forth in any one or more of SEQ ID NOS: 2613-2667 or sequences with 70-99% identity to sequences set forth in any one or more of SEQ ID NOS: 2613-2667.

[0029] In exemplary arrangements, nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets antigens of interest are described in Table 19. The sequences of nucleic acid fragments encoding backbone-1 comprising conventional CAR I and K13-vFLIP and targeting antigens of interest as described in Table 19 are set forth in SEQ ID NOs: 927-1196. The sequences of polypeptides encoding backbone-1 comprising conventional CAR I and K13-vFLIP and targeting antigens of interest as described in Table 19 are set forth in SEQ ID NOs: 2672-2941. The nucleic acid sequences in SEQ ID NOs: 927-1196 and polypeptide sequences in SEQ ID NOs: 2672-2941 also encode for a puromycin acetyl transferase (PuroR) fragment, which fragment can be used to select cells but is not essential for the function of the CAR.

[0030] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD19 (Table 19). In exemplary arrangements, the sequences of isolated nucleic acid fragments targeting CD19 and coexpressing K13-vFLIP are set forth in SEQ ID NOs: 927-941. In exemplary arrangements, the sequences of isolated polypeptide targeting CD19 and coexpressing K13-vFLIP are set forth in SEQ ID NOs: 2672-2686. In some arrangements, the scFv fragments targeting CD19 are described in Table 11 and set forth in SEQ ID NOs: 564-577 and SEQ ID NOs: 2334-2347. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD19. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD19. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD19.

[0031] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets MPL or the Thrombopoietin Receptor (Table 19). In exemplary arrangements, the sequences of isolated nucleic acid fragments targeting MPL and coexpressing K13-vFLIP are set forth in SEQ ID NOs: 1117-1124. In exemplary arrangements, the sequences of isolated polypeptide targeting MPL are set forth in SEQ ID NOs: 2862-2869. In some arrangements, the scFv fragments targeting MPL are described in Table 11 and set forth in SEQ ID NOs: 729-736 and SEQ ID NOs: 2499-2506. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets MPL. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets MPL. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets MPL.

[0032] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets Lym1 (Table 19). In exemplary arrangements, the sequence of nucleic acid fragment targeting Lym1 is set forth in SEQ ID NO: 1109. In exemplary arrangements, the sequence of isolated polypeptide fragment targeting Lym1 is set forth in SEQ ID NO: 2854. In some arrangements, the scFv fragment targeting Lym1 is described in Table 11 and set forth in SEQ ID NO: 723 and SEQ ID NO: 2493. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets Lym1. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets Lym1. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets Lym1.

[0033] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets Lym2 (Table 19). In exemplary arrangements, sequence of the isolated nucleic acid fragment targeting Lym2 is set forth in SEQ ID NO: 1110. In exemplary arrangements, the sequence of isolated polypeptide targeting Lym2 is set forth in SEQ ID NO: 2855. In some arrangements, the scFv fragment targeting Lym2 is described in Table 11 and set forth in SEQ ID NO: 724 and SEQ ID NO: 2494. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets Lym2. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets Lym2. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets Lym2.

[0034] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets GRP-78 (Table 19). In exemplary arrangements, sequence of the isolated nucleic acid targeting GRP-78 is set forth in SEQ ID NO: 1078. In exemplary arrangements, sequence of the polypeptide targeting GRP-78 is set forth in SEQ ID NO: 2823. In some arrangements, the scFv fragment targeting GRP-78 is described in Table 11 and set forth in SEQ ID NO: 703 and SEQ ID NO: 2473. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets GRP-78. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets GRP-78. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets GRP-78.

[0035] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD79b (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting CD79b are set forth in SEQ ID NOs: 995-996. In exemplary arrangements, the sequences of the polypeptide fragments targeting CD79b are set forth in SEQ ID NO: 2740-2741. In some arrangements, the scFv fragment targeting CD79b is described in Table 11 and set forth in SEQ ID NO: 628 and SEQ ID NO: 2398. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD79b. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD79b. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD79b.

[0036] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets ALK (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting ALK are set forth in SEQ ID NOs: 946-947. In exemplary arrangements, the sequences of the polypeptide fragments targeting ALK are set forth in SEQ ID NO: 2691-2692. In some arrangements, the scFv fragments targeting ALK are described in Table 11 and set forth in SEQ ID NO: 582-583 and SEQ ID NO: 2352-2353. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets ALK. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets ALK. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets ALK.

[0037] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets BCMA (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting BCMA are set forth in SEQ ID NOs: 951-957. In exemplary arrangements, the sequences of the polypeptide fragments targeting BCMA are set forth in SEQ ID NO: 2696-2702. In some arrangements, the scFv fragments targeting BCMA are described in Table 11 and set forth in SEQ ID NO: 586-592 and SEQ ID NO: 2356-2362. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets BCMA. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets BCMA. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets BCMA.

[0038] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD20 (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting CD20 are set forth in SEQ ID NOs: 964-976. In exemplary arrangements, the sequences of the polypeptide fragments targeting CD20 are set forth in SEQ ID NO: 2709-2721. In some arrangements, the scFv fragments targeting CD20 are described in Table 11 and set forth in SEQ ID NO: 596-597;599-610 and SEQ ID NO: 2366-2367;2369-2380. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD20. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD20. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD20.

[0039] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD22 (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting CD22 are set forth in SEQ ID NOs: 977-980 and SEQ ID NOs: 1192-1196. In exemplary arrangements, the sequences of the polypeptide fragments targeting CD22 are set forth in SEQ ID NOs: 2722-2725 and SEQ ID NOs: 2937-2941. In some arrangements, the scFv fragments targeting CD22 are described in Table 11 and set forth in SEQ ID NO: 598, 611-613 and SEQ ID NO: 2368, 2381-2384. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD22. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD22. Also provided herein are genetically-engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD22.

[0040] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD30 (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting CD30 are set forth in SEQ ID NOs: 981-982. In exemplary arrangements, the sequences of the polypeptide fragments targeting CD30 are set forth in SEQ ID NOs: 2726-2727. In some arrangements, the scFv fragments targeting CD30 are described in Table 11 and set forth in SEQ ID NO: 614-615 and SEQ ID NO: 2384-2385. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD30. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD30. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD30.

[0041] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD32 (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragment targeting CD32 is set forth in SEQ ID NO: 983. In exemplary arrangements, the sequence of the polypeptide fragments targeting CD32 is set forth in SEQ ID NO: 2728 In some arrangements, the scFv fragment targeting CD32 is described in Table 11 and set forth in SEQ ID NO: 616 and SEQ ID NO: 2386. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD32. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD32. Also provided herein are genetically-engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD32.

[0042] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD33 (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting CD33 are set forth in SEQ ID NOs: 984-991. In exemplary arrangements, the sequences of the polypeptide fragments targeting CD33 are set forth in SEQ ID NOs: 2729-2736. In some arrangements, the scFv fragments targeting CD33 are described in Table 11 and set forth in SEQ ID NO: 617-624 and SEQ ID NO: 2387-2394. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD33. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD33. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD33.

[0043] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD123 (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting CD123 are set forth in SEQ ID NOs: 998-1010. In exemplary arrangements, the sequences of the polypeptide fragments targeting CD123 are set forth in SEQ ID NOs: 2743-2755. In some arrangements, the scFv fragments targeting CD123 are described in Table 11 and set forth in SEQ ID NO: 630-642 and SEQ ID NO: 2400-2412. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD123. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD123. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD123.

[0044] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD138 (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragment targeting CD138 is set forth in SEQ ID NO: 1011. In exemplary arrangements, the sequence of the polypeptide fragments targeting CD138 is set forth in SEQ ID NO: 2756 In some arrangements, the scFv fragment targeting CD138 is described in Table 11 and set forth in SEQ ID NOs: 643 and 2413. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD138. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD138. Also provided herein are genetically-engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD138.

[0045] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CLL1 (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting CLL1 are set forth in SEQ ID NOs: 1024-1029. In exemplary arrangements, the sequences of the polypeptide fragments targeting CLL1 are set forth in SEQ ID NOs:2769-2774. In some arrangements, the scFv fragments targeting CLL1 are described in Table 11 and set forth in SEQ ID NO: 655-660 and SEQ ID NO: 2425-2430. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CLL1. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CLL1. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CLL1.

[0046] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CS1 (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting CS1 are set forth in SEQ ID NOs: 1031-1039. In exemplary arrangements, the sequences of the polypeptide fragments targeting CS1 are set forth in SEQ ID NOs:2776-2784. In some arrangements, the scFv fragments targeting CS1 are described in Table 11 and set forth in SEQ ID NO: 662-670 and SEQ ID NO: 2432-2440. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CS1. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD79b. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CS1.

[0047] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CSF2RA (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting CSF2RA are set forth in SEQ ID NOs: 1040-1041. In exemplary arrangements, the sequences of the polypeptide fragments targeting CSF2RA are set forth in SEQ ID NOs: 2785-2786. In some arrangements, the scFv fragments targeting CSF2RA are described in Table 11 and set forth in SEQ ID NO: 671-672 and SEQ ID NO: 2441-2442. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CSF2RA. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CSF2RA. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CSF2RA.

[0048] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets DLL3 (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting DLL3 are set forth in SEQ ID NOs: 1044-1045. In exemplary arrangements, the sequences of the polypeptide fragments targeting DLL3 are set forth in SEQ ID NOs: 2789-2790. In some arrangements, the scFv fragments targeting DLL3 are described in Table 11 and set forth in SEQ ID NO: 673-674 and SEQ ID NO: 2443-2444. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets DLL3. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets DLL3. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets DLL3.

[0049] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets GPRC5D (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting GPRC5D are set forth in SEQ ID NOs: 1070-1073. In exemplary arrangements, the sequences of the polypeptide fragments targeting GPRC5D are set forth in SEQ ID NOs: 2815-2818. In some arrangements, the scFv fragments targeting GPRC5D are described in Table 11 and set forth in SEQ ID NO: 695-674 and SEQ ID NO: 2465-2468. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets GPRC5D. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets GPRC5D. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets GPRC5D.

[0050] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets HIV1-envelop glycoprotein gp120 (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting HIV1-envelop glycoprotein gp120 are set forth in SEQ ID NOs: 959, 1089-1092. In exemplary arrangements, the sequences of the polypeptide fragments targeting HIV1-envelop glycoprotein gp120 are set forth in SEQ ID NOs: 2704, 2834-2837. In some arrangements, the scFv fragments targeting HIV1-envelop glycoprotein gp120 are described in Table 11 and set forth in SEQ ID NO: 581, 705-708 and SEQ ID NO: 2351, 2475-2478. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets HIV1-envelop glycoprotein gp120. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets HIV1-envelop glycoprotein gp120. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets HIV1-envelop glycoprotein gp120.

[0051] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets IL11Ra (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragment targeting IL 11Ra is set forth in SEQ ID NO: 1099. In exemplary arrangements, the sequence of the polypeptide fragments targeting IL11Ra is set forth in SEQ ID NO: 2844 In some arrangements, the scFv fragments targeting IL11Ra is described in Table 11 and set forth in SEQ ID NOs: 715 and 2485. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets IL11Ra. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets IL11Ra. Also provided herein are genetically-engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets IL11Ra.

[0052] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets IL13Ra2 (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting IL13Ra2 are set forth in SEQ ID NOs: 1101-1102. In exemplary arrangements, the sequences of the polypeptide fragments targeting IL13Ra2 are set forth in SEQ ID NOs: 2846-2847. In some arrangements, the scFv fragments targeting IL13Ra2 are described in Table 11 and set forth in SEQ ID NO: 716-717 and SEQ ID NO: 2486-2487. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets IL13Ra2. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets IL13Ra2. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets IL13Ra2.

[0053] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets LAMP1 (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting LAMP1 are set forth in SEQ ID NOs: 1104-1105. In exemplary arrangements, the sequences of the polypeptide fragments targeting LAMP1 are set forth in SEQ ID NOs: 2849-2850. In some arrangements, the scFv fragments targeting LAMP1 are described in Table 11 and set forth in SEQ ID NO: 719-720 and SEQ ID NO: 2489-2490. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets LAMP1. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets LAMP1. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets LAMP1.

[0054] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets NY-BR1 (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragment targeting NY-BR1 is set forth in SEQ ID NO: 1131. In exemplary arrangements, the sequence of the polypeptide fragments targeting NY-BR1 is set forth in SEQ ID NO: 2876 In some arrangements, the scFv fragments targeting NY-BR1 are described in Table 11 and set forth in SEQ ID NOs: 742 and 2512. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets NY-BR1. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets NY-BR1. Also provided herein are genetically-engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets NY-BR1.

[0055] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TIM1 / HAVCR (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting TIM1 / HAVCR are set forth in SEQ ID NOs: 1160-1161. In exemplary arrangements, the sequences of the polypeptide fragments targeting TIM1 / HAVCR are set forth in SEQ ID NOs: 2905-2906. In some arrangements, the scFv fragments targeting TIM1 / HAVCR are described in Table 11 and set forth in SEQ ID NO: 770-771 and SEQ ID NO: 2540-2541. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TIM1 / HAVCR. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TIM1 / HAVCR. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TIM1 / HAVCR.

[0056] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TROP2 (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting TROP2 are set forth in SEQ ID NOs: 1165-1166. In exemplary arrangements, the sequences of the polypeptide fragments targeting TROP2 are set forth in SEQ ID NOs: 2910-2911. In some arrangements, the scFv fragments targeting TROP2 are described in Table 11 and set forth in SEQ ID NO: 774-775 and SEQ ID NO: 2544-2545. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TROP2. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TROP2. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TROP2.

[0057] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TSHR (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting TSHR are set forth in SEQ ID NOs: 1167-1170. In exemplary arrangements, the sequences of the polypeptide fragments targeting TSHR are set forth in SEQ ID NOs: 2912-2914. In some arrangements, the scFv fragments targeting TSHR are described in Table 11 and set forth in SEQ ID NO: 776-778 and SEQ ID NO: 2546-2548. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TSHR. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TSHR. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TSHR.

[0058] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TSLPR (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragment targeting TSLPR is set forth in SEQ ID NO: 1171. In exemplary arrangements, the sequence of the polypeptide fragment targeting TSLPR is set forth in SEQ ID NO: 2916 In some arrangements, the scFv fragment targeting TSLPR is described in Table 11 and set forth in SEQ ID NOs: 779 and 2549. Also provided herein is a polypeptide encoded by a nucleic acid fragment encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TSLPR. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TSLPR. Also provided herein are genetically-engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TSLPR.

[0059] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CDH19 (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting CDH19 are set forth in SEQ ID NO: 1019 and 1180. In exemplary arrangements, the sequences of the polypeptide fragments targeting CDH19 are set forth in SEQ ID NO: 2764 and 2925 In some arrangements, the scFv fragments targeting CDH19 are described in Table 11 and set forth in SEQ ID NOs: 651, 788 and SEQ ID NOs: 2421 and 2558. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CDH19. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CDH19. Also provided herein are genetically-engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CDH19.

[0060] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CDH6 (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting CDH6 are set forth in SEQ ID NOs: 1017-1018. In exemplary arrangements, the sequences of the polypeptide fragments targeting CDH6 are set forth in SEQ ID NOs: 2761-2762. In some arrangements, the scFv fragments targeting CDH6 are described in Table 11 and set forth in SEQ ID NO: 648-649 and SEQ ID NO: 2418-2419. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CDH6. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CDH6. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CDH6.

[0061] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD324 (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting CD324 are set forth in SEQ ID NOs: 1014-1015. In exemplary arrangements, the sequences of the polypeptide fragments targeting CD324 are set forth in SEQ ID NOs: 2759-2760. In some arrangements, the scFv fragments targeting CD324 are described in Table 11 and set forth in SEQ ID NO: 646-647 and SEQ ID NO: 2416-2417. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD324. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD324. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD324.

[0062] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TCR gamma-delta (TCRgd) (Table 19). In exemplary arrangements, the sequence of the nucleic acid fragment targeting TCR gamma-delta (TCRgd) is set forth in SEQ ID NO: 1155. In exemplary arrangements, the sequence of the polypeptide fragments targeting TCR gamma-delta (TCRgd) is set forth in SEQ ID NO: 2900 In some arrangements, the scFv fragment targeting TCR gamma-delta (TCRgd) is described in Table 11 and set forth in SEQ ID NOs: 765 and SEQ ID NOs: 2535. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TCR gamma-delta (TCRgd). Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TCR gamma-delta (TCRgd). Also provided herein are genetically-engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TCR gamma-delta (TCRgd).

[0063] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TCRB1 (TCR beta 1 constant chain) (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting TCRB1 (TCR beta 1 constant chain) are set forth in SEQ ID NOs: 1151-1152. In exemplary arrangements, the sequences of the polypeptide fragments targeting TCRB1 (TCR beta 1 constant chain) are set forth in SEQ ID NOs: 2896-2897. In some arrangements, the scFv fragments targeting TCRB1 (TCR beta 1 constant chain) are described in Table 11 and set forth in SEQ ID NO: 761-762 and SEQ ID NO: 2531-2532. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TCRB1 (TCR beta 1 constant chain). Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TCRB1 (TCR beta 1 constant chain). Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TCRB1 (TCR beta 1 constant chain).

[0064] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TCRB2 (TCR beta 2 constant chain) (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting TCRB2 (TCR beta 2 constant chain) are set forth in SEQ ID NOs: 1153-1154. In exemplary arrangements, the sequences of the polypeptide fragments targeting TCRB2 (TCR beta 2 constant chain) are set forth in SEQ ID NOs: 2898-2899. In some arrangements, the scFv fragments targeting TCRB2 (TCR beta 2 constant chain) are described in Table 11 and set forth in SEQ ID NO: 763-764 and SEQ ID NO: 2533-2534. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TCRB2 (TCR beta 2 constant chain). Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TCRB2 (TCR beta 2 constant chain). Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TCRB2 (TCR beta 2 constant chain).

[0065] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets LHR (Luteinizing hormone Receptor) (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting LHR (Luteinizing hormone Receptor) are set forth in SEQ ID NOs: 1182-1183. In exemplary arrangements, the sequences of the polypeptide fragments targeting LHR (Luteinizing hormone Receptor) are set forth in SEQ ID NOs: 2927-2928. In some arrangements, the scFv fragments targeting LHR (Luteinizing hormone Receptor) are described in Table 11 and set forth in SEQ ID NO: 790-791 and SEQ ID NO: 2560-2561. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets LHR (Luteinizing hormone Receptor). Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets LHR (Luteinizing hormone Receptor). Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets LHR (Luteinizing hormone Receptor).

[0066] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets Fc region of an immunoglobulin (Table 19). In exemplary arrangements, the sequences of the nucleic acid fragments targeting Fc region of an immunoglobulin are set forth in SEQ ID NOs: 961-962. In exemplary arrangements, the sequences of the polypeptide fragments targeting Fc region of an immunoglobulin are set forth in SEQ ID NOs: 2707-2708. In some arrangements, the receptor targeting Fc region of an immunoglobulin comprises, consists of or essentially consists of extracellular ligand-binding domain of CD16 (FCGR3A) or its variant V158 or its variant F158. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets Fc region of an immunoglobulin. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets Fc region of an immunoglobulin. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets Fc region of an immunoglobulin. The genetically engineered cells comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets Fc region of an immunoglobulin can be used to enhance antibody dependent cell mediated cytotoxicity and / or enhance antibody based immunotherapy, such as cancer immunotherapy, as described in US 2015 / 0139943A1.

[0067] In exemplary arrangements, nucleic acids encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets antigens of interest are described in Table 20. The sequences of nucleic acid fragments encoding backbone-32 comprising conventional CAR II and K13-vFLIP and targeting antigens of interest as described in Table 20 are set forth in SEQ ID NOs: 1197-1466. The sequences of polypeptides encoding backbone-32 comprising conventional CAR I and K13-vFLIP and targeting antigens of interest as described in Table 20 are set forth in SEQ ID NOs: 2942-3211. The nucleic acid sequences in SEQ ID NOs: 1197-1466 and polypeptide sequences in SEQ ID NOs: 2942-3211 also encode for a puromycin acetyl transferase (PuroR) fragment, which fragment can be used to select cells and is not essential for the function of the CAR.

[0068] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD19 (Table 20). In exemplary arrangements, nucleic acid sequences of the V L -V H targeting CD19 are set forth in SEQ ID NOs: 1197-1211. In exemplary arrangements, the polypeptide sequences of the V L -V H targeting CD19 are set forth in SEQ ID NOs: 2942-2956. In some arrangements, the scFv fragments targeting CD19 are described in Table 11 and set forth in SEQ ID NOs: 564-577 and SEQ ID NOs: 2334-2347. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD19. Further provided herein are vectors encoding nucleic acids encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD19. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD19.

[0069] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets MPL (Table 20). In exemplary arrangements, nucleic acid sequences of the V L -V H targeting MPL are set forth in SEQ ID NOs: 1387-1394. In exemplary arrangements, the polypeptide sequences of the V L -V H targeting MPL are set forth in SEQ ID NOs: 3132-3139. In some arrangements, the scFv fragments targeting MPL are described in Table 11 and set forth in SEQ ID NOs: 729-736 and SEQ ID NOs: 2499-2506. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets MPL. Further provided herein are vectors encoding nucleic acids encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets MPL. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets MPL.

[0070] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets Lym1 (Table 20). In exemplary arrangements, the nucleic acid sequence of the V L -V H targeting Lym1 is set forth in SEQ ID NO: 1379. In exemplary arrangements, the polypeptide sequence of the V L -V H targeting Lym1 is set forth in SEQ ID NO: 3124. In some arrangements, the scFv fragment targeting Lym1 is described in Table 11 and set forth in SEQ ID NO: 723 and SEQ ID NO: 2493. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets Lym1. Further provided herein are vectors encoding nucleic acids encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets Lym1. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets Lym1.

[0071] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets Lym2 (Table 20). In exemplary arrangements, nucleic acid sequence of the V L -V H targeting Lym2 is set forth in SEQ ID NO: 1380. In exemplary arrangements, the polypeptide sequence of the V L -V H targeting Lym2 is set forth in SEQ ID NO: 3125. In some arrangements, the scFv fragment targeting Lym2 is described in Table 11 and set forth in SEQ ID NO: 724 and SEQ ID NO: 2494. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets Lym2. Further provided herein are vectors encoding nucleic acids encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets Lym2. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets Lym2.

[0072] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-32 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets GRP-78 (Table 20). In exemplary arrangements, the nucleic acid sequence of the V L -V H targeting GRP-78 is set forth in SEQ ID NO: 1348. In exemplary arrangements, the polypeptide sequence of the V L -V H targeting GRP-78 is set forth in SEQ ID NO: 3093. In some arrangements, the scFv fragment targeting GRP-78 is described in Table 11 and set forth in SEQ ID NO: 703 and SEQ ID NO: 2473. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets GRP-78. Further provided herein are vectors encoding nucleic acids encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets GRP-78. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets GRP-78.

[0073] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-32 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD79b (Table 20). In exemplary arrangements, the nucleic acid sequence of the V L -V H targeting CD79b are set forth in SEQ ID NO: 1381. In exemplary arrangements, the polypeptide sequence of the V L -V H targeting CD79b is set forth in SEQ ID NO: 3126. In some arrangements, the scFv fragment targeting CD79b is described in Table 11 and set forth in SEQ ID NO: 628 and SEQ ID NO: 2398. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD79b. Further provided herein are vectors encoding nucleic acids encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD79b. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets CD79b.

[0074] In one arrangement, provided herein is an isolated nucleic acid encoding backbone-1 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets Fc region of an immunoglobulin (Table 20). In exemplary arrangements, the sequences of the nucleic acid fragments targeting Fc region of an immunoglobulin are set forth in SEQ ID NOs: 1232-1233. In exemplary arrangements, the sequences of the polypeptide fragments targeting Fc region of an immunoglobulin are set forth in SEQ ID NOs: 2977-2978. In some arrangements, the receptor targeting Fc region of an immunoglobulin comprises, consists of or essentially consists of extracellular ligand-binding domain of CD16 (FCGR3A) or its variant V158 or its variant F158. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets Fc region of an immunoglobulin. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets Fc region of an immunoglobulin. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets Fc region of an immunoglobulin. The genetically engineered cells comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR II and K13-vFLIP, wherein the antigen specific domain of the CAR targets Fc region of an immunoglobulin can be used to enhance antibody dependent cell mediated cytotoxicity and / or enhance antibody based immunotherapy, such as cancer immunotherapy, as described in US 2015 / 0139943A1.

[0075] In exemplary arrangements, nucleic acids encode conventional CAR II wherein the antigen specific domain of the CAR targets antigens of interest (Table 21). The sequences of nucleic acid fragments encoding conventional CAR II and targeting antigens of interest as described in Table 21 are set forth in SEQ ID NOs: 1467-1730. The sequences of polypeptides comprising conventional CAR II and targeting antigens of interest as described in Table 21 are set forth in SEQ ID NOs: 3212-3475. The nucleic acid sequences in SEQ ID NOs: 1467-1730 and polypeptide sequences in SEQ ID NOs: 3212-3475 also encode for a puromycin acetyl transferase (PuroR) fragment, which fragment can be used to select cells and is not essential for the function of the CAR. Similarly, several CAR constructs described in Table 22 encode for PuroR or Enhanced Green Fluorescent Protein (EGFP), which are not essential for the function of these CAR constructs but may be used to select or enrich for cells expressing these CAR constructs.

[0076] In one arrangement, provided herein is an isolated nucleic acid encoding conventional CAR II, wherein the antigen specific domain of the CAR targets CD19 (Table 21). In exemplary arrangements, the sequences of isolated nucleic acid fragments targeting CD19 are set forth in SEQ ID NOs: 1470-1481, 1772. In exemplary arrangements, the sequences of isolated polypeptide targeting CD19 are set forth in SEQ ID NOs: 3215-3225, 3517. In some arrangements, the scFv fragments targeting CD19 are described in Table 11 and set forth in SEQ ID NOs: 566-577 and SEQ ID NOs: 2336-2347. Also provided herein are polypeptides encoded by nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets CD19. Further provided herein are vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets CD19. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets CD19.

[0077] In one arrangement, provided herein is an isolated nucleic acid encoding conventional CAR II, wherein the antigen specific domain of the CAR targets MPL or the Thrombopoietin Receptor (Table 21). In exemplary arrangements, the sequences of isolated nucleic acid fragments targeting MPL are set forth in SEQ ID NOs: 1657-1664, 1731-1737. In exemplary arrangements, the sequences of isolated polypeptide targeting MPL are set forth in SEQ ID NOs: 3402-3409, 3476-3484. In some arrangements, the scFv fragments targeting MPL are described in Table 11 and set forth in SEQ ID NOs: 729-736 and SEQ ID NOs: 2499-2506. Also provided herein are polypeptides encoded by nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets MPL. Further provided herein are vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets MPL. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets MPL.

[0078] In one arrangement, provided herein is an isolated nucleic acid encoding conventional CAR II, wherein the antigen specific domain of the CAR targets Lym1 (Table 21). In exemplary arrangements, the sequence of nucleic acid fragment targeting Lym1 is set forth in SEQ ID NO: 1649. In exemplary arrangements, the sequence of isolated polypeptide fragment targeting Lym1 is set forth in SEQ ID NO: 3394. In some arrangements, the scFv fragment targeting Lym1 is described in Table 11 and set forth in SEQ ID NO: 723 and SEQ ID NO: 2493. Also provided herein are polypeptides encoded by nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets Lym1. Further provided herein are vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets Lym1. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets Lym1.

[0079] In one arrangement, provided herein is an isolated nucleic acid encoding conventional CAR II, wherein the antigen specific domain of the CAR targets Lym2 (Table 21). In exemplary arrangements, sequence of the isolated nucleic acid fragment targeting Lym2 is set forth in SEQ ID NO: 1650. In exemplary arrangements, the sequence of isolated polypeptide targeting Lym2 is set forth in SEQ ID NO: 3395. In some arrangements, the scFv fragment targeting Lym2 is described in Table 11 and set forth in SEQ ID NO: 724 and SEQ ID NO: 2494. Also provided herein are polypeptides encoded by nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets Lym2. Further provided herein are vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets Lym2. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets Lym2.

[0080] In one arrangement, provided herein is an isolated nucleic acid encoding conventional CAR II, wherein the antigen specific domain of the CAR targets DLL3 (Table 21). In exemplary arrangements, the sequences of isolated nucleic acid fragments targeting DLL3 are set forth in SEQ ID NOs: 1314-1315. In exemplary arrangements, the sequences of isolated polypeptide targeting DLL3 are set forth in SEQ ID NOs: 3059-3060. In some arrangements, the scFv fragments targeting DLL3 are described in Table 11 and set forth in SEQ ID NOs: 673-674 and SEQ ID NOs: 2443-2444. Also provided herein are polypeptides encoded by nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets DLL3. Further provided herein are vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets DLL3. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets DLL3.

[0081] In one arrangement, provided herein is an isolated nucleic acid encoding conventional CAR II, wherein the antigen specific domain of the CAR targets CD324 (Table 21). In exemplary arrangements, the sequences of isolated nucleic acid fragments targeting CD324 are set forth in SEQ ID NOs: 1554-1555. In exemplary arrangements, the sequences of isolated polypeptide targeting CD324 are set forth in SEQ ID NOs: 3299-3300. In some arrangements, the scFv fragments targeting CD324 are described in Table 11 and set forth in SEQ ID NOs: 646-647 and SEQ ID NOs: 2416-2417. Also provided herein are polypeptides encoded by nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets CD324. Further provided herein are vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets CD324. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets CD324.

[0082] In one arrangement, provided herein is an isolated nucleic acid encoding conventional CAR II, wherein the antigen specific domain of the CAR targets CD276 (Table 21). In exemplary arrangements, the sequence of nucleic acid fragment targeting CD276 is set forth in SEQ ID NO: 1553. In exemplary arrangements, the sequence of isolated polypeptide fragment targeting CD276 is set forth in SEQ ID NO: 3298. In some arrangements, the scFv fragment targeting CD276 is described in Table 11 and set forth in SEQ ID NO: 645 and SEQ ID NO: 2415. Also provided herein are polypeptides encoded by nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets CD276. Further provided herein are vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets CD276. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets CD276.

[0083] In one arrangement, provided herein is an isolated nucleic acid encoding conventional CAR II, wherein the antigen specific domain of the CAR targets PTK7 (Table 21). In exemplary arrangements, the sequences of isolated nucleic acid fragments targeting PTK7 are set forth in SEQ ID NOs: 1683-1684. In exemplary arrangements, the sequences of isolated polypeptide targeting PTK7 are set forth in SEQ ID NOs: 3428-3429. In some arrangements, the scFv fragments targeting PTK7 are described in Table 11 and set forth in SEQ ID NOs: 753-754 and SEQ ID NOs:2523-2524. Also provided herein are polypeptides encoded by nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets PTK7. Further provided herein are vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets PTK7. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets PTK7.

[0084] In one arrangement, provided herein is an isolated nucleic acid encoding conventional CAR II, wherein the antigen specific domain of the CAR targets HIV1-envelop glycoprotein gp120 (Table 21). In exemplary arrangements, sequence of the isolated nucleic acid fragment targeting HIV1-envelop glycoprotein gp120 is set forth in SEQ ID NO: 1485. In exemplary arrangements, the sequence of isolated polypeptide targeting HIV1-envelop glycoprotein gp120 is set forth in SEQ ID NO: 3230. In some arrangements, the scFv fragment targeting HIV1-envelop glycoprotein gp120 is described in Table 11 and set forth in SEQ ID NO: 581 and SEQ ID NO: 2351. Also provided herein are polypeptides encoded by nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets HIV1-envelop glycoprotein gp 120. Further provided herein are vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets HIV1-envelop glycoprotein gp120. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets HIV1-envelop glycoprotein gp120.

[0085] In one arrangement, provided herein is an isolated nucleic acid encoding conventional CAR II, wherein the antigen specific domain of the CAR targets tyrosinase-derived peptides in association with MHC class I complex (Table 21). In exemplary arrangements, sequence of the isolated nucleic acid fragments targeting tyrosinase-derived peptides in association with MHC class I complex (HLA-A2) is set forth in SEQ ID NO: 1712-1714. In exemplary arrangements, the sequence of isolated polypeptides targeting tyrosinase-derived peptides in association with MHC class I complex (HLA-A2) is set forth in SEQ ID NO: 3457-3459. In some arrangements, the scFv fragments targeting tyrosinase-derived peptide in association with MHC class II (HLA-A2) complex is described in Table 11 and set forth in SEQ ID NO:780-782 and SEQ ID NO: 2550-2552. Also provided herein are polypeptides encoded by nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets tyrosinase-derived peptides in association with MHC class I complex (HLA-A2). Further provided herein are vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CARs target tyrosinase-derived peptides in association with MHC class I complex (HLA-A2). Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets target tyrosinase-derived peptides in association with MHC class I complex (HLA-A2). In exemplary arrangement, the sequence of the tyrosinase-derived peptide that is targeted by the CAR in association with MHC class I complex (HLA-A2) is -YMDGTMSQV- (Table 23).

[0086] In some arrangements, provided herein are vectors comprising nucleic acid sequences encoding the conventional CARs or novel backbones (comprising conventional CARs and accessory modules) described herein. In exemplary arrangements, the vector is any of a DNA vector, an RNA vector, a plasmid, a lentivirus vector, an adenoviral vector or a retrovirus vector. In an arrangement, the vector is a lentivirus vector as described herein. In an arrangement, the vector is pLENTI-EF1α (SEQ ID NO: 905). In another arrangement, the vector is pLenti-EF1a-DWPRE (SEQ ID NO: 906). In another arrangement, the vector is MSCV-Bg12-AvrII-Bam-EcoR1-Xho-BstB1-Mlu-Sal-ClaI.I03 (SEQ ID NO: 907). In another arrangement, the vector is a sleeping beauty transposon vector. In another arrangement, the sleeping beauty transposon vector is pSBbi-Pur (SEQ ID NO: 908). In one arrangement, the vector further comprises a promoter. Non-limiting examples of a promoter include from an EF-1 promoter, a CMV IE gene promoter, an EF-1α promoter, an ubiquitin C promoter, or a phosphoglycerate kinase (PGK) promoter. In one arrangement, the promoter is an EF-1 promoter (SEQ ID NO: 909). In one arrangement, the promoter is an inducible promoter that provides a molecular switch capable of turning on expression of the polynucleotide sequence encoding conventional CARs I to III and backbones 1-62 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 inducible promoter, a glucocorticoid inducible promoter, a progesterone inducible promoter, and a tetracycline inducible promoter. RheoSwitch ®< system (Agarwal et al, 2012, AACR-NCI-EORTC international conference on Molecular Targets and Cancer Therapeutics) represents another transcriptional regulator platform for controlling the expression of conventional CARs and backbones 1-62 of the present invention. In one arrangement, a combinatorially activated T cell circuit can be engineered in which a synthetic Notch receptor for one antigen induces the expression of a conventional CAR or backbones 1-62 targeting a second antigen as described recently for controlling the activity of CARs (Roybal KT et al., Cell, 164:1-10 2016). In one arrangement, the vector is an in vitro transcription vector, (for example, a vector that transcribes RNA from a DNA molecule) described herein. In one arrangement, the nucleic acid sequence in the vector further comprises a poly(A) tail, e.g., a poly A tail described herein, e.g., comprising about 100-150 adenosine bases. For example, contemplated herein is a poly(A) tail comprising about 150 adenosine bases (SEQ ID NO: 922). In one arrangement, the nucleic acid sequence in the vector further comprises a 3'UTR, e.g., a 3' UTR described herein, e.g., comprising at least one repeat of a 3'UTR derived from human beta-globulin.

[0087] In some arrangements, the vector comprising a nucleic acid sequence encoding a CAR may further comprise a nucleic acid sequence encoding one or more inhibitory molecules. Non-limiting examples of inhibitory molecules contemplated herein include: an inhKIR cytoplasmic domain; a transmembrane domain, e.g., a KIR transmembrane domain; and an inhibitor cytoplasmic domain, e.g., an ITIM domain, e.g., an inhKIR ITIM domain. An exemplary inhibitory molecule comprising the transmembrane and cytoplasmic domains of LAILR1 is represented by SEQ ID NOS: 849 and 2612. Inhibitory conventional CARs and backbones 1-62 targeting different antigens can be easily constructed by joining the scFv fragments described in Table 11 to the transmembrane and cytoplasmic domains of LAILR1 (SEQ ID NOS: 849 and 2612). Such inhibitory CARs can be expressed alone or in combination with the activating CARs, such as the CARs described in Tables 19-22.

[0088] In some arrangements, the ASD of the CARs described herein have a binding affinity (KD) of 10 -4< M to 10 -8< M, 10 -5< M to 10 -7< M, 10 -6< M or 10 -7< M, for the target antigen.

[0089] Also provided herein are genetically engineered cells e.g., an immune effector cell, (e.g., a population of cells, e.g., a population of immune effector cells) and / or a stem cell (e.g., a hematopoietic stem cell, a peripheral blood stem cell, a bone marrow derived stem cell, an immune stem cell, an induced pluripotent stem cell or iPSC) comprising nucleic acids, polypeptides or vectors as described herein.

[0090] In one arrangement, the cell is a human T cell. In some arrangements, the cell is an immune cell. Non-limiting examples of immune cells include T-cells and NK-cells. Further, non-limiting examples of T-cells include Tregs, CD8+ T cells, and CD4+ T cells. Immune cells, (e.g., T cells and NK 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. T cells could be tissue resident gamma-delta T cells or tumor infiltrating lymphocytes (TILs), which can be cultured and expanded in vitro prior to and / or after the expression of the CARs and the novel backbones of this invention. In some arrangements, the cell is a mammalian cell. In some arrangement, the cell is a human cell. In some arrangements, the cell is a dog cell. In some arrangements, the cell is a monkey cell.

[0091] In one arrangement, the cell is a T cell and the T cell is deficient in one or more of endogenous T cell receptor chains. T cells stably lacking expression of a functional TCR according to the invention may be produced using a, variety of approaches such as use of Zn finger nucleases (ZFN), CRISP / Cas9 and shRNA targeting the endogenous T cell receptor chains. A non-limiting exemplary method relating to shRNAs is described in US 2012 / 0321667 A1. Another non-limiting exemplary method relating to eliminating endogenous TCR expression using ZFNs targeting constant regions of α and β chains of TCRs has been described (Torikai H et al.,Blood 119:24, 2012).

[0092] In one arrangement, the cell is a stem cell and the stem cell is deficient in one or more of endogenous T cell receptor chains. In another arrangement, the cell is a stem cell in which one or more target antigens of the CAR have been deleted or mutated to a form that is no longer recognized by the CAR. In a non-limiting example, a CAR targeting CD19 is expressed in stem cells that have been made deficient in CD19 using CRISP / Cas9 or Zn finger nucleases so that the B cells produced by such stem cells are not eliminated by the T cells expressing the CD19-targeting CAR. In another non-limiting example, a CAR targeting CD19 is expressed in stem cells in which the endogenous CD19 gene has been mutated using CRISP / Cas9 or Zn finger nucleases to a form that is not targeted by CAR so that the B cells produced by such stem cells are not eliminated by the T cells expressing the CD19-targeting CAR. In another non-limiting example, the CAR is expressed in immune effector cells and the stem cells from an autologous or an allogeneic donor are genetically engineered to either lack the expression of the target antigen of the CAR or to express a mutated form of the target antigen of the CAR which is not recognized by the CAR. In another non-limiting example, a CAR targeting CD19 is expressed in T cells that are infused into a patient along with autologous or allogeneic hematopoietic stem cells that have been made deficient in CD19 using CRISP / Cas9 or Zn finger nucleases so that the B cells produced by such stem cells are not eliminated by the T cells expressing the CD19-targeting CAR. In another non-limiting example, a CAR targeting CD19 is expressed in T cells that are infused into a patient along with autologous or allogeneic hematopoietic stem cells in which the endogenous CD19 gene has been mutated using CRISP / Cas9 or Zn finger nucleases to a form that is not targeted by CAR so that the B cells produced by such stem cells are not eliminated by the T cells expressing the CD19-targeting CAR. In other non-limiting examples, CRISP / Cas9, Zn finger nucleases or siRNA / shRNA approaches are used to mutate or eliminate other endogenous antigens (e.g., MPL, CD33, CD123 etc.) in stem cells in subjects receiving CAR-T cells targeting these antigens for the treatment of specific diseases in which these antigens are expressed on disease-associated or disease-causing cells. In a non-limiting example, a CAR targeting MPL is expressed in stem cells that have been made deficient in MPL using CRISP / Cas9 or Zn finger nucleases so that the blood cells produced by such stem cells are not eliminated by the T cells expressing the MPL-targeting CAR. In another non-limiting example, a CAR targeting MPL (e.g., SEQ ID NO: 1657-1660) is expressed in stem cells in which the endogenous MPL gene has been mutated using CRISP / Cas9 or Zn finger nucleases to a form that is not targeted by MPL-directed CAR so that the blood cells produced by such stem cells are not eliminated by the T cells expressing the MPL-targeting CAR. In another non-limiting example, a CAR targeting MPL is expressed in T cells that are infused into a patient along with autologous or allogeneic hematopoietic stem cells in which the endogenous MPL gene has been mutated using CRISP / Cas9 or Zn finger nucleases to a form that is not targeted by CAR. In an exemplary arrangement the region of MPL gene that is targeted for mutation by CRISP / Cas9 or Zn finger nucleases to a form that is not targeted by MPL-directed CAR encodes for the peptide sequence -PWQDGPK-.

[0093] In various arrangements, the CARs target one, two, three, four or more antigens on the diseased-causing or disease-associated cells, such as the cancer cells. In some arrangements, the CARs comprising more than one ASD target the same antigen on the diseased-causing or disease-associated cells. In another arrangement, the CARs comprising more than one ASD target different antigens on the diseased-causing or disease-associated cells.

[0094] In some arrangements, the genetically engineered cells further comprise nucleic acids, polypeptides or vectors that encode accessory modules. In some arrangements, the accessory modules reduce or prevent toxicity associated with CARs. In some arrangements, the accessory modules are inhibitors of cytokines or chemokines, for examples, siRNA (either shRNA or Mir based shRNAs) against the involved cytokine and / or chemokine and are expressed from the same vector as the CAR. For example, any one or more of IL6, IL10 and IFNγ can be targeted. In some arrangements, the expression of the chemokine or cytokine can be blocked by expression of a blocking scFv fragment targeting the chemokine or cytokine or its receptor. Non-limiting examples of such accessory modules include IL6R-304-vHH (SEQ ID NOs: 884 and 2647), IGHSP2-IL6R-304-VHH-ALB8-VHH (SEQ ID NOs: 886 and 2649), CD8SP2-PD1-4H1-scFv (SEQ ID NOs: 889 and 2652), CD8SP2-PD1-5C4-scFv CD8SP2-CTLA4-Ipilimumab-scFv (SEQ ID NOs: 891 and 2654), CD8SP2-PD1-4H1-Alb8-vHH (SEQ ID NOs: 892 and 2655), CD8SP2-PD1-5C4-Alb8-vHH (SEQ ID NOs: 893 and 2656), CD8SP2-CTLA4-Ipilimumab-Alb8-vHH (SEQ ID NOs: 894 and 2657), IgSP-IL6-19A-scFV (SEQ ID NOs: 899 and 2662), CD8SP2-sHVEM (SEQ ID NOs: 901 and 2664), CD8SP2-sHVEM-Alb8-vHH (SEQ ID NOs: 902 and 2665), hTERT (SEQ ID NOs: 903 and 2666), Heparinase (SEQ ID NOs: 904 and 2667), IL12F (SEQ ID NOs: 863 and 2626), 41BB-L (SEQ ID NOs: 864 2627), CD40L (SEQ ID NOs: 865 2628) and shRNA targeting Brd4 (SEQ ID NO: 919). An exemplary vector expressing H1 promoter-driven shRNA targeting Brd4 that can be used to clone the conventional CARs and novel backbones 1-62 of the invention is pLenti-EF1a-shRNA-BRD4-DWPRE (SEQ ID NO: 920). An exemplary CAR in backbone 1 coexpressing shRNA targeting Brd4 is pLenti-EF1a-CD8SP-FMC63-(vL-vH)-Myc-z-P2A-K13-Flag-T2A-PAC-H1-prom-shRNA-BRD4-DWPRE (SEQ ID NO: 921).

[0095] Capillary leak syndrome is another major complication of CARs. A natural plasmin digest product of fibrin, peptide BB15-42 (also called FX06), has been shown to significantly reduce vascular leak (Groger et al., PLoS ONE 4:e5391, 2009). FX06 peptide has been shown to block capillary leak. In some arrangements, as described herein, FX06 (SEQ ID NOs: 900 and 2663) can be coexpressed with the CARs described herein from the same vector to mitigate capillary leak associated with CAR therapy. The nucleic acid and protein sequences of an exemplary CAR co-expressing FX06 are represented by SEQ ID NOs: 1764 and 3509, respectively.

[0096] A limitation of the current technology for adoptive cellular therapy with T cells is the limited life span of genetically modified T cells, such as CAR-T cells, when infused into patients. To extend the life span of genetically-modified T cells, provided herein are vectors expressing CARs in conjunction with a number of viral and cellular proteins that stimulate the proliferation of T cells and / or to protect them from activation-induced cell death. The use of these proteins, however, is not limited to CAR-expressing T cells and they can be used to extend the life-span of any immune cells that are to be used for the purpose of adoptive immunotherapy, including but not limited to any T cell (e.g., autologous T cells or allogeneic T cells), T cell subsets (e.g., CD8, CD4, TILs), T cells directed against specific tumor antigens or infectious agents, T cells expressing an exogenous T cell receptor (i.e., TCR gene therapy), and T cells expressing a synthetic or chimeric T cell receptor. The DNA and protein sequences of several proteins that can promote the proliferation and survival of CAR-expressing T cells are provided in SEQ ID NOs; 862-882 and SEQ ID NOs: 2625-2645, respectively (Table 16). In exemplary arrangements, these proteins include any one or more of viral proteins K13-vFLIP (SEQ ID NOs: 866 and 2629) andMC159-vFLIP (SEQ ID NOs: 867 and 2630) as described herein. In further arrangements, these proteins include cFLIP-L / MRIT-alpha (SEQ ID NOs: 873 and 2636) and cFLIP-p22 (SEQ ID NOs: 874 and 2637). In further arrangements, these proteins include any one, two or more of Tax and Tax-mutants from human T cell leukemia / lymphoma virus (HTLV-1 and HTLV-2) (SEQ ID NOs: 875-877 and SEQ ID NOs: 2638-2640), Tio protein from herpes virus ateles, stpC Tip from Herpes virus Saimiri. The use of the viral and cellular proteins to promote the survival and proliferation of T cells, is however, not limited to CAR-expressing T cells. In further arrangement, these viral and cellular proteins can be used to promote the survival and proliferation of any T cell, including those expressing an exogenous T cell receptor or synthetic T cell receptor.

[0097] In one arrangement, the viral and cellular proteins that are known to stimulate the proliferation of T cells and / or to protect them from activation- induced cell death can be expressed in the CAR-expressing cells constitutively. In another arrangement, the viral and cellular proteins that are known to stimulate the proliferation of T cells and / or to protect them from activation- induced cell death can be expressed in the CAR-expressing cells in an inducible manner, for example, using an inducible promoter. Examples of inducible promoters include, but are not limited to a metallothionine inducible promoter, a glucocorticoid inducible promoter, a progesterone inducible promoter, and a tetracycline inducible promoter. RheoSwitch ®< system represents another transcriptional regulator platform for controlling the expression of CAR of the present invention (Agarwal et al, 2012, AACR-NCI-EORTC international conference on Molecular Targets and Cancer Therapeutics). In another arrangement, the activity of the viral and cellular proteins that are known to stimulate the proliferation of T cells and / or to protect them from activation- induced cell death is controlled by expressing them in fusion with a dimerization domain. Non-limiting examples of dimerization domains include FKBP (SEQ ID NOs: 857-858 and SEQ ID NOs: 2620 and 2621), FKBPx2 (SEQ ID NOs: 859 and 2622) and Myr-FKBP. Non-limiting examples of several viral and cellular proteins in fusion with FKBP domains are provided in SEQ ID NOs: 878-882 and SEQ ID NOs: 2641 to 2645 (Table 16). The DNA and protein sequences of exemplary CAR constructs coexpressing a viral protein in fusion with FKBP dimerization domain(s) are provided in SEQ ID NOs: 1755 to 1758 and SEQ ID NOs: 3500 to 3503.

[0098] Further, a Src inhibitor can be used to prevent and / or treat the toxicity associated with the administration of CAR-expressing cells. In some arrangements, the Src inhibitors are particularly useful for the treatment of cytokine release syndrome and neurological complications associated with the use of genetically modified T cells (e.g., T cells expressing CAR or exogenous TCR or chimeric TCR),Bispecific T cell engagers (BiTE) and DARTs (Dual Affinity Re-targeting proteins).

[0099] Also provided herein are methods for treating a disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a nucleic acid encoding one or more conventional CARs I to III, a therapeutically effective amount of a nucleic acid encoding one or more CARs and at least one accessory module (backbones 1-62), a therapeutically effective amount of polypeptide encoded by the nucleic acid encoding one or more conventional CARs I to III, a therapeutically effective amount of polypeptide encoded by the nucleic acid encoding one or more CARs and at least one accessory module (backbones 1-62), a therapeutically effective amount of genetically modified cells, as described herein, comprising nucleic acid encoding one or more conventional CARs I to III, or a therapeutically effective amount of genetically modified cells, as described herein, comprising nucleic acid encoding one or more conventional CARs and at least one accessory module (backbones 1-62), as described herein. In one arrangement, the nucleic acids, polypeptides, vectors and genetically modified cells comprise a conventional CAR II. In one arrangement, the nucleic acids, polypeptides, vectors and genetically modified cells comprise backbone-1, comprising a conventional CAR I and K13-vFLIP. In one arrangement, the nucleic acids, polypeptides, vectors and genetically modified cells comprise backbone-32, comprising a conventional CAR II and K13-vFLIP. In one arrangement, the disease treated or prevented by CAR is a cancer. In other arrangements, the diseases treated or prevented by CARs are infectious diseases, allergic diseases, autoimmune diseases, a degenerative diseases, or a combination thereof.

[0100] In one arrangement, provided herein is a method for treating, inhibiting, reducing the severity of, preventing metastasis of and / or reducing the relapse of a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of genetically modified cells comprising nucleic acid encoding one or more conventional CARs. In one arrangement, the genetically modified cells comprise vectors encoding a conventional CAR II as described herein. In one arrangement, provided herein is a method for treating, inhibiting, reducing the severity of, preventing metastasis of and / or reducing the relapse of cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of genetically modified cells comprising nucleic acid encoding one or more CARs and at least one accessory module (backbones 1-62). In one arrangement, the genetically modified cells comprise vectors encoding backbone-1, comprising a conventional CAR I and K13-vFLIP as described herein. In one arrangement, the genetically modified cells comprise vectors encoding backbone-32, comprising a conventional CAR II and K13-vFLIP as described herein. In some arrangements, the vectors encoding CARs target two, three or more antigens on cancer cells. In one arrangement, the disease treated or prevented by administration of genetically modified cells comprising nucleic acid encoding one or more CARs is a cancer. In other arrangements, the diseases treated or prevented by administration of genetically modified cells comprising nucleic acid encoding one or more CARs are infectious disease, allergic diseases, autoimmune diseases, a degenerative diseases, or combination thereof.

[0101] In exemplary arrangements, the cancer is any of colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, primary effusion lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers, combinations of said cancers, and metastatic lesions of said cancers.

[0102] Also provided herein are methods for treating, inhibiting, reducing the severity of, preventing metastasis of and / or reducing the relapse of hematological cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of genetically modified cells, as described herein, wherein the genetically modified cells comprise vectors encoding a conventional CAR II as described herein.

[0103] Also provided herein are methods for treating, inhibiting, reducing the severity of, preventing metastasis of and / or reducing the relapse of hematological cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of genetically modified cells, as described herein, wherein the genetically modified cells comprise vectors encoding backbone-1, comprising conventional CAR I and K13-vFLIP or vectors encoding backbone-32, comprising CAR II and K13-vFLIP. In one arrangement, the ASD of the CAR targets MPL. In exemplary arrangements, conventional CAR II targeting MPL are described in Table 21 and 22 and comprise nucleic acid sequences set forth in SEQ ID NOs: 1657-1664 and 1731-1739 and comprise polypeptide sequences set forth in SEQ ID NOs: 3402-3409 and 3476-3484. In exemplary arrangements, conventional CAR I targeting MPL and coexpressing K13-vFLIP (backbone 1) are described in Table 19 and comprise nucleic acid sequences set forth in SEQ ID NOs: 1117-1124 and 1747 and comprise polypeptide sequences set forth in SEQ ID NOs: 2862-2869 and 3492. In exemplary arrangements, conventional CAR II targeting MPL and coexpressing K13-vFLIP (backbone 32) are described in Tables 20 and 22 and comprise nucleic acid sequences set forth in SEQ ID NOs: 1387-1394 and 1747 and comprise polypeptide sequences set forth in SEQ ID NOs: 3132-3139 and 3492. Non-limiting examples of hematological cancers treated or prevented by administration of cells expressing CARs targeting MPL include acute myeloid leukemia, chronic myeloid leukemia and myelodysplastic syndrome.

[0104] Also provided herein are methods for treating, inhibiting, reducing the severity of, preventing metastasis of and / or reducing the relapse of cancers expressing CD19 (e.g., B cell acute lymphocytic leukemia (B-ALL), chronic lymphocytic leukemia, diffuse large cell lymphoma, indolent lymphoma) in a subject in need thereof comprising administering to the subject a therapeutically effective amount of genetically modified cells, as described herein, wherein the genetically modified cells comprise vectors encoding a conventional CAR II targeting CD19 as described herein.

[0105] Also provided herein are methods for treating, inhibiting, reducing the severity of, preventing metastasis of and / or reducing the relapse of a CD19 expressing cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of genetically modified cells, as described herein, wherein the genetically modified cells comprise vectors encoding backbone-1, comprising conventional CAR I targeting CD19 and K13-vFLIP or vectors encoding backbone-32, comprising CAR II targeting CD19 and K13-vFLIP. In exemplary arrangements, conventional CAR II targeting CD19 are described in Table 21 and comprise nucleic acid sequences set forth in SEQ ID NOs: 1470-1480 and comprise polypeptide sequences set forth in SEQ ID NOs: 3215-3225. In exemplary arrangements, conventional CAR I targeting CD19 and coexpressing K13-vFLIP (backbone 1) are described in Table 19 and comprise nucleic acid sequences set forth in SEQ ID NOs: 927-941 and comprise polypeptide sequences set forth in SEQ ID NOs: 2672-2686. In exemplary arrangements, conventional CAR II targeting CD19 and coexpressing K13-vFLIP (backbone 32) are described in Tables 20 and 22 and comprise nucleic acid sequences set forth in SEQ ID NOs: 1197-1211 and 1759 and comprise polypeptide sequences set forth in SEQ ID NOs: 2942-2956 and 3504.

[0106] Also provided herein are methods for treating, inhibiting, reducing the severity of, preventing metastasis of and / or reducing the relapse of GRP-78 expressing cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of genetically modified cells, as described herein, wherein the genetically modified cells comprise vectors encoding backbone-1, comprising conventional CAR I and K13-vFLIP or vectors encoding backbone-32, comprising CAR II and K13-vFLIP. In one arrangement, the ASD of the CAR targets GRP-78. In exemplary arrangements, CARs targeting GRP-78 and accessory modules are described in Table 22 and comprise nucleic acid sequence set forth in SEQ ID NOs: 1771 and 1775 and comprise polypeptide sequence set forth in SEQ ID NO: 3516 and 3520.

[0107] In various arrangements, additional antigen targets, CAR constructs and accessory modules for use with the instant invention will be apparent to a person of skill in the art. Exemplary arrangements are set forth in Tables 2-22.

[0108] In some arrangements, the therapeutically effective amount of the genetically modified cells is administered at a dosage of 10 4< to 10 9< cells / kg body weight, in some instances 10 5< to 10 6< cells / kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The cells can be administered by injection into the site of the lesion (e.g., intra-tumoral injection).

[0109] In some arrangements, the therapeutic methods described herein further comprise administering to the subject, sequentially or simultaneously, existing therapies. Examples of existing cancer treatment include, but are not limited to, active surveillance, observation, surgical intervention, chemotherapy, immunotherapy, radiation therapy (such as external beam radiation, stereotactic radiosurgery (gamma knife), and fractionated stereotactic radiotherapy (FSR)), focal therapy, systemic therapy, vaccine therapies, viral therapies, molecular targeted therapies, or combinations thereof.

[0110] In some arrangements, the therapeutic methods described herein further comprise administering to the subject, sequentially or simultaneously, any one or more of a kinase inhibitor, a BTK inhibitor, an mTOR inhibitor, a MNK inhibitor, BRD4 inhibitor or a dual PI3K / mTOR inhibitor, or combinations thereof.

[0111] In one arrangement, the kinase inhibitor is a Src family kinase inhibitor, such as dasatinib.

[0112] In one arrangement, the kinase inhibitor is a CDK4 inhibitor, such as CDK4 / 6 inhibitors, selected from ribociclib, abemaciclib and palbociclib.

[0113] In one arrangement, the kinase inhibitor is an mTOR inhibitor, for example, rapamycin, a rapamycin analog, OSI-027. The mTOR inhibitor can be, for example, an mTORCl inhibitor and / or an mTORC2 inhibitor.

[0114] In one arrangement, the kinase inhibitor is a MNK inhibitor, for example, 4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-d] pyrimidine. The MNK inhibitor can be, for example, an inhibitor of MNKla, MNKlb, MNK2a and / or MNK2b. The dual PI3K / mTOR inhibitor can be, e.g., PF-04695102.

[0115] In one arrangement, the inhibitor is a Brd4 inhibitor selected from JQ1, MS417, OTXO15, LY 303511 and Brd4 inhibitor as described in US 20140256706 A1 and any analogs thereof.

[0116] In one arrangement of the methods described herein, the kinase inhibitor is a CDK4 inhibitor, e.g., palbociclib (PD0332991), and the palbociclib is administered at a dose of about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg (e.g., 75 mg, 100 mg or 125 mg) daily for a period of time, e.g., daily for 14-21 days of a 28 day cycle, or daily for 7-12 days of a 21 day cycle. In one arrangement, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of palbociclib are administered.

[0117] In one arrangement of the methods described herein, the kinase inhibitor is a BTK inhibitor selected from ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13. In one arrangement, the BTK inhibitor does not reduce or inhibit the kinase activity of interleukin- 2-inducible kinase (ITK), and is selected from GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13. In one arrangement of the methods or uses described herein, the kinase inhibitor is a BTK inhibitor, e.g., ibrutinib (PCI-32765), and the ibrutinib is administered at a dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (e.g., 250 mg, 420 mg or 560 mg) daily for a period of time, e.g., daily for 21 day cycle, or daily for 28 day cycle. In one arrangement, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of ibrutinib are administered.

[0118] In one arrangement of the methods or uses described herein, the kinase inhibitor is a BTK inhibitor that does not inhibit the kinase activity of ITK, e.g., RN-486, and RN- 486 is administered at a dose of about 100 mg, 110 mg, 110 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 100 mg, 110 mg, 220 mg, 230 mg, 240 mg, 250 mg (e.g., 150 mg, 100 mg or 250 mg) daily for a period of time, e.g., daily a 28 day cycle. In one arrangement, 1, 2, 3, 4, 5, 6, 7, or more cycles of RN-486 are administered.

[0119] In one arrangement of the methods described herein, the kinase inhibitor is an mTOR inhibitor selected from temsirolimus; ridaforolimus (lR,2R,4S)-4-[(2R)-2 [(1R,9S,12S,15R,16E,18R,19R,21R, 23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy- 19,30-dimethoxy-15, 17,21,23, 29,35-hexamethyl-2,3,10,14,20-pentaoxo-1 1,36-dioxa-4-azatricyclo[30.3.1.0] hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2- methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669; everolimus (RAD001); rapamycin (AY22989); simapimod; (5-{1,4-bis[(35")-3- methylmorpholin-4-yl]pyrido[2,3-(i]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[iran5-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3- pyridinyl)-4-methyl-pyrido[2,3-J|pyrimidin-7(8H)-one (PF04691502); and N 2< -[1,4-dioxo- 4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L- arginylglycyl-L-a-aspartylL-serine-, inner salt (SF1126); and XL765.

[0120] In one arrangement of the methods or uses described herein, the kinase inhibitor is an mTOR inhibitor, e.g., rapamycin, and the rapamycin is administered at a dose of about 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (e.g., 6 mg) daily for a period of time, e.g., daily for 21 day cycle, or daily for 28 day cycle. In one arrangement, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of rapamycin are administered. In one arrangement, the kinase inhibitor is an mTOR inhibitor, e.g., everolimus and the everolimus is administered at a dose of about 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg (e.g., 10 mg) daily for a period of time, e.g., daily for 28 day cycle. In one arrangement, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of everolimus are administered.

[0121] Also provided herein are methods for preparing genetically engineered cells comprising transfecting the cells with vectors comprising nucleic acid encoding the conventional CARs or the CARs on novel backbones described herein. In some arrangements, the cells are immune effector cells, such as human T cells or human NK cells, or stem cells that give rise to immune effector cells. In some arrangements, the cells are autologous human T cells or autologous human NK cells or autologous human stem cells. In some arrangements, the cells are allogeneic human T cells or allogeneic human NK cells or allogeneic human stem cells.

[0122] In one arrangement, the method for preparing the genetically modified cells comprise obtaining a population of cells and depleting the cells of the CD25+ T regulatory cells, for example by using antibodies specific to CD25. Methods for depleting CD25+ T regulatory cells from the population of cells will be apparent to a person of skill in the art. In some arrangements, the Treg depleted cells comprise less than 30%, 20%, 10%, 5% or less Treg cells. In some arrangements, the vectors encoding the CARs described herein are transfected into Treg depleted cells.

[0123] In some arrangements, prior to transfection, the Treg depleted cells are further depleted of cells which express checkpoint inhibitors. The checkpoint inhibitor may be any one or more of PD-1, LAG-3, TIM3, B7-H1, CD160, P1H, 2B4, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), TIGIT, CTLA-4, BTLA, and LAIR1. In some arrangements, the cells comprise less than comprise less than 30%, 20%, 10%, 5% or less cells that express checkpoint inhibitors. In some arrangements, the vectors encoding the CARs described herein are transfected into Treg depleted and checkpoint inhibitor depleted cells.

[0124] In some arrangements, methods for preparing the genetically modified cells comprise obtaining a population of cells and selecting cells that express any one or more of CD3, CD28, CD4, CD8, CD45RA, and / or CD45RO. In certain arrangements, the population of immune effector cells provided are CD3+ and / or CD28+.

[0125] In one arrangement, the method for preparing the genetically modified cells comprise obtaining a population of cells and enriching for the CD25+ T regulatory cells, for example by using antibodies specific to CD25. Methods for enriching CD25+ T regulatory cells from the population of cells will be apparent to a person of skill in the art. In some arrangements, the Treg enriched cells comprise less than 30%, 20%, 10%, 5% or less non-Treg cells. In some arrangements, the vectors encoding the CARs described herein are transfected into Treg-enriched cells. Treg enriched cells expressing a CAR may be used to induced tolerance to antigen targeted by the CAR.

[0126] In some arrangements, the method further comprises expanding the population of cells after the vectors comprising nucleic acids encoding the CARs described herein have been transfected into the cells. In arrangements, the population of cells is expanded for a period of 8 days or less. In certain arrangements, the population of cells is expanded in culture for 5 days, and the resulting cells are more potent than the same cells expanded in culture for 9 days under the same culture conditions. In other arrangements, the population of cells is expanded in culture for 5 days show at least a one, two, three or four fold increase in cell doublings upon antigen stimulation as compared to the same cells expanded in culture for 9 days under the same culture conditions. In some arrangements, the population of cells is expanded in an appropriate media that includes one or more interleukins that result in at least a 200-fold, 250-fold, 300- fold, or 350-fold increase in cells over a 14 day expansion period, as measured by flow cytometry.

[0127] In various arrangements, the expanded cells comprise one or more CARs as described herein. In some arrangements, the expanded cells comprise one CAR with one, two, three or more ASDs. In some arrangements, the expanded cells further comprise accessory modules and therapeutic controls as described herein.

[0128] Provided herein is a cell comprising nucleic acids encoding a chimeric antigen receptor (CAR) and one or more of signaling proteins selected from K13-vFLIP, MC159-vFLIP, cFLIP-L, cFLIP-p22, HTLV1-Tax and HTLV2-Tax , wherein the CAR comprises an a) extracellular antigen specific domain, b)a transmembrane domain and c) an intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM); wherein c) is located at the C-terminus of the chimeric receptor. In some arrangements, the CAR further comprises one or more co-stimulatory domains.

[0129] In some arrangements of the cell comprising nucleic acids encoding a chimeric antigen receptor (CAR) and K13-vFLIP signaling protein, wherein the CAR comprises an a) extracellular antigen specific domain, b) a transmembrane domain and c) an intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM); wherein c) is located at the C-terminus of the chimeric receptor, the CAR further comprises one or more co-stimulatory domains.

[0130] Also disclosed herein is a cell comprising nucleic acids encoding a chimeric antigen receptor (CAR) and MC159-vFLIP signaling protein, wherein the CAR comprises an a) extracellular antigen specific domain, b) a transmembrane domain and c) an intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM); wherein c) is located at the C-terminus of the chimeric receptor. In some arrangements, the CAR further comprises one or more co-stimulatory domains.

[0131] In some arrangements, the cell described herein further comprises nucleic acids encoding MC159-vFLIP signaling protein.

[0132] In some arrangements, the cell further comprises nucleic acids encoding scFvs targeting IL6 and / or IL6 receptor alpha. In some arrangements, the cell further comprises nucleic acid encoding peptide FX06 so as to mitigate capillary leak associated with CAR therapy. In some arrangements, the signaling protein is expressed in fusion with one or more copies of FKBP domain. In some arrangements, the activity of the signaling protein is controlled post-translationally by dimerization of the FKBP domain in the presence of a dimerizing agent. In some arrangements, the dimerizing agent is AP20187.

[0133] In an exemplary arrangement, wherein the antigen specific domain of the CAR targets MPL. In some arrangements, the antigen specific domain targets two antigens, wherein the two antigens are MPL and CD123.

[0134] In some arrangements, the antigen specific domain of the CAR targets CD19, CD23, Lym1, Lym2, CLEC5A, CDH179b, FLT3, GCC, Muc1, CSF2RA, GFRa4, CD32, IL11Ra, IL13Ra, NYBR1, SLea, CD200R, TGFBetaR2, CD276, TROP2, LAMP1, PTK7, DLL3, CDH1, CDH6, CDH17, CDH19, TSHR and tyrosinase.

[0135] In some arrangements, the antigen specific domain of the CAR targets MPL and comprises one or more scFv fragments selected from 161 (SEQ ID NO: 2500 and 2501), 175 (SEQ ID NO: 2499), 178 (SEQ ID NO: 2503), 111 (SEQ ID NO: 2502), AB317 (SEQ ID NO: 2404), 12E10 (SEQ ID NO: 2505) or huVB22Bw5 (SEQ ID NO: 2506) or ligands selected from extracellular receptor binding domains of hTPO (SEQ ID NO: 2323) or mTPO (SEQ ID NO: 2323).

[0136] In some arrangements, the antigen specific domain of the CAR targets CD19 and comprises one or more scFv fragments selected from CD19Bu12 or CD19MM.

[0137] In one arrangement, the cell is a T-lymphocyte (T-cell). In another arrangement, the cell is a Natural Killer (NK) cell.

[0138] Also provided herein are nucleic acids comprising a first polynucleotide encoding the CAR of claim and a second polynucleotide encoding the K13-vFLIP signaling protein. In some arrangements, the nucleic acids further comprise a third polynucleotide encoding the MC159-vFLIP signaling protein. Also provided herein are polypeptides encoded by the nucleic acids described herein and vectors encoding the nucleic acids described herein.

[0139] Further provided herein is a pharmaceutical composition comprising the cells described herein, the nucleic acids described herein, the polypeptides encoded by the nucleic acids described herein or vectors comprising the nucleic acids described herein, and a pharmaceutically acceptable carrier.

[0140] In some arrangements, provided herein is a method for treating a MPL-expressing cancer comprising administering to the subject, a therapeutically effective amount of the cell described herein, wherein the CAR targets MPL. In some arrangements, the cancer is a blood cancer, wherein the blood cancer is any one or more of acute myeloid leukemia, chronic myeloid leukemia, myelodyplastic syndrome, lymphoma, multiple myeloma and acute lymphocytic leukemia. In some arrangements, the method further comprises administering to the subject a tyrosine kinase inhibitor, wherein the inhibitor inhibits the Src family of kinases. In some arrangements, the inhibitor inhibits Lck. In some arrangements, the inhibitor is any one or more of Dasatinib, Ponatinib or A-770041.BRIEF DESCRIPTION OF THE DRAWINGS

[0141] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive. Fig. 1 depicts, in accordance with various embodiments of the invention, three generations of CARs. Depiction of first, second, and third generation chimeric antigen receptors with the scFv segments in green and the various TCR signaling components in red, blue and yellow (Casucci, Monica and Attilio Bondanza (2011). Journal of Cancer. 2: 378-382) Fig. 2A- Fig. 2D depict, in accordance with various embodiments of the invention, Fig. 2A: MPL gene expression in normal tissues. MPL has very restricted expression in normal tissues; Fig. 2B: MPL gene expression in 542 patient samples of Acute Myeloid Leukemia. MPL is expressed in most AML samples; Fig. 2C: MPL gene expression in 206 samples of Myelodysplastic syndrome (MDS). MPL is expressed in most MDS samples. Fig. 2D: MPL gene expression in 76 samples of CML. MPL is expressed in most CML samples. Fig. 3A- Fig.3B depict, in accordance with various embodiments of the invention, strong binding with 161-GGSG-NLuc-AcV5 was observed on HEL.92.1.7-Gluc-vector cells suggesting significant expression of MPL endogenously. Fig. 4A- Fig. 4B depicts in accordance with various embodiments of the invention, NLuc assay to measure expression of CAR in 293FT cells. The untransfected 293FT cells, and those transfected with CAR CD8SP-FMC63-(vL-vH)-Myc-BBz-T2A-PAC(112014-A13)[SEQ ID NO:1467] and CD8SP-MPL-161-(vL-vH)-Myc-BBz-T2A-PAC(021015-R07)[SEQ ID NO:1658] CAR were incubated with FLAG-CD19-ECD-GGSG-NLuc-AcV5[SEQ ID NO: 926] and MPL-ECD-GGSG-NLuc-AcV5[SEQ ID NO: 925]supernatants as described in material and methods section followed by washing with PBS and measurement of NLuc activity by Coeleoentrazine (CTZ; Nanolight) diluted in PBS. Luminescence was quantified using a BioTek plate reader. Data represents mean values of triplicate wells + / standard deviation (SD). Fig. 5 depicts, in accordance with various embodiments of the invention, modest binding of MPL-ECD-GGSG-NLuc-AcV5 to 293FT cells transfected with hTPO-(1-187)-Myc-CD28z-T2A-PAC(031915-U04)[SEQ ID NO:1738] and mTPO-(1-187)-Myc-CD28z-T2A-PAC(031915-V03)[SEQ ID NO: 1739] constructs and strong binding to 293FT cells transfected with CD8SP-MPL-161-(vL-vH)-Myc-BBz-T2A-PAC(021015-R07)[SEQ ID NO:1658] construct as compared to untransfected cells or cells that had been transfected with control CAR construct. Fig. 6 depicts, in accordance with various embodiments of the invention, expression of MPL CARs on the surface of T cells as determined by increased EGFP fluorescence and increased staining with APC-Myc as compared to the uninfected T cells (UI). Fig. 7 depicts in accordance with various embodiments, of the invention, strong binding of T cells expressing CD8SP-MPL-161-(vL-vH)-Myc-BBz-T2A-PAC(021015-R07)[SEQ ID NO:1658], CD8SP-175-(vL-vH)-Myc-CD28z-T2A-PAC(031615-Q04)[SEQ ID NO:1733], and CD8SP-MPL-VB22Bw4-(vL-vH)-Myc-CD28z-T2A-PAC(031615-B06)[SEQ ID NO:3536] CARs and modest binding of T cells expressing CD8SP-161-(vL-vH)-Myc-CD28z-T2A-PAC(021715-Z07)[SEQ ID NO:1731], CD8SP-AB317-(vL-vH)-Myc-CD28z-T2A-PAC(031615-T04)[SEQ ID NO:1735] and CD8SP-12E10-(vL-vH)-Myc-CD28z-T2A-PAC(031615-S03)[SEQ ID NO:1736] to MPL-GGSG-NLuc AcV5 supernatant as measured by NLuc assay. SEQ ID NO. 925 comprises MPL-ECD-GGSG-Nluc-AcV5. SEQ ID NO.: 926 comprises FLAG-CD19-ECD-GGSG-NLuc-AcV5. SEQ ID NO: 1734 comprises CD8SP-178-(vL-vH)-Myc-CD28z-T2A-PAC(031615-R04). SEQ ID NO: 3534 comprises CD8SP-4C3-(vL-vH)-Myc-CD28z-T2A-Pac(031615-WO5). Fig. 8 depicts, in accordance with various embodiments of the invention, increase in GLuc activity following co-culture with T cells expressing MPL-specific CD8SP-161-(vL-vH)-Myc-CD8TM-BBz-T2A-EGFP(090814-C03)[SEQ ID NO:1732] as compared to the uninfected T cells indicating lysis of target cells by the different MPL-CAR-T cells. Fig. 9 depicts, in accordance with various embodiments of the invention, increased killing of HEL-GLuc cells by CD8SP-MPL-161-(vL-vH)-Myc-BBz-T2A-PAC(021015-R07)[SEQ ID NO:1658] CAR expressing PBMC as compared to FMC63-(vL-vH)-Myc-BBz-T2A-PAC(112014-A13)[SEQ ID NO:1467] PBMC or uninfected PBMC. Fig. 10 depicts, in accordance with an embodiment of the invention, increased killing of HEL-GLuc cells by CD8SP-175-(vL-vH)-Myc-CD28z-T2A-PAC(031615-Q04)[SEQ ID NO:1733] CAR expressing PBMC as compared to uninfected PBMC. Fig. 11 depicts, in accordance with an embodiments of the invention, increase in GLuc activity, indicating lysis of target cells, following co-culture with T cells expressing MPL-specific CARs, which was stronger with CD8SP-161-(vL-vH)-Myc-CD28z-T2A-PAC(021715-Z07)[SEQ ID NO:1731], CD8SP-175-(vL-vH)-Myc-CD28z-T2A-PAC(031615-Q04)[SEQ ID NO:1733], and CD8SP-AB317-(vL-vH)-Myc-CD28z-T2A-PAC(031615-T04)[SEQ ID NO:1735] constructs and modest with CD8SP-178-(vL-vH)-Myc-CD28z-T2A-PAC(031615-R04)[SEQ ID NO:1734] construct as compared to the uninfected T cells. Fig. 12 depicts, in accordance with various embodiments of the invention, increase in GLuc activity, indicating lysis of target cells, following co-culture with T cells expressing MPL-specific CARs, which was stronger with CD8SP-161-(vL-vH)-Myc-CD28z-T2A-PAC(021715-Z07)[SEQ ID NO:1731], CD8SP-175-(vL-vH)-Myc-CD28z-T2A-PAC(031615-Q04)[SEQ ID NO:1733], CD8SP-AB317-(vL-vH)-Myc-CD28z-T2A-PAC(031615-T04)[SEQ ID NO:1735], and CD8SP-12E10-(vL-vH)-Myc-CD28z-T2A-PAC(031615-S03)[SEQ ID NO:1736] constructs and modest with CD8SP-178-(vL-vH)-Myc-CD28z-T2A-PAC(031615-R04)[SEQ ID NO:1734] and CD8SP-VB22Bw4-(vL-vH)-Myc-CD28z-T2A-PAC(031615-B06)[SEQ ID NO:3536] constructs as compared to the uninfected T cells. Fig. 13 depicts, in accordance with an embodiment of the invention, increase in GLuc activity, indicating lysis of target cells, following co-culture with NK92MI cells expressing MPL-specific CD8SP-161-(vL-vH)-Myc-CD8TM-BBz-T2A-EGFP(090814-C03)[SEQ ID NO:1732] CAR. Fig. 14 depicts, in accordance with an embodiment of the invention, increase in GLuc activity, indicating lysis of target cells, following co-culture with NK92MI cells expressing MPL-specific CARs. Fig. 15 depicts, in accordance with an embodiment of the invention, increased secretion of TNFα upon coculture of T cells expressing CAR targeting MPL with HEL cells as compared to uninfected T cells used as controls. Fig. 16 depicts, in accordance with an embodiment of the invention, that the median survival of mice given CD8SP-161-(vL-vH)-Myc-CD8TM-BBz-T2A-EGFP(090814-C03)[SEQ ID NO:1732] expressing NK92MI cells was 31.5 days, which was significantly higher than mice given unmodified NK92MI cells (28.5 days; p = 0.03) or those given NK92MI cells expressing a control CD8SP-4C3-(vL-vH)-Myc-BBz-T2A-EGFP(100814-K06)[SEQ ID NO:3537] CAR (28 days; p=.04). Fig. 17 depicts, in accordance with an embodiment of the invention, increased expression of the different CD19 CARs (shown with grey lines) on the surface of T cells as determined by staining with APC-Myc as compared to the uninfected T cells (UI; shown with dotted lines). Constructs depicted include CD8SP-FMC63-(vL-vH)-Myc-BBz-T2A-PAC(112014-A13)[SEQ ID NO:1467]; CD8SP-CD19Bu12-(vL-vH)-Myc-BBz-T2A-PAC(082815-P08)[SEQ ID NO:1470]; CD8SP-2-CD19MM-(vL-vH)-Myc-BBz-T2A-PAC(062915-D03)[SEQ ID NO:1471]; CD8SP-KSHV-4C3-(vL-vH)-Myc-BBz-T2A-PAC(042315-N01)[SEQ ID NO:1643]. Fig. 18 depicts, in accordance with various embodiments of the invention, strong binding of Jurkats expressing CD8SP-CD19Bu12-(vL-vH)-Myc-BBz-T2A-PAC(082815-P08)[SEQ ID NO:1470], CD8SP-2-CD19MM-(vL-vH)-Myc-BBz-T2A-PAC(062915-D03)[SEQ ID NO:1471] and CD8SP-FMC63-(vL-vH)-Myc-BBz-T2A-PAC(112014-A13)[SEQ ID NO:1467] CAR constructs to FLAG-CD19-ECD-GGSG-NLuc-AcV5 [SEQ ID NO:2671] supernatant, while no significant binding was observed on parental cells or those expressing the CD8SP-KSHV-4C3-(vL-vH)-Myc-BBz-T2A-PAC(042315-N01)[SEQ ID NO:1643] control CAR. Fig. 19 depicts, in accordance with various embodiments of the invention, strong binding of T cells expressing CD8SP-CD19Bu12-(vL-vH)-Myc-BBz-T2A-PAC(082815-P08)[SEQ ID NO:1470], CD8SP-2-CD19MM-(vL-vH)-Myc-BBz-T2A-PAC(062915-D03)[SEQ ID NO:1471] and CD8SP-FMC63-(vL-vH)-Myc-BBz-T2A-PAC(112014-A13)[SEQ ID NO:1467] CAR constructs to FLAG-CD19-ECD-GGSG-NLuc-AcV5 [SEQ ID NO:2671] supernatant, while no significant binding was observed on parental cells or those expressing CD8SP-KSHV-4C3-(vL-vH)-Myc-BBz-T2A-PAC(042315-N01)[SEQ ID NO:1643] control CAR. Fig. 20 depicts, in accordance with various embodiments of the invention, increase in GLuc activity, indicating lysis of RAJI target cells, following co-culture with T cells expressing CD19-specific CARs as compared to uninfected T cells (T-UI) or those expressing the control CAR 4C3. Constructs depicted include CD8SP-FMC63-(vL-vH)-Myc-BBz-T2A-PAC(112014-A13)[SEQ ID NO:1467]; CD8SP-2-CD19MM-(vL-vH)-Myc-BBz-T2A-PAC(062915-D03)[SEQ ID NO:1471]; CD8SP-CD19Bu12-(vL-vH)-Myc-BBz-T2A-PAC(082815-P08)[SEQ ID NO:1470]; CD8SP-KSHV-4C3-(vL-vH)-Myc-BBz-T2A-PAC(042315-N01)[SEQ ID NO:1643]; (-) indicates CAR-transduced T cells were used without puromycin selection; (+) indicates CAR-transduced T cells were used with puromycin selection. Fig. 21 depicts, in accordance with various embodiments of the invention, increase in GLuc activity, indicating lysis of RAJI target cells, following co-culture with T cells expressing CD19-specific CARs as compared to uninfected T cells (T-UI) or those expressing the control CAR 4C3. Constructs depicted include CD8SP-FMC63-(vL-vH)-Myc-BBz-T2A-PAC(112014-A13)[SEQ ID NO:1467]; CD8SP-2-CD19MM-(vL-vH)-Myc-BBz-T2A-PAC(062915-D03)[SEQ ID NO:1471]; CD8SP-KSHV-4C3-(vL-vH)-Myc-BBz-T2A-PAC(042315-N01)[SEQ ID NO:1643]; (-) indicates CAR-transduced T cells were used without puromycin selection; (+) indicates CAR-transduced T cells were used with puromycin selection. Fig. 22 depicts, in accordance with various embodiments of the invention, the survival of mice in specific groups. Fig. 23 depicts, in accordance with various embodiments of the invention, increased staining with FITC-FLAG antibody in K13-expressing T cells as compared to uninfected T cells. K13 immortalized T cells are CD8+ / CD4- (49%), CD8- / CD4+ (21%) and CD8+ / CD4+ (29%). Fig. 24 depicts, in accordance with various embodiments of the invention, that CD8SP-FMC63-(vL-vH)-Myc-BBz-T2A-PAC(112014-A13)[SEQ ID NO:1467], and CD8SP-FMC63-(vL-vH)-Myc-BBz-P2A-K13-Flag-T2A-PAC(111014-Y11)[SEQ ID NO:1197] expressing T-cells that have been selected with puromycin induce specific lysis of RS411 cells as measured by Gluc assay. Fig. 25 depicts, in accordance with various embodiments of the invention, increase in GLuc activity, indicating lysis of target cells, following co-culture with T cells expressing CD19-specific CARs as compared to uninfected T cells (T-UI) or those expressing the control CAR 4C3. Fig. 26 depicts, in accordance with various embodiments of the invention, confirms that incorporation of K13, MC159 or HTLV2-Tax (Tax2) does not negatively impact the killing ability of CARs with or without the presence of 41BB costimulatory domain. Fig. 27 depicts, in accordance with various embodiments of the invention, that the T cells expressing CAR constructs that co-expressed MC159 and HTLV2-Tax continue to exert cytotoxicity after 2 months in culture while the T cells expressing the constructs lacking MC159 and HTLV2-Tax lost this ability. Fig. 28A-Fig. 28B depict, in accordance with various embodiments of the invention, treatment with AP20187 led to increase in K13- and HTLV2-Tax RS mutant-induced NF-κB-Luc activity in all constructs in which K13 and HTLV2-Tax mutant were expressed in fusion with FKBP. Constructs depicted in Fig. 28A include FKBPX2-K13[SEQ ID NO: 879]; CD8SP-FMC63(vL-vH)-Myc-z-P2A-FKBP-K13-FLAG-T2A-EGFP(112614-B06)[SEQ ID NO:1755]; CD8SP-161-(vL-vH)-Myc-BBz-P2A-FKBP-K13-Flag-T2A-EGFP(112614-E05)[SEQ ID NO:1744]; CD8SP-FMC63(vL-vH)-Myc-z-P2A-FKBPx2-K13-FLAG-T2A-EGFP(120314-J03)[SEQ ID NO:1756]; and CD8SP-FMC63(vL-vH)-Myc-z-P2A-Myr-FKBPx2-K13-FLAG-T2A-EGFP(120314-N07)[SEQ ID NO:1757]. Constructs depicted in Fig. 28B include CD8SP-FMC63(vL-vH)-Myc-BBz-P2A-FKBPx2-FLAG-TAX2U2RS-T2A-eGFP-M03(012315-03)[SEQ ID NO:3539]; and CD8SP-FMC63(vL-vH)-Myc-BBz-P2A-FKBPx2-HTLV2-Tax-RS-T2A-EGFP(012315-O01)[SEQ ID NO:1758]. Fig. 29A- Fig. 29B depict, in accordance with various embodiments of the invention, activation of K13-mediated NF-κB activity by addition of AP20187 does not adversely affect cell killing induced by the CAR constructs that coexpress FKBP-K13. Construct depicted in Fig. 29A include CD8SP-FMC63(vL-vH)-Myc-z-P2A-FKBP-K13-FLAG-T2A-PAC(112316-S06)[SEQ ID NO:1770]. Construct depicted in Fig. 29B include CD8SP-161-(vL-vH)-Myc-z-P2A-FKBP-K13-FLAG-T2A-PAC(112916-U06)[SEQ ID NO: 1741]. Fig. 30A- Fig 30B depict, in accordance with various embodiments of the invention, induction of target cell death by different MPL-directed and CD32-directed CAR constructs that coexpress K13, MC159, HTLV2-Tax or HTLV2-Tax RS mutant. Fig. 31 depicts, in accordance with various embodiments of the invention, induction of PC3-target cell death by a TROP2-directed conventional CAR I construct that coexpresses K13. Fig. 32A- Fig 32B depict, in accordance with various embodiments of the invention, induction of target cell death by different LAMP1-directed CAR constructs that coexpress K13, Fig. 33 depicts, in accordance with various embodiments of the invention, that activation of K13 signaling in T cells expressing the FKBP-K13 fusion protein by addition of AP20187 compound does not adversely affect activation of CAR-induced NFAT signaling. Fig. 34 depicts, in accordance with various embodiments of the invention, NF-κB reporter activity by wild-type K13 and various K13 mutants. Fig. 35 depict, in accordance with various embodiments of the invention, shows inhibition of CAR CD8SP-2-CD19MM-(vL-vH)-Myc-BBz-T2A-PAC(062915-D03)[SEQ ID NO:1471]-induced cell death by 50nM and 100nM Dasatinib, while Imatinib has no effect. Fig. 36 depict, in accordance with various embodiments of the invention, increase in GLuc activity following co-culture with T cells expressing CD19-specific CARs CD8SP-FMC63-(vL-vH)-Myc-BBz-T2A-PAC(112014-A13)[SEQ ID NO: 1467] and CD8SP-2-CD19MM-(vL-vH)-Myc-BBz-T2A-PAC(062915-D03)[SEQ ID NO:1471] as compared to the uninfected T cells (T-UI) indicating lysis of target cells by the different CD19-CAR-T cells and its inhibition by Dasatinib and Ponatinib at the indicated doses. Fig. 37 depict, in accordance with various embodiments of the invention, that Dasatinib effectively blocks killing of RAJI-GLuc cells by T cells expressing the CD8SP-CD22-m971-(vL-vH)-Myc-BBz-T2A-PAC(091515-A02)[SEQ ID NO:1519] CAR as determined by GLuc assay. Fig. 38A- Fig. 38B depict, in accordance with various embodiments of the invention, that Fig. 38A: Dasatinib blocks NK92 cells mediated death of K562 cells as determined by Gluc assay and Fig. 38B: Dasatinib blocks death of RAJI-GLuc cells by CD8SP-FMC63-(vL-vH)-Myc-BBz-T2A-EGFP(070814-D06)[SEQ ID NO:3535] expressing NK92MI cells as determined by Gluc assay. Fig. 39A- Fig. 39B depict, in accordance with various embodiments of the invention, that Fig. 39A: treatment with Blinatumomab in the presence of T cells result in strong induction of death of RAJI-GLuc cells which is effectively blocked by 100nM Dasatinib and Fig. 39B: that co-culture of RAJI-GLuc cells with Blinatumomab in the presence of T cells result in strong induction of IFNγ production which is effectively blocked by 100nM Dasatinib. Fig. 40A- Fig. 40C depict, in accordance with various embodiments of the invention, Fig. 40A shows no significant toxic effect of the various drugs on RAJI-pLenti-Gluc cells that had not been co-cultured with CAR T cells. Fig. 40B shows mild to modest inhibition of CD8SP-CD19Bu12-(vL-vH)-Myc-BBz-T2A-PAC(082815-P08)[SEQ ID NO:1470] CAR-T cells-induced death of RAJI-pLenti-GLuc cells by Ruxolitinib, Fosatamatinib and Alisertib as compared to cells treated with media (Med) alone ( Fig 40A). Dasatinib was used as a positive control. Fig. 40C shows that the above compounds have only a minimal effect on Blinatumomab induced cell death mediated by T cells at the indicated concentrations. Fig. 41A- Fig. 41C depict, in accordance with various embodiments of the invention, Fig. 41A shows the release of Gluc in cells treated with the indicated drugs in the presence of media alone without any CAR-T cells. Fig.41B shows near complete inhibition of CD8SP-CD19Bu12-(vL-vH)-Myc-BBz-T2A-PAC(082815-P08)[SEQ ID NO:1470] CAR-T cells-induced death of RAJI-pLenti-GLuc cells by A-770041 at 100nM and 200nM while Saracatinib had no significant effect. Avasimibe had no significant effect on CAR-T cells induced cell death at 100nM but completely blocked it at 1µM. Fig. 41C shows that treatment with 100nM A770041 partially blocked cell death induced by combination of Blinatumomab with T cells while treatment with 200nM A770041 led to complete inhibition. Fig. 42A- Fig. 42B depict, in accordance with various embodiments of the invention, that coculturing of Jurkat cells expressing CD8SP-FMC63(vL-vH)-BBz-P2A-IgHSP-IL6R-M83(vL-vH)-Flag-T2A-PAC(011315-K04)[SEQ ID NO:1763] with RAJI cells led to increase in EGFP expression as compared to cells that had not been exposed to RAJI cells ( Fig. 42A). This indicates activation of NFAT signaling. No increase in EGFP expression was observed in parental Jurkats (J-N-G-P) without or with culturing with RAJI ( Fig. 42B). DETAILED DESCRIPTION

[0142] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Allen et al., Remington: The Science and Practice of Pharmacy 22nd ed., Pharmaceutical Press (September 15, 2012); Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3rd ed., revised ed., J. Wiley & Sons (New York, NY 2006); Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, NY 2013); Singleton, Dictionary of DNA and Genome Technology 3rd ed., Wiley-Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2nd ed., Cold Spring Harbor Press (Cold Spring Harbor NY, 2013); Köhler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 Jul, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, U. S. Patent No. 5,585,089 (1996 Dec); and Riechmann et al., Reshaping human antibodies for therapy, Nature 1988 Mar 24, 332(6162):323-7..

[0143] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of arrangements or embodiments of the invention. Indeed, the present invention is in no way limited to the methods and materials described. For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

[0144] Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular arrangements or embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0145] Chimeric antigen receptors (CAR) are artificial T cell receptors that are under investigation as a therapy for cancer, using a technique called adoptive cell transfer. Hematopoietic cells are removed from a patient and modified so that they express receptors that recognize proteins that are specific to the particular form of cancer. The cells, which can then recognize and kill the cancer cells, are reintroduced into the patient. CAR targeting the human CD19 antigen have shown impressive activity against B cell lineage human blood cancers, such as Acute Lymphocytic Leukemia, chronic lymphocytic leukemia and lymphoma and are under commercial development by several companies. However, there is a need to develop CAR targeting antigens that are expressed on non-lymphoid cells. This invention pertains to CARs targeting several such antigens.Definitions

[0146] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an arrangement or embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.).

[0147] Unless stated otherwise, the terms "a" and "an" and "the" and similar references used in the context of describing a particular arrangement or embodiment of the application (especially in the context of claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, "such as") provided with respect to certain arrangements or embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example." No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0148] As used herein, the term "about" refers to a measurable value such as an amount, a time duration, and the like, and encompasses variations of ±20%, ±10%, ±5%, ±1%, ±0.5% or ±0.1% from the specified value.

[0149] "Chimeric antigen receptor" or "CAR" or "CARs" as used herein refers to engineered receptors, which graft an antigen specificity onto cells (for example T cells such as naïve T cells, central memory T cells, effector memory T cells or combination thereof). CARs are also known as artificial T-cell receptors, chimeric T-cell receptors or chimeric immunoreceptors. In various arrangements or embodiments, CARs are recombinant polypeptides comprising an antigen-specific domain (ASD), a hinge region (HR), a transmembrane domain (TMD), co-stimulatory domain (CSD) and an intracellular signaling domain (ISD).

[0150] "Antigen-specific domain" (ASD) refers to the portion of the CAR that specifically binds the antigen on the target cell. In some arrangements or embodiments, the ASD of the CARs comprising any of the backbones described herein comprises an antibody or a functional equivalent thereof or a fragment thereof or a derivative thereof. The targeting regions may comprise full length heavy chain, Fab fragments, single chain Fv (scFv) fragments, divalent single chain antibodies or diabodies, each of which are specific to the target antigen. There are, however, numerous alternatives, such as linked cytokines (which leads to recognition of cells bearing the cytokine receptor), affibodies, ligand binding domains from naturally occurring receptors, soluble protein / peptide ligand for a receptor (for example on a tumor cell), peptides, and vaccines to prompt an immune response, which may each be used in various arrangements or embodiments of the invention. In some arrangements or embodiments, almost any molecule that binds a given antigen with high affinity can be used as an ASD, as will be appreciated by those of skill in the art. In some arrangements or embodiments, the ASD comprises T cell receptors (TCRs) or portions thereof. In exemplary embodiments, nucleic acids encoding ASDs comprising scFVs are set forth herein in SEQ ID NOs: 564-798.

[0151] "Hinge region" (HR) as used herein refers to the hydrophilic region which is between the ASD and the TMD. The hinge regions include but are not limited to Fc fragments of antibodies or fragments or derivatives thereof, hinge regions of antibodies or fragments or derivatives thereof, CH2 regions of antibodies, CH3 regions of antibodies, artificial spacer sequences or combinations thereof. Examples of hinge regions include but are not limited to CD8a hinge, and artificial spacers made of polypeptides which may be as small as, for example, Gly3 or CH1 and CH3 domains of IgGs (such as human IgG4). In some arrangements or embodiments, the hinge region is any one or more of (i) a hinge, CH2 and CH3 regions of IgG4, (ii) a hinge region of IgG4, (iii) a hinge and CH2 of IgG4, (iv) a hinge region of CD8a, (v) a hinge, CH2 and CH3 regions of IgG1, (vi) a hinge region of IgG1 or (vi) a hinge and CH2 region of IgG1. Other hinge regions will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.

[0152] "Transmembrane domain" (TMD) as used herein refers to the region of the CAR which crosses the plasma membrane. The transmembrane domain of the CAR of the invention is the transmembrane region of a transmembrane protein (for example Type I transmembrane proteins), an artificial hydrophobic sequence or a combination thereof. Other transmembrane domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. In some arrangements or embodiments, the TMD encoded CAR comprising any of the backbones described herein comprises a transmembrane domain selected from the transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 ld, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CDllb, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0153] "Co-stimulatory domain" (CSD) as used herein refers to the portion of the CAR comprising any of the backbones described herein which enhances the proliferation, survival and / or development of memory cells. The CARs of the invention may comprise one or more co-stimulatory domains. Each co-stimulatory domain comprises the costimulatory domain of any one or more of, for example, members of the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1(CD11a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40 or combinations thereof. Other co-stimulatory domains (e.g., from other proteins) will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.

[0154] "Intracellular signaling domain" (ISD) or "cytoplasmic domain" as used herein refers to the portion of the CAR comprising any of the backbones described herein which transduces the effector function signal and directs the cell to perform its specialized function. Examples of domains that transduce the effector function signal include but are not limited to the z chain of the T-cell receptor complex or any of its homologs (e.g., h chain, FceR1g and b chains, MB1 (Iga) chain, B29 (Igb) chain, etc.), human CD3 zeta chain, CD3 polypeptides (D, d and e), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5 and CD28. Other intracellular signaling domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.

[0155] "Linker" (L) or "linker domain" or "linker region" as used herein refer to an oligo- or polypeptide region from about 1 to 100 amino acids in length, which links together any of the domains / regions of the CAR of the invention. Linkers may be composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another. Linkers may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (for example T2A), 2A-like linkers or functional equivalents thereof and combinations thereof. In some arrangements or embodiments, the linkers include the picornaviral 2A-like linker, CHYSEL sequences of porcine teschovirus (P2A), Thosea asigna virus (T2A) or combinations, variants and functional equivalents thereof. In other arrangements or embodiments, the linker sequences may comprise Asp-Val / Ile-Glu-X-Asn-Pro-Gly (2A)-< Pro (2B)< motif, which results in cleavage between the 2A glycine and the 2B proline. The nucleic sequences of several exemplary cleavable linkers are provided in SEQ ID NO: 831 to SEQ ID NO: 836 and amino acid sequences of several exemplary linkers are provide in SEQ ID NO: 2598 to SEQ ID NO: 2602. Other linkers will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. In an embodiment, a Ser-Gly-Ser-Gly (SGSG) motif (SEQ ID NOs: 837-838 and SEQ ID NO: 2603) is also added upstream of the cleavable linker sequences to enhance the efficiency of cleavage. A potential drawback of the cleavable linkers is the possibility that the small 2A tag left at the end of the N-terminal protein may affect protein function or contribute to the antigenicity of the proteins. To overcome this limitation, in some arrangements or embodiments, a furine cleavage site (RAKR) (SEQ ID NO: 839-841 and SEQ ID NO: 2604) is added upstream of the SGSG motifs to facilitate cleavage of the residual 2A peptide following translation.

[0156] "Effector function" refers to the specialized function of a differentiated cell. Effector function of a T-cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.

[0157] "Enhancer", as used herein, denotes sequence elements that augment, improve or ameliorate transcription of a nucleic acid sequence irrespective of its location and orientation in relation to the nucleic acid sequence to be expressed. An enhancer may enhance transcription from a single promoter or simultaneously from more than one promoter. As long as this functionality of improving transcription is retained or substantially retained (e.g., at least 70%, at least 80%, at least 90% or at least 95% of wild-type activity, that is, activity of a full-length sequence), any truncated, mutated or otherwise modified variants of a wild-type enhancer sequence are also within the above definition.

[0158] "Accessory module" or "accessory modules" as used herein refers to an element that is co-expressed with a CAR to increase, decrease, regulate or modify the expression or activity of a CAR or CAR-expressing cells. In exemplary embodiments, accessory modules include but are not limited to examples described in Table 16. In one embodiment, the accessory module comprises K13-vFLIP (Table 16). In another embodiment, the accessory module comprises MC159-vFLIP (Table 16). In a further embodiment, the accessory module comprises an FKBP((FK506 binding protein)-fusion protein, such as FKBP-K13, in which whose activity can be controlled by the administration of a dimerizer molecule (Table 16).

[0159] "Switch domain," or a "dimerization domain" as used herein, typically refers to 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 arrangements or 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 arrangements or embodiments, the switch is intracellular. In arrangements or embodiments, the switch domain is a polypeptide-based entity, e.g., FKBP (FK506 binding protein), and the dimerization molecule is small molecule, e.g., AP20187.

[0160] "Dimerization molecule," as that term is used herein 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 arrangements or embodiments, the dimerization molecule is a small molecule, e.g., rapamycin or a rapalogue, e.g, RAD001 or AP20187.

[0161] "Therapeutic Controls" as used herein refers to an element used for controlling the activity of a CAR expressing cell.. In some arrangements or embodiments, therapeutic controls for controlling the activity of the CAR expressing cells of the invention comprise any one or more of truncated epidermal growth factor receptor (tEGFR), truncated epidermal growth factor receptor viii (tEGFRviii), truncated CD30 (tCD30), truncated BCMA (tBCMA), truncated CD19 (tCD19), thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyl transferase, human caspase 8, human caspase 9, inducible caspase 9, purine nucleoside phosphorylase, linamarase / linamarin / glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP) / indole-3-acetic (IAA), Gamma-glutamylcysteine synthetase, CD20 / alphaCD20, CD34 / thymidine kinase chimera, dox-depedent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903 / Fas system, a chimeric cytokine receptor (CCR), a selection marker, and combinations thereof. The nucleic acid sequences of several therapeutic controls are provided in SEQ ID NO: 850 to SEQ ID NO: 856 and SEQ ID NO: 883 (Table 16).

[0162] "Disease targeted by genetically modified cells" as used herein encompasses the targeting of any cell involved in any manner in any disease by the genetically modified cells of the invention, irrespective of whether the genetically modified cells target diseased cells or healthy cells to effectuate a therapeutically beneficial result. The genetically modified cells include but are not limited to genetically modified T-cells, NK cells, hematopoietic stem cells, pluripotent embryonic stem cells or embryonic stem cells. The genetically modified cells express the conventional CARs and novel backbones containing conventional CARs with accessory modules of the invention, which CARs may target any of the antigens expressed on the surface of target cells. Examples of antigens which may be targeted include but are not limited to antigens expressed on B-cells; antigens expressed on carcinomas, sarcomas, lymphomas, leukemia, germ cell tumors, and blastomas; antigens expressed on various immune cells; and antigens expressed on cells associated with various hematologic diseases, autoimmune diseases, and / or inflammatory diseases. Other antigens that may be targeted will be apparent to those of skill in the art and may be targeted by the CARs of the invention in connection with alternate embodiments thereof.

[0163] "Co-express" as used herein refers to simultaneous expression of two or more genes. Genes may be nucleic acids encoding, for example, a single protein or a chimeric protein as a single polypeptide chain. In one embodiment, simultaneous expression includes a single vector encoding a CAR and proteins that modulate an immune response. In another embodiment, simultaneous expression includes a first vector that encodes the CAR and a second vector that encodes one or more proteins that modulate an immune response.

[0164] "Autologous" cells as used herein refers to cells derived from the same individual as to whom the cells are later to be re-administered into.

[0165] "Genetically modified cells", "redirected cells", "genetically engineered cells" or "modified cells" as used herein refer to cells that express the CAR of the invention. In some arrangements or embodiments, the genetically modified cells comprise vectors that encode a CAR. In some arrangements or embodiments, the genetically modified cells comprise vectors that encode a CAR and one or more accessory molecules in the same vector. In some arrangements or embodiments, the genetically modified cells comprise a first vector that encodes a CAR and a second vector that encodes the accessory molecule. In some arrangements or embodiments, the genetically modified cells comprise a first vector that encodes a CAR and a second vector that encodes more than one accessory molecule. In some arrangements or embodiments, the genetically modified cells comprise a first vector that encodes a CAR and a second vector that encodes the first accessory molecule and a third vector that encodes a second accessory molecule.

[0166] As used herein, the term "backbone" refers to the specific combination of conventional CARs (Table 1) and accessory modules as described in Table 2. In exemplary arrangements or embodiments, specific combinations of CARs and accessory modules which comprise various backbones are described in Table 2. In one embodiment, the CAR and the accessory module are encoded by a single nucleic acid molecule. In another embodiment, the CAR is encoded by the first nucleic acid molecule and the accessory module is encoded by a second nucleic acid molecule. In some arrangements or embodiments, the accessory module is encoded by more than one nucleic acid molecule, depending on the number of components in the accessory modules.

[0167] "Immune cell" as used herein refers to the cells of the mammalian immune system including but not limited to antigen presenting cells, B-cells, basophils, cytotoxic T-cells, dendritic cells, eosinophils, granulocytes, helper T-cells, leukocytes, lymphocytes, macrophages, mast cells, memory cells, monocytes, natural killer cells, neutrophils, phagocytes, plasma cells and T-cells.

[0168] The term "immune effector function" of the CAR-containing cell refers to any of the activities shown by the CAR-expressing cell upon stimulation by a stimulatory molecule. Examples of immune effector function, e.g., in a CAR-T cell, include cytolytic activity and helper activity, including the secretion of cytokines.

[0169] "Immune effector cell" as used herein refers to the T cells and natural killer (NK) cells.

[0170] "Immune response" as used herein refers to immunities including but not limited to innate immunity, humoral immunity, cellular immunity, immunity, inflammatory response, acquired (adaptive) immunity, autoimmunity and / or overactive immunity.

[0171] As used herein, "CD4 lymphocytes" refer to lymphocytes that express CD4, i.e., lymphocytes that are CD4+. CD4 lymphocytes may be T cells that express CD4.

[0172] The terms "T-cell" and "T-lymphocyte" are interchangeable and used synonymously herein. Examples include but are not limited to naïve T cells, central memory T cells, effector memory T cells or combinations thereof.

[0173] As used herein, the term "antibody" refers to an intact immunoglobulin or to a monoclonal or polyclonal antigen-binding fragment with the Fc (crystallizable fragment) region or FcRn binding fragment of the Fc region, referred to herein as the "Fc fragment" or "Fc domain". Antigen-binding fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, inter alia, Fab, Fab', F(ab')2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single domain antibodies, chimeric antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. The Fc domain includes portions of two heavy chains contributing to two or three classes of the antibody. The Fc domain may be produced by recombinant DNA techniques or by enzymatic (e.g. papain cleavage) or via chemical cleavage of intact antibodies.

[0174] The term "antibody fragment," as used herein, refer to a protein fragment that comprises only a portion of an intact antibody, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind antigen. Examples of antibody fragments encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VH and CH1 domains; (ii) the Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CH1 domain; (iii) the Fd fragment having VH and CH1 domains; (iv) the Fd' fragment having VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (v) the Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) the dAb fragment (Ward et al., Nature 341, 544-546 (1989)) which consists of a VH domain; (vii) isolated CDR regions; (viii) F(ab')2 fragments, a bivalent fragment including two Fab' fragments linked by a disulphide bridge at the hinge region; (ix) single chain antibody molecules (e.g., single chain Fv; scFv) (Bird et al., Science 242:423-426 (1988); and Huston et al., PNAS (USA) 85:5879-5883 (1988)); (x) "diabodies" with two antigen binding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); (xi) "linear antibodies" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al. Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).

[0175] "Single chain variable fragment", "single-chain antibody variable fragments" or "scFv" antibodies as used herein refer to forms of antibodies comprising the variable regions of only the heavy (V H ) and light (V L ) chains, connected by a linker peptide. The scFvs are capable of being expressed as a single chain polypeptide. The scFvs retain the specificity of the intact antibody from which it is derived. The light and heavy chains may be in any order, for example, V H -linker-V L or V L -linker-V H , so long as the specificity of the scFv to the target antigen is retained.

[0176] "Complementarity determining region" (CDR) as used herein refers to the amino acid sequences within the variable regions of antibodies which regions confer specificity and binding affinity. In general, there are three CDRs in each of the light chain variable regions (LCDR1, LCDR2 and LCDR3) and three CDRs in each of the heavy chain variable regions (HCD1, HCDr2 and HCDR3). The boundaries of the CDRs may be determined using methods well known in the art including the "Kabat" numbering scheme and / or "Chothia" number scheme (Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Services, National Institutes of Health, Bethesda, MD; Al-Lazikani et al., (1997) JMB 273,927-948).

[0177] As used herein, the term "specific binding" means the contact between an antibody and an antigen with a binding affinity of at least 10 -6< M. In certain aspects, antibodies bind with affinities of at least about 10 -7< M, and preferably 10 -8< M, 10 -9< M, 10 -10< M, 10 -11< M, or 10 -12< M.

[0178] "Binds the same epitope as" means the ability of an antibody, scFv, antigen specific domain of a CAR or other binding agent to bind to a target and having the same epitope as the exemplified antibody, scFv, antigen specific domain of a CAR or other binding agent. As an example, the epitopes of the exemplified antibody, scFv, or other binding agent and other antibodies can be determined using standard epitope mapping techniques. Epitope mapping techniques, well known in the art include Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E.Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Patent No. 4,708,871 ; Geysen et al, (1984) Proc. Natl. Acad. Sci. USA 8:3998-4002; Geysen et al, (1985) Proc. Natl. Acad. Sci. USA 82:78-182; Geysen et al, (1986) Mol. lmmunol. 23: 709-715. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., hydrogen / deuterium exchange, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Antigenic regions of proteins can also be identified using standard antigenicity and hydropathy plots, such as those calculated using, e.g., the Omiga version 1.0 software program available from the Oxford Molecular Group. This computer program employs the Hopp / Woods method, Hopp et al, (1981) Proc. Natl. Acad. Sci USA 78:3824-3828; for determining antigenicity profiles, and the Kyte-Doolittle technique, Kyte et al, (1982) J.Mol. Bioi. 157: 1 05-132; for hydropathy plots (FROM MORPHOSYS CD19 patent WO 2013 / 024097). To determine if selected monoclonal antibodies against a target (e.g., CD19) bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, Ill.). Competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using CD19-extracellualr domain coated-ELISA plates as described above. Biotinylated mAb binding can be detected with a strep-avidin-alkaline phosphatase probe.

[0179] The epitope recognized by a CAR can be also determined from the epitope recognized by the scFv comprising the CAR. For example, since the antigen specific domain of the CAR CD8SP-MPL-161-(vL-vH)-Myc-BBz-T2A-PAC (SEQ ID NO: 1658 and SEQ ID NO: 3403) targeting MPL is comprised of scFv MPL-161-(vL-vH) (SEQ ID NO: 730 and SEQ ID NO: 2500), it is expected that the CAR would target the same epitope as the scFv and the parental antibody from which the scFv is derived. The epitopes recognized by several scFv and / or their parental antibodies used in the construction of the CARs and backbones of this invention are known in the art. Alternatively, the epitope targeted by a CAR (including the CARs that are present as parat of backbones) can be determined by generating a series of mutants of its target antigen and testing the ability of the mutants to bind to the CAR-expressing cells. As an example, the epitope recognized by the CAR CD8SP-MPL-161-(vL-vH)-Myc-BBz-T2A-PAC (SEQ ID NO: 1658 and SEQ ID NO: 3403) targeting MPL can be determined by generating a panel of mutants of the MPL-ECD-GGSG-Nluc-AcV5 fusion construct (SEQ ID NO: 925 and SEQ ID NO: 2670). The mutant constructs would be transfected into a suitable cell line (e.g., 293FT cells) and the supernatant containing the fusion protein collected and assayed for NLuc activity to assure that the different mutant MPL-ECD-GGSG-Nluc-AcV5 fusion proteins are being secreted in the supernatant. Subsequently, the fusion proteins would be tested for their ability to bind to cells (e.g., Jurkat cells or T cells) expressing the CD8SP-MPL-161-(vL-vH)-Myc-BBz-T2A-PAC (SEQ ID NO: 1658 and SEQ ID NO: 3403) CAR construct. The mutant that fails to bind to the CAR-expressing cells is a candidate for containing the epitope targeted by the MPL-specific CAR. An alternate approach to determine the epitope recognized by a particular CAR could include a functional competitive assay with different test antibodies. For example, T cells expressing the CD8SP-MPL-161-(vL-vH)-Myc-BBz-T2A-PAC (SEQ ID NO: 1658 and SEQ ID NO: 3403) CAR would be co-cultured with a cell line expressing MPL (e.g., HEL cells) in the absence and presence of increasing concentrations of different test MPL antibodies. In case the epitope recognized by a test MPL antibody overlaps with the epitope recognized by the CD8SP-MPL-161-(vL-vH)-Myc-BBz-T2A-PAC (SEQ ID NO: 1658 and SEQ ID NO: 3403) CAR, then the test antibody would be expected to block target-cell killing and cytokine production induced by T cells expressing the CD8SP-MPL-161-(vL-vH)-Myc-BBz-T2A-PAC (SEQ ID NO: 1658 and SEQ ID NO: 3403) CAR in a dose-dependent manner. A non-specific antibody of the same isotype as the test antibody would be included as a control and would be expected to have no effect on the target-cell killing and cytokine production induced by T cells expressing the CAR. Similarly, a specific CAR can be expressed in Jurkat-NFAT-EGFP cells and the ability of a test antibody to block EGFP induction by the CAR-expressing Jurkat-NFAT-GFP cells upon coculture with a target cell line can be used to determine whether the epitope recognized by the test antibody overlaps with the epitope recognized by the said CAR.

[0180] "Therapeutic agents" as used herein refers to agents that are used to, for example, treat, inhibit, prevent, mitigate the effects of, reduce the severity of, reduce the likelihood of developing, slow the progression of and / or cure, a disease. Diseases targeted by the therapeutic agents include but are not limited to infectious diseases, carcinomas, sarcomas, lymphomas, leukemia, germ cell tumors, blastomas, antigens expressed on various immune cells, and antigens expressed on cells associated with various hematologic diseases, and / or inflammatory diseases.

[0181] "Cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. The term "cancer" is meant to include 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. Examples of solid tumors include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting liver, lung, breast, lymphoid, gastrointestinal (e.g., colon), genitourinary tract (e.g., renal, urothelial cells), prostate and pharynx. Adenocarcinomas include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In one embodiment, the cancer is a melanoma, e.g., an advanced stage melanoma. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the invention. Examples of other cancers that can be treated include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin Disease, non-Hodgkin lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of said cancers. Treatment of metastatic cancers, e.g., metastatic cancers that express PD-L1 (Iwai et al. (2005) Int. Immunol. 17:133-144) can be effected using the antibody molecules described herein.

[0182] "Polynucleotide" as used herein includes but is not limited to DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (short nucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA, and / or tRNA.

[0183] It is to be inferred without explicit recitation and unless otherwise intended, that when the present disclosure relates to a polypeptide, protein, polynucleotide, antibody or fragment thereof, an equivalent or a biologically equivalent of such is intended within the scope of this disclosure. As used herein, the term "biological equivalent thereof" is intended to be synonymous with "equivalent thereof" when referring to a reference protein, antibody or fragment thereof, polypeptide or nucleic acid, intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any of the above also includes equivalents thereof. For example, an equivalent intends at least about 70% homology or identity, or at least 80% homology or identity and alternatively, or at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively at least 98% percent homology or identity and exhibits substantially equivalent biological activity to the reference protein, polypeptide, antibody or fragment thereof or nucleic acid. Alternatively, when referring to polynucleotides, an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement. Alternatively, when referring to polypeptides or proteins, an equivalent thereof is a expressed polypeptide or protein from a polynucleotide that hybridizes under stringent conditions to the polynucleotide or its complement that encodes the reference polypeptide or protein.

[0184] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. The alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0185] The term "isolated" as used herein refers to molecules or biologicals or cellular materials being substantially free from other materials. In one aspect, the term "isolated" refers to nucleic acid, such as DNA or RNA, or protein or polypeptide (e.g., an antibody or derivative thereof), or cell or cellular organelle, or tissue or organ, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, that are present in the natural source. The term "isolated" also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an "isolated nucleic acid" is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. The term "isolated" is also used herein to refer to cells or tissues that are isolated from other cells or tissues and is meant to encompass both, cultured and engineered cells or tissues.

[0186] "Naked DNA" as used herein refers to DNA encoding a CAR cloned in a suitable expression vector in proper orientation for expression. Viral vectors which may be used include but are not limited SIN lentiviral vectors, retroviral vectors, foamy virus vectors, adeno-associated virus (AAV) vectors, hybrid vectors and / or plasmid transposons (for example sleeping beauty transposon system) or integrase based vector systems. Other vectors that may be used in connection with alternate embodiments of the invention will be apparent to those of skill in the art.

[0187] "Target cell" as used herein refers to cells which are involved in a disease and can be targeted by the genetically modified cells of the invention (including but not limited to genetically modified T-cells, NK cells, hematopoietic stem cells, pluripotent stem cells, and embryonic stem cells). Other target cells will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.

[0188] The terms "T-cell" and "T-lymphocyte" are interchangeable and used synonymously herein. Examples include but are not limited to naïve T cells, central memory T cells, effector memory T cells or combinations thereof.

[0189] "Vector", "cloning vector" and "expression vector" as used herein refer to the vehicle by which a polynucleotide sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, etc.

[0190] The term "functional" when used in conjunction with "derivative" or "variant" or "fragment" refers to a polypeptide which possess a biological activity that is substantially similar to a biological activity of the entity or molecule of which it is a derivative or variant or fragment thereof.

[0191] As used herein, the term "administering," refers to the placement an agent as disclosed herein into a subject by a method or route which results in at least partial localization of the agents at a desired site.

[0192] "Beneficial results" may include, but are in no way limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the disease condition from developing, lowering the chances of a patient developing the disease condition and prolonging a patient's life or life expectancy. As non-limiting examples, "beneficial results" or "desired results" may be alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized (i.e., not worsening) state of cancer progression, delay or slowing of metastasis or invasiveness, and amelioration or palliation of symptoms associated with the cancer.

[0193] As used herein, the terms "treat," "treatment," "treating," or "amelioration" refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with, a disease or disorder. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder, such as cancer. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also a cessation of at least slowing of progress or worsening of symptoms that would be expected in absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment). In some arrangements or embodiments, treatment of cancer includes decreasing tumor volume, decreasing the number of cancer cells, inhibiting cancer metastases, increasing life expectancy, decreasing cancer cell proliferation, decreasing cancer cell survival, or amelioration of various physiological symptoms associated with the cancerous condition.

[0194] "Conditions" and "disease conditions," as used herein may include, cancers, tumors or infectious diseases. In exemplary arrangements or embodiments, the conditions include but are in no way limited to any form of malignant neoplastic cell proliferative disorders or diseases. In exemplary arrangements or embodiments, conditions include any one or more of kidney cancer, melanoma, prostate cancer, breast cancer, glioblastoma, lung cancer, colon cancer, or bladder cancer.

[0195] The term "effective amount" or "therapeutically effective amount" as used herein refers to the amount of a pharmaceutical composition comprising one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof, to decrease at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The phrase "therapeutically effective amount" as used herein means a sufficient amount of the composition to treat a disorder, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0196] A therapeutically or prophylactically significant reduction in a symptom is, e.g. at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150% or more in a measured parameter as compared to a control or non-treated subject or the state of the subject prior to administering the oligopeptides described herein. Measured or measurable parameters include clinically detectable markers of disease, for example, elevated or depressed levels of a biological marker, as well as parameters related to a clinically accepted scale of symptoms or markers for diabetes. It will be understood, however, that the total daily usage of the compositions and formulations as disclosed herein will be decided by the attending physician within the scope of sound medical judgment. The exact amount required will vary depending on factors such as the type of disease being treated, gender, age, and weight of the subject.

[0197] The phrase "first line" or "second line" or "third line" refers to the order of treatment received by a patient. First line therapy regimens are treatments given first, whereas second or third line therapy are given after the first line therapy or after the second line therapy, respectively. The National Cancer Institute defines first line therapy as "the first treatment for a disease or condition. In patients with cancer, primary treatment can be surgery, chemotherapy, radiation therapy, or a combination of these therapies. First line therapy is also referred to those skilled in the art as "primary therapy and primary treatment." See National Cancer Institute website at www.cancer.gov, last visited on May 1, 2008. Typically, a patient is given a subsequent chemotherapy regimen because the patient did not show a positive clinical or sub-clinical response to the first line therapy or the first line therapy has stopped.

[0198] "Mammal" as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.Chimeric Antigen Receptors

[0199] Provided herein are compositions comprising a CAR and an accessory module and method of using same to treat diseases, including cancer. As described herein, specific combinations of conventional CARs (Table 1) and accessory modules as described in Table 2 define a 'backbone' (Table 2).

[0200] Table 1: Conventional CAR architectures. First generation conventional CARs (Conventional CAR I) have an intracellular signaling (ISD) domain (e.g. CD3z) and no costimulatory domain. Second generation conventional CARs (Conventional CAR 2 or CAR II) have one costimulatory domain (e.g. 41BB or CD28) and an intracellular signaling (ISD) domain (e.g. CD3z). Third generation conventional CARs (Conventional CAR 3 or CAR III) have two costimulatory domains (e.g. 41BB and CD28) and an intracellular signaling (ISD) domain (e.g. CD3z). Table 1 Conventional CAR Architectures 1Conventional CAR 1 (CAR I)ASDHRTMDISD2Conventional CAR 2 (CAR II)ASDHRTMDCSDISD3Conventional CAR 3 (CAR III)ASDHRTMDCSD-ICSD-IIISD Table 2: Exemplary Backbones Backbone No. CAR Component Accessory Module NAME SEQ ID (DNA) SEQ ID (PRT) Backbone 1Conventional CAR IK13-vFLIP8662629Backbone 2Conventional CAR IMC159-vFLIP8672630Backbone 3Conventional CAR IMC160-vFLIP8682631Backbone 4Conventional CAR IE8-vFLIP8692632Backbone 5Conventional CAR IHerpesvirus-saimiri-vFLIP8702633Backbone 6Conventional CAR IMecaca-vFLIP8712634Backbone 7Conventional CAR IBHV-vFLIP8722635Backbone 8Conventional CAR IcFLIP-L / MRIT-alpha8732636Backbone 9Conventional CAR IcFLIP-p228742637Backbone 10Conventional CAR IHTLV1-TAX8752638Backbone 11Conventional CAR IHTLV2-TAX8762639Backbone 12Conventional CAR IHTLV2-TAX-RS8772640Backbone 13Conventional CAR IFKBP-K138782641Backbone 14Conventional CAR IFKBPX2-K138792642Backbone 15Conventional CAR IMyr-FKBPx2-K138802643Backbone 16Conventional CAR IFKBPx2-HTLV2-Tax-RS8812644Backbone 17Conventional CAR IFKBPx2-Flag-HTLV2-Tax-RS8822645Backbone 18Conventional CAR IIL6R-304-vHH8842647Backbone 19Conventional CAR IIGHSP2-IL6R-304-VHH-ALB8-VHH8862649Backbone 20Conventional CAR IIgSP-IL6-19A-scFV8992662Backbone 21Conventional CAR IIgSP-Fx069002663Backbone 22Conventional CAR ICD8SP2-PD1-4H1-scFv8892652Backbone 23Conventional CAR ICD8SP2-PD1-5C4-scFv8902653Backbone 24Conventional CAR ICD8SP2-CTLA4-Ipilimumab-scFv8912654Backbone 25Conventional CAR ICD8SP2-PD1-4H1-Alb8-vHH8922655Backbone 26Conventional CAR ICD8SP2-PD1-5C4-Alb8-vHH8932656Backbone 27Conventional CAR ICD8SP2-CTLA4-Ipilimumab-Alb8-vHH8942657Backbone 28Conventional CAR ICD8SP2-sHVEM-Alb8-vHH9022665Backbone 29Conventional CAR ItCD198552618Backbone 30Conventional CAR ItBCMA8562619Backbone 31Conventional CAR ICNB308522615Backbone 32Conventional CAR IIK13-vFLIP8662629Backbone 33Conventional CAR IIMC159-vFLIP8672630Backbone 34Conventional CAR IIMC160-vFLIP8682631Backbone 35Conventional CAR IIE8-vFLIP8692632Backbone 36Conventional CAR IIHerpesvirus-saimiri-vFLIP8702633Backbone 37Conventional CAR IIMecaca-vFLIP8712634Backbone 38Conventional CAR IIBHV-vFLIP8722635Backbone 39Conventional CAR IIcFLIP-L / MRIT-alpha8732636Backbone 40Conventional CAR IIcFLIP-p228742637Backbone 41Conventional CAR IIHTLV1-TAX8752638Backbone 42Conventional CAR IIHTLV2-TAX8762639Backbone 43Conventional CAR IIHTLV2-TAX-RS8772640Backbone 44Conventional CAR IIFKBP-K138782641Backbone 45Conventional CAR IIFKBPX2-K138792642Backbone 46Conventional CAR IIMyr-FKBPx2-K138802643Backbone 47Conventional CAR IIFKBPx2-HTLV2-Tax-RS8812644Backbone 48Conventional CAR IIFKBPx2-Flag-HTLV2-Tax-RS8822645Backbone 49Conventional CAR IIIL6R-304-vHH8842647Backbone 50Conventional CAR IIIGHSP2-IL6R-304-VHH-ALB8-VHH8862649Backbone 51Conventional CAR IIIgSP-IL6-19A-scFV8992662Backbone 52Conventional CAR IIIgSP-Fx069002663Backbone 53Conventional CAR IICD8SP2-PD1-4H1-scFv8892652Backbone 54Conventional CAR IICD8SP2-PD1-5C4-scFv8902653Backbone 55Conventional CAR IICD8SP2-CTLA4-Ipilimumab-scFv8912654Backbone 56Conventional CAR IICD8SP2-PD1-4H1-Alb8-vHH8922655Backbone 57Conventional CAR IICD8SP2-PD1-5C4-Alb8-vHH8932656Backbone 58Conventional CAR IICD8SP2-CTLA4-Ipilimumab-Alb8-vHH8942657Backbone 59Conventional CAR IICD8SP2-sHVEM-Alb8-vHH9022665Backbone 60Conventional CAR IItCD198552618Backbone 61Conventional CAR IItBCMA8562619Backbone 62Conventional CAR IICNB308522615

[0201] Exemplary embodiments of the CAR components are described in Tables 1-6, 8, 11, and 15.

[0202] As described herein, the various backbones comprise a CAR component and accessory modules. Exemplary embodiments of the accessory modules are described in Table 16.

[0203] In some arrangements, the compositions comprise nucleic acids encoding conventional CARs I-III (Table 1), wherein the antigen specific domain of the CAR targets one or more specific antigens as described in Tables 21 and 22. In some arrangements or embodiments, the compositions comprise nucleic acids encoding any one or more of backbones 1-62 (Table 2) where the antigen specific domain of the encoded CAR targets one or more specific antigens as described herein and in Tables 19, 20 and 22. In some arrangements or embodiments, the compositions comprise nucleic acids encoding backbone-1, wherein the antigen specific domain of the CAR in backbone-1 targets one or more cancer specific antigens as described herein and in Table 19 and 22. In some arrangements or embodiments, the compositions comprise nucleic acids encoding backbone-2, wherein the antigen specific domain of the CAR in backbone-2 targets one or more cancer specific antigens as described herein. In some arrangements, the compositions comprise nucleic acids encoding backbone-32, wherein the antigen specific domain of the CAR in backbone-32 targets one or more cancer specific antigens as described herein and in Table 20 and 22. In some arrangements, the compositions comprise nucleic acids encoding backbone-33, wherein the antigen specific domain of the CAR in backbone-33 targets one or more cancer specific antigens as described herein. In various arrangements, the nucleic acids are used in the treatment of cancer. In some arrangements, the nucleic acids encoding the CAR component of the backbones described herein comprise more than one antigen specific domain wherein each of the antigen specific domains are contiguous and in the same reading frame as the other antigen specific domains in the same CAR.

[0204] In some arrangements, the compositions comprise polypeptides encoded by nucleic acids encoding any conventional CARs 1-III, wherein the antigen specific domain of the CAR targets one or more specific antigens as described herein and in Tables 21 and 22. In some arrangements, the compositions comprise polypeptides encoded by nucleic acids encoding any one or more of backbones 1-62 (Table 2) where the antigen specific domain of the encoded CAR targets one or more specific antigens as described herein and in Tables 20 to 22. In some arrangements, the compositions comprise polypeptides encoded by nucleic acids encoding backbone-1, wherein the antigen specific domain of the CAR in backbone-1 targets one or more cancer specific antigens as described herein and in Table 19 and 22. In some arrangements, the compositions comprise polypeptides encoded by nucleic acids encoding backbone-2, wherein the antigen specific domain of the CAR in backbone-2 targets one or more cancer specific antigens as described herein. In some arrangements, the compositions comprise nucleic acids encoding backbone-32, wherein the antigen specific domain of the CAR in backbone-32 targets one or more cancer specific antigens as described herein and in Table 20 and 22. In some arrangements, the compositions comprise nucleic acids encoding backbone-33, wherein the antigen specific domain of the CAR in backbone-33 targets one or more cancer specific antigens as described herein. In various arrangements, the polypeptides are used in the treatment of cancer. In some arrangements, polypeptides encoded by the nucleic acids encoding the CAR component of the backbones described herein comprise more than one antigen specific domain wherein each of the antigen specific domains are contiguous and in the same reading frame as the other antigen specific domains in the same CAR.

[0205] In some arrangements, the compositions comprise vectors comprising nucleic acids encoding any of conventional CARs I-III, wherein the antigen specific domain of the CAR targets one or more cancer specific antigens as described herein and in Tables 21 and 22. In some arrangements, the compositions comprise vectors comprising nucleic acids encoding any one or more of backbones 1-62 (Table 2) where the antigen specific domain of the encoded CAR targets one or more specific antigens as described herein and in Tables 20 to 22. In some arrangements, the compositions comprise vectors encoding nucleic acids encoding backbone-1, wherein the antigen specific domain of the CAR in backbone-1 targets one or more cancer specific antigens as described herein and in Tables 19 and 22. In some arrangements, the compositions comprise vectors encoding nucleic acids encoding backbone-2, wherein the antigen specific domain of the CAR in backbone-2 targets one or more cancer specific antigens as described herein. In some arrangements, the compositions comprise vectors encoding nucleic acids encoding backbone-32, wherein the antigen specific domain of the CAR in backbone-32 targets one or more cancer specific antigens as described herein. In some arrangements, the compositions comprise vectors encoding nucleic acids encoding backbone-33, wherein the antigen specific domain of the CAR in backbone-33 targets one or more cancer specific antigens as described herein. In various arrangements, the vectors are used in the treatment of cancer. In some arrangements, vectors comprising nucleic acids encoding the conventional CARs I to III or CAR component of the backbones described herein comprise more than one antigen specific domain wherein each of the antigen specific domains are contiguous and in the same reading frame as the other antigen specific domains in the same CAR.

[0206] In some arrangements, the compositions comprise genetically modified cells which include vectors comprising nucleic acids encoding any one or more of conventional CARs I to III or backbones 1-62 (Table 2) where the antigen specific domain of the encoded CAR targets one or more specific antigens as described herein and in Tables 19 to 22. In some arrangements, the compositions comprise genetically modified cells which include vectors encoding nucleic acids encoding conventional CARs I to III, wherein the antigen specific domain of the CAR targets one or more cancer specific antigens as described herein and in Tables 19 and 22. In some arrangements, the compositions comprise genetically modified cells which include vectors encoding nucleic acids encoding backbone-1, wherein the antigen specific domain of the CAR in backbone-1 targets one or more cancer specific antigens as described herein. In some arrangements, the compositions comprise genetically modified cells which include vectors encoding nucleic acids encoding backbone-2, wherein the antigen specific domain of the CAR in backbone-2 targets one or more cancer specific antigens as described herein. In some arrangements, the compositions comprise genetically modified cells which include vectors encoding nucleic acids encoding backbone-32, wherein the antigen specific domain of the CAR in backbone-32 targets one or more cancer specific antigens as described herein. In some arrangements, the compositions comprise genetically modified cells which include vectors encoding nucleic acids encoding backbone-33, wherein the antigen specific domain of the CAR in backbone-33 targets one or more cancer specific antigens as described herein. In various arrangements, the genetically modified cells are used in treatment of cancer. In some arrangements, the genetically modified cells targets more than one antigen on a cancer cell.

[0207] In various arrangements, the isolated nucleic acid molecules encoding the CAR components of the backbones described herein, encode one, two, three or more antigen specific domains.

[0208] In various arrangements, the isolated nucleic acid molecules encoding the CAR components of the backbones described herein, encode one, two, three or more co-stimulatory domains.

[0209] In various arrangements, the isolated nucleic acid molecules encoding the CAR components of the backbones described herein, encode one, two, three or more intracellular signaling domain.

[0210] The nucleic acid sequences encoding for the desired components of the CARs and accessory modules described herein 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. In exemplary arrangements, the nucleic acid sequences encoding for the desired components of the CARs and accessory modules described herein are described in Tables 3-22.

[0211] In some arrangements, the genetically modified cells described herein that express the CARs and accessory components described herein also express agents that reduce toxicity of CARs. In exemplary arrangements, such agents are set forth in Table 16. In exemplary arrangements, such agents include but are not limited to any one, two, three or more of scFv against IL-6 (for example, SEQ ID NO: 899), a camelid vHH against IL-6 receptor alpha (for example, SEQ ID NO: 884), a bispecific camelid vHH targeting IL-6 receptor alpha and albumin (SEQ ID NO: 886), Fx06 peptide (SEQ ID NO: 900), or combinations thereof.

[0212] In some arrangements, the genetically modified cells described herein that express the CARs and accessory components described herein also express agents that enhance the activity of CARs. In exemplary arrangements, such agents are set forth in Table 16. In exemplary arrangements, such agents include but are not limited to any one, two, three or more of hTERT (SEQ ID NO: 903), heparinase (SEQ ID NO: 904), soluble HVEM (SEQ ID NO: 901), a fusion protein containing soluble HVEM and a camelid vHH targeting albumin (SEQ ID NO: 902), scFvs targeting PD1 (SEQ ID NOs: 889 and 890), scFv targeting CTLA4 (SEQ ID NO: 894), bispecific proteins targeting PD1 and albumin (SEQ ID NO: 892 and 893), bispecific proteins targeting CTLA4 and albumin (SEQ ID NO: 894) or combinations thereof. The rationale for targeting albumin in the bispecific proteins is to extend the half-lives of the scFvs and vHH fragments which are otherwise rapidly cleared by the kidneys due to their relatively small size.

[0213] In specific arrangements, exemplary constructs containing backbone-1, which comprises a conventional CAR I containing a CD3z activation domain and coexpresses a K13-vFLIP accessory module, are set forth in Table 19.

[0214] In specific arrangements, exemplary constructs containing backbone-32, which comprises a conventional CAR II containing a 41BB costimulatory domain, a CD3z activation domain and coexpresses a K13-vFLIP accessory module, are set forth in Table 20.

[0215] In specific arrangements, exemplary conventional CAR II constructs containing a 41BB costimulatory domain and a CD3z activation domain are described in Table 21.

[0216] In specific arrangements, exemplary CAR constructs in various arrangements of the inventions are described in Table 22. Antigen specific domain

[0217] The compositions comprising various backbones as described herein comprise CARs which comprise one or more ASD that binds specifically to a cancer associated antigen as described herein. The sequences of the ASD are contiguous with and in the same reading frame as a nucleic acid sequence encoding the remainder of the CAR.

[0218] In one arrangement, each antigen specific region comprises the full-length IgG heavy chain (specific for the target antigen) having the V H , CH1, hinge, and the CH2 and CH3 (Fc) Ig domains, if the V H domain alone is sufficient to confer antigen-specificity ("single-domain antibodies"). The full length IgG heavy chain may be linked to the co-stimulatory domain and the intracellular signaling domain via the appropriate transmembrane domain. If both, the V H and the V L domains, are necessary to generate a fully active antigen-specific targeting region, the V H -containing CAR and the full-length lambda light chain (IgL) are both introduced into the cells to generate an active antigen-specific targeting region.

[0219] In some arrangements, the antigen specific domain of the encoded CAR molecule comprises an antibody, an antibody fragment, an scFv, a Fv, a Fab, a (Fab') 2 , a single domain antibody (SDAB), a VH or VL domain, or a camelid VHH domain. In some arrangements, the antigen binding domain of the CAR is a scFv antibody fragment that is humanized compared to the murine sequence of the scFv from which it is derived.

[0220] In some instances, scFvs can be prepared according to methods known in the art (for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be produced by linking V H and V L regions together using flexible polypeptide linkers. The scFv molecules comprise a linker (e.g., a Ser-Gly linker) with an optimized length and / or amino acid composition. The linker length can greatly affect how the variable regions of a scFv fold and interact. For example, if a short polypeptide linker is employed (for example, between 5-10 amino acids) intrachain folding is prevented. Interchain folding is also required to bring the two variable regions together to form a functional epitope binding site. 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, is incorporated herein by reference.

[0221] An scFv can comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its V L and V H regions. The linker sequence may comprise any naturally occurring amino acid. In some arrangements, the linker sequence comprises amino acids glycine and serine. In another arrangement, the linker sequence comprises sets of glycine and serine repeats such as (Gly4Ser)n, where n is a positive integer equal to or greater than 1 (SEQ ID NO:252, SEQ ID NO:253). In one arrangement, the linker can be (Gly4Ser)3 (SEQ ID NO:254, SEQ ID NO:254) or (Gly4Ser)3 (SEQ ID NO:250 - SEQ ID NO:255). Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies.

[0222] 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) (references are incorporated herein by its entirety). For example, scTCR can be engineered that contains the Vα and νβ 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

[0223] In one arrangement, the antigen specific domain comprises one, two or all three heavy chain (hc) CDRs, hcCDRl, hcCDR2 and hcCDR3 of an antibody listed herein, and / or one, two or all three light chain (lc) CDRs, lcCDRl, lcCDR2 and lcCDR3 of an antibody listed herein.

[0224] In another arrangement, the antigen specific domain comprises a humanized antibody or an antibody fragment. In some aspects, 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 or fragment thereof. In one aspect, the antigen binding domain is humanized. A humanized antibody can be produced using a variety of techniques known in the art, including but not limited to, 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, each of which is incorporated herein in its entirety by reference), 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, each of which is incorporated herein by its entirety by reference), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein in its entirety by reference), 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., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267- 79 (2000), Baca et al., J. Biol. Chem., 272(16): 10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8): 1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which is incorporated herein in its entirety by reference. In some arrangements, the portion of a CAR composition of the invention that comprises an antibody fragment is humanized with retention of high affinity for the target antigen and other favorable biological properties

[0225] In some arrangements, the antigen specific domain is a T cell receptor specific for the target antigen or a fragment of the T cell receptor, wherein the fragment retains the specificity for the target antigen.

[0226] In some arrangements, the antigen specific domain of a CAR described herein is a scFv antibody fragment. In some arrangements, the antigen specific scFv antibody fragments are functional in that they bind the same antigen with the same or comparable affinity as the IgG antibody from which it is derived. In other arrangements, the antibody fragment has a lower binding affinity to the antigen compared to the antibody from which it is derived but is functional in that it provides a biological response described herein. In one arrangement, the CAR molecule comprises an antibody fragment that has a binding affinity KD of 10 -4< M to 10 -8< M, 10 -5< M to 10 -7< M, 10 -6< M or 10 -8< M, for the target antigen.

[0227] In some arrangements, antigen specific domain of a CAR described herein 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 Viral. 2011 85(5):1935-1942; Sergeeva et al., Blood, 2011117(16):4262-4272; Verma et al., Jlmmunol2010 184(4):2156-2165; Willemsen et al., Gene Ther20018(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. Exemplary CARs that are based on TCR-like antibodies targeting WT1 in association with HLA-A2 are represented by SEQ ID NO: 1176 to SEQ ID NO: 1179. In the instant invention, CARs were generated using antigen binding domain derived from TCR like antibodies against several HLA-A2 restricted intracellular peptides. The target protein antigens, the peptide fragment and the sequence of the peptide are shown in Table 23.

[0228] In some arrangements, when the CARs comprising functional fragments of antibodies (including scFv fragments) described herein bind the target antigen, a biological response is induced such as activation of an immune response, inhibition of signal-transduction originating from its target antigen, inhibition of kinase activity, and the like, as will be understood by a skilled artisan.

[0229] In some arrangements, the antigens specific for disease which may be targeted by the CARs when expressed alone or with the accessory modules as described herein include but are not limited to any one or more of CD19, CD22, CD23, MPL, CD123, CD32, CD138, CD200R, CD276, CD324, CD30, CD32, FcRH5, CD99, Tissue Factor, amyloid, Fc region of an immunoglobulin, CD171, CS-1, CLL-1 (CLECL1), CD33, EGFRvIII , GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL11Ra, Mesothelin, PSCA, VEGFR2, Lewis Y, CD24, PDGFR-beta, PRSS21, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gpl00, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLea, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, TSHR, TCR-beta1 constant chain, TCR beta2 constant chain, TCR gamma-delta, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WTl, NY-ESO-1, LAGE-la, legumain, HPV E6, E7, HTLV1-Tax, KSHV K8.1 protein, EBB gp350, HIV1-envelop glycoprotein gp120, MAGE-Al, MAGE Al, 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-l / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, DLL3, TROP2, PTK7, GCC, AFP, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, RUl, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, FITC, Leutenizing hormone receptor (LHR), Follicle stimulating hormone receptor (FSHR), Chorionic Gonadotropin Hormone receptor (CGHR), CCR4, GD3, SLAMF6, SLAMF4, FITC, Leutenizing hormone receptor (LHR), Follicle stimulating hormone receptor (FSHR), Chorionic Gonadotropin Hormone receptor (CGHR), CCR4, GD3, SLAMF6, SLAMF4, or combinations thereof.

[0230] In some arrangements, the antigens specific for a disease which may be targeted by the CARs when expressed alone or with the accessory modules as described herein include but are not limited to any one or more of 4-1BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CD123, CEA, CNTO888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin αvβ3, LAMP1, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R α, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, vimentin or combinations thereof. Other antigens specific for cancer will be apparent to those of skill in the art and may be used in connection with alternate arrangements of the invention.

[0231] In some arrangements, the antigens specific for cancer which may be targeted by the CARs when expressed alone or with the accessory modules as described herein include but are not limited to any one or more of MPL,CD19, FLT3, GRP-78, CD79b, Ly-1, Lym2, CD23, CD179b, CDH1, CDH6, CDH19, CDH17, DLL3, PTK7, TROP2, TIM1, LAMP1 or combinations thereof. In some arrangements, the antigen specific domains of the CARs specific for MPL, CD19, FLT3, GRP-78, CD79b, Lym1, Lym2, CD23, CDH1, CDH6, CDH19, CDH17, DLL3, PTK7, TROP2, TIM1, LAMP1 comprise scFv sequences set forth in Table 11.

[0232] A CAR when used alone or with accessory modules as described herein can comprise an antigen binding domain (e.g., antibody or antibody fragment) that binds to a disease-supporting antigen (e.g., a disease-supporting antigen as described herein). In some arrangements, the disease-supporting antigen is an antigen present on cells that support the survival and proliferation of disease causing cells. In some arrangements, the disease-supporting antigen is an antigen present on a stromal cell or a myeloid-derived suppressor cell (MDSC). Stromal cells can secrete growth factors and cytokines to promote cell proliferation in the microenvironment. MDSC cells can block T cell proliferation and activation. Without wishing to be bound by theory, in some arrangements, the CAR-expressing cells destroy the disease-supporting cells, thereby indirectly blocking growth or survival of disease causing cells.

[0233] In arrangements, the stromal cell antigen is selected from one or more of: bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP) and tenascin. In an arrangement, the FAP-specific antibody is, competes for binding with, or has the same CDRs as, sibrotuzumab. In arrangements, the MDSC antigen is selected from one or more of: CD33, CDllb, C14, CD15, and CD66b. Accordingly, in some arrangements, the disease supporting antigen is selected from one or more of: bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP) or tenascin, CD33, CDllb, C14, CD15, and CD66b.

[0234] In a further arrangement, each antigen specific region of the CAR may comprise a divalent (or bivalent) single-chain variable fragment (di-scFvs, bi-scFvs). In CARs comprising di-scFVs, two scFvs specific for each antigen are linked together by producing a single peptide chain with two V H and two V L regions, yielding tandem scFvs. (Xiong, Cheng-Yi; Natarajan, A; Shi, XB; Denardo, GL; Denardo, SJ (2006). "Development of tumor targeting anti-MUC-1 multimer: effects of di-scFv unpaired cysteine location on PEGylation and tumor binding". Protein Engineering Design and Selection 19 (8): 359-367; Kufer, Peter; Lutterbüse, Ralf; Baeuerle, Patrick A. (2004). "A revival of bispecific antibodies". Trends in Biotechnology 22 (5): 238-244). CARs comprising at least two antigen-specific targeting regions would express two scFvs specific for each of the two antigens. The resulting ASD is joined to the co-stimulatory domain and the intracellular signaling domain via a hinge region and a transmembrane domain. Alternatively, CARs comprising two antigen specific targeting regions would express two vHH specific for each of the two antigens or two epitopes of the same antigen. Exemplary CARs targeting two antigens are represented by SEQ ID NOs: 1042, 1043, 1049, 1050 and 1087.

[0235] In an additional arrangement, each ASD of the CAR comprises a diabody. In a diabody, the scFvs are created with linker peptides that are too short for the two variable regions to fold together, driving the scFvs to dimerize. Still shorter linkers (one or two amino acids) lead to the formation of trimers, the so-called triabodies or tribodies. Tetrabodies may also be used.

[0236] In some arrangements, the ASD of the CAR comprises V L fragments as described in Table 4.

[0237] In some arrangements, the ASD of the CAR comprises V H fragments as described in Table 6.

[0238] In some arrangements, the ASD of the CAR comprises V HH fragments (nanobodies) as described in Table 7.

[0239] In some arrangements, the ASD of the CAR comprises affibodies as described in Table 8.

[0240] In some arrangements, the ASD of the CAR comprises ligands as describes in Table 10.

[0241] In some arrangements, the ASD of the CAR comprises scFv fragments as describes in Table 11.

[0242] In one arrangement, an antigen specific domain of a CAR against a target antigen is an antigen binding portion, e.g., CDRs, of vL and vH fragments targeting this antigen as shown in Tables 4 and 6, respectively.

[0243] In one arrangement, an antigen specific domain of a CAR against a target antigen is an antigen binding portion, e.g., CDRs, of vHH fragments targeting this antigen as shown in Table 7.

[0244] In one arrangement, an antigen specific domain of a CAR against a target antigen is an antigen binding portion of a non-immunoglobulin scaffold targeting this antigen as shown in Table 8.

[0245] In one arrangement, an antigen specific domain of a CAR against a target antigen is an antigen binding portion of a receptor known to bind this target antigen.

[0246] In one arrangement, an antigen binding specific domain of a CAR against a target antigen is an antigen binding portion of a ligand known to bind this target antigen.

[0247] In one arrangement, an antigen specific domain of a CAR against a target antigen is an antigen binding portion, e.g., CDRs, of vL and vH fragments of a scFV targeting this antigen as shown in Table 11.

[0248] In another arrangement, the invention provides CARs that bind to the same epitope on the different targets described in Tables 19-22 as any of the CARs of the invention (i.e., CARs that have the ability to cross-compete for binding to the different targets with any of the CARs of the invention). In some arrangements, the antigen specific domains of these CARs could be derived from vL fragments, vH fragments or scFv fragments of antibodies. In some arrangements, the reference antibodies for cross-competition studies to determine the target-epitope recognized by a CAR of the invention described in Tables 19-22 are scFvs having sequences as shown in SEQ ID NOs: 2334-2568 (Table 11). In an exemplary arrangement, the reference scFv FMC63(vL-vH) represented by SEQ ID NO: 2334 can be used in cross-competiton studies to to determine the target-epitope recognized by FMC63-based conventional CARs and backbones of the invention (SEQ ID NOs:2672, 2942, 3212, 3495-3515) described in Tables 19-22. In some arrangements, the reference vHH fragments for cross-competition studies to determine the target-epitope recognized by a CAR of the invention described in Tables 19-22 are are vHH fragments having sequences as shown in SEQ ID NOs: 2269-2293 (Table 7). In some arrangements, the reference non-immunoglobulin antigen binding scaffolds for cross-competition studies for cross-competition studies to determine the target-epitope recognized by a CAR of the invention described in Tables 19-22 are non-immunoglobulin antigen binding scaffolds having sequences as shown in SEQ ID NOs: 2294-2298 (Table 8). ). In some arrangements, the reference ligands for cross-competition studies to determine the target-epitope recognized by a CAR of the invention described in Tables 19-22 are ligands having sequences as shown in SEQ ID NOs: 2323-2333 (Table 10). In some arrangements, the reference CARs for cross-competition studies against different targets are CARs having sequences as shown in SEQ ID NOs: 2672-2941 (Table 19).

[0249] In an arrangement, the reference antibodies for cross-competition studies to determine the target-epitopes recognized by the MPL-targeting CARs of the invention (e.g., SEQ ID NOs: 1117-1125) are antibodies mAb-1.6, mAb-1.111, mAb-1.75, mAb-1.78, mAb-1.169, and mAb-1.36 described in patent application US 2012 / 0269814 A1.

[0250] In an arrangement, the reference scFvs for cross-competition studies to determine the target-epitopes recognized by the MPL-targeting CARs of the invention (e.g., SEQ ID NOs: 1117-1125) are scFvs having sequences as shown in SEQ ID NOs: 2499-2506 (Table 11).

[0251] In an arrangement, the reference ligands for cross-competition studies to determine the target-epitopes recognized by the MPL-targeting CARs of the invention (e.g., SEQ ID NOs: 1117-1125) are ligands having sequences as shown in SEQ ID NOs: 2323-2324 (Table 10).

[0252] In an arrangement, the reference CARs for cross-competition studies to determine the target-epitopes recognized by the MPL-targeting CARs of the invention are CARs having sequences as shown in SEQ ID NOs: 1117-1125 and 1164 (Table 19).

[0253] In the preferred arrangement, an MPL-targeting CAR of the invention binds to an MPL-epitope corresponding to or overlapping with the peptide sequence -PWQDGPK-.

[0254] In an arrangement, the reference scFvs for cross-competition studies to determine the target-epitopes recognized by the CD19-targeting CARs of the invention (e.g., SEQ ID NOs: 930-941) are scFvs having sequences as shown in SEQ ID NOs: 2336-2347 (Table 11).

[0255] In an arrangement, the reference CARs for cross-competition studies to determine the target-epitopes recognized by the CD19-targeting CARs of the invention are CARs having sequences as shown in SEQ ID NOs: 930-941 (Table 19).

[0256] In an arrangement, the reference scFvs for cross-competition studies to determine the target-epitopes recognized by the CD20-targeting CARs of the invention (e.g., SEQ ID NOs: 964-977) are scFvs having sequences as shown in SEQ ID NOs: 2366-2380 (Table 11).

[0257] In an arrangement, the reference CARs for cross-competition studies to determine the target-epitopes recognized by the CD20-targeting CARs of the invention are CARs having sequences as shown in SEQ ID NOs: 964-977 (Table 19).

[0258] In the preferred arrangement, the CD20-targeting CARs of the invention bind to the epitopes corresponding to one or more of the sequences -PAGIYAPI-, - FLKMESLNFIRAHTP-, -HFLKMESLNFIRAHTPY-, -YNAEPANPSEKNSPSTQY-, - YNAEPANPSEKNSPST- and -YNCEPANPSEKNSP-.

[0259] In an arrangement, the reference scFvs for cross-competition studies against DLL3-targeting CARs of the invention (e.g., SEQ ID NOs: 1044-1045) are scFvs having sequences as shown in SEQ ID NOs: 2443-2444 (Table 11).

[0260] In an arrangement, the reference CARs for cross-competition studies against DLL3-targeting CARs of the invention are CARs having sequences as shown in SEQ ID NOs: 1044-1045 (Table 19).

[0261] In an arrangement, the reference scFvs for cross-competition studies against LAMP1-targeting CARs of the invention (e.g., SEQ ID NOs: 1104-1105) are scFvs having sequences as shown in SEQ ID NOs: 2489-2490 (Table 11).

[0262] In an arrangement, the reference CARs for cross-competition studies against LAMP1-targeting CARs of the invention are CARs having sequences as shown in SEQ ID NOs: 1104-1105 (Table 19).

[0263] In an arrangement, the reference scFvs for cross-competition studies against TROP2-targeting CARs of the invention (e.g., SEQ ID NOs: 2910-2911) are scFvs having sequences as shown in SEQ ID NOs: 2544-2545 (Table 11).

[0264] In an arrangement, the reference CARs for cross-competition studies against TROP2-targeting CARs of the invention are CARs having sequences as shown in SEQ ID NOs: 2910-2911 (Table 19).

[0265] In an arrangement, the reference scFvs for cross-competition studies against PTK7-targeting CARs of the invention (e.g., SEQ ID NOs: 1143-1144) are scFvs having sequences as shown in SEQ ID NOs: 2523-2524 (Table 11).

[0266] In an arrangement, the reference CARs for cross-competition studies against PTK7-targeting CARs of the invention are CARs having sequences as shown in SEQ ID NOs: 1143-1144 (Table 19).

[0267] In an arrangement, the reference scFv for cross-competition studies against a CD23-targeting CAR (e.g., SEQ ID NO: 2932) is a scFv having sequence as shown in SEQ ID NO: 2565 (Table 11).

[0268] In an arrangement, the reference CAR for cross-competition studies against a CD23-targeting CAR of the invention is a CAR having sequences as shown in SEQ ID NO: 2932 (Table 19).

[0269] In an arrangement, the reference scFvs for cross-competition studies against a TCR-gamma-delta-targeting CAR (e.g., SEQ ID NO: 1155) is a scFv having sequence as shown in SEQ ID NO: 2535 (Table 11).

[0270] In an arrangement, the reference CAR for cross-competition studies against TCR-gamma-delta-targeting -targeting CAR of the invention is a CAR having sequences as shown in SEQ ID NO: 1155 (Table 19).

[0271] In an arrangement, the reference scFvs for cross-competition studies against CDH6-targeting CARs of the invention (e.g., SEQ ID NOs: 1016-1017) are scFvs having sequences as shown in SEQ ID NOs: 2418-2419 (Table 11).

[0272] In an arrangement, the reference CARs for cross-competition studies against CDH6-targeting CARs of the invention are CARs having sequences as shown in SEQ ID NOs: 1016-1017 (Table 19).

[0273] In an arrangement, the reference scFvs for cross-competition studies against CDH19-targeting CARs of the invention (e.g., SEQ ID NOs: 1019 and 1180) are scFvs having sequences as shown in SEQ ID NOs: 2421 and 2558 (Table 11).

[0274] In an arrangement, the reference CARs for cross-competition studies against CDH19-targeting CARs of the invention are CARs having sequences as shown in SEQ ID NOs: 1019 and 1180 (Table 19).

[0275] In an arrangement, the reference scFvs for cross-competition studies against CD324-targeting CARs of the invention (e.g., SEQ ID NOs: 1014-1015) are scFvs having sequences as shown in SEQ ID NOs: 2416-2417 (Table 11).

[0276] In an arrangement, the reference CARs for cross-competition studies against CD324-targeting CARs of the invention are CARs having sequences as shown in SEQ ID NOs: 1014-1015 (Table 19).

[0277] In an arrangement, the reference scFv for cross-competition studies against a CD276-targeting CAR (e.g., SEQ ID NO: 2578) is a scFv having sequence as shown in SEQ ID NO: 2415 (Table 11).

[0278] In an arrangement, the reference CAR for cross-competition studies against a CD276-targeting CARs of the invention is a CAR having sequences as shown in SEQ ID NO: 2578 (Table 19).

[0279] In an arrangement, the reference scFvs for cross-competition studies against GFRa4-targeting CARs of the invention (e.g., SEQ ID NOs: 1066-1067) are scFvs having sequences as shown in SEQ ID NOs: 2461-2462 (Table 11).

[0280] In an arrangement, the reference CARs for cross-competition studies against GFRa4-targeting CARs of the invention are CARs having sequences as shown in SEQ ID NOs: 1066-1067 (Table 19).

[0281] In an arrangement, the reference scFvs for cross-competition studies against CSF2RA-targeting CARs of the invention (e.g., SEQ ID NOs: 1040-1041) are scFvs having sequences as shown in SEQ ID NOs: 2441-2442 (Table 11).

[0282] In an arrangement, the reference CARs for cross-competition studies against CSF2RA-targeting CARs of the invention are CARs having sequences as shown in SEQ ID NOs: 1040-1041 (Table 19).

[0283] In an arrangement, the reference scFvs for cross-competition studies against B7H4-targeting CARs of the invention (e.g., SEQ ID NOs: 2929-2930) are scFvs having sequences as shown in SEQ ID NOs: 2562-2563 (Table 11).

[0284] In an arrangement, the reference CARs for cross-competition studies against B7H4-targeting CARs of the invention are CARs having sequences as shown in SEQ ID NOs: 2929-2930 (Table 19).

[0285] In an arrangement, the reference scFv for cross-competition studies against a NY-BR1-targeting CAR (e.g., SEQ ID NO: 1131) is a scFv having sequence as shown in SEQ ID NO: 2512 (Table 11).

[0286] In an arrangement, the reference CAR for cross-competition studies against a NY-BR1-targeting CARs of the invention is a CAR having sequences as shown in SEQ ID NO: 1131 (Table 19).

[0287] In an arrangement, the reference scFv for cross-competition studies against a CD200R-targeting CAR (e.g., SEQ ID NO: 1190) is a scFv having sequence as shown in SEQ ID NO: 2568 (Table 11).

[0288] In an arrangement, the reference CAR for cross-competition studies against a CD200R-targeting CARs of the invention is a CAR having sequences as shown in SEQ ID NO: 1190 (Table 19).

[0289] In an arrangement, the reference scFv for cross-competition studies against a HIV1-envelop glycoprotein gp120-targeting CAR (e.g., SEQ ID NO: 945) is a scFv having sequence as shown in SEQ ID NO: 2512 (Table 11).

[0290] In an arrangement, the reference CAR for cross-competition studies against a HIV1-envelop glycoprotein gp120-targeting CAR of the invention is a CAR having sequences as shown in SEQ ID NO: 945 (Table 19).

[0291] In an arrangement, the reference ligand for cross-competition studies against a TSHR- targeting CAR (e.g., SEQ ID NOs: 1167) is a ligand having sequences as shown in SEQ ID NO: (Table 11).

[0292] In an arrangement, the reference scFvs for cross-competition studies against TSHR- targeting CARs of the invention (e.g., SEQ ID NOs: 1168-1170) are scFvs having sequences as shown in SEQ ID NOs: 2333 (Table 10).

[0293] In an arrangement, the reference CARs for cross-competition studies against TSHR- targeting CARs of the invention are CARs having sequences as shown in SEQ ID NOs: 1167-1170 (Table 19).

[0294] In some arrangements, the CARs targeting gp100, MART, Tyrosinase, hTERT, MUC1, CMV-pp65, HTLV1-Tax, HIV1-gag, NY-ESO, WT1, AFP, HPV-16-E7, PR1 bind to target peptides shown in Table 23 in complex with MHC class I (HLA-A2). Linkers

[0295] In some arrangements, two or more functional domains of the CARs as described herein, are separated by one or more linkers. Linkers are oligo- or polypeptides region from about 1 to 100 amino acids in length, that link together any of the domains / regions of the CAR of the invention. In some arrangements, the linkers may be for example, 5-12 amino acids in length, 5-15 amino acids in length or 5 to 20 amino acids in length. Linkers may be composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. Longer linkers, for example those longer than 100 amino acids, may be used in connection with alternate embodiments of the invention, and may be selected to, for example, ensure that two adjacent domains do not sterically interfere with one another. In exemplary arrangements, linkers are described in SEQ ID NOs: 250-258 (Tables 5). Hinge Region

[0296] In some arrangements, the CARs (which form part of the backbones) described herein comprise a hinge region between the antigen specific domain and the transmembrane domain. In some arrangements, the hinge region comprises any one or more of human CD8α or an Fc fragment of an antibody or a functional equivalent, fragment or derivative thereof, a hinge region of human CD8α or an antibody or a functional equivalent, fragment or derivative thereof, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence and combinations thereof. In exemplary arrangements, the hinge region comprises any one or more of (i) a hinge, CH2 and CH3 region of IgG4, (ii) a hinge region of IgG4, (iii) a hinge and CH2 region of IgG4, (iv) a hinge region of CD8α, (v) a hinge, CH2 and CH3 region of IgG1, (vi) a hinge region of IgG1, (vi) a hinge and CH2 region of IgG1, or (vii) combinations thereof.Transmembrane Domain

[0297] As described herein, the CARs (which form part of the backbones) described herein comprise a transmembrane domain. The transmembrane domain may comprise the transmembrane sequence from any protein which has a transmembrane domain, including any of the type I, type II or type III transmembrane proteins. The transmembrane domain of the CAR of the invention may also comprise an artificial hydrophobic sequence. The transmembrane domains of the CARs described herein may be selected so that the transmembrane domain do not dimerize. In some arrangements, the TMD encoded CAR comprising any of the backbones described herein comprises a transmembrane domain selected from the transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C

[0298] 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 contiguous with one of the other domains of the CAR. In one arrangement, 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.Intracellular signaling domain of chimeric antigen receptors

[0299] As described herein, the CARs (which form part of the backbones) described herein comprise an intracellular signaling domain. This domain may be cytoplasmic and may transduce the effector function signal and direct the cell to perform its specialized function. Examples of intracellular signaling domains include, but are not limited to, ζ chain of the T-cell receptor or any of its homologs (e.g., η chain, FcεR1y and β chains, MB1 (Igα) chain, B29 (Igβ) chain, etc.), CD3 polypeptides (Δ, δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5 and CD28. Specifically, the intracellular signaling domain may be human CD3 zeta chain, FcyRIII, FcεRI, cytoplasmic tails of Fc receptors, immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors or combinations thereof. Additional intracellular signaling domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. In some arrangements, the intracellular signaling domain comprises a signaling domain of one or more of a human CD3 zeta chain, FcgRIII, FceRI, a cytoplasmic tail of a Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, and combinations thereof.Co-stimulatory Domain

[0300] As described herein, the CARs (which form part of the backbones) described herein comprise a co-stimulatory domain. In exemplary arrangements, the co-stimulatory domain comprises a signaling domain from any one or more of CD28, CD137 (4-1BB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40 and combinations thereof.Cleavable Linkers

[0301] Cleavable linkers as described herein include 2A linkers (for example T2A), 2A-like linkers or functional equivalents thereof and combinations thereof. In some arrangements, the linkers include the picornaviral 2A-like linker, CHYSEL sequences of porcine teschovirus (P2A), Thosea asigna virus (T2A) or combinations, variants and functional equivalents thereof. In other arrangements, the linker sequences may comprise Asp-Val / Ile-Glu-X-Asn-Pro-Gly (2A)< -Pro (2B)< motif, which results in cleavage between the 2A glycine and the 2B proline. The nucleic sequences of several exemplary cleavable linkers are provided in SEQ ID NO: 831 to SEQ ID NO: 836 and amino acid sequences of several exemplary linkers are provided in SEQ ID NO: 2598 to SEQ ID NO: 2602. Other linkers will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. In an arrangement, a Ser-Gly-Ser-Gly (SGSG) motif (SEQ ID NOs: 837-838 and SEQ ID NO: 2603) is also added upstream of the cleavable linker sequences to enhance the efficiency of cleavage. A potential drawback of the cleavable linkers is the possibility that the small 2A tag left at the end of the N-terminal protein may affect protein function or contribute to the antigenicity of the proteins. To overcome this limitation, in some arrangements, a furine cleavage site (RAKR) (SEQ ID NO: 839-841 and SEQ ID NO: 2604) is added upstream of the SGSG motifs to facilitate cleavage of the residual 2A peptide following translation. In an arrangement, cleavable linkers are placed between the polypeptide encoding the CAR and the polypeptide encoding the accessory modules. The cleavage at the site of cleavable linker results in separation of the two polypeptides.Accessory Modules

[0302] "Accessory modules" as used herein refer to agents that enhance, reduce or modify the activity of T cells expressing the CARs or reduce toxicity associated with CARs so that the therapeutic response of the CARs is enhanced.

[0303] In some arrangements, vectors comprising polynucleotides encoding CARs further comprise polynucleotides encoding viral and cellular signaling proteins which (i) extend the life span of T cells expressing the CARs, (ii) stimulate T cell proliferation and / or (iii) protect T cells expressing the CARs from apoptosis. In exemplary embodiments, such proteins include but are not limited to vFLIP-K13 from Kaposi's sarcoma associated herpes virus (SEQ ID NO: 866), MC159 from molluscum contagiosum virus (SEQ ID NO: 867), cFLIP-L / MRITα (SEQ ID NO: 873), cFLIP-p22 (SEQ ID NO: 874) and Tax from human T cell leukemia / lymphoma virus (HTLV-1 and HTLV-2) (SEQ ID NOs: 875 and SEQ ID NO: 876) and Tax-RS mutant from HTLV-2 (SEQ ID NO: 877).

[0304] In one arrangement, vectors encoding CARs further encode vFLIP-K13. In another arrangement, vectors encoding CARs further encode MC159. In another arrangement, vectors encoding CARs further encode HTLV1-Tax. In another arrangement, vectors encoding CARs further encode HTLV2-Tax. In another arrangement, vectors encoding CARs further encode cFLIP-L / MRITα.

[0305] In some arrangements, the accessory molecules are encoded by vectors that are distinct from the vectors encoding by the CARs described herein. In some arrangements, effector cells comprising vectors encoding CARs also comprise vectors encoding accessory molecules.

[0306] In some arrangements, vectors comprising polynucleotides encoding CARs further express accessory molecule vFLIP MC160 from molluscum contagiosum virus, vFLIP E8 from equine herpes virus 2, vFLIP from human herpes virus saimiri, vFLIP from bovine herpes virus (BHV) 4, vFLIP from mecaca herpes virus and / or full length or N-terminal fragment of human cellular FLICE inhibitory protein (cFLIP-L / MRITα or cFLIP-p22). (Table 16).

[0307] In some arrangements, vectors comprising polynucleotides encoding CARs further comprise polynucleotides encoding siRNA or scFv specific for cytokines. In exemplary arrangements, the cytokines are any one or more of IL-10, IL-6, IFNy or combinations thereof. In some arrangement, the CARs are co-expressed with a secreted bispecific antibody fragment that binds to IL6 receptor α and human serum albumin. In some arrangement, the CARs are co-expressed with a secreted scFv fragment that binds to IL6. In some arrangements, the CARs are coexpressed with the peptide FX06 so as to mitigate capillary leak associated with CAR therapy.

[0308] In further arrangements, vectors comprising polynucleotides encoding CARs further comprise polynucleotides encoding siRNA or a nuclease targeting the endogenous TCR-α, TCR-β, TCR-γ, TCR-delta, CD3gamma, CD3zeta, CD3epsilon, CD3-delta.

[0309] In further arrangements, vectors comprising polynucleotides encoding CARs further comprise polynucleotides encoding a selectable marker. In exemplary arrangement, the selectable marker can encode a drug resistance gene, such as gene that confers resistance to puromycin or calcineurin inhibitors (e.g. CNB30; SEQ ID NO: 852). In some arrangements, the selectable marker may encode for extracellular and transmembrane domains of human CD30, CD20, CD19 (SEQ ID NO: 854), BCMA (SEQ ID NO: 855), EGFR (SEQ ID NO: 853), CD34, or any protein or protein fragment that is expressed on cell surface and can be recognized by an antibody that can be used to eliminate cells expressing its target antigen. In an exemplary arrangement, cetuximab, an anti-EGFR monoclonal is used to eliminate CAR-expressing cells of the invention which coexpress a truncated EGFR (SEQ ID NO: 853). The selectable marker(s) can be used to enrich for cells expressing the CAR, to select for cells that express high levels of CAR and / or to reduce the clonal diversity of the cells expressing the CAR. In further arrangements, polynucleotides encoding CARs may encode for epitope tags that are expressed on the extracellular domain of the CARs and can be used to enrich for cells expressing the CAR, to select for cells that express high levels of CAR and / or to reduce the clonal diversity of the cells expressing the CAR. Non-limiting examples of such tags are provided in SEQ ID NOs: 553-563 and 3526-3530 (Table 12). Reducing the clonal diversity of allogeneic T cells expressing the CARs will in turn lead to reduced incidence of Graft versus Host Disease (GVHD), thereby allowing the use of allogeneic T cells for CAR-T cell therapy.Polynucleotides and Polypeptides

[0310] Provided herein are one or more polypeptides encoded by one or more nucleic acid molecules encoding conventional CARs I to III or any one or more of backbones 1-62 described herein (Table 2).

[0311] Also provided herein are one or more polypeptides encoded by one or more nucleic acid molecules encoding conventional CARs I to III. In some arrangements, the antigen-specific domain of the CARs is specific to one, two, three or more antigens on target cells, such as cancer cells. As described herein, each component of the CAR is contiguous and in the same reading frame with each other components of the CAR. In some arrangements, in the CAR comprising backbone comprises more than one antigen specific domain, each of the antigen specific domains are contiguous and in the same reading frame as the other antigen specific domains in the same CAR.

[0312] Also provided herein are one or more polypeptides encoded by one or more nucleic acid molecules encoding backbone-1 comprising conventional CAR I and K13-vFLIP as described herein. In some arrangements, the antigen-specific domain of the CAR comprising backbone-1 is specific to one, two, three or more antigens on target cells, such as cancer cells. As described herein, each component of the CAR is contiguous and in the same reading frame with each other components of the CAR comprising backbone-1. In some arrangements, in the CAR comprising backbone-1 comprises more than one antigen specific domain, each of the antigen specific domains are contiguous and in the same reading frame as the other antigen specific domains in the same CAR.

[0313] Also provided herein are one or more polypeptides encoded by one or more nucleic acid molecules encoding backbone-32 which comprises conventional CAR II and K13-vFLIP as described herein. In some arrangements, the antigen-specific domain of the CAR comprising backbone-32 is specific to one, two, three or more antigens on target cells, such as cancer cells. As described herein, each component of the CAR is contiguous and in the same reading frame with each other components of the CAR. In some arrangements, in the CAR comprising backbone-32 comprises more than one antigen specific domain, each of the antigen specific domains are contiguous and in the same reading frame as the other antigen specific domains in the same CAR.

[0314] In various arrangements, the polypeptides encoded by the nucleic acid molecules encoding CARs which are part of conventional CARs I to III or part of the backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, comprise two, three or more antigen specific domains.

[0315] In various arrangements, the polypeptides encoded by the nucleic acid molecules encoding CARs which are part of conventional CARs I to III or part of the backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, comprise two, three or more co-stimulatory domains.

[0316] In various arrangements, the polypeptides encoded by the nucleic acid molecules encoding CARs which are part of conventional CARs I to III or part of the backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, comprise two, three or more intracellular signaling domain.

[0317] In various arrangements, the polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, comprise one, two, three or more viral and / or cellular signaling proteins.

[0318] The nucleic acid sequences encoding for the desired components of the CARs described herein 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.

[0319] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to target as described in Tables 19-22.

[0320] In one arrangement, an antigen specific domain of a CAR against a target antigen is an antigen binding portion, e.g., CDRs, of vL and vH fragments targeting this antigen as shown in Tables 4 and 6, respectively.

[0321] In one arrangement, an antigen specific domain of a CAR against a target antigen is an antigen binding portion, e.g., CDRs, of vHH fragments targeting this antigen as shown in Table 7.

[0322] In one arrangement, an antigen specific domain of a CAR against a target antigen is an antigen binding portion of a non-immunoglobulin scaffold targeting this antigen as shown in Table 8.

[0323] In one arrangement, an antigen specific domain of a CAR against a target antigen is an antigen binding portion of a receptor known to bind this target antigen.

[0324] In one arrangement, an antigen binding specific domain of a CAR against a target antigen is an antigen binding portion of a ligand known to bind this target antigen.

[0325] In one arrangement, an antigen specific domain of a CAR against a target antigen is an antigen binding portion, e.g., CDRs, of vL and vH fragments of a scFV targeting this antigen as shown in Table 11.

[0326] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to the targets described in Tables 19-22.

[0327] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of the backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to the thrombopoietin receptor, MPL.

[0328] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to CD19.

[0329] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to CD20.

[0330] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to CD22.

[0331] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to CD23

[0332] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to CD30, .

[0333] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to CD32.

[0334] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to CD33.

[0335] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to CD123.

[0336] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to CD138.

[0337] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to CD200R.

[0338] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to CD276.

[0339] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to CD324.

[0340] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to BCMA.

[0341] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to CS1.

[0342] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to ALK1.

[0343] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to ROR1.

[0344] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to CDH6

[0345] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to CDH16.

[0346] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to CDH17.

[0347] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to CDH19.

[0348] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to EGFRviii.

[0349] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to Her2.

[0350] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to Her3.

[0351] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to Mesothelin.

[0352] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to Folate Receptor alpha.

[0353] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to Folate Receptor beta.

[0354] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to CLL-1.

[0355] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to CLEC5A.

[0356] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to NY-ESO / MHC class I complex.

[0357] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to WT1 / MHC class I complex.

[0358] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to WT1 / MHC class I complex.

[0359] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to AFP / MHC class I complex.

[0360] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to HPV16-E7 / MHC class I complex.

[0361] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to gp100 / MHC class I complex.

[0362] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to hTERT / MHC class I complex.

[0363] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to MART1 / MHC class I complex.

[0364] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to HTLV1-Tax / MHC class I complex.

[0365] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to PR1 / MHC class I complex.

[0366] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to HIV1-gag / MHC class I complex.

[0367] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to HIV1-envelop gp120.

[0368] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to DLL3.

[0369] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to PTK7.

[0370] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to TROP2.

[0371] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to LAMP1.

[0372] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to Tim1.

[0373] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to TCR gamma-delta.

[0374] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to TCR beta1 constant chain.

[0375] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to TCR beta2 constant chain.

[0376] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to GCC.

[0377] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to B7H4.

[0378] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to LHR.

[0379] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to TSHR.

[0380] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to Tn-Muc1.

[0381] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to TSLPR.

[0382] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to Tissue Factor.

[0383] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to SSEA-4.

[0384] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to SLea.

[0385] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to Muc1 / MHC class I complex.

[0386] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to Muc16.

[0387] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to NYBR-1.

[0388] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to IL13Ra2.

[0389] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to IL11Ra.

[0390] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to L1CAM.

[0391] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to EpCAM1.

[0392] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to gpNMB.

[0393] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to GRP78.

[0394] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to GPC3.

[0395] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to GRPC5D.

[0396] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to GFRa4.

[0397] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to FITC.

[0398] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to CD79b.

[0399] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to Lym1.

[0400] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to Lym2.

[0401] In some arrangements, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARs which are part of the conventional CARs I to III or are part of backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-specific domain of the CARs is specific to Fc portion of an antibody (i.e. Ig Fc). An exemplary CAR with the antigen-specific domain specific to Ig Fc is represented by SEQ ID NO: 2708 and contains the extracellular domain of CD16-V158 as the antigen specific domain.

[0402] In some arrangements, the nucleic acid molecule encoding the CARs and / or accessory molecules described herein is provided as a messenger RNA (mRNA) transcript. In another arrangement, the nucleic acid molecule encoding the CARs and / or accessory molecules described herein is provided as a DNA construct.

[0403] Also provided herein are vectors comprising the polynucleotides described herein. In some arrangements, the vectors are viral vectors. Examples of viral vectors include but are not limited to retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, a pox virus, a herpes virus vector or a sleeping beauty transposon vector. In various arrangements, the present invention includes retroviral and lentiviral vector constructs expressing the CAR and the accessory molecules that can be directly transduced into a cell.

[0404] The present invention also includes 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...

Claims

1. A cell comprising nucleic acids encoding a chimeric antigen receptor (CAR) and Kaposi's sarcoma-associated herpesvirus-encoded viral FLICE inhibitory protein K13 (K13-vFLIP) signaling protein, wherein the CAR comprises an a) extracellular antigen specific domain, b) a transmembrane domain and c) an intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM); wherein c) is located at the C-terminus of the chimeric receptor.

2. The cell of claim 1, further comprising nucleic acid encoding Molluscum Contagiosum Virus vFLIP MC159 (MC159-vFLIP) signaling protein, or wherein the CAR further comprises one or more co-stimulatory domains, or wherein the cell further comprises nucleic acids encoding scFvs targeting IL6 and / or IL6 receptor alpha.

3. The cell of any of claims 1-2, further comprising nucleic acid encoding peptide FX06 so as to mitigate capillary leak associated with CAR therapy.

4. The cells of any of claims 1-2, wherein the signaling protein is expressed in fusion with one or more copies of FKBP domain, preferably wherein the activity of the signaling protein is controlled post-tranlationally by dimerization of the FKBP domain in the presence of a dimerizing agent, preferably wherein the dimerizing agent is AP20187.

5. The cell of claim 1, wherein the antigen specific domain of the CAR targets MPL, CD19, CD23, Lym1, Lym2, CLEC5A, CDH179b, FLT3, GCC, Muc1, CSF2RA, GFRa4, CD32, IL11Ra, IL13Ra, NYBR1, SLea, CD200R, TGFBetaR2, CD276, TROP2, LAMP1, PTK7, DLL3, CDH1, CDH6, CDH17, CDH19, TSHR or tyrosinase.

6. The cell of claim 1, wherein the antigen specific domain targets two antigens, preferably wherein the two antigens are MPL and CD123.

7. The cell of claim 5, wherein the antigen specific domain of the CAR targets MPL and comprises one or more scFv fragments selected from SEQ ID NOs:2500 and 2501 (161), SEQ ID NO:2499 (175), SEQ ID NO:2503 (178), SEQ ID NO:2502 (111), SEQ ID NO:2404 (AB317), SEQ ID NO:2505 (12E10) or SEQ ID NO:2506 (huVB22Bw5) or ligands selected from extracellular receptor binding domains of hTPO of SEQ ID NO:2323 or mTPO of SEQ ID NO:2324.

8. The cell of claim 7, wherein the antigen specific domain of the CAR targets CD19 and comprises one or more scFv fragments selected from SEQ ID NO:566 (CD19Bu12) or SEQ ID NO:567 (CD19MM).

9. The cell of claim 1, wherein the cell is a T-lymphocyte (T-cell), or a Natural Killer (NK) cell.

10. Nucleic acids comprising a first polynucleotide encoding the CAR of claim 1 and a second polynucleotide encoding the signaling protein of claim 1, and optionally further comprising a third polynucleotide encoding the MC159-vFLIP signaling protein.

11. Vectors comprising nucleic acids of claims 10.

12. A pharmaceutical composition, comprising the cell of any of claims 1-9, nucleic acid of claim 10, or a vector of claim 11, and a pharmaceutically acceptable carrier.

13. A cell of claim 7 for use in treating a MPL-expressing cancer.

14. The cell for use of claim 13, further comprising the administration of a tyrosine kinase inhibitor, wherein the inhibitor inhibits the src family of kinases, preferably wherein the inhibitor inhibits Lck, preferably wherein the inhibitor is any one or more of Dasatinib, Ponatinib or A-770041.