NZ786713A

NZ786713A - Chimeric antigen receptors targeting cancer

Info

Publication number: NZ786713A
Application number: NZ786713A
Authority: NZ
Inventors: Preet M Chaudhary
Original assignee: University Of Southern
Filing date: 2017-03-29
Publication date: 2022-04-29

Abstract

Provided herein is a composition comprising, 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 embodiments, the CAR further comprises one or mor co-stimulatory domains. Also provided herein are methods for treating diseases using the compositions described herein.

Description

CHIMERIC ANTIGEN RECEPTORS TARGETING CANCER STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with ment support under Grant No. DE019811 awarded by National Institutes of Health. The government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS The present application is a divisional ation of New Zealand Patent Application No. 746368, which is the national phase of International Application No. , which in turn claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62/314,864 filed on March 29, 2016. Each of the entioned ations are incorporated herein by cross reference in their entireties.

FIELD OF ION Provided herein are chimeric antigen receptors for treating cancers.

BACKGROUND All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description es information that may be useful in understanding the present ion. It is not an admission that any of the ation provided herein is prior art or relevant to the presently claimed invention, or that any publication ically or implicitly referenced is prior art.

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 l 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 . 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 PEA expression or proteasomal antigen processing, two mechanisms that contribute to tumor escape from diated immunity. Another feature of the broad applicability of CARs is their y to bind not only to proteins but also to carbohydrate and glycolipid structures, again expanding the range of potential targets.

Initial ﬁrst-generation CARs were ucted 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 CD34: or PC or 7 chains (Fig. 1).

Although CD34; 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-lBB, OX-40) on T cells binding to cognate ligands (i.e., CD80/86, 4-lBBL, ) 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 ing strength and persistence of the modiﬁed T cells, g 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 s the 4-IBB design s 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.

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 nes causes symptoms that include fevers, hypotension, mia, cardiac dysfunction, kidney failure and electrolyte abnormalities, collectively termed as "Cytokine release me’ (CRS). In some cases, CAR therapy can lead to ogic symptoms including tremor, seizures and can be fatal. Strategies to counteract CR5 and neurological complications include immunosuppressive agents (e.g., steroids) and Tocilizumab, a monoclonal antibody against E-6 or. However, these strategies are not uniformly sful. The instant invention provides novel approaches to control the activity of CAR-T cells so as to prevent and/or treat the above complications.

The CAR ucts in current clinical use are artiﬁcial in design as they ent fusion of several ent 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 ar activation, eventually resulting in apoptosis, ive cytokine release independent of cognate antigens, and immunologic exhaustion. Frigault et a] demonstrated that expression of some CARS containing CD28 and CD32 tandem signaling domains leads to constitutive tion and proliferation of the transduced primary human T cells which was related to inferior in vivo efﬁcacy (Frigault er al., Cancer immunology ch 7, 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 er al., Cancer immunology research 31356-67, 2015). Long er a] demonstrated that early T cell tion 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 -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 ofCAR T cells that limits antitumor cy (Long er 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 ive toxicity, such as CRS.

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.

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 ed epitope is a frequent cause of failure of the current CAR therapies. To overcome this limitation, the t invention provides CAR against multiple ns and against multiple epitopes of a single n. 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.

CD 19 is an attractive target for immunotherapy because it is uniformly expressed by the vast majority of B-cell malignancies, it is not sed 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 CDI9+ y ia 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 ered to express second-generation CARS have demonstrated sive clinical efficacy with signiﬁcant improvements in patient outcomes for a number of B-cell malignancies, the notable being the ic clinical responses observed in patients with relapsed B-cell ALL.

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 al tolerability of prolonged B-cell ion. 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 (AW), chronic myeloid leukemia (CML) and myelodysplastic syndrome MDS, has necessitated consideration of CAR-T tumor-targeting gies that may also affect normal tissues, such as bone marrow.

SUMMARY The ing embodiments and aspects thereof are described and illustrated in ction with systems, compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

In certain embodiments, the present invention provides compositions comprising genetically engineered effector cells (such as NK cells and T cells) that include polynucleotides that encode chimeric n receptors that can be used on adoptive cell therapy for ent of , infectious, autoimmune and degenerative diseases.

In some embodiments, provided herein are polynucleotides encoding ic antigen receptors (CARs) comprising an antigen speciﬁc domain (ASD), an ellular hinge or spacer region (l-IR), 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 VL and VH 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 VL and VH of MM described in Table 21 and set forth in SEQ ID NO: 1471 and SEQ ID NO: 3216.

In some embodiments, provided herein are polynucleotides encoding any of conventional CARS (Table l) 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 embodiments, the conventional CAR component and the one or more ory components of each backbone are encoded by a single polynucleotide molecule. In some embodiments, the conventional CAR component is encoded by a ﬁrst polynucleotide molecule and the one or more accessory modules are encoded by a second polynucleotide le. In some embodiments, backbones comprising a conventional CAR ent and more than one ory 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 1 and K13-vFLlP may be encoded by a single polynucleotide molecule or by two polynucleotide molecules, wherein the ﬁrst polynucleotide molecule encodes the tional CAR I and the second polynucleotide molecule encodes K13-vFLIP. In various embodiments, the cleotide molecules ng 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 ments, the antigen c domain comprises one or more VL (or vL) fragments. In some embodiments, the antigen speciﬁc domain comprises one or more VH (or vH) fragments. In some embodiments, the antigen speciﬁc domain comprises one or more scFVs (or scFvs) speciﬁc to the antigens on target cells such as cancer cells. In some embodiments, the antigen speciﬁc domain comprises one or more Fv fragments. In some embodiments, the antigen c domain comprises one or more Fab fragments. In some embodiments, the antigen speciﬁc domain comprises one or more (Fab')2 fragments. In some embodiments, the antigen ic domain comprises one or more single domain antibodies (SDAB). In some embodiments, the antigen speciﬁc domain comprises one or more camelid VHH (or vHH) domains. In some embodiments, the antigen speciﬁc domain ses one or more munoglobulin 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 , an llo repeat n, an tigen, a receptor or a ligand. In some embodiments, the antigen speciﬁc domain comprises one or more ligands.

Also ed herein are polypeptides encoded by the polynucleotides described . In various embodiments, 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 embodiments, the antigen speciﬁc domain comprises one or more VL (or vL) fragments. In some embodiments, the antigen speciﬁc domain comprises one or more VH (or vH) fragments. In some embodiments, the n speciﬁc domain comprises one or more scFVs specific to the antigens on target cells such as cancer cells. In some embodiments, the antigen speciﬁc domain comprises one or more Fv fragments. In some embodiments, the antigen speciﬁc domain comprises one or more Fab fragments. In some embodiments, the antigen speciﬁc domain comprises one or more (Fab')2 fragments. In some ments, the antigen speciﬁc domain comprises one or more single domain antibodies (SDAB). In some embodiments, the antigen speciﬁc domain comprises one or more camelid VHH (vHH) domains. In some embodiments, the antigen speciﬁc domain comprises one or more Fv fragments. In some embodiments, the antigen speciﬁc domain comprises one or more non- immunoglobulin scaffold such as a DARPIN, an afﬁbody, an , 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 llo repeat protein, an autoantigen, a receptor or a . In some embodiments, the n speciﬁc domain comprises one or more ligands.

Further provided herein are one or more vectors comprising nucleic acids encoding any of tional CARS I to 111 (Table 1) or backbones 1-62 (Table 2). In some embodiments, all the components of each backbone are encoded by a single vector. In some embodiments, each component of the backbone is d by a different vector. For examples, a backbone comprising a tional 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 ses polynucleotides ng the conventional CAR component and a second vector comprises polynucleotides encoding the accessory modules. ately, a ﬁrst vector comprises polynucleotides encoding the tional 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 embodiments, the vectors ng the polynucleotide molecule encoding the CAR component of the conventional CARS I to 111 or the nes described herein encodes one or more antigen speciﬁc domains. In some embodiments, the antigen speciﬁc domain comprises one or more VL fragments. In some embodiments, the antigen speciﬁc domain ses one or more VH fragments. In some embodiments, the antigen speciﬁc domain comprises one or more scFVs speciﬁc to the antigens on target cells such as cancer cells. In some embodiments, the antigen speciﬁc domain comprises one or more Fv fragments. In some embodiments, the antigen speciﬁc domain comprises one or more Fab fragments. In some embodiments, the antigen speciﬁc domain comprises one or more 2 fragments. In some embodiments, the antigen speciﬁc domain ses one or more single domain antibodies (SDAB). In some embodiments, the antigen speciﬁc domain comprises one or more camelid VHH domains. In some embodiments, the antigen speciﬁc domain ses one or more Fv nts. In some embodiments, the antigen speciﬁc domain comprises one or more non-immunoglobulin scaffold such as a , an afﬁbody, an afﬁlin, an adnectin, an afﬁtin, an obodies, a repebody, a r, an alphabody, an avimer, an atrimer, a centyiin, a pronectin, an anticalin, a kunitz domain, an Armadillo repeat protein, an autoantigen, a receptor or a ligand. In some embodiments, the antigen speciﬁc domain comprises one or more ligand.

Further provided herein are cally modiﬁed cells comprising vectors ng polynucleotides encoding the conventional CARS Ito III or backbones 1-62 described , wherein the CARS of the conventional CARS I to III or the backbones comprise antigen specific domains as described herein. In s embodiments, the genetically modiﬁed cells target antigen on target cells such as cancer cells via the VL fragments, VH fragments, VHH domains, scFvs, Fv fragments, Fab nts, (Fab')2 fragments, single domain antibody (SDAB) fragments, and/or non-immunoglobulin scaffold each of which are encoded by the antigen Specific domains.

In some embodiments, the antigen speciﬁc s of the CARS comprise one or more VL fragments. In exemplary embodiments, the one or more VL nts are described in Table 4. In some embodiments, the polynucleotides encoding the one more VL fragments comprise, t of or consist essentially of sequences set forth in any one or more of SEQ ID NOS: 1 1-249. In some embodiments, the polypeptides encoding the one more VL 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 embodiments, the antigen specific domains of the CARS comprise one or more VH fragments. In ary embodiments, the one or more VH fragments are described in Table 6. In some embodiments, the polynucleotides encoding the one more VH fragments comprise, consist of or consist essentially of sequences set forth in any one or more of SEQ ID NOS: 259-498. In some embodiments, the polypeptides encoding the one more VH fragments comprise, consist of or consist essentially of sequences set forth in any one or more of SEQ ID NOS: 2028-2268 or ces with 80-99% ty to sequences set forth in any one or more of SEQ ID NOS: 2028-2268 or sequences with 80-99% ty in the three complementarity determining regions (CDRS) to the sequences set forth in any one or more of SEQ ID NOS: 2028-2268 or ces 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.

In some embodiments, the antigen speciﬁc domains of the CARs comprise one or more nanobodies (VHH domains). In exemplary embodiments, the one or more VHH domains are described in Table 7. In some embodiments, the polynucleotides encoding the one more VHH nts comprise, consist of or consist essentially of sequences set forth in any one or more of SEQ ID NOS: 499-523. In some embodiments, the polypeptides encoding the one more VHH fragments comprise, t of or t 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 ns as the sequences set forth in any one or more of SEQ ID NOS: 2269-2293.

In some embodiments, the antigen specific domains of the CARS comprise one or more non-immunoglobulin antigen binding scaffolds. In ary embodiments, the one or more non-immunoglobulin antigen binding scaffolds are described in Table 8. In some embodiments, the polynucleotides encoding the one more non-immunoglobulin antigen binding scaffolds se, consist of or consist essentially of sequences set forth in any one or more of SEQ ID NOS: 524-528. In some embodiments, the polypeptides ng the one more non-immunoglobulin n 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 ces that bind to the same target antigens or the same epitopes on the target ns as the sequences set forth in any one or more of SEQ ID NOS: 2269-2293.

In some embodiments, the antigen specific domains of the CARS comprise one or more ligands. In exemplary embodiments, the ligands are described in Table 10. In some embodiments, the polynucleotides encoding the one more s se, consist of or consist essentially of sequences set forth in any one or more of SEQ ID NOS: 553-563. In some embodiments, the polypeptides ng 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.

In some embodiments, the antigen speciﬁc domains of the CARs comprise one or more scFvs. In exemplary ments, the scFv fragments are described in Table 11. In some embodiments, 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 embodiments, the polynucleotides encoding the one more scFv fragments comprise, consist of or consist essentially of ces 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 ns 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 embodiments, the ptides 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% ty 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 es on the target antigens as the sequences set forth in any one or more of SEQ ID NOS: 2334-2568.

In some embodiments, provided herein are polynucleotides ng 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 ory s as shown in Table 2. In some embodiments, the conventional CAR ent and the one or more accessory components of each backbone are encoded by a single polynucleotide molecule. In some embodiments, 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 embodiments, backbones comprising a conventional CAR component and more than one accessory , 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 embodiments, provided herein are polypeptides encoding tional CAR (Table 1) and any of nes 1-62 as described herein in Table 2, wherein each backbone comprises a conventional CAR component (Table I) and one or more accessory modules as shown in Table 2. In some embodiments, 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 embodiments, 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 embodiments, the ptide encoding the conventional CAR component and the one or more accessory module components of each backbone are encoded by a single polypeptide le. In some embodiments, 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 embodiments, backbones sing 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 cleotide molecule encodes the second accessory le. In some embodiments, backbones comprising a tional CAR ent and more than one accessory module, the ﬁrst polypeptide molecule encodes the conventional CAR ent, a second polypeptide molecule encodes the ﬁrst accessory molecule and a third polypeptide molecule encodes the second accessory molecule.

In some embodiments, 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 ments, the polynucleotides encoding the one more accessory module comprise, consist of or consist essentially of sequences that encode for polypeptide ces as set forth in any one or more of SEQ ID NOS: 2613-2667 or encode for polypeptide ces with 70-99% identity to sequences set forth in any one or more of SEQ ID NOS: 667. In some embodiments, 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: 667.

In exemplary embodiments, nucleic acids ng backbone-l comprising conventional CAR I and K13-vFLIP, wherein the antigen c 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-l comprising conventional CAR 1 and Kl3-VFLIP and targeting antigens of interest as described in Table 19 are set forth in SEQ ID N05: 2672- 2941. The nucleic acid ces in SEQ ID N03: 927-] 196 and polypeptide ces 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.

In one ment, provided herein is an isolated nucleic acid encoding backbone- 1 comprising conventional CAR I and K13-vFLIP, wherein the antigen c domain of the CAR targets CD19 (Table 19). In exemplary embodiments, the sequences of isolated nucleic acid fragments targeting CD19 and essing Kl3-VFLIP are set forth in SEQ ID NOs: 927-941. In exemplary embodiments, the sequences of isolated polypeptide targeting CD19 and coexpressing K13-VFLIP are set forth in SEQ ID NOs: 2672-2686. In some embodiments, 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 ptides encoded by nucleic acids encoding ne-1 comprising conventional CAR I and K13- vFLIP, wherein the antigen speciﬁc domain of the CAR targets CD19. Further provided herein are vectors ng c acids encoding ne-l comprising conventional CAR I and KlS-vFLH’, 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 sing c0nventional CAR I and K 13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CD19.

In one embodiment, provided herein is an isolated nucleic acid encoding ne- 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 embodiments, the sequences of isolated nucleic acid fragments targeting MPL and coexpressing K13-vFLH’ are set forth in SEQ ID NOs: 1117-1124. In exemplary embodiments, the sequences of isolated polypeptide targeting MPL are set forth in SEQ ID NOs: 869. In some embodiments, the scFv fragments targeting MPL are described in Table 11 and set forth in SEQ ID NOs: 6 and SEQ ID NOS: 506. Also provided herein are polypeptides d by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, n the antigen speciﬁc domain of the CAR targets MPL. Further provided herein are vectors encoding nucleic acids encoding backbone-l comprising conventional CAR I and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR s MPL. Also ed herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding c acids encoding backbone-1 comprising conventional CAR 1 and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets In one embodiment, 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 Lyml (Table 19). In exemplary embodiments, the sequence of nucleic acid fragment targeting Lyml is set forth in SEQ ID NO: 1109. In exemplary embodiments, the sequence of isolated ptide fragment targeting Lyml is set forth in SEQ ID NO: 2854.

In some embodiments, the scFv fragment targeting Lyml is described in Table 11 and set forth in SEQ ID NO: 723 and SEQ ID NO: 2493. Also provided herein are polypeptides d by nucleic acids encoding backbone-1 comprising conventional CAR I and K13- vFLIP, wherein the antigen speciﬁc domain of the CAR s Lyml. Further provided herein are vectors encoding nucleic acids encoding ne-l comprising conventional CAR I and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets Lyml. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-l comprising conventional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets Lyml.

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- 1 comprising conventional CAR 1 and Kl3-vFL1P, wherein the antigen c domain of the CAR targets Lym2 (Table 19). In exemplary embodiments, sequence of the isolated c acid fragment targeting Lym2 is set forth in SEQ ID NO: 1110. In exemplary embodiments, the sequence of isolated polypeptide targeting Lym2 is set forth in SEQ ID NO: 2855. In some embodiments, the scFv nt targeting Lym2 is described in Table 11 and set forth in SEQ ID NO: 724 and SEQ ID NO: 2494. Also ed 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 c acids encoding backbone-1 comprising conventional CAR I and Kl3-vFLIP, n 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 K1 3-vFLIP, wherein the n speciﬁc domain of the CAR targets Lym2.

In one embodiment, 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 s GRP-78 (Table 19). In exemplary embodiments, sequence of the isolated nucleic acid targeting GRP-78 is set forth in SEQ ID NO: 1078. In exemplary embodiments, ce of the polypeptide targeting GRP-78 is set forth in SEQ ID NO: 2823. In some embodiments, the scFv fragment ing GRP-78 is bed in Table 11 and set forth in SEQ ID NO: 703 and SEQ ID NO: 2473. Also ed herein are polypeptides d by nucleic acids encoding ne-l comprising conventional CAR I and KI3-VFLIP, wherein the antigen speciﬁc domain of the CAR s 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 s GRP-78. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids ng backbone-l comprising conventional CAR I and K13- vFLIP, wherein the antigen speciﬁc domain of the CAR targets GRP-78.

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- ] comprising conventional CAR I and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CD79b (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragments targeting CD79b are set forth in SEQ ID NOs: 995-996. In exemplary embodiments, the sequences of the polypeptide fragments targeting CD79b are set forth in SEQ ID NO: 2740-2741. In some embodiments, 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 K l3-vFLIP, n the antigen specific domain of the CAR targets CD79b. Further provided herein are s encoding nucleic acids ng backbone-I comprising conventional CAR I and K l3-vFLIP, wherein the antigen c domain of the CAR targets CD79b. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors ng nucleic acids encoding backbone-1 comprising conventional CAR I and Kl3-vFLIP, wherein the n specific domain of the CAR targets CD79b.

In one embodiment, 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 [9). In exemplary embodiments, the sequences of the nucleic acid fragments targeting ALK are set forth in SEQ ID NOs: 7. In exemplary embodiments, the sequences of the polypeptide fragments targeting ALK are set forth in SEQ ID NO: 2691-2692. In some embodiments, the scFv fragments ing 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-l comprising conventional CAR I and LIP, wherein the antigen speciﬁc domain of the CAR targets ALK. Further provided herein are vectors encoding nucleic acids encoding backbone-l comprising conventional CAR I and K l3-vFLIP, wherein the antigen specific domain of the CAR s 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 1 and Kl3-vFLIP, wherein the n speciﬁc domain of the CAR targets In one embodiment, 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 embodiments, the ces of the c acid fragments targeting BCMA are set forth in SEQ ID NOs: 951-957. In exemplary embodiments, the sequences of the polypeptide fragments targeting BCMA are set forth in SEQ ID NO: 2696-2702. In some embodiments, 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 ptides encoded by nucleic acids encoding ne-l 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-l comprising conventional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR s BCMA. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding ne-1 comprising conventional CAR I and K13-vFLlP, wherein the antigen specific domain of the CAR s BCMA.

In one embodiment, provided herein is an isolated nucleic acid ng backbone- 1 comprising conventional CAR 1 and LIP, wherein the antigen speciﬁc domain of the CAR targets CD20 (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragments targeting CD20 are set forth in SEQ ID NOs: 964-976. In exemplary embodiments, the sequences of the polypeptide fragments targeting CD20 are set forth in SEQ ID NO: 2709-2721. In some embodiments, 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 c acids encoding backbone-1 sing 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-l comprising tional CAR I and KlS-vFLIP, n the antigen speciﬁc domain of the CAR targets CD20. Also provided herein are genetically engineered cells (such as T cells, NK cells) sing vectors ng nucleic acids encoding backbone-l comprising conventional CAR 1 and K13-VFLIP, n the antigen speciﬁc domain of the CAR targets CD20.

In one embodiment, ed herein is an ed nucleic acid encoding backbone- 1 comprising conventional CAR 1 and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CD22 (Table 19). In exemplary embodiments, 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 embodiments, 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 embodiments, 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-l sing 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-l comprising conventional CAR I and Kl3-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 1 and LIP, wherein the n specific domain of the CAR targets CD22.

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- 1 comprising conventional CAR I and LIP, wherein the antigen speciﬁc domain of the CAR targets CD30 (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragments targeting CD30 are set forth in SEQ ID NOs: 981-982. In exemplary embodiments, the sequences of the ptide fragments targeting CD30 are set forth in SEQ ID NOs: 2726-2727. In some embodiments, the scFv fragments targeting CD30 are bed in Table II and set forth in SEQ ID NO: 5 and SEQ ID NO: 2384-2385.

Also provided herein are polypeptides encoded by nucleic acids ng 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 LIP, wherein the antigen speciﬁc domain of the CAR targets CD30. Also ed herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding c acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the n speciﬁc domain of the CAR targets CD30.

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- ] comprising conventional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CD32 (Table 19). In exemplary embodiments, the sequences of the c acid fragment targeting CD32 is set forth in SEQ ID NO: 983. In exemplary embodiments, the sequence of the polypeptide fragments targeting CD32 is set forth in SEQ ID NO: 2728 In some embodiments, 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 d by nucleic acids encoding backbone-1 comprising conventional CAR 1 and K13- vFLIP, wherein the antigen c domain of the CAR targets CD32. Further provided herein are vectors encoding nucleic acids encoding backbone-l 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-I comprising tional CAR I and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CD32.

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- ] comprising conventional CAR I and KI3-vFLIP, wherein the n specific domain of the CAR targets CD33 (Table 19). In exemplary ments, the sequences of the nucleic acid fragments ing CD33 are set forth in SEQ ID NOs: 984-991. In exemplary embodiments, the sequences of the ptide fragments ing CD33 are set forth in SEQ ID NOs: 2729-2736. In some embodiments, the scFv fragments targeting CD33 are described in Table II 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 K I 3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CD33. Further provided herein are vectors encoding c 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) sing vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CD33.

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- ] comprising conventional CAR I and KI3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CD123 (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragments targeting CD123 are set forth in SEQ ID NOS: 10. In exemplary embodiments, the sequences of the polypeptide fragments ing CD123 are set forth in SEQ ID NOs: 2743-2755. In some ments, the scFv fragments targeting CD123 are described in Table II 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 tional CAR I and K l3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CD123. Further provided herein are vectors encoding c acids encoding backbone-1 sing conventional CAR I and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR s 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 K l3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CD123.

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- ] comprising conventional CAR I and LIP, wherein the antigen speciﬁc domain of the CAR targets CD138 (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragment targeting CD138 is set forth in SEQ ID NO: 101 1. In exemplary embodiments, the sequence of the polypeptide nts ing CD138 is set forth in SEQ ID NO: 2756 In some embodiments, the scFv fragment targeting CD138 is described in Table 11 and set forth in SEQ ID N05: 643 and 2413. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-l comprising conventional CAR 1 and KlB-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CD138. Further ed herein are vectors encoding nucleic acids encoding backbone-l comprising conventional CAR 1 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 c acids ng backbone-l comprising tional CAR I and K13- vFLIP, wherein the antigen speciﬁc domain of the CAR s CD138.

In one ment, provided herein is an isolated nucleic acid ng backbone- 1 comprising tional CAR I and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CLLl (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragments targeting CLLI are set forth in SEQ ID NOs: 1024-1029. In exemplary ments, the sequences of the polypeptide fragments targeting CLLl are set forth in SEQ ID NOs:2769-2774. In some embodiments, the scFv fragments targeting CLLl 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 Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CLLl. Further provided herein are vectors encoding nucleic acids ng backbone-1 sing conventional CAR I and LIP, wherein the antigen speciﬁc domain of the CAR targets CLLI. 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 Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CLLI.

In one embodiment, provided herein is an ed nucleic acid encoding backbone- ] comprising conventional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CS] (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragments targeting CSl are set forth in SEQ ID NOs: 1031-1039. In exemplary embodiments, the sequences of the polypeptide fragments targeting CS] are set forth in SEQ ID 76-2784. In some embodiments, the scFv fragments targeting CSl 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 tional CAR I and Kl 3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CSl. Further provided herein are s encoding nucleic acids encoding backbone-1 comprising tional CAR I and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CD79b. Also provided herein are genetically ered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding ne-1 comprising conventional CAR 1 and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets In one embodiment, provided herein is an isolated nucleic acid encoding backbone- ] comprising conventional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR s CSFZRA (Table 19). In exemplary ments, the sequences of the nucleic acid fragments targeting CSFZRA are set forth in SEQ ID NOs: 1040-1041. In exemplary embodiments, the sequences of the polypeptide fragments targeting CSFZRA are set forth in SEQ ID NOs: 2785-2786. In some embodiments, the scFv fragments ing CSFZRA 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 sing conventional CAR I and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CSF2RA. Further provided herein are vectors ng nucleic acids encoding backbone-1 comprising conventional CAR I and LIP, wherein the n speciﬁc domain of the CAR targets CSFZRA. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising s encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CSF2RA.

In one ment, provided herein is an isolated nucleic acid encoding backbone- ] comprising conventional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets DLL3 (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragments targeting DLL3 are set forth in SEQ ID NOS: 1044-1045. In exemplary embodiments, the sequences of the polypeptide fragments targeting DLL3 are set forth in SEQ ID NOs: 2789-2790. In some embodiments, the scFv fragments targeting DLL3 are bed 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 ne-1 comprising conventional CAR I and Kl3-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 Kl3-VFLIP, n the n speciﬁc domain of the CAR s DLL3. Also provided herein are cally 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 s DLL3.

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- ] comprising conventional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets GPRCSD (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragments targeting GPRCSD are set forth in SEQ ID NOs: 1070-1073. In ary ments, the sequences of the polypeptide fragments targeting GPRCSD are set forth in SEQ ID NOs: 2815-2818. In some embodiments, the scFv fragments targeting GPRCSD are described in Table 11 and set forth in SEQ ID NO: 4 and SEQ ID NO: 2465-2468. Also provided herein are ptides encoded by nucleic acids ng backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets GPRCSD. 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 GPRCSD. Also provided herein are cally 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 s GPRCSD.

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- ] sing conventional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets HIVl-envelop glycoprotein gp120 (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragments targeting HIVl-envelop glycoprotein gp120 are set forth in SEQ ID N05: 959, 1089-1092. In exemplary embodiments, the ces of the polypeptide fragments targeting nvelop glycoprotein gp120 are set forth in SEQ ID N05: 2704, 2834-2837. In some embodiments, the scFv fragments targeting HIVl-envelop glycoprotein gp120 are described in Table 11 and set forth in SEQ ID NO: 581, 8 and SEQ ID NO: 2351, 2475-2478. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and KI3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets HIVl-envelop glycoprotein gp120. Further provided herein are vectors encoding nucleic acids encoding backbone—l comprising conventional CAR I and K l3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets HIVl-envelop glycoprotein gp120. Also ed herein are genetically ered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising tional CAR I and Kl3-vFLIP, wherein the antigen c domain of the CAR targets HIVl-envelop glycoprotein gp120.

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- 1 comprising conventional CAR I and K13-vFLIP, wherein the antigen c domain of the CAR targets ILllRa (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragment targeting ILl lRa is set forth in SEQ ID NO: 1099. In exemplary embodiments, the sequence of the polypeptide fragments targeting ILl lRa is set forth in SEQ ID NO: 2844 In some embodiments, the scFv fragments targeting HJ lRa is bed in Table 11 and set forth in SEQ ID N05: 715 and 2485. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 sing conventional CAR I and K13- vFLIP, wherein the antigen speciﬁc domain of the CAR targets ILI lRa. Further provided herein are vectors encoding nucleic acids encoding backbone-l comprising conventional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR s ILl lRa.

Also provided herein are genetically-engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids ng backbone-l sing conventional CAR I and K1 3-vFLIP, wherein the antigen c domain of the CAR targets ILI lRa.

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- ] comprising conventional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets ILl3Ra2 (Table 19). In exemplary embodiments, the sequences of the nucleic acid nts targeting ILl3Ra2 are set forth in SEQ ID NOs: 1101-1102. In exemplary embodiments, the sequences of the polypeptide fragments targeting ILl3Ra2 are set forth in SEQ ID NOs: 2846-2847. In some embodiments, the scFv fragments targeting ILl3RaZ 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-l comprising conventional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets ILl3Ra2. Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and LIP, wherein the antigen specific domain of the CAR targets ILl3Ra2. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding c acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the n speciﬁc domain of the CAR targets 2.

In one embodiment, provided herein is an ed c acid encoding backbone- ] comprising tional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR s LAMP] (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragments targeting LAMPI are set forth in SEQ ID NOs: 1104-1105. In exemplary embodiments, the sequences of the polypeptide fragments targeting LAMPI are set forth in SEQ ID NOs: 2849-2850. In some embodiments, the scFv nts targeting LAMP] are described in Table 11 and set forth in SEQ ID NO: 719-720 and SEQ ID NO: 490. 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 LAMPI. Further provided herein are vectors encoding nucleic acids ng backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets LAMP]. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding c acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, n the antigen speciﬁc domain of the CAR targets LAMP].

In one embodiment, provided herein is an isolated nucleic acid ng backbone- ] comprising conventional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets NY-BRI (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragment targeting NY-BRI is set forth in SEQ ID NO: 1131. In ary embodiments, the sequence of the polypeptide fragments targeting NY-BRI is set forth in SEQ ID NO: 2876 In some embodiments, the scFv fragments targeting NY-BRl are bed in Table 11 and set forth in SEQ ID N05: 742 and 2512. Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets NY-BR]. Further provided herein are vectors encoding nucleic acids encoding ne-l comprising conventional CAR 1 and Kl3-vFLIP, wherein the n c domain of the CAR targets NY-BRI. 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-BRl.

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- 1 comprising tional CAR I and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets THVI l/I-IAVCR (Table 19). In exemplary ments, the ces of the nucleic acid fragments targeting TIMI/I-IAVCR are set forth in SEQ ID NOS: 1 160-1 161. In exemplary embodiments, the sequences of the polypeptide fragments targeting AVCR are set forth in SEQ ID NOS: 906. In some ments, the scFv fragments targeting TIMI/HAVCR are described in Table I] and set forth in SEQ ID NO: 770-771 and SEQ ID NO: 2540-2541. Also ed herein are ptides encoded by nucleic acids encoding backbone-l comprising conventional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets AVCR. Further provided herein are vectors encoding nucleic acids encoding backbone-l comprising conventional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets TIMI/HAVCR. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-l comprising conventional CAR I and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets TIMI/HAVCR.

In one embodiment, 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 embodiments, the sequences of the nucleic acid fragments targeting TROP2 are set forth in SEQ ID NOS: 1165-1166. In exemplary embodiments, the sequences of the polypeptide nts targeting TROP2 are set forth in SEQ ID NOs: 2910-2911. In some embodiments, the scFv fragments targeting TROP2 are described in Table 11 and set foﬂh in SEQ ID NO: 774-775 and SEQ ID NO: 2544-2545.

Also ed herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and Kl3-vFLIP, n the antigen specific domain of the CAR targets TROP2. Further provided herein are vectors ng nucleic acids encoding backbone-l comprising conventional CAR I and Kl3-vFLIP, wherein the antigen specific domain of the CAR targets TROP2. Also provided herein are cally ered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and Kl3-vFLIP, wherein the antigen specific domain of the CAR targets TROP2.

In one embodiment, provided herein is an ed nucleic acid encoding backbone- ] comprising conventional CAR I and Kl3-vFLIP, wherein the n speciﬁc domain of the CAR targets TSHR (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragments targeting TSHR are set forth in SEQ ID NOS: 1167-1170. In exemplary embodiments, the sequences of the polypeptide fragments ing TSHR are set forth in SEQ ID NOs: 2912-2914. In some embodiments, the scFv fragments targeting TSHR are described in Table 11 and set fonh in SEQ ID NO: 776-778 and SEQ ID NO: 2546-2548.

Also provided herein are ptides encoded by nucleic acids encoding backbone-1 sing conventional CAR I and Kl3-vFLIP, wherein the n specific domain of the CAR targets TSHR. Further provided herein are vectors encoding nucleic acids ng backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TSHR. Also provided herein are cally engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 sing conventional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets TSHR.

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- ] comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TSLPR (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragment targeting TSLPR is set forth in SEQ ID NO: 1171. In exemplary embodiments, the sequence of the polypeptide fragment targeting TSLPR is set forth in SEQ ID NO: 2916 In some embodiments, the scFv fragment targeting TSLPR is described in Table 11 and set forth in SEQ ID N05: 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 1 and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR s TSLPR. Also provided herein are genetically-engineered cells (such as T cells, NK cells) comprising vectors encoding c acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets TSLPR.

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- 1 comprising conventional CAR 1 and K13-vFL1P, wherein the n specific domain of the CAR targets CDH19 (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragments ing CDH19 are set forth in SEQ ID NO: 1019 and 1180. In exemplary embodiments, the sequences of the polypeptide fragments targeting CDH19 are set forth in SEQ ID NO: 2764 and 2925 In some embodiments, the scFv fragments ing CDH19 are described in Table 11 and set forth in SEQ ID N05: 651, 788 and SEQ ID NOs: 2421 and 2558. Also provided herein are ptides encoded by nucleic acids ng 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 LIP, n the antigen speciﬁc domain of the CAR s 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 1 and LIP, wherein the antigen specific domain of the CAR targets CDH19.

In one embodiment, provided herein is an ed nucleic acid encoding backbone- 1 comprising tional CAR 1 and Kl3-vFL1P, wherein the antigen specific domain of the CAR targets CDH6 (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragments targeting CDH6 are set forth in SEQ ID NOs: 1017-1018. In exemplary embodiments, the sequences of the polypeptide fragments targeting CDH6 are set forth in SEQ ID NOS: 2761-2762. In some embodiments, 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 ptides d 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 s encoding nucleic acids encoding backbone-l comprising conventional CAR I and Kl3-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 ng backbone-1 comprising tional CAR I and Kl3-vFLlP, n the antigen speciﬁc domain of the CAR targets CDH6.

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- ] comprising conventional CAR I and Kl3-VFLIP, wherein the antigen speciﬁc domain of the CAR targets CD324 (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragments targeting CD324 are set forth in SEQ ID NOs: 1014-1015. In exemplary embodiments, the ces of the polypeptide fragments targeting CD324 are set forth in SEQ ID NOs: 2759-2760. In some embodiments, the scFv fragments targeting CD324 are described in Table 11 and set forth in SEQ ID NO: 646-647 and SEQ ID NO: 2416-24l7.

Also provided herein are polypeptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the n speciﬁc domain of the CAR targets CD324. Further provided herein are s encoding nucleic acids encoding backbone-l comprising conventional CAR I and Kl3-vFLIP, n 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 Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CD324.

In one embodiment, provided herein is an ed nucleic acid encoding ne- 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 embodiments, the sequence of the c acid fragment targeting TCR gamma-delta (TCRgd) is set forth in SEQ ID NO: 1155. In exemplary ments, the sequence of the polypeptide fragments targeting TCR gamma-delta (TCRgd) is set forth in SEQ ID NO: 2900 In some embodiments, the scFv fragment targeting TCR gamma-delta (TCRgd) is described in Table 11 and set forth in SEQ ID N05: 765 and SEQ ID NOs: 2535. Also provided herein are ptides encoded by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets TCR delta (TCRgd). Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and Kl3-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-I sing conventional CAR I and Kl3-vFLIP, wherein the antigen c domain of the CAR targets TCR gamma-delta (TCRgd).

In one ment, provided herein is an isolated nucleic acid encoding ne- ] comprising conventional CAR I and Kl3-VFLIP, wherein the antigen speciﬁc domain of the CAR targets TCRBI (TCR beta 1 constant chain) (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragments targeting TCRBI (TCR beta 1 constant chain) are set forth in SEQ ID NOs: 1151-1152. In exemplary embodiments, the sequences of the polypeptide fragments targeting TCRBI (TCR beta 1 constant chain) are set forth in SEQ ID NOs: 2896-2897. In some embodiments, the scFv fragments targeting TCRBI (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 d by nucleic acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen specific domain of the CAR targets TCRBI (TCR beta I constant chain). Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR I and K I3-vFLIP, wherein the n speciﬁc domain of the CAR s TCRBI (TCR beta 1 constant chain). Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising s encoding c acids encoding backbone-1 comprising conventional CAR I and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets TCRBI (TCR beta I constant chain).

In one embodiment, provided herein is an isolated nucleic acid ng ne- 1 comprising conventional CAR I and K13-VFLIP, n the antigen speciﬁc domain of the CAR targets TCRBZ (TCR beta 2 constant chain) (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragments targeting TCRBZ (TCR beta 2 constant chain) are set forth in SEQ ID NOs: 1153-1154. In exemplary ments, the ces of the polypeptide fragments targeting TCRBZ (TCR beta 2 constant chain) are set forth in SEQ ID NOs: 2898-2899. In some embodiments, 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-l comprising conventional CAR I and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets TCRB2 (TCR beta 2 constant chain). r ed herein are vectors encoding c acids encoding backbone—1 comprising tional CAR 1 and Kl 3-vFLIP, wherein the antigen c domain of the CAR targets TCRB2 (TCR beta 2 nt chain). Also ed herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-1 comprising tional CAR I and Kl3-vFLIP, wherein the antigen specific domain of the CAR targets TCRBZ (TCR beta 2 constant chain).

In one ment, provided herein is an isolated nucleic acid encoding ne- ] comprising conventional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets LHR (Luteinizing hormone or) (Table 19). In exemplary embodiments, the sequences of the c acid fragments targeting LHR (Luteinizing hormone Receptor) are set forth in SEQ ID NOS: 1 182-1 183. In exemplary embodiments, the sequences of the polypeptide fragments targeting LHR (Luteinizing hormone Receptor) are set forth in SEQ ID NOs: 2927-2928. In some embodiments, the scFv fragments targeting LHR (Luteinizing e Receptor) are described in Table 1 1 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 LI-IR (Luteinizing hormone Receptor).

Further provided herein are vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR 1 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 LIP, wherein the antigen speciﬁc domain of the CAR targets LHR (Luteinizing hormone Receptor).

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- ] comprising conventional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets Fc region of an immunoglobulin (Table 19). In exemplary embodiments, the sequences of the nucleic acid fragments targeting Fc region of an immunoglobulin are set forth in SEQ ID NOS: 961-962. In exemplary embodiments, the sequences of the polypeptide fragments targeting Fc region of an immunoglobulin are set forth in SEQ ID N05: 2707- 2708. In some embodiments, the receptor targeting Fc region of an globulin comprises, consists of or essentially ts 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 s encoding nucleic acids encoding backbone-1 comprising tional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets Fc region of an immunoglobulin. Also ed herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding ne-1 comprising conventional CAR I and KI3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets Fc region of an globulin. The genetically engineered cells comprising vectors encoding nucleic acids encoding backbone-1 comprising conventional CAR 1 and K l3-vFLIP, wherein the n speciﬁc domain of the CAR s Fc region of an immunoglobulin can be used to enhance antibody dependent cell mediated xicity and/or enhance antibody based immunotherapy, such as cancer immunotherapy, as described in US 2015/0139943A1.

In exemplary embodiments, nucleic acids encoding ne-32 comprising conventional CAR II and K13-VFLIP, wherein the antigen c domain of the CAR targets antigens of interest are described in Table 20. The sequences of nucleic acid fragments ng backbone-32 comprising conventional CAR II and Kl3-vFLIP and targeting antigens of interest as described in Table 20 are set forth in SEQ ID N05: 1197- 1466. The sequences of polypeptides encoding backbone-32 comprising conventional CAR I and LIP and targeting antigens of st 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 ptide sequences in SEQ ID NOs: 2942-3211 also encode for a puromycin acetyl transferase (PuroR) nt, which fragment can be used to select cells and is not essential for the function of the CAR.

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- 32 sing conventional CAR [1 and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CD19 (Table 20). In exemplary embodiments, nucleic acid sequences of the VL-VH targeting CD19 are set forth in SEQ ID N05: 1 1 1. In exemplary embodiments, the polypeptide sequences of the VL-VH targeting CD19 are set forth in SEQ ID N05: 2942- 2956. In some embodiments, the scFv fragments ing 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 ne-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 ne-32 comprising Tcon‘ventional CAR II and L1P, wherein the antigen speciﬁc domain of the CAR targets CD19. Also ed herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids ng backbone-32 comprising conventional CAR II and K13-VFLIP, wherein the antigen speciﬁc domain of the CAR targets CD19.

In one embodiment, provided herein is an ed nucleic acid encoding backbone- 32 comprising conventional CAR II and LIP, wherein the antigen speciﬁc domain of the CAR targets MPL (Table 20). In exemplary embodiments, nucleic acid sequences of the VL-VH targeting MPL are set forth in SEQ ID NOS: 1387-1394. In exemplary embodiments, the polypeptide sequences of the VL-VH targeting MPL are set forth in SEQ ID N05: 3132- 3139. In some embodiments, 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 LIP, wherein the antigen specific domain of the CAR targets MPL. r provided herein are vectors encoding nucleic acids encoding backbone-32 comprising conventional CAR II and Kl3-VFLIP, wherein the antigen speciﬁc domain of the CAR s 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 LIP, wherein the antigen speciﬁc domain of the CAR targets MPL.

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- 32 comprising conventional CAR [1 and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR s Lyml (Table 20). In exemplary embodiments, the nucleic acid sequence of the VL—VH targeting Lyml is set forth in SEQ ID NO: 1379. In ary embodiments, the polypeptide sequence of the VL-VH targeting Lyml is set forth in SEQ ID NO: 3124. In some embodiments, the scFv fragment ing Lyml is described in Table 11 and set forth in SEQ ID NO: 723 and SEQ ID NO: 2493. Also provided herein are polypeptides d by nucleic acids ng backbone-32 comprising conventional CAR II and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets Lyml. Further provided herein are vectors encoding nucleic acids encoding backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen c domain of the CAR targets Lyml. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding c acids encoding backbone-32 comprising conventional CAR II and K13- vFLIP, wherein the antigen speciﬁc domain of the CAR targets Lyml.

In one embodiment, 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 embodiments, nucleic acid sequence of the VL-VH targeting Lym2 is set forth in SEQ ID NO: 1380. In exemplary embodiments, the polypeptide sequence of the VL-VH targeting Lym2 is set forth in SEQ ID NO: 3125. In some embodiments, 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 ng ne-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 sing conventional CAR II and K13-vFLIP, wherein the antigen c domain of the CAR targets Lym2. Also provided herein are genetically engineered cells (such as T cells, NK cells) comprising vectors ng c acids encoding backbone-32 comprising conventional CAR II and K13- vFLIP, wherein the antigen speciﬁc domain of the CAR targets Lym2.

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- 32 comprising conventional CAR I and Kl3-vFLIP, wherein the antigen specific domain of the CAR targets GRP-78 (Table 20). In exemplary embodiments, the nucleic acid sequence of the VL-VH targeting GRP-78 is set forth in SEQ ID NO: 1348. In exemplary embodiments, the polypeptide sequence of the VL-VH targeting GRP-78 is set forth in SEQ ID NO: 3093. In some embodiments, the scFv fragment targeting GRP-78 is bed 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 ng backbone—32 comprising conventional CAR II and LIP, wherein the antigen ic domain of the CAR targets GRP—78. Further provided herein are vectors encoding nucleic acids encoding backbone-32 sing conventional CAR II and K13-vFLEP, 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 ng backbone-32 comprising conventional CAR II and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets GRP-78.

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- 32 comprising conventional CAR I and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets CD79b (Table 20). In exemplary embodiments, the nucleic acid sequence of the VL-VH targeting CD79b are set forth in SEQ ID NO: 1381. In exemplary embodiments, the polypeptide ce of the VL-VH targeting CD79b is set forth in SEQ ID NO: 3126. In some embodiments, 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 s CD79b. Further ed herein are vectors encoding nucleic acids ng backbone-32 comprising conventional CAR II and K13-VFLIP, wherein the antigen ic domain of the CAR targets CD79b. Also ed herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding backbone-32 comprising conventional CAR II and LIP, wherein the n speciﬁc domain of the CAR targets CD79b.

In one embodiment, provided herein is an isolated nucleic acid encoding backbone- ] comprising conventional CAR II and Kl3-vFLIP, wherein the antigen speciﬁc domain of the CAR targets Fc region of an immunoglobulin (Table 20). In exemplary embodiments, the sequences of the nucleic acid fragments targeting Fc region of an immunoglobulin are set forth in SEQ ID NOs: 1232-1233. In exemplary embodiments, the sequences of the polypeptide fragments targeting Fc region of an immunoglobulin are set forth in SEQ ID NOs: 2977-2978. In some embodiments, the receptor ing Fc region of an immunoglobulin ses, consists of or essentially consists of ellular -binding domain of CD16 (FCGR3A) or its variant V158 or its variant F158. Also provided herein are polypeptides encoded by nucleic acids ng backbone-1 comprising conventional CAR II and K13-vFLIP, wherein the antigen speciﬁc domain of the CAR targets Fc region of an immunoglobulin. r 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 s encoding nucleic acids encoding backbone-1 comprising conventional CAR II and Kl3-vFLlP, wherein the antigen speciﬁc domain of the CAR targets Fc region of an immunoglobulin. The genetically ered cells comprising vectors encoding nucleic acids ng backbone-1 comprising tional CAR II and LIP, n 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 e antibody based immunotherapy, such as cancer immunotherapy, as described in US 2015/0139943Al.

In exemplary embodiments, nucleic acids encode conventional CAR 11 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 ing antigens of interest as described in Table 2] 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: 475 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 on of these CAR constructs but may be used to select or enrich for cells expressing these CAR constructs.

In one embodiment, provided herein is an isolated nucleic acid encoding tional CAR II, wherein the antigen speciﬁc domain of the CAR targets CD19 (Table 21). In exemplary embodiments, the sequences of isolated nucleic acid fragments targeting CD19 are set forth in SEQ ID NOs: 1470-1481, 1772. In exemplary embodiments, the sequences of isolated ptide targeting CD19 are set forth in SEQ ID NOs: 3215-3225, 3517. In some embodiments, the scFv fragments targeting CD19 are described in Table 11 and set forth in SEQ ID NOs: 566-577 and SEQ ID NOs: 347. Also provided herein are polypeptides encoded by nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR targets CD19. Further ed herein are vectors encoding c acids encoding conventional CAR 11, 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 11, wherein the antigen speciﬁc domain of the CAR targets CD19.

In one embodiment, ed herein is an isolated c acid encoding tional CAR 11, wherein the antigen speciﬁc domain of the CAR targets MPL or the Thrombopoietin Receptor (Table 21). In exemplary embodiments, the sequences of isolated nucleic acid fragments targeting MPL are set forth in SEQ ID NOs: 1657-1664, 1731-1737.

In ary embodiments, the sequences of isolated polypeptide targeting MPL are set forth in SEQ ID NOs: 3402-3409, 3476-3484. In some embodiments, the scFv fragments ing MPL are descn'bed 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 11, wherein the antigen speciﬁc domain of the CAR targets MPL. Further provided herein are vectors encoding nucleic acids encoding conventional CAR 11, wherein the n 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 11, wherein the antigen specific domain of the CAR targets In one embodiment, ed herein is an isolated nucleic acid encoding tional CAR 11, wherein the antigen speciﬁc domain of the CAR targets Lyml (Table 21). In exemplary embodiments, the sequence of nucleic acid fragment ing Lyml is set forth in SEQ ID NO: 1649. In ary embodiments, the sequence of isolated polypeptide fragment targeting Lyml is set forth in SEQ [D NO: 3394. In some ments, the scFv fragment targeting Lyml is bed 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 11, wherein the antigen specific domain of the CAR targets Lym 1. Further provided herein are vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen speciﬁc domain of the CAR targets Lyml. Also provided herein are genetically engineered cells (such as T cells, NK cells) sing vectors encoding nucleic acids encoding conventional CAR 11, wherein the antigen specific domain of the CAR targets Lyml.

In one embodiment, 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 embodiments, sequence of the isolated nucleic acid fragment targeting Lym2 is set forth in SEQ ID NO: 1650. In ary ments, the sequence of isolated polypeptide targeting Lym2 is set forth in SEQ ID NO: 3395. In some embodiments, the scFv fragment targeting Lym2 is described in Table II 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 11, n the antigen speciﬁc domain of the CAR targets Lym2. Further provided herein are vectors encoding nucleic acids encoding conventional CAR 11, wherein the antigen specific domain of the CAR s 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 n specific domain of the CAR s Lym2.

In one ment, provided herein is an isolated nucleic acid ng conventional CAR 11, n the antigen speciﬁc domain of the CAR targets DLL3 (Table 21). In exemplary embodiments, the sequences of isolated nucleic acid fragments targeting DLL3 are set forth in SEQ ID NOs: 1314-1315. In exemplary embodiments, the sequences of isolated polypeptide targeting DLL3 are set forth in SEQ ID NOs: 3059-3060. In some embodiments, 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 ng conventional CAR II, wherein the antigen speciﬁc domain of the CAR targets DLL3. Further provided herein are vectors encoding nucleic acids encoding conventional CAR 11, wherein the antigen speciﬁc domain of the CAR s DLL3. Also provided herein are genetically engineered cells (such as T cells, NK cells) sing vectors encoding nucleic acids encoding conventional CAR [1, wherein the antigen speciﬁc domain of the CAR s DLL3.

In one embodiment, ed herein is an isolated nucleic acid encoding conventional CAR 11, wherein the antigen speciﬁc domain of the CAR targets CD324 (Table 21). In ary embodiments, the sequences of isolated nucleic acid fragments targeting CD324 are set forth in SEQ ID NOs: 1554-1555. In exemplary embodiments, the sequences of isolated polypeptide targeting CD324 are set forth in SEQ ID NOs: 3299-3300. In some embodiments, the scFv fragments targeting CD324 are described in Table II and set forth in SEQ ID NOs: 646-647 and SEQ ID NOs: 2416-2417. Also ed herein are polypeptides encoded by nucleic acids encoding conventional CAR 11, wherein the antigen speciﬁc domain of the CAR targets CD324. Further provided herein are vectors encoding nucleic acids ng conventional CAR 11, wherein the antigen c 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.

In one embodiment, provided herein is an isolated nucleic acid encoding conventional CAR 11, wherein the antigen speciﬁc domain of the CAR targets CD276 (Table 21). In ary embodiments, the sequence of nucleic acid fragment targeting CD276 is set forth in SEQ ID NO: 1553. In exemplary embodiments, the sequence of isolated polypeptide fragment targeting CD276 is set forth in SEQ ID NO: 3298. In some ments, the scFv fragment targeting CD276 is described in Table 11 and set forth in SEQ ID NO: 645 and SEQ ID NO: 2415. Also ed herein are polypeptides encoded by nucleic acids encoding conventional CAR II, wherein the antigen specific domain of the CAR s CD276. Further provided herein are vectors encoding c 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.

In one embodiment, provided herein is an ed nucleic acid encoding conventional CAR 11, wherein the antigen speciﬁc domain of the CAR s PTK7 (Table 21). In exemplary embodiments, the sequences of isolated nucleic acid fragments targeting PTK7 are set forth in SEQ ID NOs: 1683-1684. In exemplary ments, the sequences of ed polypeptide targeting PTK7 are set forth in SEQ ID NOs: 429. In some embodiments, 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 [1, wherein the antigen speciﬁc domain of the CAR targets PTK7. Further provided herein are vectors encoding nucleic acids ng conventional CAR II, wherein the n specific domain of the CAR targets PTK7. Also ed herein are genetically engineered cells (such as T cells, NK cells) comprising vectors encoding nucleic acids encoding conventional CAR II, wherein the antigen c domain of the CAR targets PTK7.

In one embodiment, provided herein is an isolated c acid encoding conventional CAR 11, wherein the antigen speciﬁc domain of the CAR s HIVl-envelop glycoprotein gp120 (Table 21). In ary embodiments, sequence of the isolated nucleic acid fragment targeting HIVI-envelop glycoprotein gp120 is set forth in SEQ ID NO: 1485.

In exemplary embodiments, the sequence of isolated polypeptide targeting HIVl-envelop glycoprotein gp120 is set forth in SEQ ID NO: 3230. In some embodiments, the scFv fragment targeting HIVl-envelop glycoprotein gp120 is described in Table 11 and set forth in SEQ ID NO: 581 and SEQ ID NO: 235]. Also provided herein are polypeptides encoded by nucleic acids encoding conventional CAR 11, wherein the antigen speciﬁc domain of the CAR targets HIVl-envelop glycoprotein gp120. Further provided herein are vectors encoding nucleic acids encoding conventional CAR 11, wherein the antigen speciﬁc domain of the CAR targets nvelop 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 HIV 1 op glycoprotein gp120.

In one embodiment, provided herein is an isolated nucleic acid encoding conventional CAR 11, wherein the antigen c domain of the CAR s nase- d peptides in ation with MHC class I complex (Table 21). In exemplary embodiments, sequence of the isolated nucleic acid fragments targeting tyrosinase-derived peptides in ation with MHC class 1 complex (HLA-A2) is set forth in SEQ ID NO: 1712-1714. In exemplary embodiments, the sequence of isolated polypeptides ing tyrosinase-derived peptides in association with MHC class I complex (PEA-A2) is set forth in SEQ ID NO: 3457-3459. In some embodiments, the scFv nts targeting tyrosinase- derived peptide in association with MHC class II (HLA-AZ) complex is described in Table 11 and set forth in SEQ ID -782 and SEQ ID NO: 2550—2552. Also provided herein are polypeptides encoded by nucleic acids ng conventional CAR 11, wherein the antigen specific domain of the CAR targets tyrosinase-den'ved peptides in association with MHC class I complex (HLA-A2). Further provided herein are vectors ng nucleic acids encoding conventional CAR 11, wherein the antigen speciﬁc domain of the CARS target tyrosinase-den'ved 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 11, wherein the antigen specific domain of the CAR targets target tyrosinase-derived peptides in association with MHC class I complex (HLA-A2). In exemplary embodiment, 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).

In some embodiments, 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 embodiments, the vector is any of a DNA vector, an RNA vector, a plasmid, a lentivirus , an adenoviral vector or a retrovirus vector. In an embodiment, the vector is a lentivirus vector as bed herein. In an embodiment, the vector is pLENTI-EFla (SEQ ID NO: 905). In another embodiment, the vector is pLenti-EFla-DWPRE (SEQ ID NO: 906). In r embodiment, the vector is MSCV-Bng-AvrII-Bam-EcoRl-Xho—BstB Sal-ClaI.IO3 (SEQ ID NO: 907). In another embodiment, the vector is a sleeping beauty transposon . In another embodiment, the sleeping beauty transposon vector is pSBbi-Pur (SEQ ID NO: 908). In one embodiment, the vector further comprises a promoter. Non-limiting examples of a promoter include from an EF-l promoter, a CMV IE gene promoter, an EF-la promoter, an ubiquitin C promoter, or a phosphoglycerate kinase (PGK) promoter. In one embodiment, the promoter is an EF-l promoter (SEQ ID NO: 909). In one embodiment, 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 111 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 terone inducible promoter, and a tetracycline inducible promoter. RheoSwitch‘é: system (Agarwal et al, 2012, AACR-NCI- EORTC international conference on Molecular Targets and Cancer Therapeutics) represents another transcriptional regulator platform for controlling the sion of tional CARS and nes l-62 of the present invention. In one embodiment, a combinatorially activated T cell circuit can be engineered in which a synthetic Notch receptor for one n induces the expression of a conventitonal CAR or backbones 1-62 targeting a second n as described recently for controlling the ty of CARS (Roybal KT et a/., Cell, 164: 1—10 2016). In one embodiment, the vector is an in vitro transcription vector, (for example, a vector that nbes RNA from a DNA molecule) described herein. In one embodiment, the c acid ce in the vector r comprises a poly(A) tail, e.g., a poly A tail bed herein, e.g., comprising about 100-150 adenosine bases. For example, plated herein is a poly(A) tail sing about 150 adenosine bases (SEQ ID NO: 922). In one embodiment, 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.

In some embodiments, the vector comprising a nucleic acid ce encoding a CAR may further comprise a nucleic acid sequence encoding one or more inhibitory molecules. Non-limiting examples of inhibitory molecules contemplated herein e: 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 asmic domains of LAILRl 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 LAILRl (SEQ ID NOS: 849 and 2612). Such inhibitory CARS can be expressed alone or in combination with the activating CARS, such as the CARS bed in Tables 19-22.

In some embodiments, 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"" M or 10'7 M, for the target antigen.

Also provided herein are genetically engineered cells e.g., an immune effector cell, (e.g., a tion 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 d stem cell, an immune stem cell, an induced pluripotent stem cell or iPSC) comprising c acids, polypeptides or vectors as described herein.

In one embodiment, the cell is a human T cell. In some embodiments, 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 clear cells, bone marrow, lymph node , 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 embodiments, the cell is a mammalian cell. In some embodiment, the cell is a human cell. In some embodiments, the cell is a dog cell. In some embodiments, the cell is a monkey cell.

In one embodiment, 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 ion may be produced using a, variety of approaches such as use of Zn ﬁnger nucleases (ZFN), CRISP/Cas9 and shRNA ing the endogenous T cell receptor chains. A non-limiting exemplary method relating to shRNAs is described in US 2012/0321667 A1. r non-limiting exemplary method ng to eliminating endogenous TCR expression using ZFNs targeting constant regions of a and B chains of TCRs has been described (Torikai H et al.,Blood , 2012).

In one embodiment, the cell is a stem cell and the stem cell is deficient in one or more of endogenous T cell receptor chains. In another embodiment, 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, 21 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 nous 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 ated by the T cells expressing the CD l9-targeting CAR. In another non-limiting example, the CAR is expressed in immune effector cells and the stem cells from an autologous or an neic 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 gous 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 gous 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 ate 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 ns 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 nt 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 rgeting CAR. In another non-limiting example, a CAR targeting MPL (e.g., SEQ ID NO: 1657-1660) is sed in stem cells in which the endogenous MPL gene has been mutated using CRISP/Cas9 or Zn ﬁnger ses 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 d into a patient along with autologous or neic hematopoietic stem cells in which the endogenous MPL gene has been mutated using Cas9 or Zn finger nucleases to a form that is not ed by CAR. In an exemplary embodiment the region of MPL gene that is targeted for mutation by CRISP/Cas9 or Zn finger ses to a form that is not targeted by MPL-directed CAR encodes for the peptide sequence —PWQDGPK—.

In various embodiments, 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 embodiments, the CARS comprising more than one ASD target the same n on the diseased-causing or disease-associated cells. In another embodiment, the CARs comprising more than one ASD target different antigens on the diseased-causing or disease-associated cells.

In some embodiments, the genetically engineered cells further comprise nucleic acids, polypeptides or vectors that encode accessory modules. In some embodiments, the accessory modules reduce or t toxicity associated with CARS. In some embodiments, the accessory s 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 1L6, ILIO and IFNy can be targeted. In some embodiments, 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. miting examples of such accessory modules include H.6R- 304-vHH (SEQ ID N05: 884 and 2647), IGHSP2-1L6RVHH-ALB8-VHH (SEQ ID N03: 886 and 2649), CDSSP2-PD1-4Hl-scFv (SEQ ID N03: 889 and 2652), CD8SP2- PD1-5C4-scFv CD8SP2-CTLA4-Ipilimumab-scFv (SEQ ID N03: 891 and 2654), CD83P2- PD1-4Hl-Alb8-VHH (SEQ ID N03: 892 and 2655), CD88P2-PD1-5C4-Alb8-VHH (SEQ ID N05: 893 and 2656), CDSSP2-CTLA4-Ipilimumab-Ale-VHH (SEQ ID N05: 894 and 2657), IgSP-IL6-19A-SCFV (SEQ ID NOS: 899 and 2662), CD8SP2-SHVEM (SEQ ID N05: 901 and 2664), CD8SP2-sHVEM-Alb8-VHH (SEQ ID N05: 902 and 2665), hTERT (SEQ ID N05: 903 and 2666), Heparinase (SEQ ID N05: 904 and 2667), ILIZF (SEQ ID N05: 863 and 2626), 41BB-L (SEQ ID N05: 864 2627), CD40L (SEQ ID N05: 865 2628) and shRNA ing 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-EFIa-shRNA-BRD4-DWPRE (SEQ ID NO: 920).

An exemplary CAR in backbone l coexpressing shRNA targeting Brd4 is pLenti-EFla- CD8SP-FMC63-(vL-vH)—Myc-z-P2A-K 1 3-Flag-T2A-PAC-H l -prom-shRNA-BRD4- DWPRE (SEQ ID NO: 921).

Capillary leak syndrome is r major complication of CARS. A natural plasmin digest t of ﬁbrin, peptide BB 1 5-42 (also called FX06), has been shown to signiﬁcantly reduce vascular leak (Groger et al., PLoS ONE 42e539l, 2009). FXO6 peptide has been shown to block capillary leak. In some embodiments, as described herein, FX06 (SEQ ID N05: 900 and 2663) can be essed 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.

A limitation of the current technology for ve cellular therapy with T cells is the limited life span of cally modiﬁed T cells, such as CAR-T cells, when infused into patients. To extend the life span of genetically-modiﬁed T cells, ed herein are vectors expressing CARS in conjunction with a number of viral and cellular proteins that stimulate the eration 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 s (e.g., CD8, CD4, TILs), T cells directed against speciﬁc tumor antigens or infectious agents, T cells expressing an exogenous T cell or (i.e., TCR gene therapy), and T cells expressing a synthetic or chimeric T cell or. 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, tively (Table 16). In exemplary embodiments, these proteins include any one or more of viral ns K13-vFLlP (SEQ ID NOs: 866 and 2629) andMClS9-VFL1P (SEQ ID NOS: 867 and 2630) as described herein. In further embodiments, these proteins include cFLIP- L/MRIT-alpha (SEQ ID NOS: 873 and 2636) and cFLIP-p22 (SEQ ID NOS: 874 and 2637).

In further embodiments, these proteins include any one, two or more of Tax and Tax-mutants from human T cell leukemia/lymphoma virus (HTLV-I and HTLV-2) (SEQ ID N05: 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 r embodiment, these viral and cellular ns can be used to promote the survival and proliferation of any T cell, including those expressing an exogenous T cell or or synthetic T cell receptor.

In one embodiment, 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 embodiment, the viral and ar 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 d to a metallothionine inducible promoter, a glucocorticoid inducible promoter, 3 progesterone inducible promoter, and a tetracycline inducible promoter. RheoSwitch"? system represents another transcriptional regulator platform for controlling the expression of CAR of the present ion al et al, 2012, AACR-NCI-EORTC international conference on Molecular Targets and Cancer Therapeutics). In another embodiment, 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 s include FKBP (SEQ ID NOs: 8 and SEQ ID N05: 2620 and 2621), FKBPx2 (SEQ ID N05: 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: 2 and SEQ ID NOs: 2641 to 2645 (Table 16). The DNA and protein sequences of ary CAR constructs coexpressing a viral protein in fusion with FKBP dimerization domain(s) are provided in SEQ ID N05: 1755 to 1758 and SEQ ID N05: 3500 to 3503. r, a Src tor can be used to prevent and/or treat the toxicity associated with the administration of CAR-expressing cells. In some embodiments, 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).

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 c acid ng 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 tional 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 ones 1-62), a therapeutically effective amount of genetically ed cells, as described herein, comprising c 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 embodiment, the nucleic acids, polypeptides, vectors and cally modiﬁed cells comprise a conventional CAR II. In one embodiment, the nucleic acids, polypeptides, vectors and genetically modified cells comprise backbone-1, comprising a conventional CAR I and K13-vFLIP. In one ment, the nucleic acids, polypeptides, vectors and genetically ed cells comprise ne-32, sing a conventional CAR II and K1 3-vFLIP. In one embodiment, the disease treated or prevented by CAR is a cancer. In other embodiments, the diseases treated or ted by CARS are infectious diseases, allergic diseases, autoimmune diseases, a degenerative diseases, or a combination thereof.

In one embodiment, 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 f, comprising administering to the subject a therapeutically effective amount of genetically modiﬁed cells comprising nucleic acid encoding one or more conventional CARS.

In one embodiment, the genetically modified cells comprise vectors encoding a conventional CAR 1] as described herein. In one embodiment, ed herein is a method for treating, inhibiting, reducing the severity of, preventing metastasis of and/or reducing the relapse of cancer in a t in need f, comprising administering to the subject a therapeutically effective amount of cally modiﬁed cells sing nucleic acid encoding one or more CARS and at least one accessory module (backbones 1-62). In one embodiment, the genetically modiﬁed cells se vectors encoding backbone-l, comprising a tional CAR I and K13-vFLIP as described herein. In one embodiment, the genetically d cells comprise vectors encoding backbone-32, sing a conventional CAR II and K13- vFLIP as described herein. In some embodiments, the vectors encoding CARS target two, three or more antigens on cancer cells. In one embodiment, the disease treated or prevented by administration of genetically modiﬁed cells comprising nucleic acid encoding one or more CARS is a cancer. In other embodiments, 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.

] In exemplary embodiments, 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, ma, bone cancer, pancreatic cancer, skin , cancer of the head or neck, cutaneous or intraocular malignant melanoma, e 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 , oma 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, y effusion lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, moid cancer, squamous cell cancer, T-cell ma, environmentally induced cancers, combinations of said cancers, and metastatic lesions of said cancers.

] Also provided herein are methods for treating, inhibiting, reducing the ty 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, n the genetically modiﬁed cells comprise vectors encoding a conventional CAR II as described herein.

Also provided herein are methods for treating, inhibiting, reducing the ty of, preventing metastasis of and/or reducing the relapse of logical cancer in a subject in need thereof comprising administeﬁng to the subject a therapeutically effective amount of genetically modiﬁed cells, as described herein, wherein the genetically d cells comprise vectors encoding backbone-1, comprising conventional CAR 1 and K13-vFLIP or vectors encoding backbone-32, comprising CAR II and Kl3-vFLIP. In one embodiment, the ASD of the CAR targets MPL. In exemplary embodiments, conventional CAR II targeting MPL are described in Table 21 and 22 and se nucleic acid sequences set forth in SEQ ID NOs: 664 and 1731-1739 and comprise polypeptide ces set forth in SEQ ID NOs: 3402-3409 and 3476-3484. In exemplary embodiments, conventional CAR I targeting MPL and coexpressing K13-vFLIP (backbone 1) are described in Table 19 and se 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 embodiments, conventional CAR II targeting MPL and essing K13-vFLIP one 32) are described in Tables 20 and 22 and comprise c 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.

Also provided herein are methods for treating, ting, reducing the severity of, preventing metastasis of and/or reducing the e of cancers expressing CD 19 (e.g., B cell acute lymphocytic ia (B-ALL), chronic lymphocytic leukemia, diffuse large cell lymphoma, nt ma) 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.

Also provided herein are methods for treating, inhibiting, reducing the severity of, preventing asis of and/or reducing the relapse of a CD19 expressing cancer in a subject in need f comprising administering to the subject a therapeutically effective amount of genetically modiﬁed cells, as described herein, wherein the genetically modiﬁed cells comprise s encoding backbone-l, comprising conventional CAR I targeting CD19 and Kl3-vFLIP or vectors encoding backbone-32, comprising CAR II targeting CD19 and K13- vFLIP. In exemplary embodiments, 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 ces set forth in SEQ ID NOs: 3215-3225. In exemplary embodiments, conventional CAR I targeting CD19 and coexpressing Kl3-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 ary embodiments, conventional CAR II targeting CD19 and coexpressing LIP (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.

Also provided herein are methods for treating, ting, 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 eutically effective amount of genetically modiﬁed cells, as described herein, wherein the genetically modiﬁed cells comprise vectors encoding ne- l conventional CAR I and K l3-vFLIP , sing or vectors encoding ne-32, comprising CAR II and LIP. In one embodiment, the ASD of the CAR targets GRP-78. In exemplary embodiments, 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.

In s embodiments, additional antigen targets, CAR constructs and accessory modules for use with the instant invention will be nt to a person of skill in the art.

Exemplary embodiments are set forth in Tables 2-22.

In some embodiments, the therapeutically effective amount of the genetically modiﬁed cells is administered at a dosage of 104 to 109 kg body weight, in some instances 105 to 106 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 on techniques that are ly 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., tumoral injection).

In some embodiments, the therapeutic methods described herein further comprise administering to the subject, tially or simultaneously, ng 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 urgery (gamma knife), and fractionated stereotactic radiotherapy (FSR)), focal y, systemic therapy, vaccine therapies, viral therapies, molecular targeted therapies, or combinations thereof.

In some ments, 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.

In one embodiment, the kinase inhibitor is a SI'C family kinase inhibitor, such as dasatinib. in one embodiment, the kinase inhibitor is a CDK4 inhibitor, such as (IT'DKMG inhibitors, ed from ribociciib, abemaciciib and palbociciib.

In one embodiment, the kinase inhibitor is an mTOR inhibitor, for example. rapamycin, a rapamycin analog, OSI~027. The m'I'OR inhibitor can be, for example, an mTORCl inhibitor and/or an mTORC'ZZ inhibitor. in one embodiment, the kinase inhibitor is a MNK inhibitor, for example, 4—amino— —i4—iluoroanilino_)—pyrazolo 13,441] dine. The MNK, inhibitor can be, for example, an tor of MNKEa, MNKlb, MNKZa aridz’or MNKZb. The dual PI3K/mTOR inhibitor can be, eg, PF-04695102. in one ment, the inhibitor is a Brd4 inhibitor selected from JQl, M8437, OTXOIS, LY 303511 and Brd4 tor as bed in US 20140256706 Al and any analogs thereof. in one embodiment of the methods described herein, the kinase inhibitor is a CDK4 inhibitor, eg, iclib (P00332991), and the palbociciib is stered at a dose of about 50 mg, 60 mg, 7t:I mg, 75 mg, 80 mg, 90 mg, 100 mg, 105 mg, 110 mg, HS mg, 120 mg, 125 mg, 130 mg, 135 mg (eg, 75 mg, 100 mg or 125 mg) daily for a period ot‘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 embodiment, l, 2, 3, 4, 5, 6, 7, 8, ‘3), 10, ll, 12 or more cycles of palbociclib are administered.

In one embodiment of the methods described herein, the kinase inhibitor is a BTK inhibitor selected from ibmtinib (PG—32765); CDC-0834, R.N~486; CGI~560; GUI—1764‘, HM~71224; CCn292; ONO-4059; CNX-774; and LFM—All in one embodiment. the BTK inhibitor does not reduce or inhibit the kinase activity of interleukin— 2—inducibie kinase (ITK), and is ed from 34; RN-486; (Iii-560; CGl-l764; HM-"l 224; ETC-292.; ONO-40.59; CNK-77I-‘l; and LF‘M-A l 3. in one embodiment of the methods or uses described herein, the kinase inhibitor is a B’I‘K inhibitor, e.g., ibmtinib (PCI~32765), and the ibmtinib 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, 5.20 mg, 540 mg, 560 mg, 580 mg, 600 mg (eg, 250 mg, 420 mg or 560 mg) daily for a period of time, e.g., daily for 21 day cycle, or daiiy for 28 day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, IO, 1 1, 12 or more cycles ot‘ibrutinib are administered. ] in one embodiment of the methods or uses bed herein, the kinase inhibitor is a BTK inhibitor that does not inhibit the kinase activity of ITK, e.g., Rid-486, and RN- 486 is administered at a. dose of about 100 mg, no mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, l80 mg, 190 mg, 200 mg, 210 mg, 220 m 2, 230 mg, 240 mg, 250 mg (eg, 150 mg, 200 mg or 250 mg) daily for a period of time, eg daiiy a 28 day cycle. in one embodiment, 1, 2, 3, 4, 5, 6, 7, or more cycles of BIN-486 are administered. in one embodiment of the s described herein, the kinase inhibitor is an mTOR inhibitor selected from temsirolimns; ridaforoiimus (lR,2R,4S)—4-[(2R)—2 [(lR,95,l 2S,l 5R,i6E,18R,19R,Zl R, 23S,24E,26E,282’.,308,32$,3 5R)~1,l 8—dihydroxy— l9,30- dimethoxy-l 5, i 7,2 t ,23, hexamethyl-2,3, l O, 14,20-pentaoxo-i 1,36-dioxa cyclo[30.3.l .0] hexatriaconta—l 6,24,26,28—tetraen—l 2~yl}propyl]~2— methoxycyclobexyl dimethyiphosphinate, also known as Al’23573 and MK8669; everolimus (RADOOl); rapamycin (AY22989); simapimod, (5—{2,4—bis[(35")~3~ methylmorpholin~4~yl]pyrido[2,3- (i]pyrimidin—7-yl }methoxyphenyl nol OSS); 2—smino—8-[iran5-4—( 2- hydroxyethoxy)cyclohexyl](6-methoxy-3— pyridinyl)methyl-pyridoiZj ~3 pyrimi di 11- 7(SI-I)~one (PF04691502); and Nz—[l,4«dioxo- 4—[{4~(4~ox0phenyl-4I-I-l«benmpyi‘amz- yl)morpholinium—él-yl]methoxy]butyl]-L- arginylglycyl—L-a—aspartylE..-serine-, inner salt (SFl126); and X34765.

In one embodiment of the methods or uses described herein, the kinase inhibitor is an mTOR inhibitor, e.g., rapamycin, and the cin 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 embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, , 11, 12 or more cycles of cin are administered. In one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g., everolimus and the everolimus is administered at a dose ofabout 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 embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of everolimus are administered.

] Also ed 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 embodiments, 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 embodiments, the cells are autologous human T cells or autologous human NK cells or autologous human stem cells. In some embodiments, the cells are allogeneic human T cells or allogeneic human NK cells or allogeneic human Stem cells.

In one embodiment, the method for preparing the genetically modiﬁed cells comprise obtaining a tion of cells and depleting the cells of the CD25+ T tory 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 embodiments, the Treg depleted cells comprise less than 30%, 20%, 10%, 5% or less Treg cells. In some embodiments, the vectors encoding the CARS described herein are transfected into Treg depleted cells.

In some embodiments, prior to ection, the Treg depleted cells are further depleted of cells which express checkpoint inhibitors. The checkpoint inhibitor may be any one or more of PD-l, LAG-3, T1M3, B7-Hl, CD160, P 1H, 2B4, CEACAM (e.g., CEACAM- 1, CEACAM-3, and/or CEACAM-S), TIGIT, CTLA-4, BTLA, and LAIR]. In some embodiments, the cells comprise less than comprise less than 30%, 20%, 10%, 5% or less cells that express checkpoint inhibitors. In some embodiments, the vectors encoding the CARS described herein are ected into Treg depleted and checkpoint tor depleted cells.

In some embodiments, methods for ing the genetically modiﬁed cells comprise ing a population of cells and selecting cells that express any one or more of CD3, CD28, CD4, CD8, CD45RA, and/or CD45RO. In certain embodiments, the tion of immune effector cells provided are CD3+ and/or CD28+.

In one embodiment, the method for preparing the genetically modiﬁed cells comprise obtaining a population of cells and enriching for the CD25+ T tory cells, for e 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 at. In some embodiments, the Treg enriched cells comprise less than 30%, 20%, 10%, 5% or less non- Treg cells. In some ments, 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.

In some embodiments, 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 ments, the population of cells is expanded for a period of 8 days or less. In certain embodiments, the population of cells is expanded in culture for 5 days, and the resulting cells are more potent than the same cells ed in culture for 9 days under the same culture conditions. In other embodiments, 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 n stimulation as compared to the same cells expanded in culture for 9 days under the same e conditions. In some embodiments, the population of cells is expanded in an appropriate media that includes one or more interleukins that result in at least a ZOO-fold, 250-fold, 300— fold, or 350-fold increase in cells over a 14 day expansion period, as measured by flow cytometiy.

In various embodiments, the expanded cells comprise one or more CARs as described herein. In some embodiments, the expanded cells comprise one CAR with one, two, three or more ASDs. In some embodiments, the expanded cells further comprise accessory modules and therapeutic controls as described herein. [00l28] Provided herein is a cell comprising c acids encoding a ic antigen or (CAR) and one or more of signaling proteins selected from Kl3-vFLIP, MC159- vFLIP, cFLIP-L, cFLIP-p22, HTLVl-Tax and HTLVZ-Tax wherein the CAR comprises an a) extracellular antigen speciﬁc domain, b)a transmembrane domain and c) an intracellular ing domain comprising an immunoreceptor tyrosine-based activation motif (ITAM); wherein c) is located at the C-terminus of the chimeric or. In some embodiments, the CAR further comprises one or more co-stimulatory s.

] Also provided herein is a cell comprising nucleic acids encoding a chimeric antigen receptor (CAR) and Kl3-vFLIP signaling protein, n the CAR comprises an a) extracellular n speciﬁc domain, b) a transmembrane domain and c) an intracellular signaling domain comprising an receptor tyrosine-based activation motif (ITAM); wherein c) is located at the C-terminus of the chimeric receptor. In some embodiments, the CAR r ses one or more co-stimulatory domains.

Also provided herein is a cell comprising nucleic acids encoding a chimeric antigen receptor (CAR) and MC 159-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 embodiments, the CAR r comprises one or more co—stimulatory domains.

In some ment, the cell described herein further comprises nucleic acids encoding MC159-vFLIP signaling protein.

In some embodiments, the cell further comprises nucleic acids encoding scFvs targeting E6 and/or 1L6 receptor alpha. In some embodiments, the cell further comprises nucleic acid encoding peptide FXO6 so as to mitigate capillary leak associated with CAR therapy. In some embodiments, the signaling protein is expressed in fusion with one or more copies of FKBP domain. In some embodiments, the activity of the signaling protein is controlled post-translationally by dimerization of the FKBP domain in the presence of a zing agent. In some embodiments, the dimerizing agent is AP20187.

In an exemplary embodiment, n the antigen speciﬁc domain of the CAR targets MPL. In some embodiments, the antigen c domain s two antigens, wherein the two antigens are MPL and CD123.

In some embodiments, the antigen speciﬁc domain of the CAR targets CD19, CD23, Lyml, Lym2, , CDH179b, FLT3, GCC, Mucl, CSF2RA, GFRa4, CD32, ILl lRa, IL13Ra, NYBRI, SLea, CD200R, TGFBetaR2, CD276, TROP2, LAMP], PTK7, DLL3, CDHl, CD1-I6, CDH17, CDH19, TSHR and tyrosinase.

In some embodiments, the antigen speciﬁc domain of the CAR targets MPL and comprises one or more scFv fragments selected from 161 (SEQ ID NO: 2500 and 250]), 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 huVBZZBWS (SEQ ID NO: 2506) or ligands selected from extracellular receptor binding domains of hTPO (SEQ ID NO: 2323) or mTPO (SEQ ID NO: 2323).

In some embodiments, the antigen speciﬁc domain of the CAR targets CD19 and comprises one or more scFv fragments selected from CD19Bu12 or CD 1 9MM.

] In one embodiment, the cell is a T-Iymphocyte (T-cell). In another embodiment, the cell is a Natural Killer (NK) cell.

Also provided herein are nucleic acids comprising a ﬁrst polynucleotide encoding the CAR of claim and a second polynucleotide encoding the LIP signaling protein. In some embodiments, 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.

Further ed herein is a pharmaceutical composition sing the cells described herein, the nucleic acids described herein, the polypeptides encoded by the c acids described herein or vectors comprising the nucleic acids described herein, and a ceutically acceptable carrier.

In some embodiments, provided herein is a method for treating a MPL-expressing cancer comprising stering to the subject, a therapeutically effective amount of the cell described herein, wherein the CAR targets MPL. In some embodiments, the cancer is a blood , wherein the blood cancer is any one or more of acute myeloid leukemia, chronic myeloid leukemia, myelodyplastic syndrome, lymphoma, multiple myeloma and acute cytic leukemia. In some embodiments, the method further comprises administering to the t a tyrosine kinase inhibitor, wherein the inhibitor ts the Src family of kinases.

In some embodiments, the inhibitor inhibits Lck. In some embodiments, the inhibitor is any one or more of Dasatinib, Ponatinib or A-77004l.

BRIEF DESCRIPTION OF THE DRAWINGS ] 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 ments of the ion, 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— 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 s; Fig. 28: 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 s embodiments of the invention, strong binding with l6l-GGSG-NLuc-ACV5 was observed on HEL.92.1.7-Gluc- vector cells ting significant sion 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 CDSSP-FMC63-(vL-vH)-Myc-BBz-T2A- 2014-A13)[SEQ ID 7] and CDSSP-MPL-lé1-(vL-vH)-Myc-BBz-T2APAC (021015-R07)[SEQ ID NO:1658] CAR were incubated with FLAG-CDl9-ECD-GGSG- NLuc-AcV5[SEQ ID NO: 926] and MPL-ECD-GGSG-NLuc-AcV5[SEQ ID NO: 925]supematants as described in material and methods section followed by washing with PBS and measurement of NLuc activity by Coeleoentrazine (CTZ; Nanolight) diluted in PB S.

Luminescence was quantiﬁed using a BioTek plate reader. Data ents mean values of triplicate wells +/- standard deviation (SD).

Fig. 5 depicts, in accordance with s ments of the ion, modest binding of MPL-ECD-GGSG-NLuc-AcVS to 293FT cells ected with hTPO-(l- 187)-Myc-CD28z-T2A-PAC(O31915-UO4)[SEQ 1r) NO:1738] and mTPO-(l-187)-Myc- CD28z-T2A-PAC(O31915-V03)[SEQ ID NO:1739] constructs and strong binding to 293FT cells transfected with MPL-l6l-(vL-vH)-Myc-BBz-TZA-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 ion, sion 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 (Ul).

Fig. 7 depicts in accordance with various embodiments, of the invention, strong binding of T cells expressing CDSSP-MPL(vL-VH)-Myc-BBz-T2A- PAC(021015-R07)[SEQ ID NO:1658], CDSSP(vL-vH)-Myc-CD28z—T2A- PAC(O31615-QO4)[SEQ ID NO:1733], and CD8SP-MPL-VB22Bw4-(vL-vH)-Myc-CD282- T2A-PAC(031615-B06)[SEQ ID NO:3536] CARS and modest binding of T cells expressing CD8SP(vL-vH)-Myc-CD282-T2A-PAC(021715-ZO7)[SEQ ID NO:1731], CD8SP- AB3l7-(vL-vH)-Myc-CD28z-T2A-PAC(O3I615-T04)[SEQ ID NO:1735] and CD8SP- 12El0-(vL-VH)-Myc-CD282—T2A-PAC(031615—SO3)[SEQ ID 6] to MPL—GGSG- NLuc AcVS supernatant as measured by NLuc assay. SEQ ID NO. 925 comprises MPL- ECD-GGSG-Nluc-AcVS. SEQ ID NO.: 926 comprises FLAG-CDl9-ECD-GGSG-NLuc- AcV5. SEQ ID NO: 1734 comprises CD8SP(vL-vH)-Myc-CD282—T2A-PAC(031615- R04). SEQ ID NO: 3534 comprises CD8SP-4C3-(vL-vH)-Myc-CD28z-T2A-Pac(03l6]5- Fig. 8 depicts, in accordance with various embodiments of the invention, increase in GLuc activity ing co-culture with T cells expressing MPL-specific CDSSP- 161-(vL-vH)-Myc-CD8'I‘M-BBz-T2A-EGFP(O908l4-CO3)[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, sed killing of I-IEL-GLuc cells by CDSSP-MPL-léI-(vL-vH)-Myc-BBz-T2A- PAC(021015-RO7)[SEQ ID 8] CAR expressing PBMC as compared to FMC63-(vL- VH)-Myc-BBz-T2A-PAC(I l2014-A13)[SEQ ID NO:1467] PBMC or uninfected PBMC.

Fig. 10 depicts, in accordance with an embodiment of the invention, increased g of HEL-GLuc cells by CDSSP(vL-vH)-Myc-CD282-T2A-PAC(O31615- Q04)[SEQ ID NO:1733] CAR sing PBMC as compared to cted 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-l6l-(vL-vH)-Myc-CD282—T2APAC (021715-ZO7)[SEQ ID NO:1731], CD8SP- 1 75-(vL-vH)—Myc-CD282-T2A- PAC(031615-Q04)[SEQ ID NO:1733], and AB317-(vL-vH)-Myc-CD28z-T2APAC (031615-T04)[SEQ ID NO:1735] constructs and modest with CD8SP-l78-(vL-VH)- Myc-CDZSz—TZA-PAC(031615-R04)[SEQ ID NO:1734] construct as compared to the cted T cells.

Fig. 12 s, 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 CDSSP-l61-(vL-vH)—Myc-CD28z- T2A-PAC(021715-ZO7)[SEQ ID NO:1731], CDSSP(vL-vH)-Myc-CD28z-T2A- PAC(031615-Q04)[SEQ ID 3 , CD8SP-AB3l7-(vL-vH)—Myc-CD282-T2APAC (031615-TO4)[SEQ ID NO:1735], and CD8SP-12ElO-(vL-vH)-Myc-CD28z-T2A- 1615-SO3)[SEQ ID NO:1736] constructs and modest with CDSSP-l78-(vL-VH)- Myc-CDZSz-T2A-PAC(O31615-R04)[SEQ ID NO:1734] and CD8SP-VB22Bw4-(vL-VH)- Myc-CD28z-T2A-PAC(O31615-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 CDSSP-I 6| -(vL-vl-l)-Myc-CD8TM-BBz-T2A-EGFP(O908 l 4- 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, ing co-culture with NK92MI cells expressing MPL-specific CARS.

Fig. 15 depicts, in accordance with an embodiment of the invention, increased secretion of TNFa 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(vL-vH)-Myc-CD8TM-BBz-T2A-EGFP(090814- CO3)[SEQ ID N011732] 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(l008l4- KO6)[SEQ ID N023537] CAR (28 days; .

Fig. 17 depicts, in accordance with an embodiment of the invention, sed 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 (Ul; shown with dotted . Constructs depicted include CD8SP-FMC63-(vL-vH)-Myc-BBz—T2A- PAC(112014-A13)[SEQ ID NO: 1467]; CDSSP-CD19Bul2-(vL-vH)-Myc-BBz-T2A- PAC(082815-P08)[SEQ ID NO:1470]; CDSSP-Z-CDI9lvﬂVI-(vL-vH)-Myc-BBz-T2A- PAC(O629] 5-D03)[SEQ ID NO: 1471]; CD8SP-KSHV—4C3-(vL-vH)-Myc-BBz-T2A- PAC(042315-N01)[SEQ ID NO: 1643].

] Fig. 18 s, in ance with various embodiments of the invention, strong g of Jurkats expressing CD8SP-CD19BulZ-(vL-vH)-Myc-BBz-T2A- PAC(082815-P08)[SEQ ID 0], CD8SPCD19MM-(vL-vH)-Myc-BBz-T2A- PAC(O62915-D03)[SEQ ID 1] and CDSSP-FMC63-(vL-vH)-Myc-BBz-T2A- PAC(l lZOl4-Al3)[SEQ ID NO:1467] CAR ucts to FLAG-CDl9-ECD-GGSG-NLuc- AcVS [SEQ ID 1] supernatant, while no signiﬁcant binding was observed on parental cells or those expressing the KSHV-4C3-(vL-vH)-Myc-BBz-TZA-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(O82815-PO8)[SEQ ID NO:1470], CD8SPCDl9MM-(vL-VH)-Myc-BBz-T2A- PAC(062915-D03)[SEQ ID NO:1471] and CD8SP-FMC63-(vL-vH)-Myc-BBz-T2A- PAC(1 12014-A13)[SEQ ID NO:1467] CAR ucts to FLAG-CDl9-ECD-GGSG-NLuc- AcVS [SEQ ID NO:2671] supernatant, while no significant binding was observed on parental cells or those sing CD8SP-KSI-IV-4C3-(vL-vH)-Myc-BBz-TZA-PAC(O42315- EQ 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 CDl9-speciﬁc CARS as compared to uninfected T cells (T-Ul) or those expressing the control CAR 4C3. Constructs depicted include FMC63-(vL-VH)- z-TZA-PAC(112014-A13)[SEQ ID NO:1467]; CD8SPCD19l\/ﬂ\/I-(vL-VH)-IVch- A-PAC(062915—D03)[SEQ 1D NO:1471]; CDSSP-CDI9Bu12-(vL-vH)—Myc—BBz— T2A-PAC(0828|5-P08)[SEQ ID NO:l470]; CD8SP-KSHV-4C3-(vL-vH)-Myc-BBz—T2A- PAC(O42315-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 ture 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 CD88P-FMC63-(vL-VH)- Myc-BBz-TZA-PAC(112014-A13)[SEQ ID NO:1467]; CD8SPCD]9MM-(vL-vH)-Myc- BBz-T2A-PAC(062915—D03)[SEQ ID NO:1471]; CD8SP-KSHV-4C3-(vL-vH)—Myc—BB2- T2A-PAC(O42315-N01)[SEQ ID NO:1643]; (-) indicates CAR-transduced T cells were used t 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 dy 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-TZA-PAC(l12014-A13)[SEQ ID NO:]467], and CD8SP-FMC63-(vL-vH)-Myc-BBz-P2A-K 1 3-Flag-T2A-PAC(1 1 1014-Y1 1)[SEQ ID N01 197] expressing s that have been selected with cin induce speciﬁc lysis of RS4] 1 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 ance with various embodiments of the invention, s that incorporation of K13, MC159 or 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 MC 1 59 and I-ITLVZ-Tax continue to exert cytotoxicity after 2 months in culture while the T cells expressing the constructs lacking MC 1 59 and HTLV2-Tax lost this ability.

Fig. 28A-Fig. 28B depict, in accordance with various ments of the invention, treatment with AP20187 led to se in K13- and HTLVZ-Tax RS mutant- d NF-KB-Luc activity in all constructs in which K13 and Tax mutant were expressed in fusion with FKBP. Constructs depicted in Fig. 28A include FKBPXZ-Kl3[SEQ ID NO: 879]; CD8SP-FMC63(vL-vH)-Myc-z-P2A-FKBP-K13-FLAG-T2A-EGFP(112614- B06)[SEQ ID N021755]; CD8SP-l6l-(vL-vH)—Myc-BBz-P2A-FKBP-K13-Flag-T2A- EGFP(1 12614-E05)[SEQ ID NO:1744]; CD8SP-FMC63(vL-VH)-Myc-z-P2A-FKBPx2-K13- FLAG-T2A-EGFP(1203l4-JO3)[SEQ ID NO:1756]; and FMC63(vL-vH)-Myc-z- P2A-Myr-FKBPx2-K13-FLAG—T2A-EGFP(120314-N07)[SEQ ID N011757]. Constructs depicted in Fig. 28B e CD8SP-FMC63(vL-vH)-Myc-BBz-P2A-FKBPx2-FLAG- TAX2U2RS-T2A-eGFP-MO3(O12315-O3)[SEQ ID N013539]; and CDSSP-FMC63(vL-VH)- Myc-BBz-P2A-FKBPx2-HTLV2-Tax—RS-T2A-EGFP(O12315-OOl)[SEQ ID NO:1758].

Fig. 29A-Fig. 29B , in accordance with various embodiments of the invention, activation of K13-mediated NF-KB activity by addition of 7 does not ely affect cell killing induced by the CAR constructs that coexpress FKBP-Kl3.

Construct depicted in Fig. 29A include CD8SP-FMC63(vL-vH)-Myc-z-P2A-FKBP-K13- 2A-PAC(112316-806)[SEQ ID NO:1770]. Constmct depicted in Fig. 298 include CD8SP(vL-vI-I)-Myc-z-P2A-FKBP-K13-FLAG-T2A-PAC(112916-U06)[SEQ ID NO: 1741].

Fig. 30A-Fig 3GB depict, in accordance with various embodiments of the invention, ion of target cell death by ent MPL-directed and CD32-directed CAR constructs that coexpress K13, MC159, HTLVZ-Tax or HTLVZ-Tax RS mutant.

Fig. 31 s, in accordance with various embodiments of the invention, induction of PC3-target cell death by a TROPZ-directed conventional CAR I construct that coexpresses K13.

Fig. 32A-Fig 32B depict, in accordance with s embodiments of the invention, induction of target cell death by different LAMPl-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 sing 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- KB reporter activity by wild—type K13 and various K13 mutants.

Fig. 35 , in accordance with various embodiments of the invention, shows inhibition of CAR CD8SPCD19MM-(VL-VH)—Myc-BBZ-T2A-PAC(0629l5- DO3)[SEQ ID NO:147l]-induced cell death by SOnM and 100nM Dasatinib, while Imatinib has no effect.

Fig. 36 depict, in accordance with various embodiments of the invention, se in GLuc activity following co-culture with T cells expressing CDl9-specific CARS CD8SP-FMC63-(vL-vH)-Myc-BBz-TZA-PAC(112014-A13)[SEQ ID NO:1467] and CD8SPCDl9MM-(VL-vH)-Myc-BBz-TZA-PAC(O629l5-DO3)[SEQ ID NO:l47l] 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 Luc cells by T cells expressing the CD8SP- 971-(vL-vH)-Myc-BBz-TZA-PAC(09l515-A02)[SEQ ID NO:1519] CAR as determined by GLuc assay.

Fig. g. 38B depict, in accordance with various embodiments of the invention, that Fig. 38A: nib blocks NK92 cells mediated death of K562 cells as determined by Gluc assay and Fig. 388: Dasatinib blocks death of RAJI-GLuc cells by CD8SP-FMC63-(vL-vH)—Myc—BBz-T2A-EGFP(0708l4-DO6)[SEQ ID NO:3535] expressing NK92MI cells as determined by Gluc assay.

Fig. 39A-Fig. 39B , in accordance with various embodiments of the invention, that Fig. 39A: treatment with Blinatumomab in the ce of T cells result in strong induction of death of RAJI-GLuc cells which is ively 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 IFNy production which is effectively blocked by 100nM Dasatinib.

Fig. 40A-Fig. 40C depict, in accordance with various embodiments of the ion, Fig. 40A shows no significant toxic effect of the various drugs on RAJI-pLenti- Glue 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). nib was used as a positive l. 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 d with the indicated drugs in the presence of media alone without any CAR-T cells. Fig.4lB shows near complete inhibition of CD8SP-CDl9Bul2-(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 d cell death at 100nM but completely blocked it at luM. Fig. 41C shows that treatment with IOOnM A77004l partially d cell death induced by combination of Blinatumomab with T cells while ent with 200nM A77004l led to complete inhibition.

Fig. 42A-Fig. 42B depict, in accordance with various embodiments of the invention, that coculturing of Jurkat cells expressing CDSSP-FMC63(vL-vH)—BBz-P2A- lgHSP-lL6R-M83(VL-VH)—Flag-T2A-PAC(Ol I315-KO4)[SEQ ID NO:l763] with RAJI cells led to increase in EGFP sion as compared to cells that had not been exposed to RAJI cells (Fig. 42A). This tes activation of NFAT ing. No increase in EGFP expression was observed in parental Jurkats (J—N-G-P) without or with culturing with RAJI (Fig. 4213).

DETAILED DESCRIPTION All references cited herein are incorporated by reference in their entirety as though fully set forth. 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 ofPharmacy 22'"! ed., Pharmaceutical Press mber 15, 2012); Hornyak et (1]., Introduction to Nanoscience and Nanotechnology, CRC Press ; 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 7'" ed. J. Wiley & Sons (New York, NY 2013); Singleton, Dictionary ofDNA and Genome Technology 3", ed., Wiley-Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning: A tory 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 e antibodies, see Greenﬁeld, Antibodies A Laboratory Manual 2"d ed., Cold Spring Harbor Press (Cold Spring Harbor NY, 2013); Kohler and Milstein, Derivation ofspeciﬁc antibody-producing tissue culture and tumor lines by cell , Eur. J. Immunol. 1976 Jul, 6(7):51 1-9; Queen and Selick, zed immunoglobulii1s, U. S. Patent No. 089 (1996 Dec); and Riechmann et al., Reshaping human antibodiesfor therapy, Nature 1988 Mar 24, 332(6162):323-7._ One skilled in the art will recognize many methods and materials similar or lent to those described herein, which could be used in the practice of the present invention. Other es and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the anying drawings, which illustrate, by way of example, various features of embodiments of the ion. Indeed, the present invention is in no way limited to the methods and materials described. For convenience, certain terms employed herein, in the cation, examples and appended claims are collected here.

Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or nt 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 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.

Chimeric antigen ors (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 ize 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 n 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 [00l88] 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 embodiment, yet open to the inclusion of unspecified ts, r 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 des" should be interpreted as "includes but is not limited to," etc.).

] Unless stated otherwise, the terms "a" and "an" and "the" and similar references used in the context of describing a ular embodiment of the application (especially in the context of claims) can be construed to cover both the singular and the . The recitation of ranges of values herein is merely ed 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 orated into the specification as if it were dually recited herein. All methods bed herein can be med 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 embodiments herein is intended merely to better illuminate the application and does not pose a tion on the scope of the application otherwise claimed. The abbreviation, "eg." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting e. Thus, the abbreviation "eg." 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.

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 i20%, :th%, i5%, i196, i0.5% or :t0. 1% from the specified value.

"Chimeric n receptor" or "CAR" or "CARS" as used herein refers to engineered receptors, which graft an n speciﬁcity onto cells (for e T cells such as naive 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 ors or chimeric immunoreceptors. In s embodiments, CARs are recombinant polypeptides sing an antigen-speciﬁc domain (ASD), a hinge region (HR), a embrane domain (TMD), co-stimulatory domain (CSD) and an intracellular signaling domain (ISD).

"Antigen-speciﬁc domain" (ASD) refers to the portion of the CAR that specifically binds the antigen on the target cell. In some 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 ition 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 embodiments of the invention. In some 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 embodiments, the ASD comprises T cell receptors (TCRs) or portions thereof. In exemplary embodiments, nucleic acids encoding ASDs sing scFVs are set forth herein in SEQ ID NOS: 564-798.

"Hinge region" (HR) as used herein refers to the hilic region which is between the ASD and the TMD. The hinge s include but are not limited to Fe fragments of antibodies or fragments or derivatives thereof, hinge regions of antibodies or fragments or derivatives thereof, CH2 regions of antibodies, CH3 regions of dies, artiﬁcial spacer sequences or combinations thereof. Examples of hinge regions include but are not d to CD8a hinge, and artificial spacers made of polypeptides which may be as small as, for example, Gly3 or CH] and CH3 domains of IgGs (such as human IgG4). In some 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 IgG], (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.

"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 ce 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 embodiments, the TMD encoded CAR comprising any of the backbones bed 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, K1RDS2, 0X40, CD2, CD27, LFA-l (CD1 la, CD18), ICOS ), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), , NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGAl, VLAl, CD49a, ITGA4, 1A4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CDI la, LFA-l, ITGAM, CDI lb, ITGAX, CD1 10, ITGBI, CD29, ITGBZ, CD18, LFA-l, ITGB7, TNFR2, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAMl, CRT AM, Ly9 (CD229), CD160 (BYSS), PSGLl, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMFI, CD150, IPO-3), BLAME (SLAMFS), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKGZD, and/or NKGZC.

"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 pment of memory cells. The CARS of the invention may comprise one or more mulatory 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 (0X40), DaplO, CD27, CD2, CD5, lCAM-l, CD1 la/CD18), Lck, , TNFR-II, Fas, CD30, CD40 or combinations thereof. Other mulatory domains (e.g., from other proteins) will be apparent to those of skill in the art and may be used in tion with alternate embodiments of the invention.

"Intracellular signaling domain" (ISD) or "cytoplasmic domain" as used herein refers to the portion of the CAR sing 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 2 chain of the T-cell receptor complex or any ofits homologs (e.g., h chain, Fceng and b chains, MBl (Iga) chain, B29 (1gb) 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 tion with alternate embodiments of the invention. r" (L) or "linker " or "linker region" as used herein refer to an oligo- or polypeptide region from about I to 100 amino acids in , 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 s include 2A s (for example T2A), 2A-like linkers or functional equivalents thereof and combinations thereof. In some embodiments, the linkers include the picornaviral 2A-like linker, CHYSEL sequences of porcine teschovirus (P2A), ’Ihosea asigna virus (T2A) or combinations, variants and functional equivalents thereof. In other ments, the linker sequences may se Asp-Val/Ile- Glu-X-Asn-Pro-Gly‘lAWro(2B) motif, which results in cleavage between the 2A e and the 2B proline. The nucleic sequences of l exemplary cleavable linkers are provided in SEQ ID NO: 831 to SEQ ID NO: 836 and amino acid ces 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 ate 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 ial 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 embodiments, a fun'ne cleavage site (RAKR) (SEQ ID NO: 839-841 and SEQ ID NO: 2604) is added upstream of the SGSG motifs to tate cleavage of the residual 2A e following translation.

"Effector function" refers to the specialized function of a differentiated cell.

Effector function of a T-cell, for e, may be cytolytic activity or helper activity including the secretion of cytokines. [00l99] "Enhancer", as used herein, s 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 onality 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 ype er sequence are also within the above definition.

"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 ty 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-Kl3, in which whose activity can be controlled by the administration of a dimerizer molecule (Table "Switch ," 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 ng of a first entity linked to, e.g., fused to, a first switch domain, and a second entity linked to, e.g., fused to, a second switch domain. A ﬁrst and second switch domain are collectively referred to as a dimerization switch. In embodiments, the first and second switch domains are the same as one another, e.g., they are polypeptides having the same primary amino acid sequence, and are ed to collectively as a homodimerization switch. In embodiments, the switch is intracellular. In embodiments, the switch domain is a polypeptide-based entity, e.g., FKBP (FK506 g protein), and the dimerization molecule is small molecule, e.g., AP20187.

"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 ments, the dimerization molecule does not naturally occur in the subject, or does not occur in trations that would result in signiﬁcant dimerization. In embodiments, the dimerization molecule is a small molecule, e.g., rapamycin or a rapalogue, eg, RADOO] or AP20187. peutic Controls" as used herein refers to an element used for controlling the activity of a CAR expressing cell. . In some 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, ne- guanine phosphoribosyl transferase, human caspase 8, human caspase 9, ble caspase 9, purine nucleoside phosphorylase, linamarase/linamarin/glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP)/indoleacetic (1AA), glutamylcysteine synthetase, CD20/alphaCD20, CD34/thymidine kinase chimera, dox-depedent caspase-2, mutant thymidine kinase KSR39), AP1903/Fas system, a chimeric cytokine or (CCR), a selection marker, and combinations thereof. The nucleic acid sequences of several therapeutic ls are provided in SEQ ID NO: 850 to SEQ ID NO: 856 and SEQ ID NO: 883 (Table 16).

"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 ion, irrespective of whether the genetically modiﬁed cells target diseased cells or healthy cells to uate 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 s the conventional CARS and novel backbones containing conventional CARS with accessory modules of the invention, which CARS may target any of the antigens sed on the surface of target cells. Examples of ns which may be targeted include but are not limited to ns 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 es. 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.

"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, aneous 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.

"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.

"Genetically modiﬁed cells", "redirected cells", "genetically ered cells" or "modiﬁed cells" as used herein refer to cells that express the CAR of the invention. In some embodiments, the cally modiﬁed cells comprise vectors that encode a CAR. In some embodiments, the genetically modiﬁed cells comprise s that encode a CAR and one or more accessory molecules in the same vector. In some embodiments, the cally modiﬁed cells comprise a ﬁrst vector that encodes a CAR and a second vector that s the accessory molecule. In some embodiments, the genetically modiﬁed cells se a ﬁrst vector that encodes a CAR and a second vector that s more than one accessory molecule. In some ments, the genetically d cells comprise a ﬁrst vector that encodes a CAR and a second vector that encodes the first accessory molecule and a third vector that s a second accessory molecule.

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 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 embodiments, the accessory module is encoded by more than one nucleic acid molecule, depending on the number of components in the accessory modules.

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

The term "immune effector on" 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.

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

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

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

The terms "T-cell" and "T-lymphocyte" are interchangeable and used mously herein. Examples include but are not limited to naive T cells, central memory T cells, effector memory T cells or combinations thereof.

] 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 , referred to herein as the "Fc fragment" or "PC 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 dies, chimeric antibodies, diabodies and polypeptides that contain at least a n of an immunoglobulin that is sufficient to confer speciﬁc antigen binding to the polypeptide. The Fc domain includes portions of two heavy chains buting to two or three classes of the antibody.

The Fc domain may be produced by recombinant DNA techniques or by tic (e.g. papain cleavage) or via chemical cleavage of intact antibodies.

The term "antibody nt," as used herein, refer to a protein fragment that ses only a portion of an intact antibody, generally including an antigen g site of the intact antibody and thus retaining the y 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 nt having one or more ne residues at the C-terminus of the CHI domain; (iii) the Fd fragment having VH and CH1 domains; (iv) the Fd' nt 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' nts 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 ); (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-CHl-VH-CHI) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et a1. Protein Eng. 8(10): 1057-1062 (1995); and US.

Pat. No. 5,641,870).

"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 (VH) and light (VL) 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, VH-linker-VL or ker-VH, so long as the speciﬁcity of the scFv to the target antigen is retained. ementarity determining " (CDR) as used herein refers to the amino acid sequences within the variable regions of antibodies which regions confer speciﬁcity and binding y. In general, there are three CDRs in each of the light chain le regions (LCDRl, LCDR2 and LCDR3) and three CDRs in each of the heavy chain variable regions (HCDl, 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, 5‘11 Ed. Public Health Services, National utes of Health, Bethesda, MD; Al-Lazikani et al., (1997) JMB 273,927-948).

As used herein, the term "speciﬁc binding" means the contact between an antibody and an n with a binding afﬁnity of at least 10'6 M. In certain aspects, antibodies bind with afﬁnities of at least about 10—7M, and preferably 10—8 M, 10'9 M, 10'10 M, 10—11 M, or "2M.

"Binds the same epitope as" means the ability of an dy, 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 c 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 y, Vol. 66 (Glenn is, 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 dies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., US. Patent No. 871 ; Geysen et al, (1984) Proc. Natl. Acad. Sci. USA 823998-4002; 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 ols, 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 ble from the Oxford Molecular Group. This computer program employs the Hopp/Woods , Hopp et a1, (1981) Proc. Natl. Acad. Sci USA 783824- 3828; for ining 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 CD19) bind to unique epitopes, each antibody can be biotinylated using cially available reagents (Pierce, Rockford, 111.). 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.

The e 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(vL-vH)-Myc-BBz-T2A-PAC (SEQ ID NO: 1658 and SEQ ID NO: 3403) targeting MPL is comprised of scFv MPL(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 ized by several scFv and/or their al antibodies used in the uction 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 g 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—AcVS 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 ning the fusion protein collected and assayed for NLuc activity to assure that the different mutant MPL-ECD-GGSG-Nluc-AcVS fusion proteins are being secreted in the supernatant. uently, the fusion proteins would be tested for their ability to bind to cells (e.g., Jurkat cells or T cells) expressing the CD8SP-MPL(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 ized by a particular CAR could include a ﬁJnctional itive assay with different test antibodies. For example, T cells sing the CD8SP-MPL(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 sing concentrations of different test MPL antibodies. In case the epitope recognized by a test MPL antibody overlaps with the epitope recognized by the CD8SP-Ivﬂ’L(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 CDSSP-MPL-l6l-(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 - 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 e recognized by the said CAR.

"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 eutic agents include but are not limited to infectious diseases, carcinomas, sarcomas, lymphomas, leukemia, germ cell tumors, blastomas, antigens sed on various immune cells, and antigens expressed on cells ated with various hematologic diseases, and/or inﬂammatory diseases.

"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 nic processes, metastatic tissues or malignantly transformed cells, tissues, or , ective of histopathologic type or stage of invasiveness. Examples of solid tumors include malignancies, e.g., sarcomas, adenocarcinomas, and omas, 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 ine and cancer of the esophagus. In one embodiment, the cancer is a melanoma, e.g., an advanced stage melanoma. Metastatic lesions of the entioned s 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 ma, 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 d gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, c or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, c lymphocytic leukemia, solid tumors of childhood, cytic lymphoma, cancer of the bladder, cancer of the kidney or , carcinoma of the renal pelvis, sm of the central nervous system (CNS), primary CNS ma, 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 s that express PD-Ll (Iwai et al. (2005) Int. Immunol. 17:133-144) can be ed using the antibody molecules described .

"Polynucleotide" as used herein es 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, tic DNA, synthetic RNA, and/or tRNA.

It is to be inferred without explicit recitation and unless otherwise ed, 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 ty 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 gy 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 f is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement. atively, when ing 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 nce polypeptide or protein.

A polynucleotide or polynucleotide region (or a ptide or polypeptide region) having a n percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, that tage 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 ms known in the art, for example those described in Current Protocols in Molecular y (Ausubel et al., eds. 1987) Supplement , section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A red alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and , using the following default parameters: Genetic code=standard; ﬁltanone; 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.nihgov/cgi-bin/BLAST.

The term "isolated" as used herein refers to les or biologicals or ar materials being ntially free from other materials. In one aspect, the term "isolated" refers to nucleic acid, such as DNA or RNA, or protein or ptide (e.g., an antibody or derivative thereof), or cell or cellular lle, 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 ally 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 ed 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 s that are isolated from other cells or tissues and is meant to ass both, cultured and engineered cells or tissues.

"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 te embodiments of the ion will be apparent to those of skill in the art.

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

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

] "Vector", "cloning vector" and "expression vector" as used herein refer to the vehicle by which a polynucleotide sequence (eg. a n 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.

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.

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 l localization of the agents at a desired site.

"Beneﬁcial results" may include, but are in no way d to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease ion, preventing the e 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 s" 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 .

As used herein, the terms "treat," "treatment," "treating," or oration" refer to therapeutic treatments, wherein the object is to reverse, ate, ameliorate, inhibit, slow down or stop the progression or ty of a condition associated with, a e 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 tive" if one or more symptoms or al markers are reduced. Alternatively, treatment is "effective" if the progression of a disease is reduced or halted. That is, ment" 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, ation of one or more symptom(s), shment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, ration 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 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 s physiological symptoms associated with the cancerous condition.

"Conditions" and "disease conditions," as used herein may include, cancers, tumors or infectious diseases. In exemplary embodiments, the conditions include but are in no way limited to any form of ant neoplastic cell proliferative disorders or es. In ary embodiments, ions e any one or more of kidney cancer, melanoma, prostate cancer, breast cancer, glioblastoma, lung cancer, colon cancer, or r cancer.

The term "effective " 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 f, 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 . The phrase "therapeutically effective " as used herein means a sufﬁcient amount of the composition to treat a disorder, at a reasonable benefit/risk ratio able to any l treatment.

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 s such as the type of disease being treated, gender, age, and weight of the subject.

The phrase "ﬁrst line" or "second line" or "third line" refers to the order of ent 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 y, 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.cancergov, last visited on May 1, 2008.

Typically, a patient is given a subsequent chemotherapy n 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.

] "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 ] 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 bed in Table 2 deﬁne a ‘backbone’ (Table 2).

Table 1: Conventional CAR architectures. First generation conventional CARS (Conventional CAR 1) have an ellular signaling (ISD) domain (eg. CD32) and no costimulatory . Second generation tional CARS (Conventional CAR 2 or CAR II) have one costimulatory domain (e.g. 41BB or CD28) and an intracellular signaling (ISD) domain (e.g. CD32). Third generation conventional CARs (Conventional CAR 3 or CAR 111) have two costimulatory domains (e.g. 4lBB and CD28) and an intracellular signaling (ISD) domain (e.g. CD32).

Table 1 Conventional CAR Architectures tional CAR 1 ASD HR TMD ISD l_------CAR11) CAR Ill Table 2: Exemplary Backbones Accesson Module ——_-—(DNA (PRT Conventional CARI MCl60-vFLlP 2631 Backbone4 Conventional CARI E8-vFLIP \‘FLIP Mvr-FKBPxZ-KB Backbonc16 Conventional CARI FKBPxZ—HTLVZ-Tax- 2644 Backbone 17 Conventional CARI FKBPxZ-Flag-HTLVZ- Tax-RS Backbone 18 Conventional CARI lL6RvHI-I 2647 Backbone 19 Conventional CARI IGHSP2-1L6R 886 2649 VHH-ALBS-VHH Backbone 20 Conventional CARI l-SP-lL6-l9A-scFV 2662 ne 21 Conventional CARI I SP-Fx06 m— m-scFv —_I_-mlilimumab-SCFV Ale-VHH I ilimumab-Ale-vI-lI-l Accessory Module ———--(DNA) (PRT) Alb8-VHH Backbone 36 Conventional CAR 11 2633 vFLIP ——m__ Backbone 47 Conventional CARII FKBPxZ-HTLVZ-Tau- w 2644 Backbone 48 tional CARII FKBPxZ-Flag-HTLVZ- Tax-RS Backbone 49 Conventional CAR n 04-VHH 2647 Backbone 50 Conventional CAR [1 lGHSPZ-[L6R 886 2649 VHH-ALBS-VHH Backbone 51 Conventional CARII 1 SP-lL6-l9A-scFV 2662 Backbone 52 tional CARII I-SP-Fx06 m2663 m-scFv I ilimumab-SCFV Ale-VHH Alb8-vHH I ilimumab-Ale-VHH Alb8—vHI-I Backbone 60 Conventional CAR II 2618 Backbone 61 Conventional CARII tBCMA 2619 Backbone 62 Conventional CARII CNB30 Exemplary embodiments of the CAR components are described in Tables 1-6, 8, 11, and 15.

As descn'bed , the various nes comprise a CAR component and accessory modules. Exemplary embodiments of the accessory modules are described in Table 16.

In some embodiments, the compositions comprise nucleic acids encoding conventional CARs I-III (Table 1), wherein the antigen speciﬁc domain of the CAR s one or more speciﬁc antigens as described in Tables 21 and 22. In some embodiments, the compositions comprise nucleic acids encoding any one or more of backbones 1-62 (Table 2) where the n c domain of the encoded CAR targets one or more speciﬁc antigens as described herein and in Tables 19, 20 and 22. In some embodiments, the compositions compn'se nucleic acids encoding backbone-1, wherein the antigen speciﬁc domain of the CAR in backbone-l targets one or more cancer speciﬁc antigens as described herein and in Table 19 and 22. In some embodiments, the compositions se 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 embodiments, 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 ments, 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 embodiments, the nucleic acids are used in the treatment of cancer. In some ments, the nucleic acids encoding the CAR component of the backbones described herein comprise more than one n speciﬁc domain wherein each of the antigen speciﬁc domains are contiguous and in the same g frame as the other antigen speciﬁc domains in the same CAR.

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

In some embodiments, the compositions comprise vectors comprising nucleic acids encoding any of conventional CARS I-III, n the antigen speciﬁc domain of the CAR targets one or more cancer speciﬁc ns as described herein and in Tables 21 and 22. In some embodiments, the compositions se 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 ns as described herein and in Tables 20 to 22. In some embodiments, the compositions se 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 bed herein and in Tables 19 and 22. In some embodiments, 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 . In some embodiments, the compositions comprise vectors encoding nucleic acids encoding backbone-32, n the antigen c domain of the CAR in backbone-32 targets one or more cancer speciﬁc antigens as described herein.

In some embodiments, the compositions comprise vectors encoding nucleic acids encoding backbone-33, wherein the antigen speciﬁc domain of the CAR in ne-33 targets one or more cancer speciﬁc antigens as described . In various embodiments, the vectors are used in the treatment of cancer. In some embodiments, vectors comprising nucleic acids encoding the conventional CARs I to III or CAR ent 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.

In some embodiments, the compositions comprise genetically modiﬁed cells which include vectors comprising nucleic acids encoding any one or more of conventional CARs I to 111 or backbones 1-62 (Table 2) where the n c domain of the encoded CAR targets one or more speciﬁc antigens as described herein and in Tables 19 to 22. In some embodiments, the compositions compn'se genetically modiﬁed cells which include vectors encoding nucleic acids encoding conventional CARS I to III, wherein the antigen c domain of the CAR targets one or more cancer speciﬁc antigens as described herein and in Tables 19 and 22. In some embodiments, the compositions comprise genetically d 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 embodiments, the itions comprise genetically modiﬁed cells which e vectors encoding nucleic acids encoding backbone-2, wherein the antigen c domain of the CAR in backbone-2 targets one or more cancer speciﬁc antigens as described . In some embodiments, the compositions comprise genetically modified cells which include vectors encoding nucleic acids ng backbone-32, n the antigen speciﬁc domain of the CAR in backbone-32 targets one or more cancer speciﬁc antigens as described herein. In some embodiments, the compositions comprise cally modiﬁed cells which include vectors encoding nucleic acids encoding backbone-33, n the antigen speciﬁc domain of the CAR in backbone-33 targets one or more cancer speciﬁc antigens as described herein. In various embodiments, the genetically modiﬁed cells are used in treatment of cancer. In some embodiments, the genetically modiﬁed cells targets more than one antigen on a cancer cell.

In various embodiments, the isolated c acid molecules encoding the CAR components of the nes described herein, encode one, two, three or more antigen speciﬁc domains.

In various embodiments, the isolated c acid molecules encoding the CAR components of the backbones described , encode one, two, three or more co- stimulatory domains.

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

The nucleic acid sequences encoding for the desired ents 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 le, 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. atively, the c acid of interest can be produced synthetically, rather than cloned.

In ary embodiments, the nucleic acid sequences encoding for the d components of the CARS and accessory modules described herein are described in Tables 3-22.

In some ments, 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 embodiments, such agents are set forth in Table 16. In exemplary ments, 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 bispecifrc camelid vHH targeting IL-6 receptor alpha and n (SEQ ID NO: 886), Fx06 peptide (SEQ ID NO: 900), or combinations thereof.

In some embodiments, 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 embodiments, such agents are set forth in Table 16. In exemplary embodiments, such agents e 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 PDl (SEQ ID NOS: 889 and 890), scFv targeting CTLA4 (SEQ ID NO: 894), bispecif‘rc proteins targeting PD] and albumin (SEQ ID NO: 892 and 893), bispeciﬁc proteins targeting CTLA4 and n (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.

In speciﬁc embodiments, exemplary constructs containing backbone-1, which comprises a conventional CAR I containing a CD32 activation domain and coexpresses a K 1 3-vFLIP accessory module, are set forth in Table 19.

In speciﬁc embodiments, exemplary constructs containing ne-32, which comprises a conventional CAR 11 containing a 41BB ulatory domain, a CD32 activation domain and coexpresses a K 13-vFLIP accessory module, are set forth in Table 20.

In speciﬁc embodiments, exemplary conventional CAR II constructs ning a 41BB costimulatory domain and a CD32 tion domain are described in Table 21.

In speciﬁc embodiments, exemplary CAR constructs in various embodiments of the ions are described in Table 22.

Antigen c domain 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.

In one embodiment, each n speciﬁc region comprises the full-length IgG heavy chain (speciﬁc for the target antigen) having the VH, CH1, hinge, and the CH2 and CH3 (Fc) lg domains, if the VH domain alone is sufﬁcient to confer n-speciﬁcity ("single-domain antibodies"). The full length IgG heavy chain may be linked to the co- stimulatory domain and the ellular signaling domain via the appropriate transmembrane domain. If both, the VB and the VL domains, are necessary to generate a fully active n- speciﬁc targeting region, the VH-containing CAR and the full-length lambda light chain (lgL) are both introduced into the cells to generate an active antigen-speciﬁc targeting region.

In some embodiments, the antigen c domain of the encoded CAR molecule comprises an antibody, an dy fragment, an scFv, a Fv, a Fab, a (Fab')2, a single domain antibody , a VH or VL domain, or a camelid VI-II-I domain. In some embodiments, the antigen binding domain of the CAR is a scFv dy fragment that is humanized compared to the murine sequence of the scFv from which it is derived.

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., (I988) tl. Acad.

Sci. USA 85:5879-5883). ScFv molecules can be produced by g VH and VL 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 a1. 1993 Proc Natl Acad. Sci. USA. 90:6444-6448, US. Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCT ation Nos.

W02006/020258 and W02007/024715, is incorporated herein by reference.

An scFv can comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, 12, l3, [4, , 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its VL and VH regions. The linker sequence may comprise any naturally occurring amino acid. In some ments, the linker sequence comprises amino acids glycine and serine. In another embodiment, the linker sequence ses 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 ). In one embodiment, 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 ty, giving rise to or efﬁcacy in activity studies.

In another aspect, the antigen binding domain is a T cell receptor ("TCR"), or a fragment thereof, for example, a single chain TCR (scTCR). s 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 1]: 487-496 (2004); Aggen et al, Gene Ther, 19(4):365- 74 (2012) (references are orated herein by its entirety). For example, scTCR can be engineered that contains the Va and VB genes from a T cell clone linked by a linker (e.g., a ﬂexible e). 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 In one embodiment, the antigen specific domain comprises one, two or all three heavy chain (hc) CDRs, thDRl, thDR2 and thDR3 of an antibody listed herein, and/or one, two or all three light chain (lc) CDRs, , lcCDR2 and lcCDR3 of an antibody listed herein.

In another embodiment, the n 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 nt 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 d to, CDR-grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91/09967; and US. Pat. Nos. ,225,539, 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 6; Padlan, 1991, Molecular logy, 28(4/5):489-498; Studnicka et al., [994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 911969-973, each of which is incorporated herein by its entirety by reference), chain shufﬂing (see, e. g., US. Pat.

No. 332, which is incorporated herein in its ty by reference), and ques disclosed in, e.g., US. Patent Application Publication No. /0042664, US. Patent Application Publication No. US2005/00486l7, US. Pat. No. 213, US. Pat. No. ,766,886, International ation No. W0 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 (I996), 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 embodiments, 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 In some embodiments, 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.

In some embodiments, the antigen speciﬁc domain of a CAR described herein is a scFv dy fragment. In some embodiments, the antigen speciﬁc scFv antibody fragments are onal in that they bind the same antigen with the same or comparable afﬁnity as the IgG antibody from which it is derived. In other embodiments, the antibody fragment has a lower binding afﬁnity to the n compared to the antibody from which it is derived but is onal in that it provides a biological response bed herein. In one embodiment, the CAR molecule comprises an antibody fragment that has a binding afﬁnity KD of 10" M to '8 M, 10'5 M to 10'7 M, 10'6 M or 10'8 M, for the target antigen.

In some embodiments, n speciﬁc domain of a CAR described herein binds to a MHC presented e. Normally, peptides derived from endogenous ns 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 tutively expressed by all nucleated cells. In cancer, virus-Speciﬁc and/or tumor-speciﬁc peptide/MHC complexes represent a unique class of cell surface s for immunotherapy.

TCR-like antibodies targeting peptides derived from viral or tumor antigens in the context of human leukocyte antigen A1 or HLA-A2 have been described (see, e. g., Sastry et al., J Viral. 201 1 85(5):]935-1942; Sergeeva et al., Blood, 20] I l :4262-4272; Verma et al., Jlmmun012010 :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 yed library. Exemplary CARS that are based on TCR-like antibodies targeting WT] in association with HLA-A2 are represented by SEQ ID NO: 1 176 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 nt and the sequence of the peptide are shown in Table 23.

In some embodiments, 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 ating from its target antigen, inhibition of kinase activity, and the like, as will be understood by a d n.

In some embodiments, 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, FcRHS, CD99, Tissue Factor, amyloid, Fc region of an immunoglobulin, CD171, CS-l, CLL-l (CLECLl), CD33, II , GD2, GD3, BCMA, Tn Ag, PSMA, RORI, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, ILllRa, Mesothelin, PSCA, VEGFRZ, Lewis Y, CD24, PDGFR-beta, PRSS21, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2/neu), MUCl, EGFR, NCAM, Prostase, PAP, ELFZM, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, Fucosyl GMl, sLea, GM3, TGSS, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEMl/CD248, TEM7R, CLDN6, TSHR, TCR-betal constant chain, TCR beta2 nt chain, TCR gamma-delta, GPRCSD, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLACI, GloboH, NY-BR-l, UPK2, HAVCRI, ADRB3, PANX3, GPR20, LY6K, 0R51E2, TARP, WT], NY-ESO-l, LAGE-la, legumain, HPV E6, E7, HTLVl-Tax, KSHV K8.l protein, EBB gp350, HIVl-envelop glycoprotein gpl20, MAGE-Al, MAGE A], ML, sperm protein 17, XAGEI, Tie 2, MAD-CT-l, MAD-CT-Z, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-l/Galectin 8, MelanA/MARTI, Ras mutant, hTERT, DLL3, TROP2, PTK7, GCC, AFP, sarcoma ocation breakpoints, ML-IAP, ERG (TMPRSSZ ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYPlBl, BORIS, SART3, PAXS, OY-TESI, LCK, AKAP-4, SSX2, RAGE- 1, RUl, RU2, intestinal carboxyl se, mut hsp70-2, CD79a, CD79b, CD72, LAIRl, FCAR, LILRA2, CD3OOLF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRLS, IGLLl, FITC, Leutenizing hormone receptor (LI-IR), Follicle stimulating hormone receptor (FSHR), Chorionic tropin Hormone receptor (CGHR), CCR4, GD3, , SLAMF4, FITC, Leutenizing hormone receptor (LHR), Follicle stimulating hormone receptor (FSHR), nic tropin Hormone receptor (CGHR), CCR4, GD3, SLAMF6, SLAMF4, or combinations thereof.

] In some embodiments, the antigens speciﬁc for a disease which may be targeted by the CARS when expressed alone or with the accessory modules as described herein e 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-IVLET, 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, DRS, 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-l receptor, IGF-I, IgGl, Ll-CAM, IL-1 3, IL- 6, insulin-like growth factor I receptor, integrin (15B1, integrin avB3, LAMP], MORAb-009, MS4A1, MUCl, mucin CanAg, N-glycolylneuraminic acid, NPC-lC, PDGF-R a, PDL192, phosphatidylserine, tic carcinoma cells, RANKL, RON, RORl, SCH , SDCl, SLAMF7, , tenascin C, TGF beta 2, TGF-B, TRAl-Rl, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-l, VEGFRZ, vimentin or ations f. Other antigens speciﬁc for cancer will be nt to those of skill in the art and may be used in connection with alternate embodiments of the ion.

In some embodiments, the antigens speciﬁc for cancer which may be targeted by the CARs when sed 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-l, Lym2, CD23, CD179b, CDHl, CDH6, CDH19, CDH17, DLL3, PTK7, TROP2, TIM], LAMP] or combinations thereof. In some embodiments, the n speciﬁc domains of the CARS speciﬁc for MPL, CD19, FLT3, GRP-78, CD79b, Lyml, Lym2, CD23, CDHI, CDH6, CDH19, CDH17, DLL3, PTK7, TROP2, TIMI, LAMPl comprise scFv sequences set forth in Table 11.

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 embodiments, the disease-supporting antigen is an antigen present on cells that support the survival and proliferation of e g cells. In some embodiments, 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. NUDSC cells can block T cell proliferation and activation. Without wishing to be bound by theory, in some embodiments, the CAR- expressing cells destroy the disease-supporting cells, thereby indirectly blocking growth or al of disease causing cells.

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

] In a r embodiment, each antigen speciﬁc region of the CAR may comprise a divalent (or bivalent) single-chain variable fragment Fvs, bi—scFvs). In CARS comprising di-scFVs, two scFvs speciﬁc for each antigen are linked er by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs. (Xiong, Cheng- Yi; Natarajan, A; Shi, XB; Denardo, GL; Denardo, SJ (2006). "Development of tumor targeting anti-MUC-l multimer: effects of di-scFv unpaired cysteine location on PEGylation and tumor binding". Protein Engineering Design and Selection 19 (8): 359—367; Kufer, Peter; btlse, Ralf; Baeuerle, Patrick A. . "A revival of bispeciﬁc antibodies".

Trends in Biotechnology 22 (5): 238—244). CARs comprising at least two antigen-speciﬁc ing regions would express two scFvs speciﬁc for each of the two antigens. The ing 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 n. Exemplary CARS targeting two ns are represented by SEQ ID N05: 1042, 1043, 1049, 1050 and 1087.

In an onal embodiment, 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 ies. Tetrabodies may also be used.

In some embodiments, the ASD of the CAR comprises VL fragments as described in Table 4.

In some embodiments, the ASD of the CAR comprises VH fragments as described in Table 6.

In some embodiments, the ASD of the CAR comprises VHH nts (nanobodies) as described in Table 7.

] In some embodiments, the ASD of the CAR ses afﬁbodies as described in Table 8.

In some embodiments, the ASD of the CAR comprises ligands as describes in Table 10.

In some embodiments, the ASD of the CAR comprises scFv fragments as bes in Table 11.

In one ment, 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.

In one embodiment, an antigen specific domain of a CAR against a target antigen is an antigen binding portion, e.g., CDRs, of vI-IH fragments targeting this antigen as shown in Table 7.

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

In one embodiment, an antigen speciﬁc domain of a CAR against a target antigen is an n g portion of a receptor known to bind this target antigen.

In one embodiment, 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.

In one embodiment, an n specific domain of a CAR against a target antigen is an n binding portion, e.g., CDRS, of VL and vH fragments of a scFV targeting this antigen as shown in Table 11.

In r embodiment, 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 embodiments, the antigen c domains of these CARS could be derived from vL fragments, vH fragments or scFv fragments of antibodies. In some embodiments, the reference dies for cross-competition studies to determine the target-epitope ized by a CAR of the invention described in Tables 19-22 are scFvs having ces as shown in SEQ ID NOs: 2334-2568 (Table 11). In an exemplary embodiment, 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 ments, the reference vI-IH fragments for cross-competition studies to determine the target-epitope recognized by a CAR of the ion described in Tables 19-22 are are vI-IH fragments having sequences as shown in SEQ ID NOS: 2269-2293 (Table 7). In some embodiments, the reference non- immunoglobulin antigen binding lds for cross-competition studies for cross- competition studies to determine the -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 embodiments, 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 embodiments, the reference CARS for cross- competition studies against different s are CARS having sequences as shown in SEQ ID NOS: 2672-2941 (Table 19).

In an embodiment, the reference antibodies for cross-competition studies to determine the target-epitopes ized by the MPL-targeting CARS of the invention (e.g., SEQ ID NOS: 1117-1125) are antibodies mAb-1.6, mAb-1.111, mAb-l.75, mAb-l.78, mAb- 1.l69, and mAb-1.36 described in patent application US 2012/0269814 A1.

In an embodiment, 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: 1 1 17-1 125) are scFvs having ces as shown in SEQ ID NOS: 2499-2506 (Table In an embodiment, the reference ligands for competition s to determine the -epitopes recognized by the MPL-targeting CARS of the ion (e.g., SEQ ID NOS: 1117-1125) are ligands having sequences as shown in SEQ ID NOS: 2323-2324 (Table In an embodiment, 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: 125 and l 164 (Table 19).

In the preferred embodiment, an MPL-targeting CAR of the invention binds to an MPL-epitope corresponding to or overlapping with the peptide sequence —PWQDGPK—.

] In an embodiment, the reference scFvs for cross-competition Studies to determine the target-epitopes recognized by the CDl9-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).

] In an embodiment, the reference CARS for cross-competition studies to determine the target-epitopes recognized by the CDI9-targeting CARS of the invention are CARS having sequences as shown in SEQ ID NOS: 930—941 (Table 19).

In an embodiment, the nce 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).

In an embodiment, the reference CARS for cross-competition studies to determine the target-epitopes recognized by the CDZO-targeting CARS of the invention are CARS having sequences as shown in SEQ ID NOS: 964-977 (Table 19).

In the preferred embodiment, the CD20-targeting CARS of the invention bind to the epitopes ponding to one or more of the sequences —PAGIYAPI—, — FLKMESLNFIRAHTP: —HFLKMESLNFIRAHTPY—, —YNAEPANPSEKNSPSTQY—, — YNAEPANPSEKNSPST— and ANPSEKNSP—.

] In an embodiment, the reference scFvs for cross-competition studies t 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 II).

In an embodiment, 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).

In an embodiment, the reference scFvs for competition studies t LAMPl-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).

In an embodiment, the reference CARS for cross-competition studies against targeting CARS of the invention are CARS having sequences as shown in SEQ ID NOS: 1104-1105 (Table 19).

In an embodiment, the reference scFvs for cross-competition studies against TROPZ-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).

In an embodiment, the reference CARS for cross—competition Studies against TROPZ-targeting CARS of the invention are CARs having sequences as shown in SEQ ID NOS: 2910-2911 (Table 19).

In an embodiment, the nce scFvs for cross-competition studies against PTK7- ing CARS of the invention (e.g., SEQ ID NOS: 1 l43-1 144) are scFvs having sequences as shown in SEQ ID NOS: 2523-2524 (Table 11).

In an embodiment, 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).

In an embodiment, the nce scFv for cross-competition studies t a CD23- targeting CAR (e.g., SEQ ID NO: 2932) is a scFv having sequence as shown in SEQ ID NO: 2565 (Table 11).

In an embodiment, the nce 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).

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

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

In an embodiment, 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).

] In an embodiment, 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).

In an embodiment, 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 N05: 2421 and 2558 (Table 11).

In an embodiment, the reference CARS for cross-competition studies against targeting CARS of the invention are CARS having sequences as shown in SEQ ID N05: 1019 and 1180 (Table 19).

In an embodiment, 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).

In an embodiment, the reference CARS for competition studies against CD324-targeting CARS of the invention are CARS having ces as shown in SEQ ID NOS: 1014-1015 (Table 19).

In an embodiment, 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).

In an embodiment, 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).

In an embodiment, the reference scFvs for 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).

In an embodiment, the reference CARS for cross-competition s against GFRa4-targeting CARS of the invention are CARS having sequences as shown in SEQ ID NOS: 1066-1067 (Table 19).

In an embodiment, 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).

In an embodiment, the reference CARS for competition studies against CSF2RA-targeting CARS of the invention are CARS having sequences as Shown in SEQ ID NOS: 1040-1041 (Table 19).

In an ment, 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).

In an ment, the reference CARS for cross-competition studies t B7H4- targeting CARS of the invention are CARS having sequences as shown in SEQ ID NOS: 2929-2930 (Table 19).

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

In an embodiment, the reference CAR for cross-competition studies against a NY- BRI-targeting CARS of the invention is a CAR having sequences as shown in SEQ ID NO: 1131 (Table 19).

In an embodiment, 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 ).

] In an embodiment, the reference CAR for cross-competition studies against a CDZOOR-targeting CARS of the invention is 3. CAR having sequences as shown in SEQ ID NO: 1 190 (Table 19).

In an ment, the reference scFv for cross-competition studies against a HlVl- envelop rotein gplZO-targeting CAR (e.g., SEQ ID NO: 945) is a scFv having sequence as shown in SEQ ID NO: 2512 (Table 11).

In an embodiment, the reference CAR for cross-competition studies t a HlVl- envelop glycoprotein gpIZO-targeting CAR of the invention is a CAR having sequences as shown in SEQ ID NO: 945 (Table 19).

In an ment, the reference ligand for cross-competition studies against a TSHR-targeting CAR (e.g., SEQ ID N05: 1167) is a ligand having sequences as shown in SEQ ID NO: (Table 11).

In an embodiment, the reference scFvs for cross-competition studies against TSHR- targeting CARS of the invention (e.g., SEQ ID NOS: 1 168-1170) are scFvs having sequences as shown in SEQ ID N05: 2333 (Table 10).

In an embodiment, the reference CARS for cross-competition studies t TSHR- targeting CARS of the invention are CARS having sequences as shown in SEQ ID NOs: 1167-1170 (Table 19).

In some embodiment, the CARs ing gplOO, MART, nase, hTERT, lVIUCl, CMV—pp65, HTLVl-Tax, HIVl-gag, NY-ESO, WTl, AFP, HPV-l6-E7, PR] bind to target peptides shown in Table 23 in complex with MHC class I (I-ILA-A2). Linkers In some embodiments, two or more functional s 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 embodiments, 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 s are free to move relative to one r. 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 embodiments, linkers are described in SEQ ID NOs: 250-258 (Tables 5).

Hinge Region In some ments, the CARs (which form part of the backbones) described herein se a hinge region between the antigen specific domain and the transmembrane domain. In some embodiments, the hinge region comprises any one or more of human CD80L or an Fc fragment of an antibody or a functional equivalent, fragment or derivative thereof,I a hinge region of human CD801 or an dy or a onal equivalent, fragment or derivative thereof, a CH2 region of an dy, a CH3 region of an antibody, an artificial spacer sequence and combinations thereof. In exemplary embodiments, 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 CD80, (v) a hinge, CH2 and CH3 region of IgG 1, (vi) a hinge region of IgG1, (vi) a hinge and CH2 region of IgGl, or (vii) combinations thereof. embrane Domain 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 ce. The transmembrane domains of the CARS described herein may be selected so that the transmembrane domain do not dimerize. In some embodiments, the TMD encoded CAR comprising any of the backbones described herein comprises a transmembrane domain ed 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, , 0X40, CD2, CD27, LFA-l (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, [LZR beta, IL2R gamma, IL7R a, ITGAl, VLA], CD49a, ITGA4, 1A4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 la, LFA-l, ITGAM, CD1 lb, ITGAX, CDl lc, ITGBl, CD29, ITGB2, CD18, LFA-l, ITGB7, TNFR2, DNAMl(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAMI, CRT AM, Ly9 (CD229), CD160 (BY55), PSGLI, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF 1, CD150, [PO-3), BLAME (SLAMF8), SELPLG ), LTBR, p, NKp44, NKp30, NKp46, NKGZD, and/or NKGZC 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 ellular region of the protein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 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 embrane protein is derived (e.g., l, 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 embodiment, the transmembrane domain may be from the same protein that the signaling domain, costimulatory domain or the hinge domain is derived from.

In r aspect, the transmembrane domain is not derived from the same protein that any other domain of the CAR is derived from.

Intracellular signaling domain qfchimeric antigen receptors As described herein, the CARs (which form part of the nes) described herein comprise an intracellular signaling domain. This domain may be cytoplasmic and may transduce the or function signal and direct the cell to perform its specialized function.

Examples of intracellular signaling domains include, but are not limited to, C chain of the T- cell receptor or any of its homologs (e.g., 11 chain, chR17 and B chains, MBl (Igor) chain, B29 (IgB) chain, etc), CD3 polypeptides (A, 5 and a), syk family tyrosine kinases (Syk, ZAP 70, etc), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and other molecules involved in T- cell uction, such as CD2, CD5 and CD28. Speciﬁcally, the intracellular signaling domain may be human CD3 zeta chain, FcyRIII, FcaRI, 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 embodiments, the intracellular signaling domain ses a signaling domain of one or more of a human CD3 zeta chain, chRIII, FceRI, a cytoplasmic tail of a Fc receptor, an immunoreceptor ne-based activation motif (ITAM) beating cytoplasmic receptors, and ations thereof.

Co-stimulatory n As described herein, the CARS (which form part of the backbones) described herein comprise a co-stimulatory domain. In exemplary embodiments, the co-stimulatory domain comprises a signaling domain from any one or more of CD28, CD137 (4-lBB), CD134 (0X40), DaplO, CD27, CD2, CD5, ICAM-l, LFA-l, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40 and combinations thereof.

Cleavable Linkers Cleavable linkers as described herein include 2A linkers (for example T2A), 2A- like linkers or functional equivalents thereof and ations thereof. In some ments, the linkers include the picornaviral 2A-like linker, CHYSEL ces of porcine teschovirus (P2A), Thosea asigna virus (T2A) or ations, variants and functional equivalents thereof. In other embodiments, the linker sequences may se Asp-Val/Ile-Glu-X-Asn-Pro-Gly(ZN-Prom" motif, which results in cleavage between the 2A glycine and the 2B proline. The nucleic sequences of several exemplary Cleavable linkers are ed in SEQ ID NO: 831 to SEQ ID NO: 836 and amino acid ces of several exemplary s 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 ion. In an embodiment, a y-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 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. In an embodiment, cleavable linkers are placed between the polypeptide ng 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 "Accessory modules" as used herein refer to agents that enhance, reduce or modify the ty of T cells expressing the CARS or reduce toxicity associated with CARS so that the therapeutic response of the CARS is enhanced.

In some embodiments, vectors comprising polynucleotides ng CARS ﬁlrther 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 sing the CARS from apoptosis. In exemplary embodiments, such proteins e but are not limited to vFLIP-K13 from 's sarcoma associated herpes virus (SEQ ID NO: 866), MC159 from molluscum contagiosum virus (SEQ ID NO: 867), cFLIP- L/MRITa (SEQ ID NO: 873), cFLIP-p22 (SEQ ID NO: 874) and Tax from human T cell leukemia/lymphoma virus (HTLV-I and HTLV-Z) (SEQ ID NOS: 875 and SEQ ID NO: 876) and Tax-RS mutant from HTLV-Z (SEQ ID NO: 877).

In one embodiment, vectors encoding CARS further encode vFLIP-Kl3. In another embodiment, s encoding CARS further encode MC159. In r embodiment, vectors encoding CARS further encode HTLVl-Tax. In another embodiment, vectors encoding CARS r encode HTLV2-Tax. In another embodiment, vectors encoding CARS further encode cFLIP-L/MRITa.

In some embodiments, the accessory molecules are d by vectors that are distinct from the vectors encoding by the CARS described herein. In some embodiments, or cells comprising vectors encoding CARS also comprise vectors encoding accessory molecules.

] In some embodiments, 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 -L/NﬂUTa or p22).

(Table 16).

In some ments, vectors comprising polynucleotides encoding CARS further comprise polynucleotides encoding siRNA or scFv speciﬁc for cytokines. In exemplary embodiments, the cytokines are any one or more of IL-10, IL-6, IFNy or ations thereof. In some ment, the CARS are co-expressed with a secreted bispecific antibody fragment that binds to IL6 receptor a and human serum albumin. In some embodiment, the CARS are co-expressed with a secreted scFv fragment that binds to H.6. In some embodiments, the CARS are coexpressed with the peptide FX06 so as to mitigate capillary leak associated with CAR therapy.

In further embodiments, vectors comprising polynucleotides ng CARS r comprise polynucleotides encoding siRNA or a nuclease ing the endogenous TCR-a, TCR-B, TCR-y, TCR-delta, CD3gamma, CD3zeta, CD3epsilon, CD3-delta.

In further embodiments, vectors comprising polynucleotides encoding CARS r comprise polynucleotides encoding a selectable marker. In exemplary embodiment, 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 embodiment, the selectable marker may encode for ellular 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 embodiment, cetuximab, an anti-EGFR monoclonal is used to eliminate CAR- expressing cells of the invention which coexpress a tmncated 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 embodiments, 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: 3 and 3526-3530 (Table 12). Reducing the clonal diversity of allogeneic T cells sing the CARS will in turn lead to reduced incidence of Graft versus Host Disease , thereby allowing the use of allogeneic T cells for CAR-T cell Polynucleotides and Polypeptides Provided herein are one or more polypeptides encoded by one or more nucleic acid molecules encoding conventional CARS I to 111 or any one or more of backbones 1-62 described herein (Table 2).

Also provided herein are one or more polypeptides encoded by one or more nucleic acid molecules encoding conventional CARS I to 111. In some embodiments, 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 uous and in the same reading frame with each other components of the CAR. In some embodiments, 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 c s in the same CAR.

Also provided herein are one or more polypeptides encoded by one or more nucleic acid molecules encoding backbone-l sing conventional CAR I and K13-vFLIP as described herein. In some embodiments, 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 , each component of the CAR is contiguous and in the same reading frame with each other ents of the CAR comprising backbone-1. In some embodiments, in the CAR comprising backbone-l comprises more than one antigen speciﬁc , each of the antigen speciﬁc domains are contiguous and in the same reading frame as the other antigen speciﬁc domains in the same CAR.

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 embodiments, the n-speciﬁc domain of the CAR comprising backbone-32 is c 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 ments, in the CAR sing backbone-32 comprises more than one antigen speciﬁc domain, each of the n speciﬁc domains are contiguous and in the same reading frame as the other antigen speciﬁc domains in the same CAR.

In various embodiments, 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 ne-1, backbone-2, backbone-32 or backbone-33, comprise two, three or more antigen speciﬁc domains.

In various embodiments, the polypeptides encoded by the nucleic acid molecules encoding CARs which are part of conventional CARs I to 111 or part of the backbones described herein, such as backbone-1, backbone-2, backbone-32 or ne-33, comprise two, three or more co-stimulatory domains.

In s ments, the polypeptides d by the c acid molecules encoding CARS which are part of conventional CARS I to 111 or part of the backbones described herein, such as ne-1, backbone-2, backbone-32 or backbone-33, comprise two, three or more intracellular signaling domain.

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

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 ning the same, using standard techniques. Alternatively, the nucleic acid of interest can be produced synthetically, rather than cloned.

In some embodiments, 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, ne-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is Speciﬁc to target as described in Tables 19-22.

In one embodiment, an antigen speciﬁc domain of a CAR t a target n is an antigen binding n, e.g., CDRs, of vL and vH fragments ing this antigen as shown in Tables 4 and 6, respectively.

In one embodiment, 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.

In one embodiment, an antigen speciﬁc domain of a CAR against a target antigen is an antigen binding n of a non-immunoglobulin scaffold targeting this antigen as shown in Table 8.

] In one embodiment, an antigen c domain of a CAR against a target n is an antigen binding n of a receptor known to bind this target antigen.

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

In some embodiments, provided herein are polypeptides encoded by the c acid molecules encoding CARs which are part of the conventional CARS I to 111 or are part of backbones described herein, such as backbone-l, 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.

] In some embodiments, provided herein are polypeptides d 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-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to the thrombopoietin receptor, In some ments, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARS which are part of the conventional CARS l to 111 or are part of backbones described , such as backbone-l, backbone-2, backbone-32 or backbone-33, n the antigen-speciﬁc domain of the CARS is Speciﬁc to CD19.

In some embodiments, 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-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to CD20.

In some embodiments, 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-l, backbone-2, ne-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is c to CD22.

In some embodiments, provided herein are polypeptides encoded by the nucleic acid molecules ng CARS which are part of the conventional CARS l to 111 or are part of backbones bed herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to CD23 In some embodiments, 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 ne-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is c to CD30, .

In some embodiments, provided herein are polypeptides encoded by the nucleic acid les encoding CARS which are part of the conventional CARS I to III or are part of backbones bed herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to CD32.

In some embodiments, 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-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to CD33.

In some embodiments, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARS which are part of the conventional CARS I to 111 or are part of backbones described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to CD 123.

In some embodiments, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARS which are part of the conventional CARS I to 111 or are part of backbones described herein, such as backbone-l, ne-2, ne-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to CD138.

In some embodiments, provided herein are ptides encoded by the c acid molecules encoding CARS which are part of the conventional CARS I to III or are part of backbones described herein, such as backbone-l, backbone-2, ne-32 or ne-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to CD200R.

] In some embodiments, 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-l, ne-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to CD276.

] In some embodiments, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARS which are part of the conventional CARS I to 111 or are part of nes described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, n the n-speciﬁc domain of the CARS is speciﬁc to CD324.

In some embodiments, 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 ne-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to BCMA.

In some ments, provided herein are polypeptides encoded by the nucleic acid les encoding CARS which are part of the conventional CARS I to III or are part of backbones described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, n the antigen-speciﬁc domain of the CARS is speciﬁc to CS1.

In some ments, provided herein are ptides encoded by the nucleic acid molecules encoding CARS which are part of the conventional CARS I to 111 or are part of backbones described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to ALKl.

In some embodiments, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARS which are part of the conventional CARS I to 111 or are part of backbones described , such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to RORl.

In some ments, ed herein are polypeptides d 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-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to CDH6 In some embodiments, 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-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to CDH16.

In some embodiments, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARS which are part of the conventional CARS I to 111 or are part of backbones described herein, such as ne-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to CDH17.

In some embodiments, provided herein are polypeptides encoded by the c acid molecules ng CARS which are part of the conventional CARS I to III or are part of backbones described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to CDHl 9.

In some embodiments, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARS which are part of the tional CARS I to III or are part of backbones described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to EGFRviii.

In some embodiments, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARS which are part of the conventional CARS I to 111 or are part of backbones described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to Her2.

In some ments, ed herein are polypeptides encoded by the nucleic acid molecules encoding CARS which are part of the conventional CARS I to 111 or are part of backbones bed herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, n the antigen-speciﬁc domain of the CARS is speciﬁc to Her3.

In some embodiments, provided herein are polypeptides encoded by the nucleic acid molecules ng CARS which are part of the conventional CARS I to III or are part of backbones described herein, such as backbone-l, backbone-2, ne-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to Mesothelin.

In some embodiments, 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-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is c to Folate Receptor alpha.

In some embodiments, provided herein are polypeptides encoded by the c acid molecules encoding CARS which are part of the conventional CARS I to 111 or are part of backbones described herein, such as backbone-l, backbone-2, ne-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to Folate Receptor beta.

] In some embodiments, 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-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to CLL-l.

] In some embodiments, provided herein are polypeptides encoded by the nucleic acid molecules ng CARS which are part of the conventional CARS I to III or are part of backbones described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is Speciﬁc to CLECSA.

In some embodiments, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARS which are part of the conventional CARS I to 111 or are part of backbones described herein, such as ne-l, backbone-2, backbone-32 or ne-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to NY-ESO/MHC class I complex.

] In some embodiments, 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-l, backbone-2, backbone-32 or backbone-33, wherein the n-speciﬁc domain of the CARS is speciﬁc to WTl/MHC class I complex.

In some embodiments, provided herein are polypeptides encoded by the c acid molecules encoding CARS which are part of the conventional CARS I to III or are part of backbones described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the n-speciﬁc domain of the CARS is Speciﬁc to WTl/MHC class I complex.

] In some embodiments, provided herein are polypeptides encoded by the nucleic acid les encoding CARS which are part of the conventional CARS l to 111 or are part of backbones described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to AFP/MHC class I complex.

In some embodiments, 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 nes described , such as ne-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is speciﬁc to HPV16-E7/MHC class I complex.

In some embodiments, provided herein are polypeptides encoded by the nucleic acid les encoding CARS which are part of the conventional CARS I to III or are part of backbones described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is Speciﬁc to gplOO/MHC class I complex.

In some embodiments, 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, n the antigen-speciﬁc domain of the CARS is specific to hTERT/MHC class 1 complex.

In some embodiments, ed herein are polypeptides encoded by the nucleic acid molecules encoding CARS which are part of the conventional CARS I to 111 or are part of backbones described herein, such as backbone-1, ne-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARs is speciﬁc to MARTl/MHC class I complex.

In some embodiments, provided herein are polypeptides encoded by the nucleic acid molecules ng CARS which are part of the tional CARS I to 111 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 HTLVl-Tax/MHC class I In some embodiments, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARS which are part of the conventional CARS I to 111 or are part of backbones described , such as backbone-1, backbone-2, backbone-32 or ne-33, wherein the antigen-speciﬁc domain of the CARS is Speciﬁc to PRl/MHC class I complex.

] In some embodiments, provided herein are polypeptides d 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 c to HIVl-gag/MHC class I complex.

In some embodiments, provided herein are polypeptides encoded by the nucleic acid molecules ng CARS which are part of the conventional CARS I to III or are part of backbones described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is Speciﬁc to nvelop gp120.

I] In some embodiments, 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 bed herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is Specific to DLL3.

In some embodiments, provided herein are ptides 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, ne-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is speciﬁc to PTK7.

] In some embodiments, 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-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is Speciﬁc to TROPZ. [004l4] In some embodiments, ed 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 LAMP].

In some ments, provided herein are polypeptides encoded by the c 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 Timl.

In some embodiments, ed herein are polypeptides encoded by the nucleic acid les encoding CARS which are part of the conventional CARS I to 111 or are part of backbones described herein, such as backbone-l, backbone-2, ne-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is Speciﬁc to TCR gamma-delta.

In some embodiments, 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-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is speciﬁc to TCR beta] constant chain.

In some embodiments, provided herein are ptides encoded by the nucleic acid les encoding CARS which are part of the conventional CARS I to III or are part of backbones described herein, such as ne-1, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is Specific to TCR beta2 constant chain.

In some embodiments, 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 ne-33, wherein the antigen-Speciﬁc domain of the CARS is speciﬁc to GCC.

In some embodiments, 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 , such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the n-Speciﬁc domain of the CARS is Speciﬁc to B7H4.

] In some ments, provided herein are polypeptides encoded by the nucleic acid molecules ng CARS which are part of the conventional CARS I to III or are part of backbones bed herein, such as backbone-1, backbone-2, backbone-32 or ne-33, wherein the antigen-speciﬁc domain of the CARS is c to LHR.

In some embodiments, 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.

In some embodiments, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARS which are part of the conventional CARS I to 111 or are part of backbones described , such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is Speciﬁc to Tn-Mucl.

In some embodiments, 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-l, ne-2, backbone-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is Speciﬁc to TSLPR.

In some ments, ed herein are polypeptides encoded by the nucleic acid molecules encoding CARS which are part of the tional CARS I to III or are part of nes 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.

In some embodiments, 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.

In some embodiments, provided herein are ptides 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-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is Speciﬁc to SLea.

In some embodiments, ed 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 Mucl/MHC class I complex.

In some ments, 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, ne-2, backbone-32 or backbone-33, n the antigen-speciﬁc domain of the CARS is speciﬁc to Muc16.

In some embodiments, provided herein are ptides encoded by the nucleic acid molecules encoding CARS which are part of the conventional CARS I to 111 or are part of backbones described herein, such as ne-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is Speciﬁc to NYBR-l.

In some embodiments, provided herein are polypeptides encoded by the nucleic acid les encoding CARS which are part of the conventional CARS I to III or are part of backbones bed herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is Speciﬁc to IL13Ra2.

In some embodiments, provided herein are polypeptides encoded by the nucleic acid molecules ng CARS which are part of the conventional CARS I to III or are part of backbones described , such as backbone-1, backbone-2, ne-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is Specific to ILl lRa.

In some embodiments, provided herein are polypeptides encoded by the nucleic acid les ng 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 LlCAM.

In some embodiments, 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-l, ne-2, backbone-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is Speciﬁc to EpCAMl.

In some embodiments, provided herein are polypeptides d 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, ne-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is c to gpNMB.

In some embodiments, 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 n-speciﬁc domain of the CARS is speciﬁc to GRP78.

In some embodiments, provided herein are polypeptides encoded by the nucleic acid les encoding CARS which are part of the conventional CARS I to 111 or are part of backbones described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is Speciﬁc to GPC3.

In some embodiments, 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 nes described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is speciﬁc to GRPCSD.

In some embodiments, provided herein are polypeptides encoded by the nucleic acid molecules encoding CARS which are part of the tional 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.

In some embodiments, provided herein are polypeptides d 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, n the antigen-Speciﬁc domain of the CARS is speciﬁc to FITC.

In some embodiments, 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 , such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is speciﬁc to CD79b.

In some ments, 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, ne-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to Lym 1.

In some embodiments, provided herein are ptides 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 ne-1, ne-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to Lym2.

In some ments, provided herein are polypeptides encoded by the nucleic acid les encoding CARS which are part of the conventional CARS I to 111 or are part of backbones described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the n-Speciﬁc domain of the CARS iS speciﬁc to Fc portion of an antibody (ie lg Fc). An exemplary CAR with the antigen-speciﬁc domain c to lg Fe is represented by SEQ ID NO: 2708 and contains the extracellular domain of CDl6-V158 as the antigen speciﬁc domain.

In some embodiments, the nucleic acid molecule encoding the CARS and/or ory molecules described herein is provided as a messenger RNA (mRNA) transcript.

In another embodiment, the nucleic acid molecule encoding the CARS and/or accessory molecules described herein is provided as a DNA uct.

Also provided herein are vectors comprising the polynucleotides described herein.

In some embodiments, the s are viral s. Examples of viral s include but are not limited to retrovirus, an adenovirus, an associated virus, a lentivirus, a pox virus, a herpes virus vector or a sleeping beauty transposon vector. In various embodiments, the present invention includes retroviral and lentiviral vector constructs expressing the CAR and the accessory molecules that can be directly transduced into a cell.

The present ion 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 ce ("UTR") (e.g., a 3‘ and/or 5' UTR bed herein), a 5' cap (e.g., a 5' cap described herein) and/or Internal Ribosome Entry Site (IRES) (e.g., an IRES described herein), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length (SEQ ID NO:922). RNA so produced can ntly transfect different kinds of cells. In one embodiment, the template includes sequences for the CAR. In an embodiment, an RNA CAR vector is transduced into a cell, e.g., a T cell or a NK cell, by oporation. In another embodiment, an RNA CAR vector is transduced into a cell, e.g., a T cell or a NK cell, by causing transient perturbations in cell membrane using a microﬂuid device as described in patent application WO 2013/059343 Al (PCT/U82012/060646). The polynucleotide sequences coding for the d molecules can be obtained using inant methods known in the art, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.

The present invention also es vectors in which a DNA encoding the CARs of the present invention is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable ation of a transgene and its propagation in daughter cells. Lentiviral s have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. Exemplary lentiviral vectors are provided in SEQ ID NOs: 905-906. A retroviral vector may also be, e.g., a gammaretroviral vector. A gammaretroviral vector may include, e.g., a promoter, a packaging signal (\y), a primer binding site (PBS), one or more (e.g., two) long al repeats (LTR), and a transgene of interest, e.g., a gene encoding a CAR. A gammaretroviral vector may lack viral ural gens such as gag, pol, and env. Exemplary gammaretroviral vectors include Murine Leukemia Virus (MLV), Spleen-Focus Forming Virus (SFFV), and Myeloproliferative Sarcoma Virus (MPSV), and vectors derived therefrom. Other gammaretroviral s are described, e.g., in Tobias Maetzig et al., "Gammaretroviral Vectors: Biology, Technology and Application" Viruses. 2011 Jun; 3(6): 677-713. An exemplary retroviral vector is provided in SEQ ID NO: 907. In another embodiment, the vector comprising the c acid encoding the desired CAR of the invention is an adenoviral vector (AS/35).

] The expression of natural or synthetic nucleic acids encoding CARS is typically ed by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to a promoter, and orating the construct into an expression vector. The vectors can be suitable for ation and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. The expression constructs of the present invention may also be used for nucleic acid immunization and gene therapy, using rd gene delivery ols. Methods for gene delivery are known in the art. See, e.g., US. Pat. Nos. 5,399,346, 5,580,859, 466, incorporated by reference herein in their entireti es.

Cloning and expression methods will be apparent to a person of skill in the art and may be as bed in WO 42675; Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1 -4, Cold Spring Harbor Press, NY; June et al. 2009 Nature Reviews Immunology 9.10: 704-716; WO 01/96584; WO 01/29058; US. Pat. No. 193, the contents of each of which are herein incorporated by reference in their entirety as though set forth herein.

Physical s for introducing polynucleotides of into host cells such as calcium phosphate transfection and the like are well known in the an and will be apparent to a person of skill in the art. In exemplary embodiments, such methods are set forth in ok et al., 2012, MOLECULAR CLONING: A TORY MANUAL, s 1 -4, Cold Spring Harbor Press, NY); US. Pat. Nos. 5,350,674 and 5,585,362, the contents of each of which are herein incorporated by reference in their entirety as though set forth herein. In another embodiment, 3 CAR vector is transduced into a cell, e.g., a T cell or a NK cell, by causing transient perturbations in cell membrane using a microfluid device as described in patent application Eng. 1, 0039 (2017) the ts of each of which are herein incorporated by reference in their entirety as though set forth herein.

Genetically Engineered Cells In various embodiments, the cells for modiﬁcations with CARS described herein, including T cells or NK cells may be obtained from a subject desiring therapy. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of ion, ascites, pleural effusion, spleen , and tumors. T cells could be tissue resident gamma-delta T cells, which can be cultured and expanded in vitro prior to expression of the CAR.

In one aspect, the invention provides a number of chimeric antigen receptors (CAR) comprising an n binding domain (e.g., antibody or antibody fragment, TCR or TCR fragment) engineered for specific binding to a disease-associated antigen, e.g., a tumor antigen described herein. In one , the invention provides an immune effector cell (e.g., T cell, NK cell) engineered to express a CAR, wherein the engineered immune effector cell exhibits a therapeutic ty. In one aspect, the invention provides an immune effector cell (e.g., T cell, NK cell) engineered to s a CAR, wherein the engineered immune effector cell exhibits an anticancer property In one aspect, a cell is transformed with the CAR and the CAR is expressed on the cell surface. In some embodiments, the cell (e.g., T cell, NK cell) is transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral . In some embodiments, the Viral vector is a lentiviral vector. In some such ments, the cell may stably express the CAR. In another embodiment, the cell (e. g., T cell, NK cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a CAR.

In some such embodiments, the cell may transiently express the CAR.

The present invention provides immune effector cells (e.g., T cells, NK cells) that are engineered to contain one or more CARS that direct the immune effector cells to diseased cells or disease-associated cells, such as cancer cells. This is achieved through an antigen binding domain on the CAR that is speciﬁc for a cancer associated antigen. There are two classes of cancer associated antigens (tumor antigens) that can be targeted by the CARS of the instant invention: (1) cancer associated antigens that are expressed on the surface of cancer cells; and (2) cancer associated antigens that itself is intracellular, however, a fragment of such antigen (peptide) is ted on the surface of the cancer cells by MHC (major histocompatibility complex).

Furthermore, the present invention es CARS and CAR—expressing cells and their use in medicaments or methods for treating, among other diseases, cancer or any malignancy or autoimmune diseases or infectious disease or degenerative e or allergic disease involving cells or tissues which express a tumor antigen or disease associated antigen as described herein.

In one aspect, the invention provides an immune effector cell (e.g., T cell, NK cell) engineered to express a chimeric antigen receptor (CAR), wherein the engineered immune effector cell exhibits an anti-disease ty, such as mor property. A preferred antigen is a cancer associated antigen (i.e., tumor antigen) described herein. In one aspect, the antigen g domain of the CAR comprises a partially humanized antibody fragment. In one aspect, the n binding domain of the CAR comprises a partially zed scFv.

Accordingly, the invention provides CARS that comprises a humanized n binding domain and is engineered into a cell, e.g., a T cell or a NK cell, and methods of their use for adoptive therapy.

Further ed herein are genetically engineered cells, comprising the cleotides and/or the chimeric antigen receptors described herein. In some embodiments, the cell is a T-lymphocyte (T-cell). In some embodiment the cell is a naive T cells, a central memory T cells, an effector memory T cell, a regulatory T cell (Treg) or a combination thereof. In some embodiments, the cell is a natural killer (NK) cell, a hematopoietic stem cell (HSC), an embryonic stem cell, or a pluripotent stem cell.

Genetically engineered cells which may comprise and express the CARs of the invention include, but are not limited to, T-lymphocytes (T-cells), naive T cells (TN), memory T cells (for e, central memory T cells (TCM), effector memory cells (TEM)), natural killer cells, hematopoietic stem cells and/or pluripotent embryonic/induced stem cells e of giving rise to therapeutically relevant progeny. In an ment, the genetically engineered cells are autologous cells. In an embodiment, the genetically engineered cells are allogeneic cells. By way of e, dual T-cells of the invention may be CD4+/CD8-, CD4- /CD8+, CD4-/CD8- or CD4+/CD8+. The T-cells may be a mixed population of CD4+/CD8- and CD4-/CD8+ cells or a tion of a single clone. CD4+ T-cells of the invention may produce lL-Z, IFNy, TNFOL and other T-cell effector cytokines when co-cultured in vitro with cells expressing the target antigens (for example CD20+ and/or CD19+ tumor cells). CD8+ T-cells of the invention may lyse n-speciﬁc target cells when co-cultured in vitro with the target cells. In some embodiments, T cells may be any one or more of CD45RA+ CD62L+ naive cells, CD4SRO+ CD62L+ central memory cells, CD62L— effector memory cells or a combination thereof (Berger et al., Adoptive transfer of Virus-speciﬁc and tumor- speciﬁc T cell immunity. Curr Opin Immunol 2009 24-232), cally modiﬁed cells may be produced by stably transfecting cells with DNA encoding the CAR of the invention.

Various methods produce stable transfectants which express the CARS of the invention. In one embodiment, a method of stably transfecting and ecting cells is by electroporation using naked DNA. By using naked DNA, the time required to produce redirected cells may be signiﬁcantly reduced. Additional methods to genetically engineer cells using naked DNA ng the CAR of the invention include but are not limited to chemical transformation s (e.g., using m phosphate, dendrimers, liposomes and/or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical transformation, gene electrotransfer, transient bation in cell membranes and/or hydrodynamic delivery) and/or particle-based methods (e.g., impalefection, using a gene gun and/or magnetofection). The transfected cells demonstrating presence of a single integrated un-rearranged vector and sion of the CAR may be expanded ex vivo. In one embodiment, the cells selected for ex vivo expansion are CD8+ and demonstrates the ty to speciﬁcally recognize and lyse antigen-speciﬁc target cells.

Viral transduction methods may also be used to te cted cells which express the CAR of the invention. Cell types that may be used to generate genetically modiﬁed cells expressing the CAR of the invention e but are not limited to T- lymphocytes (T-cells), natural killer cells, poietic stem cells and/or pluripotent embryonic/induced stem cells capable of giving rise to therapeutically relevant progeny.

Stimulation of the T-cells by an antigen under proper conditions results in proliferation (expansion) of the cells and/or production of IL-2. The cells comprising the CAR of the invention will expand in number in response to the binding of one or more antigens to the n-speciﬁc ing regions of the CAR. The invention also provides a method of making and expanding cells sing a CAR. The method comprises transfecting or transducing the cells with the vector expressing the CAR and stimulating the cells with cells expressing the target antigens, recombinant target antigens, or an antibody to the receptor to cause the cells to proliferate, so as to make and expand T-cells. In an embodiment, the cells may be any one or more of T-lymphocytes (T-cells), natural killer (NK) cells, hematopoietic stem cells (HSCs) or pluripotent embryonic/induced stem cells e of giving rise to eutically relevant progeny.

In some embodiments, genetically engineered cells described herein express the various backbones described herein, wherein the CAR component of the backbone determines target speciﬁcity based on the antigen specific domain of the CAR.

In one embodiment, the genetically engineered cells comprise nucleic acid molecules encoding conventional CARS I to III or conventional CARs I to 111 which are part of the backbones described herein, such as ne-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to MPL.

In one embodiment, the genetically engineered cells comprise nucleic acid molecules encoding conventional CARS I to 111 or conventional CARS Ito III which 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 CD19.

In one embodiment, the genetically engineered cells comprise nucleic acid molecules encoding conventional CARS I to 111 or conventional CARS I to III which are part of the backbones described herein, such as backbone-l, backbone-2, backbone-32 or ne-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to CD20.

In one embodiment, the genetically engineered cells comprise c acid molecules encoding conventional CARS I to 111 or conventional CARS Ito III which are part of the nes bed herein, such as backbone-1, ne-2, backbone-32 or backbone-33, n the n-speciﬁc domain of the CARS is speciﬁc to BCMA.

In one embodiment, the genetically engineered cells comprise nucleic acid molecules encoding conventional CARS I to 111 or conventional CARS I to 111 which are part of the backbones described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to CD22.

] In one embodiment, the genetically engineered cells comprise nucleic acid molecules encoding conventional CARS I to 111 or conventional CARS I to III which are part of the backbones described herein, such as ne-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to CD30.

In one embodiment, the genetically engineered cells comprise nucleic acid molecules encoding tional CARS I to 111 or conventional CARS I to 111 which are part of the backbones described herein, such as backbone-1, backbone-2, backbone-32 or backbone-33, n the antigen-speciﬁc domain of the CARS is speciﬁc to CD33.

In one embodiment, the cally ered cells comprise nucleic acid molecules encoding conventional CARS I to 111 or conventional CARS I to 111 which are part of the backbones described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to CD123.

In one embodiment, the genetically engineered cells comprise nucleic acid molecules encoding tional CARS I to 111 or conventional CARS I to 111 which are part of the backbones described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to CD138.

In one embodiment, the genetically ered cells comprise nucleic acid molecules encoding conventional CARS I to 111 or conventional CARS I to III which are part of the backbones described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to CLLl.

] In one embodiment, the genetically engineered cells comprise nucleic acid molecules encoding tional CARS I to 111 or conventional CARS I to 111 which 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 TCR-betal constant chain.

In one embodiment, the cally engineered cells comprise nucleic acid molecules encoding conventional CARS I to 111 or conventional CARS I to 111 which 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 TCR-beta2 constant chain.

In one embodiment, the genetically engineered cells se nucleic acid molecules encoding conventional CARS I to 111 or conventional CARS I to 111 which 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 ALK.

In one ment, the genetically engineered cells comprise c acid molecules encoding conventional CARS I to 111 or conventional CARS I to III which are part of the backbones described herein, such as ne-1, backbone-2, ne-32 or backbone-33, wherein the n-speciﬁc domain of the CARS is speciﬁc to PTK7.

In one embodiment, the genetically engineered cells comprise nucleic acid molecules encoding conventional CARS I to 111 or conventional CARS I to 111 which are part of the backbones described herein, such as backbone-l, backbone-2, ne-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is speciﬁc to DLL3.

In one embodiment, the genetically engineered cells comprise nucleic acid molecules encoding conventional CARS I to III or conventional CARS I to 111 which are part of the backbones described herein, such as backbone-l, backbone-2, ne-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is speciﬁc to TROPZ.

In one embodiment, the genetically engineered cells comprise nucleic acid molecules encoding conventional CARS I to III or tional CARS I to III which are part of the backbones described herein, such as ne-l, backbone-2, ne-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is Specific to Tim 1.

In one ment, the genetically engineered cells comprise nucleic acid molecules encoding conventional CARS I to 111 or conventional CARS I to 111 which are part of the backbones described herein, such as backbone-1, ne-2, backbone-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is speciﬁc to LAMPl.

In one ment, the genetically engineered cells comprise nucleic acid molecules encoding conventional CARS I to 111 or conventional CARS I to 111 which are part of the backbones described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS iS speciﬁc to CS].

In one embodiment, the genetically engineered cells comprise nucleic acid molecules encoding conventional CARS I to III or conventional CARS I to III which are part of the backbones described herein, such as backbone-l, ne-2, backbone-32 or ne-33, wherein the antigen-Speciﬁc domain of the CARS is Specific to Lyml.

In one embodiment, the genetically engineered cells comprise nucleic acid molecules encoding conventional CARS I to 111 or conventional CARS I to 111 which are part of the nes 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.

In one embodiment, the genetically engineered cells comprise c acid molecules encoding conventional CARS I to 111 or conventional CARS I to 111 which are part of the backbones described herein, such as backbone-l, backbone-2, backbone-32 or ne-33, wherein the antigen-Speciﬁc domain of the CARS iS c to TSHR.

In one embodiment, the genetically engineered cells comprise nucleic acid molecules encoding conventional CARS I to 111 or conventional CARS I to 111 which are part of the backbones described herein, such aS backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is speciﬁc to NY-ESO/IVHIC class I complex.

In one embodiment, the genetically engineered cells comprise nucleic acid molecules encoding tional CARS I to III or conventional CARS I to 111 which are part of the backbones described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen-speciﬁc domain of the CARS is specific to WT]/ MHC class I complex.

In one embodiment, the genetically engineered cells comprise nucleic acid molecules encoding conventional CARS 1 to 111 or conventional CARS 1 to 111 which are part of the backbones described herein, such as ne-1, backbone-2, ne-32 or backbone-33, wherein the antigen-Speciﬁc domain of the CARS is speciﬁc to HIVl envelop glycoprotein gp120.

In one embodiment, the genetically engineered cells se c acid molecules encoding conventional CARS I to 111 or tional CARS I to III which are part of the backbones described herein, such as backbone-l, backbone-2, backbone-32 or ne-33, wherein the n-speciﬁc domain of the CARS is speciﬁc to Fc region of an immunoglobulin.

In various embodiments, the genetically engineered cells comprise nucleic acid molecules encoding conventional CARS I to 111 or conventional CARS I to III which are part of the backbones described herein, such as backbone-l, backbone-2, backbone-32 or backbone-33, wherein the antigen Specific domain targets n described in Tables 4, 7, 8, 11,19, 20, 21 or 22.

Immune effector cells such as T cells and NK cells comprising CARS and/or enhancers as described herein may be activated and expanded generally using methods as described, for example, in US. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; ,858,358; 6,887,466; 6,905,681; 575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; ,883,223; 6,905,874; 6,797,514; 041; and US. Patent Application Publication No. 20060121005.

Therapeutic Methods ed herein are methods for ng a disease associated with expression of a disease-associated antigen or a cancer associated antigen. in one embodiment, provided herein are methods for treating a disease in a subject in need thereof by administering to the t a therapeutically effective amount of genetically modiﬁed cells bed herein (such as T cells, NK cells) that are engineered to express an n—speciﬁc CAR alone or an antigen specific CAR and an accessory molecule, wherein the antigen is a disease speciﬁc antigen as described herein, and wherein the disease causing or disease-associated cells express the said disease~specitic antigen.

] In one embodiment, provided herein are methods for treating cancer in a subject in need thereof by administering to the subject a therapeutically effective amount of genetically modiﬁed cells described herein (such as T cells, NK cells) that are engineered to express an antigen-speciﬁc CAR alone or an antigen speciﬁc CAR and an accessory le, wherein the antigen is a cancer specific antigen as described , and wherein the cancer cells express the said tumor antigen. in one embodiment, the cancer speciﬁc antigen is expressed on both normal cells and cancers cells but is expressed at lower levels on normal cells. in one embodiment, the method further ses selecting a CAR that binds the cancer speciﬁc antigen of interest with an afﬁnity that allows the antigen specific CAR to bind and kill the cancer cells. in some embodiments, the antigen speciﬁc CAR kills cancer cells but kills less than 30%, 259/, %. 15%, 10%, 5% or less of the normal cells expressing the cancer antigen. In exemplary embodiments, the percentage of cells killed by the antigen speciﬁc CARS may be ined using the cell death assays bed . in some embodiment, the present invention provides methods of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the genetically modified cells (eg, T cells, NK cells) that are engineered to express conventional CARs I to ill, wherein the ASD of the CARS is c to the antigen that is expressed on cancer cells (for example, the antigen is expressed at lower levels on normal cells relative to cancer cells). in some embodiment, the present invention es methods of treating, cancer in a subject in need thereof comprising administering to the subject a therapeutically ell‘ective amount of the genetically modiﬁed cells (eg, T cells, NK cells) that are engineered to express backbone-l sing the conventional CARS I and the accessory module K13- vFLlP, wherein the ASI) of the (ITARs is specific to the antigen that is expressed on cancer cells (for example, the antigen is expressed at lower levels on normal cells relative to cancer cells). in some embodiment, the present invention provides methods of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the genetically ed cells (cg. T cells, NK cells) that are engineered to comprising backbone-2 comprising the conventional CARS I and the accessory module MClSQ-VFLH’, wherein the ASD of the CARs is c to the antigen that is sed on cancer cells (for example, the antigen is expressed at lower levels on normal cells relative to cancer cells). in some embodiment, the present invention es s of ng cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the genetically modiﬁed cells (cg, T cells, NK cells) that are engineered to comprising backbone-32 comprising the conventional CAR ll and the accessory module KlS-VFLIP, wherein the ASD of the CARS is speciﬁc to the antigen that is expressed on cancer cells (for example, the antigen is expressed at lower levels on normal cells ve to cancer cells). in some embodiment, the present invention provides methods oftreating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the genetically modiﬁed cells (eg, '1‘ cells, NK cells) that are engineered to sing backbone-33 comprising the conventional CAR II and the accessory module MClS9-vFl..lP, wherein the ASI) of the CARs is speciﬁc to the antigen that is expressed on cancer cells (for example, the antigen is expressed at lower levels on normal cells relative to cancer cells). in exemplary embodiments, the antigens that may be targeted for the therapeutic methods described herein include but are not limited to any one, two, three, four or more of: C2319; CDl23; CD22; C1323, CD30; CD32, CD99; Tissue Factor; MPL; C0171; CS-l (also referred to as CD2 subset l, CRACC, SLAMF7, CD319, and 19A24)‘, C~type -like molecule-1 (CLL-l or CLECLI); CD33; epidermal growth factor receptor variant Ill (EGFRVIII); ganglioside GP. (GDZ); ganglioside GD3(aNeuSAc(2n8)aNeuSAGO-3 )bDGalptl- 4)bI)Glcp(l-l)Cer); 'I'NF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or ca—Ser/Thr'»; prostate—speciﬁc membrane n (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (RORl); Fms- Like Tyrosine Kinase ‘3 (FLT3); Tumor- associated glycoprotein 72 (TAG72); CD38; CD44V6; Carcinoembryonic n (CEA); Epithelial cell adhesion molecule (EPCAM); B7113 (CD276); KIT (CD117); eukin—lit receptor subunit alpha—2 (IL-l3Ra2 or CD2 IBAZ); Mesothelin; Interleukin 11 or alpha (IL-l lRa); prostate stem cell antigen ; Protease Serine 21 (Testisin or PRSSZl); vascular endothelial growth factor receptor 2 (VEC‘iFR'Z); Lewis(Y) antigen; (1)24; Platelgt- derived growth factor receptor beta —beta); Stage—specific embryonic antigen-4 (SS!§EA~4); CDZO; 'Folate receptor alpha; Receptor tyrosine-protein kinase ERBIEZ (HerZIneu); Mucin 1, cell surface associated (MUCl); epidermal growth factor receptor (EGFR); neural cell adhesion molecule ; Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (PAP); insulin-like growth factor 1 receptor (16F?! receptor), carbonic anhydrase IX (CAFX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (It..MP2); glycoprotein 100 (gplOO); oncogene fusion protein consisting of hreakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog l (Abl) (bcr—abl); tyrosinase; eplirin type-A receptor 2 (EphAZ); Pucosyl GMl; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeuSA.c(Z—3)bDGalptl— 4)bDGlcp(l-l)Cer); transglutaminase 5 (TGSS); high molecular weight—melanoma associated antigen {HMWMAA}; yl-GDZ ganglioside (OAcGD’Z); Folate receptor beta; tumor endothelial marker 1 (TEMl/CDZ48); tumor endothelial marker ted ('I'EM7R‘); claudin 6 (CLDNO'); thyroid stimulating hormone receptor ); G n- coupled or class C group 5; member D (GPRCSD); chromosome X open reading frame 61 (CHIRP-‘61); CD97; (3037921; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific t (PLACI); hexasaccharide portion of globoH eramide (Globol-I); mammary gland differentiation antigen (’NY-BR‘l); uroplakin 2 (UPKZ); Hepatitis A virus cellular receptor 1 (HAVCRl); adrenoceptor beta 3 (ADRB3); in 3 ); G protein-coupled receptor 20 (GPRZO); lymphocyte antigen 6 complex, locus K 9 (LY'6K); Olfactory receptor Sl E2 (ORSIVEZ); 'l'CR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WTl); Cancer/testis antigen 1 (NY- ESO-l); Cancer/testis antigen 2 -l-la); Melanoma-associated antigen 1 {MAGE—Al); ETS translocation-variant gene 6, located on chromosome lZp -ANE); sperm protein 17 (SPAN); X Antigen Family, Member 1A (XAGEI); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen— 1 (MAD—CT—l); melanoma cancer testis antigen-2 T—Z); Fos—related antigen .1; tumor protein p53 (p53); p.53 ; prostein; surviving; telomerase; prostate carcinoma tumor antigen- 1 (PC’I‘A—l or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human 'I'elomerase reverse riptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML—LAP); ERG (transmembrane protease. serine 2 lPRSSZ) li'I'S fusion gene); N-Acetyl glucosaminyl-transferase V (NAN); paired box protein Fax—3 (PAX3); Androgen receptor; Cyclin Bl; v-myc avian myeiocytomatosis viral ne neuroblastoma derived homolog (hil‘i’tilN'); Ras olog Family Member C (RhoC); 'l‘yrosinase—related protein 2 ("l‘RP—Z); Cytochrome P450 lBl (CY‘l’lBl); CCCTC- Binding Factor (Zinc Finger Protein)~Like (BORIS or Brother of the tor of imprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SAR’l‘3); Paired box n Pax~5 (PA'XS); proacrosin binding protein spBZ (OY—TESl); lymphocyte- speciﬁc n tyrosine irinase ; A kinase anchor protein 4 (AKAP—ii); synoyial sarcoma, X breakpoint 2 (SSXZ); Receptor for Advanced Glycation Endproducts (RAGE—l); renal ubiquitous 1 mm); renal ubiquitous 2 (R12); legumain; human papilloma virus E56 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp’IO-Z); CD79a; CD79b; CD72; I...eukocyte-associated immunoglobulin— like receptor 1 (LAlRl); Fc fragment of lgA receptor (FCAR or CD89); Leukocyte immunoglolmlin-like receptor ily A member 2 (l...lI..RA.2); CD300 cule-lilte family member f (CD3OOLF); C~type lectin domain family 12 member A (CLEClZA); bone marrow stromal cell antigen 2 ; EGF—like module-containing mucin— like hormone receptor- like 2 (IEi'MRZ); lymphocyte antigen 75 (LY75); Glypican-3 (1(iPCS); Fc receptor- like 5 (FCRLS); and immunoglobulin lambda—like polypeptide l (IGLLl), PTK7, LAMPI, 'l'ROP2, ".l‘iml and HIV 1 envelop glycoprotein gpth).

] In exemplary embodiments, the antigens that may be targeted for the therapeutic s described herein include but are not limited to any one, two, three, four or more of: TSHR, CD171, CS-t, CLL-i, GD3, '"i‘n Ag, 131.713, CD38, CD44V6, B7H3, KET, IL-13Ra2, 1L-liRa, PSCA, PRSSZE, VEGFRZZ, LewisY, CD24, PDGFR-beta, SSEA-éi, MUCI, EGFR, NCAM, CAEX, LMPZ, EphAZ, Fucosyi GM}, sLe, (3M3, , HMWMAA, o—aeetyl—GDZ, Folate receptor beta, TEMl/CDZ48, TEM7R, CLDN6, GPRCSD, CXORF61, CD97, (3017951, ALK, Polysialic acid, PLACI, bol-i, NY-BR—l, UPK2, t-lAVCRi, ADRB3, PANX3, GPRIZO, LY6K, ORSiEB, TARP, W11, ETVé—AIVIL, sperm protein 17, XAGEi, Tie 2, MAD—CT—l, MAD—C'ILZ, lies-related antigen 1, 953 mutant, h'l’IER'I’, sarcoma translocation breakpoints, i‘vt'L—IAP, ERG (’i‘MPRSSQ E'I‘S fusion gene), NA'E7, PAX3, Androgen receptor, Cyclin Bi, MYCN, RhoC, , BORIS, SART3, PAXS, ()Y—TESi, LCK, i, SSXZ, CD793, CD79b, CD72, LAIRI, FCAR, LILRAZ, CD300LF, CLEC 12A, BSTZ, EMR2, LY75. GPCS, FCRLS, and IGLL'E.

In exemplary embodiments, the antigens that may be targeted for the therapeutic methods described herein include but are not d to any one, two, three, four or more of: WT-l, hTERT, FRAME, HMMR/Rhamm, PR1, CGl, CD33, Cyclin-Al, NY-ESO-l, MAGE, HA-l, ACCl, T4A, LB-LY7S-IK, BCR-ABL, FLT3-ITD, B-cell receptor idiotype, HTLV-I Tax protein, CD19, Lewis Y, CD22 and RORl.

In exemplary embodiments, the antigens that may be targeted for the therapeutic methods described herein include but are not d to any one, two, three, four or more of the targets described in Tables 4, 7, 8, ll, 19, 20, 21 or 22. in one embodiment, the present invention provides methods of treating cancer by providing to the subject in need ot‘immune effector cells (eg, T cells, NK cells) that are engineered to express conventionai CARS I to 111 or backbones (for example, ne-i, backbone—2. backbone-32 or backbone—33) specific to MPL, wherein the cancer cells express MP1,. In one embodiment, the cancer to be treated is acute myeloid leukemia, chronic myeloid leukemia, and myelodysplastic syndrome. In one ment, the antigen speciﬁc domain of the MP1. speciﬁc CAR comprises VL nts as described in SEQ 11) N05: 180- 187, Va fragments as described in SEQ ID NOS: 427-434 and/or scFV as described in SEQ 11) N03: 729~736. In some embodiments, the activity of CAR-T cells may be lled using Dasatinib, nib and/or A-770041, concurrently or sequentiaiiy with the immune effector cetls described herein. in one embodiment, the present invention provides methods of treating cancer, autoimmune or ic disease by ing to the subject in need thereof immune etl‘ector cells (e.g., '1' cells, NK cells) that are engineered to express conventional CARS l to ill or backbones (for example, backbone-l, backbone~2, ne—32 or backbonen33) speciﬁc to (3019, wherein the cancer cells express (3019. in one embodiment, the cancer to be treated is acute lyniphoblastic leukemia, chronic lymphocytic leukemia, B cell , malignancy, non— kins lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, or, multiple myeloma. In one embodiment, the antigen c domain of the CD19 speciﬁc CAR comprises VL fragments as described in SEQ [D NOs: 40-55, V}; fragments as described in SEQ ID NOS: 288-303 and/or scFV as described in SEQ ID NOS: 564—577. In some embodiments, the activity of CAR—T cells may be controlled using Dasatinib, nib and/or A-770041, concurrently or sequentially with the immune effector cells bed herein. in one embodiment. the present invention provides methods of treating cancer by providing, to the t in need thereof immune effector cells (e.g., T cells, NK cells) that are ered to express backbones (for example, ne~l, backboneu2, backbonev32 or backbone~33) speciﬁc to GRP-78, wherein the cancer cells express GRP—7r . In one embodiment, the cancer to be treated is breast cancer. in one embodiment, when the immune effector cells expressing the GRP-78 CAR. also expresses an MP1. CAR, the cancer to be treated is acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, diffuse large B-cell lymphoma, mantle cell lymphoma, myelodysplastic syndrome or multiple myeloma. In one embodiment, the antigen speciﬁc domain of the CERF-"8 c CAR ses scFV as bed in SEQ ID NO: 7'03. In one embodiment. the antigen speciﬁc domain of the MP}... speciﬁc CAR comprises VL fragments as bed in SEQ 1D NOs: lilo-187, VH fragments as described in SEQ ID NOs: 427-434 and/or scFV as described in SEQ ID NOS: 729—736, In some embodiments, the activity of CAR—T cells may be controlled using, Dasatinib, l’onatinib and/or A-77004l, rently or sequentially with the immune effector cells described herein.

In one embodiment, the present invention provides methods of treating cancer, autoimmune or allergic disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are ered to express backbones (for example, backbone-1, backbone-2, backbone-32 or backbone-33) speciﬁc to CD79b, wherein the cancer cells express CD79b. ln one embodiment. the cancer to be treated is acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeioid leukemia, diffuse large Bncell lymphoma, mantle cell lymphoma, myelodysplastic syndrome or le myeloma. In one embodiment, the antigen speciﬁc domain of the CD79b speciﬁc CAR comprises VL fragments as bed in SEQ ID NOs: 92-93, VH fragments as described in SEQ ID NOs: 1 and/or scFV as described in SEQ ID NO: 628. In some embodiments, the activity of CAR—'1' cells may be controlled using Dasatinib, l’onatinib and/or ilk—770041, concurrently or sequentially with the immune effector cells described herein.

In one embodiment, the t invention provides methods of treating cancer, autoimmune or allergic disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to s tional CARS I to III or backbones (for example, backbone-1, backbone-2, backbone-32 or backbone-33) speciﬁc to ROR-l (Receptor tyrosine kinase-like orphan receptor 1), wherein the disease associated or disease causing cells express ROR-l .11} some embodiments, cancer to be d is chronic cytic leukemia or solid organ . In one embodiment, the antigen speciﬁc domain of the ROR-l speciﬁc CAR comprises VH fragments as bed in SEQ ID NOs: 454—455 and/or scFV as described in SEQ ID NO: 755-756. In some embodiments, the acrivity of CAR—T cells may be controlled using Dasatinib, Ponatinib and/or A—77004l, concurrently or sequentially with the immune effector cells bed herein.

] In one ment, the present invention provides methods of treating cancer, autoimmune or allergic a disease by ing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express backbones (for example, backbone-1, backbone-2, backbone-32 or backbone-33) speciﬁc to Lym-l. In one embodiment, the cancer to be treated or prevented is a B cell malignancyln one embodiment, the antigen speciﬁc domain of the Lym-l speciﬁc CAR comprises VL fragments as bed in SEQ ID NO: 175, V1.1 fragments as described in SEQ ID NO: 421 and/or scFV as described in SEQ ID NO: 723. In some embodiments, the activity of CAR-3i" cells may be controlled using Dasa'tinib, Ponatinib and/or A—77004l, concurrently or sequentially with the immune effector cells described herein.

In one embodiment, the present invention es methods of treating , autoimmune or allergic disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express conventional CAR I to III or backbones (for example, backbone-1, backbone-2, backbone-32 or backbone-33) c to Lym-2. In some embodiments, the cancer to be treated is blood cancer, n cancer, prostate cancer or pancreatic . In one embodiment, the antigen speciﬁc domain of the Lym-2 speciﬁc CAR comprises VL fragments as described in SEQ ID NO: 176, VH fragments as described in SEQ ID NO: 422 and/or scFV as described in SEQ ID NO: 724. In some embodiments, the activity ofCA R-T cells may be controlled using Dasa'tinib, Penatinib and/or A—77004’i, concurrently or sequentially with the immune effector celis described herein.

In one aspect, the present invention provides methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express a CD123-specif1c conventional CAR or backbones 1-62 (for example, backbone-l, backbone- 2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells express CD123. In one embodiment, the disease to be treated or prevented is a cancer. In one embodiment, the cancer to be d or prevented is AML or myelodysplastic syndrome (MDS). in some en'ibodinients. the activity of CAR-T cells may be controlled using Dasatinib, Ponatinib and/0r A—77004I, concurrently or sequentially with the immune effector cells described herein.

In one aspect, the present invention es methods of treating or preventing a disease by providing to the t in need f immune or cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express a CD22-specific conventional CAR or backbones 1-62 (for example, backbone-l, backbone-2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells express CD22. In one embodiment, the disease to be treated or prevented is a cancer. In one embodiment, the cancer to be d or prevented is B cell ancies. In one embodiment, the disease to be treated or prevented is an immune disease or allergic disease.

In some embodiments, the activity of CAR-T ceils may be controlled using Dasatinib, Ponatinih and/or A-?7004l, concurrently or sequentially with the immune effector cells bed herein In one aspect, the present invention provides methods of treating or preventing a disease by providing to the subject in need f immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express a CD23-specif1c conventional CAR or backbones 1-62 (for example, backbone-1, backbone-2, backbone-32 or backbone-33), n the disease causing or disease associated cells express CD23. In one embodiment, the disease to be treated or prevented is a cancer. In one embodiment, the cancer to be d or prevented is B cell ancies. In one embodiment, the disease to be d or prevented is an immune disease or allergic disease.

In some embodiments, the activity of CAR-T cells may be controlled using Dasatinib, Ponatinib and/or A-‘I'IOOAE !, concurrently or sequentially with the administration of immune effector cells described herein, In one aspect, the present invention provides s of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express a CS-l-specific conventional CAR or nes 1-62 (for example, backbone-1, ne-2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells express CS-l. In one embodiment, the disease to be treated or prevented is an immune disease or allergic disease. In one embodiment, the disease to be treated or prevented is a cancer. In one embodiment, the cancer to be treated or prevented is a plasma cell malignancy or multiple myeloma or primary effusion lymphoma. In some embodiments, the activity of CAR—T cells may be controiled using nib, Ponatinib and/or It l, concurrently or sequentially with the administration ofimmune effector cells described herein.

In one aspect, the present invention provides methods of ng or preventing a disease by providing to the subject in need f immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are ered to express a BCMA-CAR, wherein the disease causing or disease associated cells express BCMA. In one embodiment, the disease to be treated or prevented is an immune disease or allergic disease.

In one embodiment, the disease to be treated or prevented is a cancer. In one ment, the cancer to be d or prevented is a plasma cell malignancy or multiple myeloma or primary effusion lymphoma. In one embodiment, the disease to be treated or ted is an immune disease. in some embodiments, the activittr of CA R—T cells may be controlled using Dasatinib, Ponatinib and/or A-77004 l, concurrently or sequentially with the administration of immune effector cells described herein.

] In one aspect, the present invention es methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e. g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express a CLL-l-speciﬁc conventional CAR or backbones 1-62 (for example, backbone- l, backbone-2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells express CLL-l. In one ment, the disease to be treated or prevented is a cancer. In one embodiment, the cancer to be treated or prevented is AML or MDS. In some embodiments, the activity of CAR—T cells may be lled using Dasatinib, Pcnatinib and/or A-7700ill, concurrently or sequentially with the administration of immune effector cells bed herein.

] In one aspect, the t invention provides s of ng or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express a pecific conventional CAR or backbones 1-62 (for e, backbone-1, backbone-2, backbone-32 or ne-33), wherein the disease causing or disease ated cells express CD30. In one embodiment, the disease to be treated or prevented is a cancer. In one embodiment, the cancer to be treated or prevented is lymphoma or leukemia. in some ments, the activity of CAR—T cells may be controlled using Dasatinib, Penatinib and/or A3700", concurrently or sequentially with the stration of immune effector cells described herein.

In one aspect, the present invention provides methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express a CD33-speciﬁc conventional CAR or backbones 1-62 (for example, backbone-l, backbone-2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells express CD33. In one embodiment, the disease to be treated or prevented is a cancer. In one embodiment, the cancer to be treated or prevented is AML or MDS. In some embodiments, the activity of CAR-T cells may be controlled using Dasatinib, l‘onatinih and/or A-C’7004l, concurrently or sequentially with the administration of immune effector cells described herein.

In one aspect, the present invention es methods of treating or preventing a disease by providing to the subject in need f immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express a IL-13Ra2-speciﬂc conventional CAR or backbones 1-62 (for example, backbone-l, backbone-2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells express IL-I3Ra2. In one ment, the disease to be treated or prevented is a cancer.

In one ment, the cancer to be treated or prevented is glioblastoma. In some ments, the activity of CAR~T cells may be controlled using Dasatinib, Ponatinib and/or xix-77004}, concurrently or tially with the administration of immune effector cells described herein In one aspect, the present invention provides methods of treating or preventing a e by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are ered to express a IL—l lRa-CAR, wherein the disease causing or disease associated cells s lL—l lRa. In one embodiment, the e to be treated or prevented is a cancer. In one embodiment, the cancer to be treated or prevented is osteosarcoma. In some embodiments, the activity of CAR—T cells may be controlled using Dasatinib, Ponaiinib and/or A—‘7’70041, concurrently or sequentially with the administration of immune effector cells described herein.

In one aspect, the t invention provides methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express CD20-CAR, wherein the disease causing or e associated cells express CD20. In one embodiment, the e to be treated or prevented is a cancer. In one embodiment, the cancer to be treated or prevented is a B cell malignancy, such as chronic lymphocytic leukemia, acute cytic leukemia, diffuse large cell lymphoma, follicular lymphoma or mantle cell lymphoma. In one embodiment, the disease to be treated or prevented is an immune disease or allergic disease. in some embodiments, the. activity of CAR—T cells may be controlled using Dasatinib, Ponatinib and/or A-C’7004 l, concurrently or sequentialiy with the administration ofimmune effector cells described .

In one aspect, the present invention provides methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express folate or alpha-specific conventional CAR or backbones 1-62 (for example, backbone- 1, backbone-2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells express folate receptor alpha. In one embodiment, the disease to be treated or prevented is a cancer. In one embodiment, the cancer to be treated or prevented is ovarian cancer, NSCLC, tn'al , renal cancer, or other solid . In some embodiments, the activity of CAR~T cells may be controlled using Dasatinib, Ponatinib and/or A-77()04i, concurrently or sequentially with the administration of immune effector cells described herein.

In one aspect, the t ion provides methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express TSHR-speciflc conventional CAR or backbones 1—62 (for example, backbone-1, backbone—2, backbone-32 or backbone-33), wherein the disease causing or disease ated cells express TSHR (Thyroid Stimulating e Receptor). In one embodiment, the disease to be treated or prevented is cancer. In one embodiment, the cancer to be treated or prevented is thyroid cancer, or le myeloma or T cell leukemia or T cell lymphoma. in some embodiments, the activity of CAR—T celis may be controlled using Dasatinib, Ponatinib and/or A3700", concurrently or sequentially with the stration of immune effector cells described herein.

In one aspect, the present invention provides methods of treating or ting a disease by ing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express GPRCSD-specific conventional CAR or backbones 1-62 (for example, backbone-l, backbone-2, backbone—32 or ne-33), wherein the disease causing or disease associated cells express GPRCSD. In one ment, the disease to be treated or prevented is cancer, immune or allergic e. In one embodiment, the cancer to be treated or prevented is a plasma cell disorder, multiple myeloma or primary effusion lymphoma. In some embodiments, the activity of CAR~T cells may be controlled using Dasatinib, Poriatinih and/or A-77()()4l, concurrently or sequentially with the stration of immune effector cells described herein.

In one aspect, the present ion es methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e. g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express ALK-specifrc conventional CAR or backbones 1-62 (for example, backbone-l, backbone-2, backbone-32 or backbone-33), n the disease causing or disease associated cells express ALK. In one embodiment, the disease to be d or ted is a cancer. In one embodiment, the cancer to be treated or prevented is a NSCLC, ALCL (anaplastic large cell lymphoma), IMT (inﬂammatory myoﬁbroblastic tumor), or neuroblastoma. In some embodiments, the activity of ‘ cells may be controlled using Dasatinib, Ponatinib andl'or A‘770041, concurrently or sequentially with the administration of immune effector cells described .

] In one aspect, the present invention provides methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express a HAVCRI—specifrc conventional CAR or backbones 1-62 (for example, backbone-l, backbone-2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells express I-IAVCRl. In one embodiment, the disease to be treated or ted is a cancer.

In one embodiment, the cancer to be treated or prevented is renal . In some embodiments, the activity of CARJI' cells may be controlled using Dasatinib, Ponatinib and/or zit-770043, concurrently or sequentially with the administration of immune erfector cells bed herein.

In one , the present invention provides methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune or cells that are engineered to express a WTl/MHC I complex—speciﬁc CAR, wherein the disease causing or disease associated cells express WTl in association with MHC class I complex. In one embodiment, the disease to be treated or prevented is a cancer. In one embodiment, the cancer to be treated or prevented is AML or myeloma. In some embodiments, the activity of CAR-'1' cells may be controlled using Dasatinib, Ponatinib and/or A—770041, concurrently or sequentially with the administration of immune effector cells described herein.

In one aspect, the present invention provides methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e,g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express a NY-ESO-l/MHC 1 complex-speciﬁc conventional CAR or backbones 1-62 (for example, backbone-l, backbone-2, backbone-32 or ne-33), wherein the disease causing or disease associated cells express NY-ESO-I in association with MHC class I complex. In one embodiment, the disease to be d or prevented is a cancer. In one embodiment, the cancer to be treated or prevented is myeloma. In some embodiments, the activity of CAR-l" cells may be controlled using nib, Ponatinib and/or A-77004l. rently or sequential!y with the administration of immune or cells described herein.

In one aspect, the present invention provides s of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune or cells that are engineered to express PTK7-specif1c conventional CAR or backbones 1-62 (for example, backbone-1, backbone-2, backbone-32 or backbone-33), wherein the disease causing or e associated cells express PTK7. In one embodiment, the disease to be treated or prevented is a cancer. In one embodiment, the cancer to be treated or prevented is melanoma, lung cancer or ovarian cancer. in some embodiments, the ty of ' cells may be controlled using Dasatinib, Ponatinib and/or A—7700till, concurrently or sequentially with the administration of immune effector cells described .

In one aspect, the present invention provides s of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e. g, T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express DLL3-specific conventional CAR or backbones 1-62 (for example, backbone-1, ne-2, backbone-32 or ne-33), wherein the disease causing or disease associated cells express DLL3. In one embodiment, the disease to be treated or ted is a cancer. In one embodiment, the cancer to be treated or prevented is ma, lung cancer or ovarian cancer. In some embodiments, the activity of CAR—T cells may be controlled using Dasatinib, nib and/or ill-770043, concurrently or tially with the administration of immune effector ceils described herein.

In one aspect, the present ion provides methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express GCC—speciﬁc conventional CAR or backbones 1-62 (for e, backbone-l, backbone-2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells express GCC. In one embodiment, the disease to be d or prevented is a cancer. In one embodiment, the cancer to be treated or ted is gastrointestinal cancer. In some embodiments, the activity of CAR—T cells may be lled using nib, nib and/or A-770041, concurrently or sequentially with the administration of immune effector cells described .

In one aspect, the present invention provides methods of treating or preventing a e by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express CCR4-speciflc conventional CAR or backbones 1-62 (for example, backbone-l, backbone-2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells express CCR4. In one embodiment, the disease to be treated or prevented is a cancer. In one embodiment, the cancer to be treated or prevented is T cell leukemia or ma. In some embodiments, the activity of CAR-T cells may be controlled using Dasatinib, Ponaiinib and/or A--770041, concurrently or sequentially with the administration of immune effector cells described .

In one aspect, the present invention provides methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express CDHl/CD324-speciﬂc conventional CAR or backbones 1-62 (for e, backbone-l, ne-2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells express CDI-Il/CD324. In one embodiment, the disease to be treated or prevented is a cancer. In some embodiments, the activity ofCAR—T cells may be controlled using Dasatinib, Ponatinib and/or 4l, concurrently or sequentially with the administration of immune effector cells described herein.

In one aspect, the present invention es s of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are ered to express CDZOOR-specific conventional CAR or nes 1-62 (for example, ne-l, backbone- 2, backbone-32 or ne-33), wherein the disease causing or disease associated cells express . In one embodiment, the e to be treated or prevented is a cancer. In some embodiments, the activity ot‘CAR-T cells may be controlled using Dasatinib, Ponatinib and/or its—770043, concurrently or sequentially with the administration of immune effector cells descn‘ bed herein.

In one aspect, the present invention provides methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e. g., T cells, NK cells) that are ered to express a speciﬁc conventional CAR or backbones 1-62 (for example, backbone-1, backbone-2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells express CDH19. In one embodiment, the disease to be treated or prevented is a cancer. In one embodiment, the cancer to be treated or prevented is a solid tumor. In some embodiments, the activity of CAR~'I‘ cells may be controlled using Dasatinih, POIléltinil) and/or 04 l, concurrently or sequentially with the administration of immune effector cells described herein.

In one aspect, the present invention provides methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (eig, T cells, NK cells) that are engineered to express a CDH17-specific conventional CAR or backbones 1-62 (for e, backbone-1, backbone-2, backbone-32 or backbone-33), wherein the disease g or disease associated cells s CDHl7. In one embodiment, the disease to be treated or prevented is a cancer. In one embodiment, the cancer to be treated or prevented is a solid tumor. In some embodiments, the activity of CAR-'1‘ cells may be controlled using Dasatinih, Ponatinih and/or A—77004 l or sequentially with the administration of , concurrently immune effector cells described herein.

] In one aspect, the present invention provides methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a pecific conventional CAR or backbones 1-62 (for example, backbone-l, backbone-2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells express CDH6. In one embodiment, the disease to be d or ted is a cancer. In one ment, the cancer to be treated or prevented is a solid tumor. in some embodiments, the activity of CAR—T cells may be controlled using Dasatinib, Ponatinib and/or A-77004l. concurrently or tially with the administration of immune effector ceils described herein.

In one aspect, the present invention es methods of treating or ting a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a GFRa4—specific conventional CAR or backbones 1-62 (for e, backbone-l, backbone-2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells express GFRa4. In one embodiment, the disease to be treated or prevented is a cancer. In one embodiment, the cancer to be d or prevented is a thyroid medullary cancer. In some embodiments, the activity of CAR-T cells may be controlled using nib, Ponatinib and/or A-770{)4l, concurrently or tially with the administration ofimmune effector cells described herein.

In one aspect, the present ion provides methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express LHR-specific conventional CAR or backbones 1-62 (for example, backbone-1, ne-2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells express LHR (Leutanizing Hormone Receptor). In one embodiment, the disease to be treated or prevented is a cancer. In one embodiment, the cancer to be treated or prevented is prostate cancer, ovarian cancer or breast cancer.

In one aspect, the present invention provides methods of treating or preventing a disease by ing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express FHR-speciﬂc conventional CAR or nes 1-62 (for e, backbone-1, backbone-2, ne-32 or backbone-33), wherein the disease causing or disease associated cells express FHR (Follicular Hormone Receptor). In one embodiment, the disease to be treated or prevented is a cancer. In one embodiment, the cancer to be treated or prevented is prostate cancer, ovarian cancer or breast cancer.

In one aspect, the present invention provides methods of ng or preventing a disease by providing to the subject in need thereof immune or cells (egr, T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express GR-speciﬁc conventional CAR or backbones 1-62 (for example, backbone-1, backbone-2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells express GR (Gonadotropin Receptor). In one embodiment, the disease to be treated or prevented is a cancer. In one ment, the cancer to be treated or prevented is prostate cancer, ovarian cancer or breast cancer.

In one aspect, the t invention es methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express CD45-speciﬁc conventional CAR or backbones 1-62 (for e, backbone-l, backbone-2, backbone-32 or backbone-33), n the disease causing or disease associated cells express CD45, In one embodiment, the disease to be treated or prevented is a cancer. In one embodiment, the cancer to be treated or prevented is a blood cancer.

In one aspect, the present invention provides methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express CD32-speciﬁc conventional CAR or backbones 1-62 (for example, backbone-1, backbone-2, backbone-32 or backbone-33), n the disease causing or disease associated cells express CD3 2. In one embodiment, the disease to be treated or prevented is a . In one embodiment, the cancer to be treated or prevented is a blood cancer.

In one aspect, the present invention provides methods of treating or ting a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express specific tional CAR or backbones 1-62 (for e, backbone-1, backbone- 2, backbone-32 or backbone-33), wherein the e g or disease ated cells express TROPZ. In one embodiment, the disease to be treated or prevented is a cancer. In one embodiment, the cancer to be treated or prevented is breast or prostate cancer.

In one aspect, the present invention provides methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express LAMP-specific conventional CAR or backbones 1-62 (for example, backbone-1, backbone-2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells express LAIvH’l. In one embodiment, the disease to be treated or prevented is a cancer. In one embodiment, the cancer to be treated or prevented is a leukemia, lymphoma or myeloma.

] In one aspect, the present ion provides methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune or cells that are engineered to express CD32-specif1c tional CAR or backbones 1-62 (for e, backbone-1, backbone-2, backbone-32 or ne-33), n the disease causing or disease associated cells express CD32. In one embodiment, the e to be treated or prevented is a cancer.

In one aspect, the present invention provides methods of treating or ting a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express specific conventional CAR or nes 1-62 (for example, backbone-1, backbone- 2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells express TCRBl (T cell receptor Betal constant chain). In one embodiment, the disease to be treated or prevented is a cancer or immune disease. In one ment, the cancer to be treated or prevented is T cell leukemia or T cell lymphoma. In one ment, the immune disorder to be treated or ted is multiple sclerosis, rheumatoid arthritis, sing spondylitis, inflammatory Bowel Disease, Diabetes Mellitus, Graft vs host disease or autoimmune Thyroiditis.

In one aspect, the present invention provides methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune or cells that are engineered to express TCRB2-specific conventional CAR or backbones 1-62 (for example, backbone-l, backbone- 2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells express TCRB2 (T cell receptor Beta2 constant chain). In one embodiment, the disease to be treated or prevented is a cancer or immune disorder. In one embodiment, the cancer to be treated or prevented is T cell leukemia or T cell lymphoma. In one embodiment, the immune disorder to be treated or prevented is multiple sclerosis, rheumatoid tis, ankylosing spondylitis, inﬂammatory Bowel Disease, Diabetes Mellitus, Graft vs host disease or autoimmune ditis.

In one aspect, the present invention provides methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e. g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express T cell receptor gamma-delta-specific tional CAR or backbones 1-62 (for example, backbone-1, backbone-2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells express T cell or gamma-delta. In one embodiment, the disease to be treated or prevented is a cancer or immune disorder. In one ment, the cancer to be treated or prevented is T cell leukemia or T cell lymphoma. In one embodiment, the immune disorder to be treated or prevented is multiple sclerosis, rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, diabetes mellitus, Graft vs host disease or autoimmune Thyroiditis.

In one aspect, the present invention provides methods of treating or preventing a e by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express HLA—AZ—speciﬁc conventional CAR or backbones 1-62 (for example, backbone-l, backbone-2, backbone-32 or ne-33). In one embodiment, the disease to be d or prevented is Graft vs Host e.

In one , the present invention provides methods of treating or preventing a disease by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express HIVl-CAR on backbones 1-62 (for e, backbone-l, backbone-2, backbone-32 or backbone-33) directed against the HIV envelop glycoprotein gp120, wherein the disease causing or disease ated cells express the HIVl envelop glycoprotein gp120. In one embodiment, the e to be treated or ted is HIVI/AIDS.

In one aspect, the present invention provides methods of treating or preventing CMV (Cytomegalovirus) infection or associated disease by providing to the subject in need f immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express CMV-specific CAR or backbones 1-62 (for example, backbone-l, backbone-2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells are infected with CMV. In one aspect the CMV CAR and backbones are directed against the CMV pp65 protein. In one aspect, the CMV-associated e is pneumonia, colitis or meningoencephalitis.

In one aspect, the present invention provides methods of treating or preventing EBB infection or diseases associated with EBB infection by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are ered to express an EBB-specific conventional CAR or backbones 1-62 (for example, backbone-1, backbone-2, backbone-32 or ne-33), wherein the disease causing or disease associated cells are infected with EBB. In one aspect the EBB-speciﬁc CARs and backbones are directed against the EBB EBNA3c protein. In one aspect, the EBB-speciﬁc CARS and backbones comprise a broadly neutralizing antibody against EBB. In one aspect, the EBB-associated disease is lymphoma.

In one aspect, the present invention provides s of treating or preventing KSHV ion or diseases associated with KSHV infection by providing to the subject in need thereof immune effector cells (e. g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are ered to express an KSHV-speciﬁc conventional CAR or nes I-62 (for example, backbone-l, backbone-2, backbone-32 or backbone-33 wherein the e causing or disease associated cells are infected with KSHV. In one aspect, the KSHV associated disease is Kaposi’s sarcoma or primary effusion ma or multicentric castleman’s disease.

In one aspect, the present invention provides methods of treating or preventing HTLVl infection or diseases ated with HTLVl infection by providing to the subject in need f immune effector cells (e. g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express HTLVl-specific tional CAR or nes 1-62 (for example, backbone-l, backbone-2, backbone-32 or backbone-33), wherein the disease causing or disease associated cells are infected with HTLVl. In one aspect, the HTLVl-speciflc conventional CAR or backbones target HTLVl Tax protein/MI-IC class I complex. In one , the HTLVI disease is human T cell leukemia or lymphoma.

In one aspect, the present invention provides methods of treating or preventing Inﬂuenza A infection or diseases associated with Inﬂuenza A infection by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express InﬂuenzaA-speciﬂc conventional CAR or backbones 1-62 (for example, backbone-1, ne-2, backbone-32 or backbone-33), wherein the disease g or disease associated cells are infected with Inﬂuenza A. In one aspect, the InﬂuenzaA-speciﬁc tional CAR or backbones comprise n binding domains derived from a broadly neutralizing antibody against Inﬂuenza A hemagglutinin (HA), such as MEDI8852.

In one aspect, the present invention provides s of treating or preventing a cancer, ion, autoimmune or ic diseases by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express a universal conventional CAR or nes 1-62 (for example, backbone-l, backbone-2, backbone-32 or backbone-33) encoding CD16V158 or a deletion— or point-mutant fragment thereof along with an antibody or an antibody fragment that binds to the CD16 domain of the CAR through its Fc region and on an antigen expressed on the disease associated cells through its variable (vL and/or vH) regions. In one aspect the disease associated cell is a cancer cell, an infected cell, or a plasma cell or a B cell or a T cell.

In one aspect, the present invention provides methods of ng or preventing a cancer, infection, autoimmune or allergic diseases by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) or stem cells that can give rise to immune effector cells that are engineered to express a pecific conventional CAR or nes 1-62 (for example, ne-1, backbone-2, backbone-32 or backbone-33) along with a FITC-labeled antibody or an antibody fragment that binds to an antigen expressed on the disease associated cells. In one aspect the disease associated cell is a cancer cell, an infected cell, or a plasma cell or a B cell or a T cell. ary cancers whose growth can be inhibited include s typically responsive to immunotherapy. miting examples of cancers for ent include melanoma (e.g., metastatic malignant melanoma), renal cancer (eg. clear cell carcinoma), prostate cancer (eg. hormone refractory prostate adenocarcinoma), breast , colon cancer and lung cancer (cg. non-small cell lung cancer). Additionally, refractory or recurrent malignancies can be treated using the molecules described herein. in exemplary embodiments. cancers treated by the methods bed herein include solid tumors such as sarcomas, adcnocarcinomas, and carcinomas, of the s organ systems, such as those affecting liver, lung. breast, lymphoid, gastrointestinal (cg, colon), genitourinary tract (eg, renal, urothelial cells), prostate and pharynx. Adenocarcinomas include malignancies such as most colon cancers, rectal cancer, cell oma, liver cancer, non~small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In one ment, 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 s that can be treated include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant ma, 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, nonal-lodgkin 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, c myeloid leukemia, acute lymphohlastic ia, 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), y CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T~cell lymphoma, nmentally induced s including those induced by asbestos, and combinations of said cancers. Treatment of metastatic cancers, e..,g metastatic cancers that express PD-Ll (lwai et al. (2005) int. Immunol. l7: E33444) can be effected using the antibody molecules described . Further a disease associated with a cancer associate antigen as described herein expression include, but not limited to, e.g., atypical and/or non—classical cancers, malignancies, precancerous ions or proliferative es associated with expression of a cancer associate antigen as described herein. in some embodiments, a CAR—expressing '1' cell or NK cell as bed herein reduces the quantity, number, amount or percentage of cells and/or cancer cells by at least 2. ‘34), at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% in a subject with hematological cancer or another cancer associated Mth a cancer associated antigen as described herein, expressing cells relative to a negative control. in one embodiment, the subject is a human.

In one embodiment, the present invention provides methods of treating hematological cancer by providing to the subject in need thereof genetically modiﬁed cells (e.g., T cells, NK cells) that are engineered to express the conventional CARs or backbones comprising antigen speciﬁc CARS speciﬁc to thrombopoietin receptor (MPL). In some embodiments, the hematological s include chronic enous leukemia (CMl_.), Acute Myeloid ia and Myelodysplastic syndrome. In some embodiments, a cancer that can be treated using the compositions and methods of the t invention is multiple myeloma. Multiple myeloma is a cancer of the blood, characterized by accumulation of a plasma cell clone in the bone marrow Current ies for multiple a include, but are not limited to, treatment with lenalidomide, which is an analog of thalidomide.

Lenalidomide has activities which include anti~tumor activity, angiogenesis inhibition, and immunomodulation. Generally, myeloma cells are thought to be negative for a cancer associate antigen as described herein expression by flow cytometry. Thus, in some embodiments, a BCMA CAR (SEQ II) Nos: 951—957), may be used to target myeloma cells.

In some embodiments, the CARS of the present invention therapy can be used in combination with one or more onal therapies, cg, lenalidomide treatment. in one aspect, the invention pertains to a method of inhibiting growth of a e (eg, cancer, autoimmune e, infectious disease or ic disease or a degenerative disease), sing contacting the disease causing or disease associated cell with a genetically modiﬁed cell of the present invention expressing a conventional CAR or a CA R with accessory s (i.e., backbones 1—62) such that the CAR—T is activated in response to the antigen and targets the disease causing or e associated cell, wherein the growth of the disease causing or disease associated cell is inhibited, In one aspect, the invention pertains to a method of preventing a disease, sing stering to a patient at risk of disease a CAR—expressing cell or a cell that is capable of generating a CAR-expressing cell of the present invention such that the ' is activated in response to the antigen and targets the disease causing or disease associated cell, wherein the growth of the disease causing or disease associated cell is prevented. In one aspect the disease is a cancer, an infectious disease, an immune disease, an allergic disease, or a degenerative disease.

Embodiments of the instant invention includes a type of cellular therapy where effector cells (such as T cells and NK cells) or stem cells that can give rise to effector cells are genetically modiﬁed to s a CAR as described herein and the CAR-expressing, T cell or NK cell is infused to a recipient in need thereof. The infused cell is able to kill tumor cells in the recipient. in various aspects, the immune effector cells (eg, '1‘ cells, NK cells) stered to the patient, or their progeny, persist in the patient for at least four months. ﬁve months, six months, seven months, eight months, nine months, ten , eleven months, twelve , thirteen , en month, ﬁfteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one. months, —two months, twenty— three months, two years, three years, four years, or five years after stration of the T cell or NK cell to the patient.

The invention also includes a type of cellular therapy where immune effector cells (e.g., '1' cells, NK cells) are modiﬁed, e.g., by in vitro ribed RNA, to transiently express a tional CAR or a conventional CAR with accessory modules (e.g backbones 1452). '1‘ cells or NK cells are infused to a recipient in need thereof. The infused cells are able to kill disease associated cells (on, tumor cells or vitally infected cells) in the recipient. Thus, in various aspects, the CAR-expressing immune effector cells leg, T cells, NK cells) persist for less than one month, eg, three weeks, two weeks, one week, after administration of the T cell or 'NK cell to the patient.

The invention also includes a type of cellular therapy where stem cells (eg, poietic stem cell or lymphoid stem cells or embryonic stem cells, or induced pluri potent stem cells) that are capable of giving rise to immune effector cells (cg, T cells or NK cells) are modiﬁed to express a conventional CAR or a conventional CAR with accessory modules (eg, backbones 1-62.) and are administered to a recipient in need f. The administered stem cells give rise to immune effector cells (cg, T cells or NK cells) after transplantation into the recipient, which (i.e. the immune effector cells) are able to kill disease ated cells in the recipient. Thus, in various aspects, the immune effector cells (6.9,, T cells, NK cells) that are produced in the patient after administration of CAR- expressing stem cells persist in the patient for at least one week, 2 weeks, 3 weeks. one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen month, ﬁfteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty—one months, twenty-two months, twenty three months, two years, three years. four years, five years, ten years or twenty years after administration of the cally modiﬁed stem cells to the patient. The invention also includes a type of cellular therapy where stem cells that are capable of giving rise to immune effector cells (cg, T cells or NK cells) are modiﬁed to express a tional CAR or a tional (TAR with accessory modules (cg. backbones l. —62) and are differentiated in vitro to generate immune effector cells that are infused to a recipient in need thereof The infused immune effector cells (cg, T cells or NK cells) after on into the recipient are able to kill disease associated cells in the recipient. Thus, in various aspects, the immune effector cells (cg, T cells, NK cells) that are administered to the patient persist in the patient for at least i day, 2 days, 3 days, ii days, 5. days, 6 days, one week, 2 weeks, 3 weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine , ten months, eleven months, twelve , thirteen months, fourteen month, fifteen months, sixteen months, seventeen months, eighteen months, en , twenty months, twenty—one months, twenty—two months, twenty three months, two years, three years, four years, five years, ten years or twenty years.

] The invention also includes a. type of ar therapy where regulatory immune effector cells leg, "l‘REG, or CD25+ '1' Cells) are d to express a conventional CAR or a conventional CAR with accessory modules (cg, backbones 1-62) targeting a speciﬁc antigen. Such CAR-'l‘RlE-ZG are administered to a t to suppress immune response t the speciﬁc antigen. The CAR—TREG can be used to prevent and treat autoimmune diseases and to enhance immune tolerance.

] The anti-tumor immunity response elicited by the CAR-modiﬁed immune effector cells (cg, T cells, NK cells) may he an active or a passive immune response, or alternatively may be due to a direct vs indirect immune response. In one aspect, the CAR-transduced immune effector cells (cg, T cells. NK cells) exhibit speciﬁc pro-inﬂammatory cytokine secretion and potent cytolytic activity in response to human diseased cells (e.g., cancer or infected cells) sing the disease associate n as described herein, resist soluble disease associate antigen as described herein, mediate bystander killing and mediate regression of an established human disease, including .

The invention also includes a type of cellular therapy where immune effector cells (cg, T cells and NK cells) or stem cells that are capable of giving rise to immune effector cells (cg, T cells or NK cells) are modiﬁed to express a conventional CAR or a conventional CAR with accessory modules (cg, backbones #62) and are used ex vivo to purge the bone marrow or peripheral blood poietic stem cells of disease‘associated cells (eg. cancer cells). As an example, T cells expressing a CD19—spcciiic CAR are cocultured with bone marrow or peripheral blood stem cell sample taken from a patient with acute lymphocytic leukemia or nonuHodgkin lymphoma so as to kill off any leukemia or lymphoma cells present in the bone marrow or peripheral blood stem cell preparation. After a suitable duration of e in vitro (ex vivo), which may range from a 6 hours to several days, the purged bone marrow and peripheral blood sample is used for autologous transplant in the patient.

] Ex vivo expansion ot‘hematopoietic stem and progenitor cells has been described in US Pat. No. 5, l99,942, and is incorporated herein by reference, and can he applied to the cells of the present ion. However, the present ion is not limited to any particular method of ex vivo expansion of the cells and other suitable methods known in the art can be utilized. Briefly, ex vivo culture and expansion of hematopoietic stem cells comprises: (l) collecting (3034+ hematopoietic stem and progenitor cells from a mammal from peripheral blood harvest or bone marrow explants; and ('2) expanding such cells ex vivo. in addition to the cellular growrh factors described in US. Pat. No 5,199,942, other s such as llt3-L, lL—l, IL-3 and c—kit ligand, can he used for ing and expansion of the cells. in addition to using a cell-based vaccine in terms of ex VlV'O immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response ed against an antigen in a patient.

In some embodiments, the human CAR-modified geneticaliy d ceils (such as T cells, NK cells) of the invention may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal (for example, . With respect to ex vivo immunization. at least one of the foliowing occurs in vitro prior to administering the cell into a mammal: i) expansion ofthe ceiis, ii.) introducing a nucieic acid encoding a CAR to the cells or iii) cryopreservation of the cells. Ex vivo ures are wet! known in the art. for e, as described in US. Pat. No. 5,199,942, incorporated herein by reference in addition to using a. cei1~based vaccine in terms of ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to eiicit an immune response directed against an antigen in a patient.

Further described herein are methods for controlling the activity of CAR-T cells and iﬁc T cell Engager (BiTE) (such as Blincyto or Blinatumomab) when administered to the patients. In some embodiment these methods can be used to control the side effects of CAR-T cells and BiTE, such as ne release syndrome, capillary leak syndrome and ogical complications. In majority of patients who d to engineered CAR T cells and umomab, 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). A number of these patients require admission to intensive care units for pressure support and artiﬁcial ventilation. In signiﬁcant number of cases, CAR T cells and Blinatumomab therapy can lead to neurologic symptoms including , es and can be fatal. Although a number of treatment approaches, ing steroids and an 1L6 receptor antibody, are used for the treatment of these disorders, a number of ts are refractory to such therapies. The instant invention provides a novel method for the treatment and prevention of complications, such as cytokine release syndrome and neurological complications, of adoptive cellular therapies, including but not limited to CAR-T cell and Bispecifrc T cell engagers (BiTE). In some embodiment the method involves stration of inhibitors of tyrosine kinases, particularly Scr family kinase, and in particular Lck kinase. In one embodiment, the method involves administration of Dasatinib, an oral small molecule inhibitor of Abl and Src family tyrosine kinases (SFK), including p56Lck (Lck) (Lee KC et a1, Leukemia (2010) 24, 896—900).

Dasatinib has been previously shown to block antigen-speciﬁc effector T cell functions (Weichsel R et al, Clin Cancer Res 2008; 14(8), 2484). r, it has never been tested for the ability to block functions of CAR-T cells and Bispeciﬁc T cell engagers. CAR-expressing T cells have been detected in the cerebrospinal ﬂuid (CSF) of patients ing from neurological complications (Hu, Y et al, Journal of Hematology & Oncology20169:70). A particularly important advantage of Dasatinib for the treatment and prevention of cytokine release syndrome and neurological complications caused by CAR-T cells and BiTE is its ability to cross the blood-brain barrier and achieve effective concentration in the brain when stered at clinically acceptable doses (Porkka K et al., Blood. 2008 Aug ;] 12(4)21005-12). ore, Dasatinib is expected to block the effector functions, including cytokine release, of CAR-T cells that have crossed the blood brain barrier.

In one embodiment, the Src kinase inhibitor is administered to the patient after the stration of CAR-expressing cells to control or terminate the activity of CAR- expressing cells. In one embodiment, an Lck inhibitor is administered to the patient after the administration of pressing cells to control or terminate the activity of CAR- expressing cells. In one embodiment, Lck inhibitor is 4 I.

In one embodiment, Dasatinib is administered to the patient after the administration of CAR-expressing cells to control or terminate the activity of pressing cells. In one embodiment, dasatinib is administered orally at a dose of at least 10 , 20 mg/day, 40mg/day, 60mg/day, 70mg/day, 90 mg/day, lOOmg/day, l40mg/day, day, 210mg/day, 250mg/day or day‘ In one embodiment, Ponatinib is administered to the patient after the administration of CAR-expressing cells to control or ate the ty of CAR-expressing cells. In one embodiment, ponatinib is administered orally at a dose of at least 15 mg/day, 30 mg/day, 45mg/day, 60mg/day.

T lymphocytes have a limited replicative life span until they reach the terminally differentiated state and then enter into a replicative senescence phase due to progressive loss of telomeres with age. Human T lymphocytes display a limited life-span of about 30—50 population doublings when cultured in vitro.

Further contemplated herein are methods for promoting the survival and proliferation of peripheral blood mononuclear cells and T cells and preventing their replicative senescence to extend the life span of immune cells (e.g., lymphocytes and NK cells) for the purpose of ve cell therapy. The method entails ectopic expression of viral and cellular ns that promote survival and proliferation and block activation induced cell death. The exemplary viral proteins suitable for this purpose include viral FLICE Inhibitory Protein (vFLIP) K13 encoded by the Kaposi’s sarcoma associated herpesvirus (also known as human herpesvirus 8), MC159-vFLIP and MC160 vFLIP from the molluscum contagiosum virus (MCV), E8-vFLIP from the equine virus 2 (EHV2), bovine herpesvirus 4 encoded vFLIP (BHV—vFLIP), vFLIP encoded by herpesvirus mecaca, vFLIP encoded by herpesvirus saimiri and HTLVl-encoded Tax, HTLVZ-encoded Tax and HTLV2-encoded Tax-RS mutant. Exemplary ar proteins suitable for this purpose include cFLIP-L m or MRIT-a, cFLIP-p22 deletion mutant or MRIT-p22 (Bertin er al., Proc Natl Acad Sci U S A 94:1172-6, 1997; Hu et a/., J Biol Chem 272:9621-4, 1997; Thome er al., Nature 386:517-21, 1997). In some embodiments, the above viral and cellular proteins are expressed in the immune cells (e.g., T cells and NK cells) in their native state or carrying small e tags and are functionally active in a constitutive manner. In other embodiments, the above viral and ar proteins are expressed in the immune cells (e.g., T cells and NK cells) in fusion with one or more copies of a switch domain (or a dimerization domain), such as FKBP (SEQ ID NOs; 2620 and 2621) and FKBPx2 (SEQ ID NO; 2622). In other ment, the FKBP or the FKBP-x2 domain may additionally carry an N-terminal myrisotylation (Myr) sequence to anchor the l proteins to the cell membrane. The sequence of an exemplary FKBPx2-K13 fusion protein carrying a Myr sequence is presented in SEQ ID NO: 2623. The fusion proteins carrying the switch domains are functionally inactive in their basal state but are activated upon addition of a dimerizer agent, such as In an embodiment, an immune effector cell, e.g., a T cell, ectopically expresses a viral or cellular signaling protein selected from the group of K13-vFLIP (SEQ ID NO: 2629), MC159-vFLIP (SEQ ID NO: 2630), MC LIP (SEQ ID NO: 2631), E8-vFLIP (SEQ ID NO: 2632), BHV-vFLIP (SEQ ID NO: 2635), mecaca vFLH’ (SEQ ID NO: 2634), virus saimiri vFLIP (SEQ ID NO: 2633), cFLIP—L/MRITa (SEQ ID NO: 2636), cFLIP-p22 (SEQ ID NO: 2637), HTLVl-Tax (SEQ ID NO: 2638), HTLV2-Tax (SEQ ID NO: 2639), HTLVZ-Tax-RS mutants (SEQ ID NO: 2640) or proteins with 70-99% identity to amino acid sequences of the above proteins (SEQ ID N05: 2629 to 2640).

In an embodiment, an immune effector cell, e.g., a T cell, ectopically expresses a fusion protein containing one or more switch domains, e.g, FKBP (SEQ ID NO: 2620-2621), FKBPx2 (SEQ ID NO: 2622) or Myr-FKBP, and one or more viral or cellular signaling protein selected from the group of K13-vFLIP (SEQ ID NO: 2629), MC159-vFLIP (SEQ ID NO: 2630), MC160-VFLIP (SEQ ID NO: 2631), E8-VFLIP (SEQ ID NO: 2632), BHV- vFLIP (SEQ ID NO: 2635), mecaca vFLIP (SEQ ID NO: 2634), herpesvirus saimiri vFLIP (SEQ ID NO: 2633), cFLIP-L/MRITa (SEQ ID NO: 2636), cFLIP-p22 (SEQ ID NO: 2637), HTLVl-Tax (SEQ ID NO: 2638), HTLV2-Tax (SEQ ID NO: 2639), HTLV2-Tax—RS mutants (SEQ ID NO: 2640) or proteins with 70-99% identity to amino acid sequences of SEQ ID N05: 2629 to 2640. Exemplary fusion proteins containing one or more switch domains and viral signaling proteins are represented by SEQ ID NOS: 645.

In some aspects, this disclosure provides a method of producing an immune or cell suitable for ve cellular y, comprising contacting the cell with a nucleic acid encoding one or more of a viral or cellular signaling protein selected from the group of K13-vFL1P (SEQ ID NO: 2629), MC159-vFLIP (SEQ ID NO: 2630), MC160- vFLIP (SEQ ID NO: 2631), E8-vFLIP (SEQ ID NO: 2632), BHV-VFLIP (SEQ ID NO: 2635), mecaca vFLIP (SEQ ID NO: 2634), herpesvirus saimiri VFLIP (SEQ ID NO: 2633 , L/MRITa (SEQ ID NO: 2636), cFLIP-p22 (SEQ ID NO: 2637), HTLVl-Tax (SEQ ID NO: 2638), HTLV2-Tax (SEQ ID NO: 2639), HTLVZ-Tax-RS mutants (SEQ ID NO: 2640) or ns with 70-99% identity to amino acid sequences of SEQ ID N05: 2629 to 2640.

In some aspects, this disclosure provides a method of producing an immune effector cell suitable for ve cellular therapy, comprising contacting the cell with a nucleic acid encoding expresses a fusion protein ning one or more switch domains, e.g., FKBP (SEQ ID NO: 2620-2621), FKBPxZ (SEQ ID NO: 2622) or BP, and one or more viral or cellular signaling protein selected from the group of K 1 3-vFLIP (SEQ ID NO: 2629), MC159-vFLIP (SEQ H) NO: 2630), MC160-vFLIP (SEQ ID NO: 2631), E8-vFLIP (SEQ ID NO: 2632), BHV-vFLIP (SEQ ID NO: 2635), mecaca vFLIP (SEQ ID NO: 2634), virus saimiri VFLIP (SEQ ID NO: 2633), cFLIP-L/MRITd (SEQ ID NO: 2636), cFLIP-p22 (SEQ ID NO: 2637), HTLVl-Tax (SEQ ID NO: 2638), HTLV2-Tax (SEQ ID NO: 2639), HTLV2-Tax-RS mutants (SEQ ID NO: 2640) or ns with 70-99% identity to amino acid sequences of SEQ ID NOS: 2629 to 2640.

In an embodiment, the cell suitable for adoptive cell therapy expresses a natural or synthetic immune receptor. Exemplary such immune receptors include a chimeric n receptor (CAR), a T cell receptor (TCR), a chimeric T cell receptor (cTCR), a tic T cell receptor and a synthetic notch receptor. In an embodiment, the cell may be contacted with the nucleic acid encoding the viral and cellular signaling proteins before, simultaneous with, or after being contacted with a construct encoding a natural or synthetic immune receptor. In an embodiment, the cell may be contacted with the nucleic acid encoding the viral and cellular signaling proteins containing a switch or dimerization domain before, simultaneous with, or after being contacted with a construct ng a natural or synthetic immune receptor.

In one aspect, the sure features a method of making a population of immune effector cells (e.g., T cells, NK cells). In an embodiment, the method comprises: providing a population of immune effector cells (e.g., T cells or NK cells), contacting the population of immune effector cells with a nucleic acid encoding an immune receptor (e.g., CAR, TCR, synthetic TCR) and contacting the population of immune effector cells with a c acid encoding a viral or cellular signaling protein of SEQ ID NOs: 645, under conditions that allow for immune receptor and viral or cellular signaling protein co-expression.

In an ment, the nucleic acid encoding the viral or cellular signaling protein of SEQ ID NOs: 2629-2645 is DNA. In an embodiment, the nucleic acid encoding the viral or cellular signaling n of SEQ ID NOs: 2629-2645 contains promoter capable of driving expression of the viral and cellular signaling proteins. In an embodiment, the c acid encoding the viral or cellular signaling protein of SEQ ID NOs: 2629-2645 and the nucleic acid ng the immune receptor is expressed from the same vector. In an embodiment, the nucleic acid encoding the viral or cellular signaling protein of SEQ ID NOs: 2629-2645 and the nucleic acid encoding the immune or is expressed from separate vectors. In an embodiment, the nucleic acid encoding the viral or cellular signaling n of SEQ ID NOS: 2629-2645 (subunit 1) and the c acid encoding the immune receptor (subunit 2) is sed from the same polynucleotide nt containing an internal ribosomal entry site (IRES) that allows the translation of the second subunit. In an embodiment, the nucleic acid encoding the viral or cellular signaling protein of SEQ ID NOs: 2629-2645 (subunit 1) and the c acid ng the immune receptor (subunit 2) is expressed from a single polynucleotide fragment that encodes and the different subunits are separated by a cleavable linker (e.g., SEQ ID NOs: 831-836).

In an embodiment, the nucleic acid encoding the viral or cellular signaling protein of SEQ ID NOs: 2629-2645 is an in vitro transcribed RNA. In an ment, the viral or cellular signaling protein of SEQ ID NOs: 2629-2645 (subunit 1) and the immune receptor (subunit 2) is expressed from the same RNA ning an al ribosomal entry site (IRES) that allows the ation of the second subunit. In an embodiment, the viral or cellular signaling protein of SEQ ID NOs: 2629-2645 (subunit 1) and the immune receptor it 2) is expressed from a single RNA and the different ts are separated by cleavable linkers (e.g., SEQ ID NOs: 831-836). ation therapies Therapeutic methods described herein comprise using compositions comprising genetically modified cells comprising nucleic acids encoding CARS described . In various embodiments, the therapeutic methods described herein may be combined with existing therapies and agents. The therapeutic compositions described herein, comprising genetically modiﬁed cells comprising c acids encoding the CARS described , are administered to the subject with at least one additional known therapy or therapeutic agent.

In some ments, the compositions described herein and the additional therapy or therapeutic agents are administered sequentially. In some ments, the compositions described herein and the additional therapy or therapeutic agents are administered simultaneously. The optimum order of administering the compositions described herein and the existing therapies will be apparent to a person of skill in the art, such as a physician.

A CARmxpressing celE described herein and the at ieast one additional therapeutic agent can. be. administered simuitanemsEy, in the same or in separate compositicns, or sequentiaily. For sequential ariministratinn, {he {TAR—expressing eelE described herein can be administered ﬁrst, and the additional agent can be administered second, or the order of administratitm can be reversed, Combinations therapies may be administered to the subject over the duration of the disease. Duration of the disease es from diagnosis until conclusion of treatment, wherein the treatment results in ion of symptoms and/or elimination of symptoms. In various embodiments, the effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the ﬁrst treatment delivered is still detectable when the second is delivered.

The CAR therapy and/or other therapeutic agents, procedures or modalities can be administered during periods of active disorder, or during a period of remission or less active disease. The CAR therapy can be administered before the other ent, concurrently with the treatment, post-treatment, or during remission of the disorder.

When administered in combination, the CAR therapy and the additional agent (e.g., second or third , or all, can be administered in an amount or dose that is higher, lower or the same than the amount or dosage of each agent used individually, e.g., as a monotherapy. In certain ments, the administered amount or dosage of the CAR therapy, the additional agent (e.g., second or third agent), or all, is lower (e. g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, e. g., as a monotherapy. In other embodiments, the amount or dosage of the CAR therapy, the additional agent (e.g., second or third agent), or all, that results in a desired effect (e.g., treatment of cancer) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dosage of each agent used individually, e.g., as a erapy, required to achieve the same therapeutic .

] Further method aspects relate administering to the subject an effective amount of a cell, e.g., an immune effector cell, or a population thereof, each cell sing a CAR molecule, optionally in combination with an agent that ses the efficacy and/or safety of the immune cell. In further aspects, the agent that increases the efficacy and/or safety of the immune cell is one or more of: (i) a protein phosphatase inhibitor; (ii) a kinase inhibitor; (iii) a cytokine; (iv) an inhibitor of an immune inhibitory molecule; or (v) an agent that decreases the level or activity of a TREG cell; vi) an agent that increase the proliferation and/or persistence of CAR-modiﬁed cells vii) a ine viii) an agent that increases the expression of CAR ix) an agent that allows regulation of the sion or activity of CAR x) an agent that allows control over the survival and/or persistence of CAR-modified cells xi) an agent that controls the side effects of CAR-modiﬁed cells xii) a Brd4 tor xiii) an agent that delivers a therapeutic (e.g. sHVEM) or prophylactic agent to the site of the disease xiv) an agent that increases the expression of the target antigen against which CAR is ed; xv) an adenosine AZa receptor antagonist in some embodiments, a the geneticaiiy modified ceiis Liesmibed herein may be used in a treaiment regimen in combination with surgery, chemotherapy, ion, immimosnppressive agents, such as cyciespenn, azathieprine. methetrexate, heneie’re. and Oé, antibodies, or ether iminnneabiative agents such as CAEViPA‘i‘i-i, anti—CD} ai-itibcdies 01' other antibody thetapiesr n. fiuderabine. cyclcsperin, FKSOé, rapamycin, niycophenoiic acid, steroids, FR90E228, cytokines, and ation, peptide vaccine, such as that described in iznmetn et al. 2098 .i‘ Neu-rcsurg 108:963~971. in ene embodiment, a CAR~ expressing celi described herein can be used in combination with a chemmherapentie. agent Exempiary herapeutic agents include an anthracyeiine (cg, doxomiiicin (cg, iipnsomal dexcmiﬁcin», a vinca aikalnid (eﬂg vinhiastine, vincristine, vindesine, VitiOi‘t‘:ii)l§1€}, an aikylating agent (eg, cyeii‘iphcsphainidc, azine, meipbalan, ifosfamidc, ten‘iezeininide), an immune ceil dy (cg. zamab, gemtuzumab, ritnxiinab, i‘it‘atnmnmab, t‘i‘isitiimemab, brentuximab}, an antimetabi‘aiite (including, eg, feiic acid antagenists, pyrimidine analogs, purine. anaiegs and adenosine deaminase inhibitors teg. lindaraiiime», an mTOR. inhibitor, 2. TNFR glneneertieeid induced TNFR reinted pretein (GI'E'R) t, a pi‘eteaseme inhibitor (eg, nomycin A, giiotexin or eniib). an inmatineeinduia'tcr such as tiiaiidemide er 3, tbaiidmnide derivative (cg, ienalidemide).

In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with cyclophosphamide and ﬂudarabine.

In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with bendamustine and rituximab. In embodiments, the subject has In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with rituximab, cyclophosphamide, doxorubicin, stine, and/or a corticosteroid (e.g., prednisone). In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with n'tuximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). In embodiments, the subject has diffuse large B- celllymphoma (DLBCL).

In embodiments, a CAR-expressing cell described herein is stered to a subject in combination with etoposide, prednisone, vincn'stine, cyclophosphamide, doxorubicin, and/or rituximab. In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and tituximab -R). In embodiments, a CAR- expressing cell described herein is administered to a subject in combination with dose adjusted EPOCH—R (DA-EPOCH-R). In embodiments, the subject has a B cell lymphoma, e. g, a Myc-rearranged aggressive B cell lymphoma.

In ments, a CAR-expressing cell described herein is administered to a subject in ation with brentuximab. Brentuximab is an dy-drug conjugate of anti- CD30 antibody and monomethyl auristatin E. In embodiments, the subject has Hodgkin's lymphoma (HL), e.g., relapsed or refractory HL. In embodiments, the subject comprises CD30+ HL. In embodiments, the subject has undergone an autologous stem cell transplant (ASCT).

In some embodiments, a CAR-expressing cell described herein is administered to a subject in combination with a CD20 tor, e.g., an D20 antibody (e.g., an anti- CD2O mono- or bispeciﬁc antibody) or a fragment thereof.

In one ment, a CAR expressing cell described herein is administered to a subject in combination with an mTOR inhibitor, e.g., an mTOR inhibitor described , e.g., a rapalog such as everolimus. In one embodiment, the mTOR inhibitor is administered prior to the CAR-expressing cell. For example, in one embodiment, the mTOR inhibitor can be administered prior to apheresis of the cells. In one embodiment, the subject has CLL.

In one ment, a CAR-expressing cell described herein can be used in combination with a kinase inhibitor. In one ment, the kinase inhibitor is a CDK4 inhibitor, e.g., a CDK4 tor described herein, e.g., a CD4/6 inhibitor, such as, e.g., 6- Acetylcyclopentylmethyl(S-piperazin- l -yl-pyridinylamino )-8H-pyrido[2,3- d]pyn'midinone, hydrochloride (also referred to as iclib or PD0332991). In one embodiment, the kinase inhibitor is a BTK inhibitor, e.g., a BTK inhibitor described herein, such as, e.g., ibrutinib. In some embodiments, ibrutinib is stered at a dosage of about 0 mg/day (e.g., about 300-350, 350-400, 400-450, 450-500, 500-550, or 550-600 mg/day, e.g., about 420 mg/day or about 560 mg/day), e.g., orally. In embodiments, 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 embodiment, l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles ofibrutinib are administered. Without being bound by theory, it is thought that the on of ibrutinib enhances the T cell proliferative response and may shift T cells from a T-helper-2 (Th2) to T- helper-l (Th1) phenotype. Th1 and Th2 are phenotypes of helper T cells, with Th1 versus Th2 directing different immune response pathways. A Th1 phenotype is associated with proinﬂammatory responses, e.g., for killing cells, such as intracellular pathogens/viruses or cancerous cells, or perpetuating autoimmune responses. A Th2 ype is associated with eosinophil lation and anti-inﬂammatory ses. In one ment, the kinase inhibitor is an mTOR inhibitor, e.g., an mTOR inhibitor described herein, such as, e.g., rapamycin, a rapamycin analog, 081- 027. The mTOR inhibitor can be, e.g., an mTORCl tor and/or an mTORC2 inhibitor, e.g., an mTORC 1 inhibitor and/or mTORC2 inhibitor described herein. In one embodiment, the kinase inhibitor is a MNK inhibitor, e.g., a MNK inhibitor described herein, such as, e.g., o(4-ﬂuoroanilino)-pyrazolo [3,4-d] pyrimidine. The MNK inhibitor can be, e.g., a MNKla, MNKlb, MNK2a and/or MNK2b tor. In one embodiment, the kinase inhibitor is a dual PI3K/mTOR inhibitor described herein, such as, e.g., PF-04695102. In one embodiment, the kinase inhibitor is a Src kinase inhibitor. In one embodiment, the kinase inhibitor is Dasatinib. In one embodiment, the Src kinase inhibitor is administered to the t after the administration of CAR expressing cells to control or terminate the activity of CAR-expressing cells. In one embodiment, Dasatinib is administered to the patient after the administration of CAR expressing cells to control or terminate the activity of CAR-expressing cells. In one embodiment, dasatinib is administered orally at a dose of at least 10 mg/day, 20 mg/day, 40mg/day, 60mg/day, 70 mg/day, 90 mg/day, 100mg/day, 140 mg/day, 180 mg/day or 210 mg/day.

In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with an anaplastic lymphoma kinase (ALK) inhibitor. Exemplary ALK s include but are not d to crizotinib (Pfizer), cen'tinib (Novartis), alectinib (Chugai), brigatinib (also called AP26113; Ariad), entrectinib (Ignyta), PF-06463922 (Pﬁzer), TSR-OII (Tesaro) (see, e.g., Clinical Trial Identiﬁer No. NCT02048488), CEP- 37440 (Teva), and X-396 ry). In some embodiments, the subject has a solid , e.g., a solid cancer described herein, e.g., lung cancer.

Drugs that inhibit either the calcium dependent phosphatase calcineurin ( cyclosporine and FK506) or inhibit the p7OS6 kinase that is important for growth factor induced signaling (rapamycin). (Liu et al., Cell66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993) can also be used. In a further aspect, the cell compositions of the t invention may be administered to a patient in conjunction with (e. g., before, simultaneously or following) bone marrow lantation, T cell ablative therapy using chemotherapy agents such as, fludarabine, extemal-beam radiation therapy (XRT), cyclophosphamide, and/or antibodies such as OKT3 or CAMP ATH. In one aspect, the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, in one embodiment, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects e an infusion of the ed immune cells of the present invention. In an additional embodiment, expanded cells are administered before or following surgery.

In embodiments, a pressing cell described herein is administered to a subject in combination with an gous stem cell transplant, an allogeneic stem cell transplant, an autologous bone marrow transplant or an allogeneic bone marrow transplant.

In embodiments, a CAR-expressing cell bed herein is stered to a subject in combination with microtransplant or HLA mismatched neic cellular therapy (Guo M et al, J Clin Oncol. 2012 Nov 20;30(33):4084-90).

In ments, a CAR-expressing cell described herein is administered to a subject in combination with an indoleamine 2,3-dioxygenase (IDO) inhibitor.

In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with a modulator of myeloid-derived suppressor cells (MDSCs).

MDSCs accumulate in the periphery and at the tumor site of many solid tumors. These cells suppress T cell responses, thereby hindering the efﬁcacy of CAR-expressing cell therapy.

Without being bound by , it is thought that stration of a MDSC modulator enhances the efﬁcacy of a CAR-expressing cell described . In an embodiment, the subject has a solid tumor, e.g., a solid tumor bed herein, e.g., glioblastoma. Exemplary modulators of MDSCS include but are not d to MCSllO and BLZ945. MCSllO is a monoclonal antibody (mAb) against macrophage colony-stimulating factor (M-CSF). See, e.g., Clinical Trial Identiﬁer No. NCT00757757. BLZ945 is a small molecule inhibitor of colony stimulating factor 1 or (CSFlR). See, e.g., Pyonteck et al. Nat. Med. 19(2013):IZ64-72.

In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with a Brd4 or BET (bromodomain and extra-terminal motif) inhibitor. BET protein BRD4 directly regulated expression of the transcription factor BATF in CD8+ T cells, which was associated with differentiation of T cells into an effector memory phenotype. JQl, an inhibitor of omain and extra-terminal motif (BET) proteins, maintained CD8+ T cells with functional properties of stem cell-like and central memory T cells. Exemplary Brd4 inhibitors that can be administered in combination with CAR- expressing cells include but are not limited to JQI, MS4I7, OTXOIS, LY 3035]] and Brd4 inhibitor as bed in US 20140256706 A1 and any analogs thereof.

In some embodiments, a pressing cell described herein is administered to a subject in combination with a interleukin-15 (IL-15) polypeptide, a interleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination of both a 1L-15 polypeptide and a 1L-15Ra ptide e.g., hetiL-15 (Admune Therapeutics, LLC). hetiL- 15 is a dimeric non- covalent complex of IL-IS and 1L-15Ra. lS is described in, e.g., US. 8,124,084, US. 2012/0177598, US. 2009/0082299, US. 2012/0141413, and US. 201110081311, incorporated herein by reference. In embodiments, het-IL-IS is stered subcutaneously.

In embodiments, the subject has a cancer, e.g., solid , e.g., melanoma or colon cancer.

In embodiments, the subject has a metastatic .

In one embodiment, the subject can be administered an agent which reduces or ameliorates a side effect associated with the administration of a CAR-expressing cell. Side effects associated with the administration of a CAR-expressing cell include, but are not limited to CRS, and hemophagocytic lymphohistiocytosis (HLH), also termed Macrophage Activation Syndrome (MAS). Symptoms of CRS include high fevers, nausea, transient hypotension, hypoxia, and the like. CRS may include al constitutional signs and symptoms such as fever, fatigue, anorexia, myalgias, arthalgias, nausea, ng, and headache. CRS may include clinical skin signs and symptoms such as rash. CRS may e clinical gastrointestinal signs and symptoms such as nausea, vomiting and diarrhea. CRS may include clinical respiratory signs and symptoms such as tachypnea and mia. CRS may include clinical cardiovascular signs and symptoms such as tachycardia, widened pulse pressure, hypotension, increased cardiac output (early) and potentially diminished cardiac output (late). CRS may include clinical coagulation signs and symptoms such as elevated d- dimer, hypoﬁbrinogenemia with or without bleeding. CRS may include clinical renal signs and symptoms such as azotemia. CRS may include clinical hepatic signs and symptoms such as transaminitis and hyperbilirubinemia. CRS may include clinical neurologic signs and symptoms such as he, mental status changes, confusion, delirium, word finding difﬁculty or frank a, hallucinations, tremor, ia, d gait, and seizures.

Accordingly, the methods described herein can comprise administering a CAR- expressing cell described herein to a subject and r administering one or more agents to manage elevated levels of a soluble factor resulting from treatment with a CAR-expressing cell. In one embodiment, the soluble factor elevated in the t is one or more of IFN-y, TNFa, IL-2 and IL-6. In an embodiment, the factor elevated in the t is one or more of lL-l, GM-CSF, lL-lO, lL-8, l-S and lkine. Therefore, an agent administered to treat this side effect can be an agent that neutralizes one or more of these soluble factors. In one embodiment, the agent that neutralizes one or more of these soluble forms is an antibody or antigen binding fragment thereof. Examples of such agents include, but are not d to a steroid (e.g., corticosteroid), Src inhibitors (e.g., Dasatinib) an inhibitor of TNFa, and an inhibitor of H46. An example of a TNFa inhibitor is an anti-TNFa antibody molecule such as, inﬂiximab, adalimumab, certolizumab pegol, and golimumab. Another example of a TNFa inhibitor is a fusion protein such as entanercept. An example of an lL-6 inhibitor is an anti- IL-6 antibody molecule or an anti-IL-6 receptor antibody molecule such as tocilizumab (toe), sarilumab, elsilimomab, CNTO 328, ALD5 lS/BMS-945429, CNTO I36, CPSI-2364, 8, VX30, ARGX-109, FE301, and FMlOl. In one embodiment, the anti-IL-6 receptor antibody le is tocilizumab. In one ment, the IL-6 inhibitor is a camelid bispeciﬁc antibody that binds to IL6R and human serum albumin (e.g., IL6RAlb8) (SEQ ID NO: 2649). An example of an UAR based inhibitor is anakinra. In one embodiment, an agent administered to treat the side effects of CAR-expressing cells is a Src inhibitor (e.g., Dasatinib). In one embodiment, an agent administered to treat the side effects of CAR- expressing cells is the Src inhibitor Dasatinib. In ments, Dasatinib is administered at a dose of about 10 mg/day to 240 mg/day (e.g., 10mg/day, 20mg/day, 40mg/day, 50mg/day, 70 mg/day, 80mg/day, lOOmg/day, 110mg/day, 120mg/day, l40mg/day, 180mg/day, 210 mg/day, 240mg/day or 300mg/day).

In one embodiment, the subject can be administered an agent which enhances the activity of a CAR-expressing cell. For e, in one ment, the agent can be an agent which inhibits an inhibitory le. Inhibitory molecules, e.g., Programmed Death 1 (PD-l), can, in some ments, decrease the ability of a CAR-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD-l, PDLl, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIRI, CD160, 2B4 and TGFR beta. Inhibition of an inhibitory molecule, e.g., by inhibition at the DNA, RNA or protein level, can optimize a CAR-expressing cell performance. In embodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, a clustered rly interspaced short palindromic repeats (CRISPR), a transcription-activator like effector nuclease ), or a zinc ﬁnger clease (ZFN), e.g., as described herein, can be used to inhibit sion of an inhibitory molecule in the CAR-expressing cell. In an embodiment the tor is an shRNA.

In an embodiment, the inhibitory molecule is inhibited within a CAR-expressing cell. In these embodiments, a dsRNA molecule that inhibits expression of the inhibitory molecule is linked to the nucleic acid that encodes a component, e. g., all of the components, of the CAR. In one embodiment, the inhibitor of an inhibitory signal can be, e.g., an antibody or antibody fragment that binds to an tory molecule. For example, the agent can be an antibody or antibody fragment that binds to PD-l, PD-Ll, PD-LZ or CTLA4 (e.g., ipilimumab (also referred to as MDX-OIO and MDX- 101, and marketed as Yervoy®; Bristol-Myers Squibb; Tremelimumab (IgG2 monoclonal antibody available from Pﬁzer, formerly known as mumab, CP-675,206).). In an embodiment, the agent is an antibody or dy fragment that binds to TIM3. In an embodiment, the agent is an dy or antibody fragment that binds to CEACAM (CEACAM-l, CEACAM-3, and/or CEACAM-5). In an embodiment, the agent is an antibody or antibody fragment that binds to LAG3.

PD-l is an inhibitory member of the CD28 family of receptors that also includes CD28, CTLA-4, ICOS, and BTLA. PD-I is expressed on ted B cells, T cells and myeloid cells. Two ligands for PD-I, PD-Ll and PD-L2 have been shown to down regulate T cell activation upon binding to PD-l . PD-LI is abundant in human cancers. Immune suppression can be ed by ting the local interaction of PD-I with PD-Ll.

Antibodies, antibody fragments, and other inhibitors of PD-l, PD-LI and PD-L2 are available in the art and may be used combination with a CAR of the present invention described herein. For e, nivolumab (also referred to as EMS-936558 or NHDX1106; Bristol-Myers Squibb) is a fully human IgG4 monoclonal antibody which specifically blocks PD-l. Nivolumab (clone 5C4) and other human monoclonal antibodies that speciﬁcally bind to PD-l are disclosed in US 8,008,449 and W02006/121168. Pidilizumab (CT-011; Cure Tech) is a humanized Igle onal antibody that binds to PD-I. Pidilizumab and other zed anti-PD-l monoclonal antibodies are disclosed in W02009/1016l l.

Pembrolizumab (formerly known as lambrolizumab, and also referred to as MK03475; Merck) is a humanized IgG4 onal antibody that binds to PD-l. Pembrolizumab and other zed D-l antibodies are disclosed in US 8,354,509 and W02009/l 14335.

MEDI4736 (Medimmune) is a human monoclonal antibody that binds to PDLI, and inhibits interaction of the ligand with PD]. MDPL3280A (Genentech I Roche) is a human Fe optimized IgGl monoclonal antibody that binds to PD-Ll. MDPL3280A and other human monoclonal antibodies to PD-Ll are disclosed in US. Patent No.: 7,943,743 and US Publication No.: 20120039906. In other embodiments, the agent that enhances the activity of a CAR-expressing cell is a CEACAM inhibitor (e.g., CEACAM-l, CEACAM-3, and/or CEACAM-5 inhibitor).

In one embodiment, the agent which enhances activity of a CAR-described herein is another agent that ses the sion of the target antigen t which the CAR is directed. The agents that can be administered to the subject receiving a CAR-expressing cell described herein include: Arsenic trioxide, ATRA (all-trans-retinoic acid), compounds 27, 40, 49 of (Du et al, Blood; Prepublished online October 12, 2016), IDH2 inhibitors (e.g., AG- 221) or a combination thereof. In an embodiment, the agents are stered prior to, concurrently or after administration of CAR-expressing cells. In preferred embodiments these agents are administered prior to administration of CAR-expressing cells. In preferred embodiment, the CAR expressing cells that are administered with the above agents target a B cell antigen (e.g., CD19, CD20, or CD22 etc.) In one ment, the agent which es activity of a CAR described herein is a soluble receptor. Soluble receptor that can be administered to the subject receiving a CAR- expressing cell bed herein include: sl-IVEM (SEQ ID NO: 2664), sI-IVEM-Alb8-vHI-I fusion protein (SEQ H) NO: 2665), or a combination f. The soluble receptor can be administered once a day or more than once a day, e.g., twice a day, three times a day, or four times a day. The soluble or can be administered for more than one day, e.g. the soluble receptor is administered for 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. For example, the soluble receptor is administered once a day for 7 days.

In one embodiment, the t can be administered an agent which protects against the toxicity of a pressing cell on normal tissues. One of the limitations of CAR-T cell therapy could be toxicity on normal tissue. For example, CAR targeting CD19 could lead to long-term depletion of normal B cells, which also express CD19 antigen. In one embodiment, CD19 CAR-T cell therapy can be combined with knock-out or mutation of endogenous CD19 in normal hematopoietic stem cells. In one ment, the knock out or mutation of the endogenous CD19 is achieved using CRIPS/Cas9, Talons or other suitable gene editing methods which are known in the art. The epitope of CD19 bound by the CD19 CAR-T cells in current clinical use has been mapped to exon2-4. In one embodiment, missense or nonsense mutations are generated in exon 2 (or other suitable exons/regions that are recognized by CD19 ed CAR T-cells) of autologous or allogeneic hematopoietic stem cells using CRISP/Cas9, Zn ﬁnger nucleases, Talons or other methods known in the art. In one ment, the subject is given CD19 CART cells infusion to control his/her disease and an autologous or allogeneic stem cell transplant using CD19 deleted/mutated hematopoietic stem cells. As the B cells that will originate from the modiﬁed stem cells will not be ed by CD19-CAR-T cells, the patient will escape B cell aplasia which is a common side effect of CD19 CAR-T cells. In another embodiment, MPL CAR-T cell therapy is combined with knock-out or on of endogenous MPL in normal hematopoietic stem cells. In another embodiment, CD123 CAR-T cell therapy is combined with knock-out or mutation of endogenous CD123 in normal hematopoietic stem cells. In another embodiment, CD33 CAR-T cell y is combined with knock-out or mutation of endogenous CD33 in normal hematopoietic stem cells. In another embodiment, CD20 CAR-T cell therapy is combined with knock-out or mutation of nous CD20 in normal hematopoietic stem cells. In another embodiment, CD22 CAR-T cell therapy is combined with knock-out or mutation of endogenous CD22 in normal hematopoietic stem cells. In another embodiment, CS1 CAR-T cell therapy is combined with knock-out or mutation of endogenous C81 in normal poietic stem cells. In another embodiment, BCMA CAR-T cell therapy is ed with knock-out or mutation of endogenous BCMA in normal hematopoietic stem cells. In r embodiment, CD45 CAR-T cell therapy is combined with knock-out or mutation of endogenous CD45 in normal hematopoietic stem cells or immune effector cells (e.g., T cells or NK cells). Essentially, a similar approach could be used to mitigate the toxicity of CAR-T cells against normal tissue where the antigen ed by the CAR is also expressed on normal hematopoietic stem cells or one ofits progenies.

] In another ment, CAR-T cell therapy is combined with out or mutation of endogenous gene or n targeted by the CAR in the immune effector cell (e.g., T cells or NK cells) or stem cells that give rise to immune effector cells. For example, since CD45 is expressed on all hematopoietic cells, CAR-T cells targeting CD45 would be difﬁcult to generate as they would be killed off by neighboring CD45-CART cells. However, such cells can be generated if expression of CD45 CAR in T cells is combined with knock- down or deletion of endogenous CD45 in the T cells in which CD45 CAR is being expressed.

Essentially a similar approach can be used to generate CAR targeting other antigens that are expressed on immune effector cells. Exemplary such antigens include, but are not limited to, CD5, TCRa, TCRBI, TCRBZ, TCRy, TCR5, preTCRa and various receptors expressed on NK cells.

Cytokines that can be administered to the subject receiving a CAR-expressing cell described herein include: IL-2, IL-4, IL-7, IL-9, IL-15, IL- 18, LIGHT, and IL-21, or a combination thereof. In preferred embodiments, the ne administered is IL—7, IL-15, or IL-21, ILlZF, or a combination thereof. The cytokine can be administered once a day or more than once a day, e.g., twice a day, three times a day, or four times a day. The cytokine can be administered for more than one day, e.g. the cytokine is administered for 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. For e, the cytokine is administered once a day for 7 days. Administration of the cytokine to the subject that has sub-optimal response to the CAR-expressing cell therapy improves CAR-expressing cell efficacy or anti-cancer activity. In a preferred embodiment, the cytokine administered after administration of CAR-expressing cells is E-7.

In one embodiment, the agent which enhances activity of a pressing cell described herein is a Brd4 inhibitor or an siRNA or an shRNA targeting BRD4 as described in (Tolani, B et al., ne, 29; 33(22):2928-37. PMID: 23792448)(Tolani, Gopalakrishnan, Punj, Matta, & Chaudhary, 2014).

Pharmaceutical Compositions Also provided herein are pharmaceutical compositions comprising any one or more of the chimeric antigen receptors, the polynucleotides, the ptides, the s, the viruses, and/or the genetically engineered cells and/or chemical compounds described herein and a pharmaceutically acceptable carrier. Such compositions may se buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present invention are in one aspect formulated for intravenous stration.

Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated. The quantity and frequency of stration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, gh appropriate dosages may be determined by clinical trials.

When a peutically effective amount" is indicated, the precise amount of the itions of the t invention to be administered can be determined by a ian with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient. (subject). It can generally be stated that a ceutical composition comprising the genetically modiﬁed cells (T cells, NK cells) described herein may be administered at a dosage. of 10" to 109 cells/kg body weight, in some instances 10510 ltl'3 cells/kg body weight, including all r 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, eg, Rosenberg et al, New Eng J. of Med. 3191676, 3988).

In some embodiments, it may be desired to administer activated genetically modified cells (T cells, NK cells) to a t and then subsequently redraw blood (or have an apberesis perforated), activate the genetically modiﬁed cells rom and reint‘use the patient with these activated and expanded genetically modified cells. This process can be carried out multiple times every few weeks. In certain aspects, immune effector cells (eg, 1‘ cells, NK cells) can be activated from blood draws of from lilac to 400cc. In certain aspects, immune effector cells (eg, ’1‘ cells, NK cells) are activated from blood draws of 20cc, 3000. 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc...

"Pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical ition that is lly safe, non-toxic, and desirable, and includes ents that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an l composition, gaseous.

In various embodiments, the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration. "Route of administration" may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, intravenous, intramuscular, eritoneal, tion, transmucosal, transdermal, parenteral, implantable pump, continuous infusion, topical ation, capsules and/or injections.

] The pharmaceutical compositions according to the invention can also contain any pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For e, the carrier may be a liquid or solid filler, diluent, excipient, t, or encapsulating material, or a combination thereof.

Each ent of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in t with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic beneﬁts, The pharmaceutical itions according to the invention can also be encapsulated, tableted or prepared in an on or syrup for oral administration.

Pharmaceutically acceptable solid or liquid carriers may be added to enhance or ize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid rs include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or yl distearate, alone or with a wax The pharmaceutical preparations are made ing the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and ﬁlling for hard gelatin e forms. When a liquid r is used, the preparation will be in the form of syrup, elixir, on or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft n capsule.

The pharmaceutical compositions according to the invention may be delivered in a therapeutically effective amount. The precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of y of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, e type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One d in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine mentation, for instance, by monitoring a subject’s response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: Llhe Science and Practice qf Pharmacy ro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

The stration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, transfusion, implantation or transplantation. The itions described herein may be stered to a patient trans- arterially, aneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one aspect, the T cell compositions of the present invention are administered to a t by intradermal or subcutaneous injection. In one aspect, the T cell compositions of the present invention are administered by i. v. ion. The compositions of immune effector cells (e.g., T cells, NK cells) may be injected directly into a tumor, lymph node, or site of infection.

In a particular exemplary aspect, subjects may undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate the cells of st, e.g., T cells. These T cell isolates may be expanded by methods known in the an and treated such that one or more CAR constructs of the invention may be introduced, thereby ng a CAR T cell of the invention. Subjects in need f may subsequently undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain aspects, following or concurrent with the transplant, subjects receive an infusion of the expanded CAR T cells of the present invention. In an additional aspect, expanded cells are administered before or following surgery.

In one embodiment, the CAR is introduced into immune effector cells (e.g., T cells, NK cells), e.g., using in vitro transcription, and the subject (e.g., human) receives an initial administration of CAR immune effector cells (e.g., T cells, NK cells) of the invention, and one or more subsequent administrations of the CAR immune effector cells (e.g., T cells, NK cells) of the invention, n the one or more subsequent administrations are administered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous stration. In one embodiment, more than one administration of the CAR immune effector cells (e.g., T cells, NK cells) of the invention are administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of the CAR immune or cells (e.g., T cells, NK cells) of the invention are administered per week. In one embodiment, the t (e.g., human subject) receives more than one stration of the CAR immune effector cells (e.g., T cells, NK cells) per week (e. g., 2, 3 or 4 administrations per week) (also referred to herein as a cycle), followed by a week of no CAR immune effector cells (e.g., T cells, NK cells) administrations, and then one or more additional administration of the CAR immune or cells (e.g., T cells, NK cells) (e.g., more than one administration ofthe CAR immune effector cells (e.g., T cells, NK cells) per week) is stered to the subject. In another embodiment, the subject (e.g., human subject) receives more than one cycle of CAR immune effector cells (e.g., T cells, NK cells), and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one ment, the CAR immune effector cells (e.g., T cells, NK cells) are stered every other day for 3 administrations per week. In one embodiment, the CAR immune effector cells (e.g., T cells, NK cells) of the invention are administered for at least two, three, four, five, six, seven, eight or more weeks.

] A potential issue that can arise in patients being treated using transiently expressing CAR immune or cells (e.g., T cells, NK cells) (particularly with murine scFv bearing CAR-Ts) is anaphylaxis after multiple treatments.

Without being bound by this theory, it is believed that such an anaphylactic response might be caused by a patient ping humoral anti -CAR response, i.e., anti- CAR antibodies having an anti-IgE isotype. It is thought that a patient's antibody producing cells undergo a class switch from lgG isotype (that does not cause anaphylaxis) to IgE isotype when there is a ten to fourteen day break in exposure to antigen.

If a patient is at high risk of developing an anaphylactic response to CAR y or generating an IgE type CAR antibody response during the course of CAR therapy, then omalizumab (Xolair) can be administered before or during the CAR therapy.

If a patient is at high risk of generating an anti-CAR dy response during the course of transient CAR therapy (such as those generated by RNA transductions), CART infusion breaks should not last more than ten to fourteen days.

Kits to practice the invention are also provided. For example, kits for treating a cancer in a subject, or making a CAR T cell that expresses one or more of the CARS disclosed herein. The kits may include a c acid molecule or a polypeptide molecule ng a CAR or a vector encoding a CAR along with a method to introduce the nucleic acid into the immune effector cells. The kit may include a virus comprising a nucleic acid encoding a CAR and chemicals, such as polybrene, to enhance the virus transduction. The kit may n components for isolation of T cells for sing a CAR. Alternatively, the kit may contain immune or cells (e. g., T cells or NK cells) or stem cells expressing a CAR.

More than one of the disclosed CAR can be included in the kit. The kit can include a container and a label or package insert on or associated with the container.

] Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container typically holds a composition including one or more of the nucleic acid les, viruses, vectors, T cells expressing a CAR. In several ments the container may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). A label or package insert indicates that the composition is used for treating the particular ion. The label or package insert typically will further include instructions for use of a sed nucleic acid molecules, CARS or T cells expressing a CAR, for example, in a method of treating or preventing a tumor or of making a CAR T cell. The package insert typically includes instructions customarily ed in commercial packages of eutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. The instructional materials may be written, in an electronic form (such as a computer diskette or compact disk) or may be visual (such as video ﬁles).

The kits may also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally contain means for measuring the expression of CAR on T cells or of determining the number or percentage of T cells that express the CAR or of determining the functionality of CART cells. The kits may additionally include buffers and other reagents routinely used for the practice of a ular method. Such kits and appropriate contents are well known to those of skill in the art.

Table 3: Exemplary signal es CD8-SIGNAL-PEPTIDE 1781 IH Sinal Petide 1782 I H Si nal Petide _ 1782 NAME SEO ID (DNA) SEO ID(PRT) I H-Si Hal-Petidc 1782 IH SinalPetide _ I782 Amyloid158- "83 Sinal- etide hCDl9 Sinal Petide 1784 Table 4: Exemplary VL nts (DNA) (PRT) ___I_— —l__— —I__BCMA-huCI2A3-vL BCMA-J6M0-vL BCMA-hucw-Flz-x-L ——ma-— CDIZ3-DARTvL CD123-9F6-vL b CDlz3-13RBz-vL b) ' —__CD123-2BS-vL1))'JI-L- —_Im_CD123-9D7-vLLA) —_IEI-CDIz3-3BIO-vL9) ——m-—_ DNA PRT CD19-huSJZSCl-vL CDI9-hB4-vL AI9-vL CD20—BM-CA-I925~x--4~v-L —E__— —E__— —l__— CD324-hSCl0vL CD33-AF5-vL —E__— CD33-Him3vL —_lm-— —I__— DNA PRT CDH6—NOV710-vL CLLl-hulO75-vl-\-'L —IE__— —IlI-_— CSl-HuZ7A-vL —II__— CSFZRA-Abl-vL —Il__— EGFRviiivL l—_I_- ——mmFLT3-NC7-vL HIVI-N6-vL DNA PRT HIVl-gag (77-85)/MHC class [-1928 HIVl-ES-vL comlcx HIVI-VR-COI-vL TAX-T3E3-vL 9HU3-vL Mesothelin-m912-x-L —IE.-_— DNA PRT ___mMucI-D6-MsBx-vL —m_—— NKGzo-MS-vL —IE__PDL1-SPI42-vL PSMA-Jsm-vL ___R0R1-4A5-vL __ma-R0R1-4CI0~vL BI-vL ___Im-TERT-3G3-T865-vL TERT—4A9-T540-vL TROPz-ARA47-HV3KV3-vL __IES-TROPz-h7Eé-SVG-vL TSHR-5C9-vL 233 VEGFR3-Ab1-vL DNA PRT WTl-AbIS-vL WTl-Abl-vL LHRvL LHR-5F4vL u1D11-v-L IE-omalizumab-vL CD200R-hqu182-vL Table 5: Exemplary Linkers SEQ ID SEQ ID Table 6: Exemplary VH fragments DNA PRT AFP/MHC classlcom lex AFPvH AIkvH AmvloidvH DNA PRT uCI1.05.3L1H3-vH BCMA-huCB-Flz-vH CDlzs-husEs-vH CD19-Medrex-24DI-vH CD20-Ubn-v4-vH DNA PRT CD30-5FI I-vH CD33-ISGISvH CD5vH CDHl9-I6A4-vH CDH6-NOV712-vH CLLl-lm|075-v2-VH /MHC class 1 355 2124 CMVpp65-FS-vH com lex DNA PRT EBNA3c/MHC class I 369 2138 EBNA3cVH com 010x FlTC-4MvH FLT3-NC7-vH GFRAlha4-P4vH GPRCSD-ETISO-S-VH nveIo lvco-rotein HIVl-gag (77-85) MHC class I 400 2169 HIVl-ES-vI-l complex HIV 1 -envelop ‘ lvco . rotein HLA-A2-3PBZ-vH SEQ ID SEQ ID CONSTRUCT NAME DNA PRT com-lcx ——— HPVl6-E7/MI-IC class 1407 2176 HPVl6vH comlex ILllRa-8E2-vH 2-hulo7-V-‘H LAMPl-Immabl-Z-vH MPL-huVBZZBWS-VH Muc16-4Hll-vI-I . class 2209 NYEso—Tl—VH . class 2210 NY-Eso-Tz-VH comlex PSMA-159l-vH DNA PRT PTK7-hSC6vH _——RORl-4A5-vH l-4C10-vH TCRBl (TCR-betal constant 2227 TCRBl-E09—vH chain TCRBl 2228 TCRB l-Jovil-vH TCRB2 (TCR-betaz constant 2229 TCRBZ-CPOl-DOS-VH chain) TCRd-GSvH TERT-3G3-T865-vH TERT-4A9-T540-vH TGFBRZ-Abl-VH TROPZ-h7E6-SVG-vH TSHR-5C9-vH TSHR-KBl-vH lvm ho oictin rccc tor comlex com-lex com-lcx VEGFRS-Abl-vH WTl-Ab13-vH WTl/MHC class I comlex lS-VH WTl-AbI-vH LHR (Lutcinizing hormone LHR-SB7-vH Rccc tor LHR-5F4vH B7H4 2260 B7H4-thZC10-VH SEQ ID SEQ ID CONSTRUCT NAME DNA PRT 871-14 2261 B7H4-hulDll-VH 2262 IE—omalizumab-vH CD23 2263 CD23— 51384111 GCC-5F9-vH Table 7: Exemplary VHH fragments (Nanobodies) DNA PRT —Her2 499 2269 Her2-2D3-VHH —Her2 500 2270 Her2-5F7-\-’HH —Her2 501 2271 Her2-47D5-v1-1H —Her3 502 2272 Her3-17BOSSo-VHH —Her3 503 2273 Her3-2 lFO6-v1—IH —CEA 504 2274 CEA 1-\-'HH —CEA 505 2275 CEAS-VHH —EGFR 506 2276 EGFR 1 -vHI-I —EGFR 507 2277 EGFR33-VHH —cMet 508 2278 7 I —CXCR4 509 2279 CXCR4VHH —CXCR4 510 2280 CXCR4—l-v1-IH —Mcsothclin 5 l l 2281 SD l-vHH —Mesothelin 5 12 2282 SDZ-VHH —Albumin 513 2283 A1b8-VHH —CD123 514 2284 1-vH1-1 —CD123 J —' J 2285 CD123VHH L6R 516 2286 IL6RVHH EGFR & 517 2287 EGFR 1 -v1*1H-Gly-Ser-Linker-CEA l-vHH EGFR & 5 18 2288 EGFR33-vHH-Gly-Ser-Linker—CEAS-VHH —Her2 UI '— 9 Herz—sF7-vHH-le-Ser-Linker—Herz—47D5«+111 —Her2 UI N0 HerZ-Hu4D5-VL-Glv-Ser-Linker-HerZ-Hu4D5-VH —Her3 & Her2 UI N — Her3-l7B05So-vHH-le-Ser-Linker-Her2-2D3-vHH cMet & ’JI N N 2292 cMET—171«KHH-Gly-Ser-Linker-Her3-21F06-VHH —Mesothelin -23JI 2293 SD 1 —vI-1H-G1\'-Ser-Linker-SDZ-vI—IH Table 8: Exemplary non-immunoglobulin n binding scaffolds (afﬁbodies, darpins).

DNA) PRT) EGFR EGFR-afﬁ Table 9: Exemplary receptor extracellular domain SEQ ID SEQ [D CD19Extracellular-Domain-minus-s-ignal- e tide(6l-867) tracellular-Domain-xx1th-si - 53 PD] ECD-w1thnative-Si-nal-Pe tide CTLA4--o t-ECD "ltll si mi e tide _—PTK7-ECD 2311 CD33ECD —:_—CD22-v5-ECD Th)roid Stimulating Hormone Receptor (TSHR)- NKGZD-ECD-minus-si'anal-e tide Table 10: Exemplary ligands DNA PRT) CGH-al v'ha-minus-Si nal-Pe tide CGH-beta-with-Sinal-Pe tide 3 2327 ta-minus-Si nal-Petide LH-beta-with-Si . nal-Pe ntide SP-CGHb-Glxr-Scr-Linkcr-CGHa SP-Ll—Ib-le-Ser-Linker-CGHa SP-TSHb-GlV-Ser-Linker-CGHa Table 11: Exemplary scFv fragments cow-Hosv-qu-m-vm CDI9-hu8125CI-wL-vH) CstP-Ritx-CD19-hB4-(vL-vH) 2356 BCMA-J6MO-(vL-vl—I) BCMA—Er-w-(vL-vm BCMA-ET—03- BCMA-huCl l.D5.3LlH3-(\IL-VH) CDS(vL-vH) -98 CDZZ-hl0F4v2-(vL-VH) cmo-BM-CAv4—(vL—vH) —m_—CDzo-AME(x-rL-vH) cmz-mmm-vm -u-L-vm CD8SP-RitxZ-BC33-Boehr2800308- CD8SP-Ritx2-C033-Him3~4-(vL-vH) coas-lscls-ss-wL-vw covo—me-wL-vm CDlz3(vL-vH) ovvlo- TCRgd-Gs(V—L-VH) TERT—3G3-T865- Table 12: Epitope Tags AVI-TAG-delta-GSG b.) ‘JI b.) RITX-TAG 9) UI DJ l RITXZ-TAG L») UI 9) RITX4-TAG b 'JI GA-TAG Table 13: Exemplary reporter fragments SEQ" SEQ"— GLuc Gaussia nurinces Luc Minus Secretorv Si_nal 2531 NLuc NanoLuc 2582 TLuc (TurboLucl6 Minus Sccrctorx’ Si nal m-MLuc7-(Metrida longa) Luc M43L/Ml 10L Variant Minus Secretory‘ Si nal IE__— MaLucl ia asymmetrica minus secretorv si nal MoLucl Metridia okhotensis minus orvsi-nal MoLch 'Metridia okhotensis minus secretory si-nal MLuc39 Metridialon-a minus secretorvsinal PsLucl Pleuromamma scutullata minus secretorvsinal -3 Lucicutia ovaliformis minus secreton'sinal HtLch Hotcrorhabdus tanncri minus sccrctorvsinal a Luc Table 14: Exemplary cleavable linkers and fun'ne cleavage site SEQ ID SEQ ID Table 15: Exemplary CAR components SEQ ID SEQ ID hCDx-Hine-TM 2606 hCD8-Hine-TM-BBZ 2607 hCD8TM-Hine-BB 2608 4-1BB-cvtosolic-domain 2609 CD3z-cxtosolic-domain 2610 CD3z-cxtosolic-domain 848 261 1 CD28'H‘"ge'TM' chosollc-domam 2612 LAILRl-TM-CP ] Table 16: Exemplary Therapeutic Controls,Accessory modules and their components SEQ ID SEQ ID DNA) (PRT 850 2613 PuroR Variant-(PAC) 851 2614 BlastR 852 2615 CNB3O 853 2616 GMCSF-SP-tEGFR MC LIP oesvirus-saimiri-VFLIP cFLlP-L/MRIT-alha SEQID SEQ ID FKBPxZ-Fl —Tax-RS 04-vI-IH IGHSP2-1L6RVHH-ALB8-VHH CD88P2-CTLA4-l ilimumab-scFv CD8SP2-PDl-4Hl-Alb8-VHH Table 17: Exemplary vectors, components and polyA sequence SEQ ID Lenti-EFla Lcnti-EFla-DWPRE MSCV-Bng-AvrlI-Bam-EcoRl-Xho-BstBl-Mlu-Sal- ClaI.[03 LENTI-NLuc-AcVS-B1asticidin-Pa08 LENTI-TurboLuc- 1 6-X3 Fla -C04 Lenti-EFIa—Pac-TZA-Gluc—BO7 MSCV-hvro-NLuc-AcVS-D07 SEQ ID _18 LENTI-Gluc-Fl -blast-BO7 Hl-shRNABRD4-4 Lenti—EFla-shRNA—BRDAI-DWPRE 921 pLenti-EFla-CD8SP-FMC63-(vL-vH)-Myc-z-PZA-Kl3- Fla -T2A-PAC-H 1 - hRNA-BRD4-DWPRE Table 18: : Exemplary GGS-Luc reporter proteins SEQ ID SEQ ID CD8SP-FMC63 vL-VH -GGSG-NLuc-AcV5 CD8SP-l6l- vL-vl-I -GGSG-NLuc-AcV5 MPL-ECD-GGSG-Nluc-ACVS 2671 FLAG-CDl9-ECD-GGSG-NLuc-AcV5 Table 19: Exemplary CAR constructs containing CD32 activation domain and coexpressing K13.

SEQ ID SEQ ID CD19 CD8SP-FMC63-(vL-vH)-l\/lyc-z-P2A- "1614305 2672 CD8SP-huFMC63-l l-N203Q-(vL-VH)- M\c-z--P-2AKl3-Fla CD8SP-CD19-MEDl(vL-VH)- Mvc-z-PZA-K 1 3-Fla ~T2A~PAC CDSSP-CDl9-Medrex-24Dl-(vL-vH)- Mvc-z-PZA-K l3-F1a -T2A-PAC CDSSP-Ritx-CDl9-MOR0028-(VL- vH)-Mvc-z-P2A-Kl3-Fl AC CDl9-HD37-H2Ll-(vL-vH)- Mvc-z-PZA-K 1 3-Fla -T2A-PAC CDSSP-CDl9-huBly3-(vL-vH)-Myc-z- 3-Fla —T2A-PAC CDSSP-CDl9-huSJZSCl-(vL-vH)- Mvc-z-PZA-Kl3-Flaw-T2A-PAC _--2684CD 19 . CDSSP-Ritx-CD l9-hB4-(vL-vH)-Myc- z-PzA-KI3-F1a~-T2A-PAC CDl9 CD8SP-CDl9-hu-mROOS(\-'L-vH)- 940 2685 PZA-KB-Fla -T2A-PAC CD19 CDl9-hA19-(vL-vH)—Myc-z- 941 2686 P2A-K l3-Fla - -T2A-PAC AFP/MHC class I 942 2687 CDSSP-AFP-é l -(vL-vH)-M_vc-z-P2A- con'lplex ag-T2A-PAC AFP/MHC class I 943 2688 CD8SP-AFP(vL-vH)-M_\rc-z-P2A- complex K 1 3-Flag-T2A-PAC AFP/MHC class I 944 2689 CD8SP-AFP(vL-vH)-Myc-z-P2A- ex K 1 3-Flag-T2A-PAC HIV 1 op 945 2690 CD8SP-HIV l -N6-(vL-vH)-1V1_V'c-z- glycoprotein P2A-K l3-Flag-T2A-PAC ALK (Anaplastic 051916-D04 946 2691 CD8SP-Alk(\v'L-VH)-M_\'c-z-P2A- Lymphoma Kinase) K 1 3-Flag-T2A-PAC ALK (Anaplastic 947 2692 CDSSP-Alk-S 8-(vL-vI-I)-l\/ch-z-P2A- Lymphoma Kinase) KlS-Flag-TZA-PAC Amyloid SP-Amyloid—l 5 8-(vL-vH)-Myc—z-P2A- Biotin CD8SP-dc-Avidin-Myc-z-P2A-Kl 3- CD45 - - CD8SP-BC8-CD45-(vL-vH)-Myc-z- BCMA CDSSP-BCMA-J6MO-(vL-vI-I)-l\/ch-z- 95‘ l 2696 PZA-K l3-Fla~ -T2A-PAC BCMA -- CDSSP-BCMA-huC12A3-L3H3-(vL- ‘ 2697 vH)—Mvc—z-P2A-K l 3-Fl -T2A-PAC BCMA CD8SP-BCMA-ET(vL-vH)-Myc-z- 95 3 2698 PZA-K l3-Fla -T2A-PAC BCMA CD8SP-BCMA-ET(vL-vH)-M_\rc-z- 954‘ 2699 P2A-K l3-Fla - -T2A-PAC BCMA CD8SP-BCMA-ET-O3-(vL-vH)-Myc-z- 955 2700 P2A-K l3-Fla- -T2A-PAC BCMA -- BCMA-huCl l.D5.3LlH3-(vL- vH)-Mvc-z-P2A-K l 3-Fl . . AC BCMA CDSSP-BCMA-huC13-F12-(vL-VH)- 957 "7702 Mvc-z-PZA-K l 3-Fla -T2A-PAC CCR4 CDSSP-CCR4-humAblS67-(vL-vl—l)- 958 2703 Mvc-z-PZA-K l 3-Fla -T2A-PAC HIVlJcnvelop - - - CD8SP-CD4-ECD—GlV-Ser-Linker-DC- - 7 7 CD5 - CD8SP-CD5(vL-vH)-Myc-z-PZA- CD5 CD8SP-CD5- l 8-(vL-vH)—Myc-z-P2A- ———2m wasp-cu1eA-vIsa-Ecn-vz-Myc-z- ——__PZA-K13-F1a--T2APAC CD8SPCD16A-V158ECD-vl -M\c-z- CDSSP-CDZO-BM-CA-l925-v4-(VL- vH)-Mw-z-P2A-K l 3-Fl - 0 976 2721 CD8SP-CD20-AME(vL-\«'H)-Mycm z-P2A-Kl3-Fla -T2A-PAC CD22 CDSSP-CD22-h10F4v2-(vL-x-H)-Myc- 977 2722 z-P2A—Kl3—Fla- -T2A-PAC CD22 CD8SP-CD22-H22R110V2ACDRKA- -H05 978 2723 (v L-\‘H)-M_\'c-z-P2A-K ] 3-Flag-T2A- _--2724CD22 CD8SP-CD22-m97 l -(\-’L-VH)-Myc-Z- PzA-K13-F1a-T2A-PAC 980 2725 CD88P-CD22-m971—HL-(VH-VL)-Myc- z-P2A-Kl3-Fla -T2A-PAC _0606M»CD30 - CD8SP-CD30-5Fl vH)-Myc-z— pZA-K13-F1a mm CD30 CD8SP-CD30-Ac10-(\'L-VH)-M_\’c-z- _0609‘6'A02CD32 a CD8SP-CD32-Med9-(vL-vH)-M_VC-z- PzA-Kls-Fla-TzA-PAC _--2729CD33 , CD85P-CD33-AF5-(VL-\5H)-Myc-z- pzA-m3-na-TzA-pAc CD33 CD33-huMyc9-(vL-vH)-M_\'c- 98‘V 27.;0‘ z-PZA-Kl3-Fla -T2A-PAC CD33 _- 2731 CDSSP-CD33-Boehr2800308-(vL-VH)- ——__M1c--z--P2AK13-Fla -T2A-PAC 8SPCD33Him3-4(VL-v1-I)M\_'-cz- —-111l- _--2743CD 123 CDSSP-CD123-CSL362-(vL-VH)-Myc- z-pzA-ms-na - _-CD123 - CD8SP-CD123-DART-l-(vL-VH)- Mvc-z-P2A-K13-Fla -T2A-PAC CD 123 CDSSP-CD123-DART(vL-VH)- 1001 2746 MVc—z-PZA-K l 3-Fla . -T2A-PAC _-CD123 CD8SP-CD123-13RB18-(\~'L-VH)-M_\_"c- 1002 2747 2.1%msma _---p21K13-CD123 CD8SP-CDI23-hu3E3-(vL-\H)--M\c-z- CD123 - " ' ' ' " ' ' 1004 2749 CDSSP-CD123-l l76-(vL-vH)-Myc-z- PZA-K l3-Fla-T2A-PAC CDSSP-RitxZ-CD 123-881 1-(vL-vH)- PZA-Kl3-Fla — CDSSP-CDl23(vL-vH)-Myc-z- P2A-Kl3-Fl --T2A-PAC CD138 - CDSSP-CDl38-(VL-\'H)-Myc-Z-P2A- _03‘7'6'N03CD 1 79b - - ""2 . CD8SP-CD179b-(V-‘L-V'H)-M)'C-z-P2A- K13-F1 -T2A-PAC _---pzA-K13-F1a—TzA-mcCD276 ., - CDSSP-CD276- l 7-(V’L-\-’H)-M}'c-z- _-""4CD324 - CD324 lO(vL-vH)-Myc-Z- P2A-K13-F1a-T2A-PAC CD324 CD8SP-CD324-hSC 10-1 7-(vL-vH)- 1 i' 2760 M\-'c-z-P2A-Kl3-Fla -T2A-PAC 6-105CDH6 - CDSSP-CDH6—NOV7 l 0—(vL-vH)-M}'c- 1016 2761 z-P2A-Kl3-F]a--T2A-PAC _031716-J03CDH6 _ - CD8SP-CDH6-NOV712-(vL-VH)-Myc- 1017 2762 z-PZA-Kl3-Fla .112A4,AC CDH17 051716- CDSSP-CDHI7-PTA001A4-(VL-VH)- 1018 2763 M06 ~ PZA-K l 3-Fla -T2A-PAC _031716-V04CDH19 - CD8SP-CDH19— l6A4-(VL-V'H)-M)-'c—z- . 1019 2764 p2A_K13_Fla._T2A_PAC _-EGFR - CD8SP-Cetuximab-(vL-vH)-M_V‘c-z- 1211—1113121 121-1% _CLECSA _ _ CD8SP-CLECSA-8H8F5-(vL-VH)- 11122—121113111-nmc 6106CLECSA - CDXSP-CLECSA-3El2A2-(VL-VH)- 1022 2767 111.021% GR/LHR SP-CGHb-Gly-Ser-Linker-CGHa-Myc- (Gonadotropin 102"3 2768 z-P2A-Kl3-Flag-T2A-PAC Receptor) _-CLLl CDSSP-CLLl-M26-(vL-vH)—Myc-z- ")2" 276° PzA-KI3-FIa-T2A-PAC CLLl - CDSSP-CLLl-M32-(vL-vH)-M_vc-z- CLLl CDSSP-CLLl-ZlC9-L2H3-(vL-VH)- 1026 2771 Mvc-z-PZA-K 1 3-F1a -T2A-PAC _-CLLl CDSSP-CLLl-6E7L4Hle-(vL-vH)- 1027 2772 Mxv'c-z-PzA-KB-Fla-T2A-PAC _-CLLl CDSSP-CLLl-hulO75-vl-(vL-vH)- Mxvvc-z-PzA-KB-Fla -T2A-PAC _ CDSSP-CLLl-hulO75-x12-(vL-VH)- CMVpp65/MHC --- —CMVpp65—F5—(vL—vH)—Myc—z— class I complex ‘ ‘ P2A—Kl3-Flag-T2A-PAC CSl (SLAMF7) - CSl-huLuc63-(vL-vH)-Myc-z- 103] 2776 CSI (SLAMF7) -- CD8SP-CSl-HuLuc64-(vL-vH)-Myc-z- ~ 2777 P2A-KI3-Fla -T2A-PAC €31 (SLAMF7) -- 2778 CDSSP-CSl-huLuc90-(vL-vI—I)-M_vc-z- P2A-Kl3-Fla-T2A-PAC CSl(SLAMF7) _ 1034 CDSSP-CSl—PDL24l-(\'L-vH)-Myc-z- ——--PZA-K13-F1a--T2APAC CSl (SLAMF7) -- CD8SP-CS] H1127A-(\L-VH)-M\C-z- CSl (SLAMF7) - ' ' 1036 2731 _ _ "1 "LAW" -- "SLAM" -- CS‘ ("W -- CSFZRA 051616-H05 1040 CSFZRA 05l6l6-GO] 1041 2786 CXCR4 and CD123 CD8SP-CXCR4- l -v-HHG1)—Ser— 1042 2787 Linker-CD123— l—VHH-Myc-z—-P—2A- CXCR4 and CD123 CDSSP-CXCR4-VHH-Gly-Ser— 1043 2788 Linker-CD123VHH-Myc-z-P2A- Kl3-Fl -T2A-PAC DLL3 (Delta Like CD8SP-DLL3-hSC16(vL-vH)- 1044 2739 Ligand 3) Myc-z-PZA-K l3-Flag-T2A-PAC DLL3 (Delta Like CDSSP-DLL3-hSCl6(vL-\-’H)- 104; 2790 Ligand 3) ' Myc-z-PZA-Kl3-Flag-T2A-PAC EBNA3c/MHC CDSSP-EBNA3c-3 lS-(vL-vH)-MyC-z- 1046 7791 class I complex " P2A-Kl3-Flag-T2A-PAC _-EBV-gp350 CD8SP-EBV-gp350-(V’L-VH)-M_\'C-Z- "’47, 2792 PzA-K -T2A-PAC EGFR CDSSP-EGFR l -vHH-Myc-z-P2A-K l 3- EGFR & CEA CDSSP—EGFRI-vI-II-I—Gly—Ser—Linker— 040716-008 1049 2794 CEA l -vHH-Myc-z—P2A-K 13-Flag- T2A-PAC EGFR & CEA CD8SP-EGFR33-vI-H-I-Gly-Ser-Linker- T2A-PAC P2A—K l3-Fla- -T2A—PAC z-PZA-K l 3-Fla -T2A-PAC Epcam l-MM l-(vL-vH)—Myc—z- PZA-K l3-Fla -T2A-PAC Epcam l-DSKS-(vL-vH)-M_V'c- z-PZA-K l 3-Fla -T2A-PAC CDSSP-FLT3-NC7-(V’L-V’H)-M_VC-Z- P2A-K l3-Fla - AC FITC 0510161304 280! CD8SP-FITC-(vL-vH)-Myc-z-P2A- ——__K13-Fl -T2A-PAC FITC - CD8SP-FITC-4M(vL-vH)-Myc-z- FlTC - CD8SP-FITC-EZ-HL-(VH-vL)-Myc-z- Inﬂuenza A HA CD8SP-FLU-MEDI-8852—(vL-vH)— [0‘9‘ 2804 Mvc-z-PZA-Kl3-Fla -T2A-PAC FRI (Folate CDSSP-FRl-huMov19-(vL-vl—I)-Myc- 1060 2805 Receptor alpha) z-PZA-K l 3-Flag-T2A-PAC FSHR (Follicle CDSSP FSHb G]- - S L' k CGH Stlmulatmg- - y- cr— 1n or- a- 1061 2806 PZA-Kl3-F1ag-T2A-PAC Hom1one Receptor) ' GAD (Glutamic Acid CD8SP-GAD-G3H8-(vL-vH)-Myc-z- 1062 2 807 Decarboxylase)/M P2A-K l3-Flag-T2A-PAC HC class I complex , CD8SP-GD2-hul4(vL-vH)-M_Vc-z- GD2 CD8SP-GD2-hu3F8-(vL-vH)-l\/l-\_a'c-z- GD3 - - CDSSP-GD3-KM-64 l -(vL-vI-l)-Myc-z- GFRa4 LAMP] (L5'S°5°‘"al' CD8SP LAMP! Mb4( L- - - v assoc1ated- -vH) M- _V‘C-Z- 051916-307 l 105 . P2A-KI3-FIag-T2A-PAC membrane protem 1:"isY CD8SP-LewisY-huS l 93-(VL-vI-I)-l\/lyc- l 106 z-PZA-K13-Fla - LlCAM 1107852CDSSP-LlCAM3-HU3-(vL—vH)-‘ Mvc-z-PZA-K l3-Fla - - LHR - SP-LHb-Gly-Ser—Linker—CGHa-Myc-z- —---— LymZ - - CDSSP——L_\m2—-(-VLvI-I)—Myc—z-P2A- MARTI/MHC class CDSSP-MARTl-CAGIO-(vL-vH)- 1 1 12 2857 I complex Myc-z-PZA-Kl3-Flag—T2A-PAC MARTl/MHC class CDSSP-MARTl-CLAlZ-(vL-vH)- 1 1 laJ 2858 I complex ‘ PZA-KlS-Flag-TZA-PAC elin CDSSP-Mesothelin-m9l2-(vL-vH)— 1 1 14 28 59‘ Mvc-z-PZA-KB-Fla-T2A-PAC cMet - CD8SP-cMet-l7l-vl-lH-Myc-z-PZA- cMct and I-Icr3 CDSSP-cMET-l7 l-vI-II-l—Gly-Scr- 041 1 16-805 1 1 16 2861 Linker-Her3-21F06-vHH-Myc-z-P2A- K 1 3-F1 o -T2A-PAC ("Eimbol’o'etm - "17 CD8SP-MPL—(vL-vI—l)-M)c-z- P2A-Kl3-Flag-T2A-PAC receptor) CDSSP MPL- v( L-vH) M- (Thromboporctm-,- - - ~V'c-z- 1 1 1316-N03 1 1 18 2863 P2A-K l3-Flag-T2A-PAC MPL 040716- ’ MPL CDSSP-MPL-AB3 1 7-(vL-vH)-Myc-z- MPL a CDSSP-MPL-12E10-(\-’L-vH)-Myc-z- . CD8SP-MPL-huVBZZBwS-(vL-VH)- Mucl/MHC class I CDSSP-Muc1-D6-M3B8-(vL-vH)- 1 12; complex ' 2870 Myc-z-PZA-K l -T2A-PAC HC class I CDSSP-MUCl-D6-M3Al-(vL-VH)- 1126 2871 complex PZA—K l 3-Flag-T2A-PAC _-"27Muc l 6 CDSSP-Muc 16-4Hl l-(\:L-vH)-1\/lyc-z- PzA-K13-F1a-T2A-PAC EGFR CD8SP-Nimotuzumab-(vL-VH)-Myc-z- l 128 2873 P2A-K13-F1a -T2A-PAC _030316404NKGZD Ligand - - , CD88P-NKG2D-(GGGGS-GGGGD)- "29 2874.

Mvc-z-PZA-Kl3-F1a-T2A-PAC NKGZD CDSSP-N KG).D-MS-(VL-VH)-M}-'c-z- l 130 2875 P2A-K13-Fla -T2A-PAC NY-BRl - - a CDSSP-NYBRI-(vL-v1-I)-l\/ch-z-P2A- NY-ESO/MHC CD8SP-NYESO-T1-(vL-vH)-Myc-z- 1 1423 2877 class 1 complex PZA-K l3-Flag-T2A-PAC NY-ESO/MHC CD8SP-NYESO-TZ-(vL-vH)-Myc-z- 1 13 q) 2878 class 1 complex ‘ ‘ P2A-K13-F1ag-T2A-PAC PDl ligand (eg" PDI-ECD-Myc-z-PZA-KB- PDL I) Flag-TZA-PAC CDSSP-PDL l -Atezoli-(vL-vH)-M§r'c-z- PZA-K l3-Fla- -T2A-PAC PSCA (Prostate CD8SP-PSCA-Ha14-12l-(vL-vH)- 1138 2883 stem cell antigen) Myc-z-PZA-K l 3-Flag-T2A-PAC PSCA (Prostate CD8SP-PSCA-Hal4-l l7-(vL-vH)- 1 139 '7884 stem cell antigen) Myc-z-PZA-Kl3-Flag-T2A-PAC PRl/MHC class I CDSSP-PRI-(vL-vH)—Myc-z-P2A-Kl3- 1140 7885 complex ' Flag-TZA-PAC PSMA ate CD8SP-PSMA(vL-vH)-Myc-z- Spe.°'ﬁ° Membme P2A-K l3-Flag-T2A-PAC Antigen) PSMA (Prostate CD8SP-PSMA-JS9 l-(vL-vH)—M_Vc-z- Speciﬁc Membrane l 142 P2A-K 13-Flag-T2A-PAC Antigen) PTK7 (Tymsmc' cossp PTK7 hSC6 23- - - - V - ' . . . protein kmase like l 143A ( L-‘.-'H) M.V'c- z-PZA-Kl 3-Flag-T2A-PAC PTK7 (Tyrosine- _ _ _ - v w - ' CDSSP pTK76 10 2 1;;ote1n k"inase ie-l'k 1144' ( L H) Mlye-Z- pzA-K13-Flag-T2A-PAC 7 890 CDSSP-RORI-4A5-(VL-VH)-M}v’c-z- 1146 2891 Mesothelin CD8SP-SD 1 -vl-l]-l-Gly-Ser-Linker— l 147 2892 -lH-Myc-z-P2A-Kl3-Flag-T2A- _0:1716-X0:SLea - - CD8SP-SLea-5B l -(vL-vH)-Myc-z- 1149, 2894.

P2A-Kl3-Fla-T2A-PAC SSE/M (Stage' CDSSP SSEA4- - . . _ ( L‘1 -‘7 p H) M_ },'c_z-P2A- speciﬁc embryomc 1 130 K l3-Flag-T2A-PAC antigen 4) TCRBl (TCR beta TCRBl-CPOl-E09-(\'L—VH)- 1 nt chain) Myc-z-PZA-Kl3-F1ag-T2A-PAC TCRBl (TCR beta TCRBl-Jovi l-(vL-vI-l)-l\/lyc—z- l constant chain) P2A-K13-Flag-T2A-PAC TCR32 (TCR beta CD8SP-TCRBZ-CP0l-DOS—(vL-VH)- 115.3 2 constant chain) Myc-z-P2A-Kl3-Flag-T2A-PAC TCRB2 (TCR beta CDSSP-TCRB2-CP0l-EOS-(vL-vH)- 2 constant chain) Myc-z-PZA-K l 3-Flag-T2A-PAC TCRgd (TCR _ _ _ _ CD8SP-TCRgd—GS(VL-vI-I)-Myc-z— 0317164103 gamma/delta) P2A-K13-Flag-T2A-PAC hTERT/MHC class CDXSP-TERT-4A9-T540-(vL-VH)- 1 1i6 I complex " PZA-Kl3-Flag-T2A-PAC CD8SP-TERT-3G3-T865-(vL-VH)- Myc-z-PZA-K l 3-Flag-T2A-PAC —--—5123:2211-2er{ram-V")- Sgéiz:3211221<€§.12w:1$1c Tn-Mucl CDSSP-TnMucl-huSES-RHAS-RKA- l 163 2908 2-(vL-vH)-Myc-z-P2A-Kl 3-Flag-T2A- ("111:r0mbopoietin - CDSSP—hTPO—Myc-z-PZA-Kl3-Flag- T2A-PAC receptor) TROPZ CD8SP-TROP2-ARA47-HV3KV3-(vL- (TmphOblag CCH' 031716003 M_\c---z-P2AKl3---Flag-T2APAC surface antrgen-Z) TROPZ CD88P‘TROP2‘h7E6‘SVG‘WL-VH)‘ (Trophoblast cell- 052616-D04 M3c 2r - -P2A-Kl3-Fl AC e antlgen-2).

TSHR (Thyrotropin 1 167 2917 SP-TSHb-Gly-Ser-Linker-CGHa-Myc- receptor) " z-PZA-K l 3-Flag-T2A-PAC TSHR (Thyrotropin Cstp-TSHR-K 1 (\'L-vH)-Myc-z- 1 168 291% receptor) ‘ ‘ PZA-K g-T2A-PAC TSHR (Thyrotropin _ CDSSP-TSHR-KB I -(vL-\-'H)-l\/lyc-z- TSHR (Thyrotmpin CD85P-TSHR-5C9-(vL-vH)-Myc-z- 1 170 2915 receptor) P2A-Kl3-Flag-T2A-PAC TSLPR (Thymic stromal CD8SP-TSLPR-(vL-vH)-Myc-z-P2A- l 17 l 291 6 poietin K13—Flag—T2A—PAC receptor) Tyrosinase/MHC CD8SP-Tyros-B2-(vL-vH)-Myc-z- 1 172 2917 class I complex PZA-K l3-Flag-T2A-PAC Tyrosinase/MHC 1172 2918 CDSSP-Tyros-MCI-(vL-\-’l-I)-Myc-z- class 1 complex PZA-K l3-Flag-T2A-PAC nase/MHC CD8SP-Tyros-TAZ-(vL-VH)-Myc-z- 1 174 2919 class 1 complex P2A-Kl3-Flag-T2A-PAC VEGFR3 CDSSP-VEGFR3-Abl-(vL-\V=H)-Myc-z- 060616_P04 2920 WTl/MHC class I CDSSP-WTI-Ab1-(vL-vH)-M_\,-'c-z- 1176 2921 complex P2A-K l3-Flag-T2A-PAC WTl/MHC class1 _ 1177 2922 CDSSP-WTI-Ab5-(vL-\-'H)-M)-'c-z- —_——PzA-Kls-nag-TzA-PAC WTl/MHC class 1 CDSSP-MYC3-WTl-Abl3-(vL-vH)- 1 178 2923 complex Myc-z-PZA-K l3-F1ag-T2A-PAC CcIassl - CD8SP-MYC3-WTl-Ab15-(vL-vH)— 1179 2924 x Myc-z-PZA-K l 3-Flag-T2A-PAC _°°'7'6'U°°CDH19 - - - CDSSP-CDH19-4B lO-(vL-vH)-Myc-z- PzA-K13-F1a-T2A-PAC Folatc Receptor CD8SP-FRbeta-m923-(vL-vI-I)-M_vc-z- 1 181 2926 beta PZA-K l3-Flag-T2A-PAC LHR (Lutcinizing CDSSP-LHR—8B7-(\-'L-\'H)-Myc-z- l 182 2927 hormone Receptor) PZA-K I3-Flag-T2A-PAC LHR nizing CDSSP-LHR-S F4-2 1 -(vL-vI-I)-M)«'c-z- 1 183 2928 honnone Receptor) P2A-Kl3-Flag-T2A-PAC 371—14 1 184 2929 CD8SP-B7H4-h1122Cl0-(vL-vH)-Myc- z-P2A-K 1 3-Fla- -T2A-PAC B7H4 . CDSSP-B7H4-hu 1 D1 l-(V‘ Myc- IgE CD8SP-IgE-omalizumab-(vL-vH)- l 186 29?' * l Mvc-z-PZA-KB-Fla -T2A-PAC _-"87c023 2932 CD8SP-CDZ3-p5E8-(vL-vI-I)-l\/lyc-z- PzA-K13-F1a--T2A-PAC GCC (Guanylyl CD8SP-GCC-5F9-(\v-'L-\:H)-Myc-z- cyclase C) PZA-K I3-FIag-T2A-PAC GCC(Guanylyl - CD8SP-GCC-Ab229-(vL-vH)-Myc—z- 1139 2934 cyclase C) P2A-Kl3-Flag-T2A-PAC CDZOOR CDSSP-CDZOOR-hqu182-(vL-vH)- 1 190 2935 MVc-z—PZA-K 1 3-Fla- -T2A-PAC c1322 -- 2937 fl-gglzZ-D-HL-(vH-vL)-Myc-z- _--2938CD22 . CDSSP-CDZZ-lO-HL-(x-‘H-vLyMyc-z- PzA-K13-Fla-T2A-PAC c022 - CD8SP-CDZZ-S3-HL-(vH-vL)-Myc-z- c1322 CD8SP-CD22HL-(vH-vL)-Myc-z- 1 196 2941 P2A-Kl3-Flao-T2A-PAC Table 20: Exemplary CAR constructs containing 41BB costimulatory domain, CD32 tion domain and coexpressing K13 Clone SEQ ID SEQ ID CD 19 l l 1014- CD8SP-FMC63-(VL-VH)-M}-'c-BBz-P2A- l 197 2942 Yll Kl3-Fl -T2A-PAC CD 19 CDSSP-huFMC63-l l-(vL-vH)-Myc-BBZ- 294.3 P2A-K13-Fl. -T2A-PAC CDl9 huFMC63-l l-N203Q-(VL-VH)- MV'C-BBz-PZA-K l 3-Fl ~ -T2A-PAC _-"00CD19 - , CD8SP-CDl9BulZ-(vL-vH)-M}'c-BBZ- PzA-K13-FI AC —-CD 19 , CDXSP-Z-CD19MM-(VL-VH)-Myc-BBZ- '2‘" 2946 PzA-KI3-FI AC _-CD 19 CDSSP-CD (V'L-VH)-Myc-BBZ- '202 2947 P2A-Kl3-Fla -T2A-PAC CD 19 CD8SP-CD l9-MEDI(VL-VH)-M_\g'c- 1203 2948 BBz-PZA-Kl3-Fla -T2A-PAC _-CD 19 CDSSP-CD l9-Mcdrcx-24D l-(vL-VH)- 1204, 2949, -p2A-K13-F...-TZA-pAc CD 19 CD8SP-Ritx-CD l9-MOR0028-(VL-VH)- 1205 29")- MV’C-BBZ-PZA-KB-Fl. -T2A-PAC CD 19 CDXSP-CD l9-HD37-H2L l-(VL-VH)-M_\;‘C- 1206 29"' BBz-PZA-Kl3-Fla -T2A-PAC CD 19 1207 - CDSSP-CD l9-huBly3-(\-'L-VH)-Myc-BBZ- P2A-Kl3-Fl ~ -T2A-PAC CD 19 CDSSP-CD l9-huSJ25C vH)-Myc- 1208 9%"9") BBZ-PZA-Kl3-Fla -T2A-PAC _-CD 19 CDBSP-RitX-CD l9-hB4-(VL-VH)-M_\-'c- A—m—na ‘ AFP/MHC class I CD8SP-AFP(vL-vH)-M)-'c-BBz-P2A- complex K l3-Flag-T2A-PAC AFP/MHC class I CD8SP-AFP(\-’L-vH)-M_\-'c-BBz-P2A- l 2 13 29; 8 complex ' K l3-Flag-T2A-PAC AFP/MI-IC class I CD8SP-AFP(vL-vH)-M_\;'c-BBz-P2A— 1214 2959 complex ' K l3-Flag-T2A-PAC HIV 1 -envelop CD8SP-I-IIV l-N6-(vL-\-‘H)-Myc-BBz- 12 15 2960 glycoprotein ' P2A-K l 3-Flag-T2A-PAC ALK (Anaplastic CD8SP-Alk(vL-vH)-Myc-BBz-P2A- 12 I (3 296' Lymphoma Kinase) K l3-Flag-T2A-PAC ALK (Anaplastic CD8SP—Alk(\'L-vH)-Myc-BBz-P2A- 1217 2962 Lymphoma Kinase) K l3-Flag-T2A-PAC —_1218 SP-Amyloid-l58-(\=‘L-\-'H)-Myc-BBz~P2A~ —____ Biotin , CD88P-dc-Avid1n-M_vc-BBz-P2A-Kl3- CD45 - CD8SP-BC8-CD45-(vL-vI-I)-l\/ch-BBz- P2A-Kl3-Fl -T2A-PAC _--CDBSP-BCMA-huClZA3-L3H3-(vL-vH)-Mvc-BBz-PZA-Kl3-Fl -T2A-PAC BBz-PZA—Kl 3-Fla —T2A-PAC BCMA CD8SP-BCMA-ET(vL-vH)-Myc- * BBz-PZA-K13-Fla-T2A-PAC —--CD8SP-BCMA-huCll.D5.3LlI—I3-(VL-vH)-M\_-'c-BBz-P2A-Kl3-Fl -T2A-PAC _--CDSSP-BCMA-huC13-F12-(vL-vH)-Myc-BBz-PZA-K13-Fla -T2A-PAC CCR4 CCR4-humAb1567-(vL-vH)—Myc- 1228 2972’ BBz-PZA-K13-Fla-T2A-PAC HIV 1 ﬁve/10p CD85P-CD4-ECD-Gly-Ser-Linker-DC- 1229 2974 glycoprotcin yc-BBz-PZA-K l 3-Flag-T2A-PAC CD5 CD8SP-CD5(vL-vH)-Myc-BBz-P2A- '230 2973- Kl3-Fl -T2A-PAC CD5 . CD8SP-CD5- l 8-(vL-vH)-Myc-BBz-P2A- 12" 2976 Kl3-Fl o-TZA-PAC 1g Fe CD8SP-CDl6A-V158-ECD-v2-Myc-BBz- 1232 2977 P2A-K13-Fl AC lg Fc CDXSP-CDI6A-Vl58-ECD-vl-Myc-BB2- '233 2978 P2A-Kl3-Fl AC —--53:21-523.1.215111278Bl- —--3:51:2302211;:w-Mw-BBZ- —--$3351.33.362113511 —--51:21-52:-.1:::1:r:H>-Mw-BBZ- —--gmﬁwmm _--$913522?0-?3157-Mw-BBZ- _--cps-SP-Con-TaMfA»;11-(1’L-VH1M\c BBz PZA K13 Fl T2A PAC CD20 -1242 2987 CDXSP-CDZO-Ubli-v4-(vL-vH)-l\71yc- —___BBz—PzA-K13-FIa-T2A-PAC CD20 CD8SP-CD20-2H7-(vL-vH)-MyC-BBZ- 1243 2988 P2A-Kl3-Fl -T2A-PAC _-CD20 '2‘", , CD8SP-CD20-hlF5-(vL-\.’H)-l\'1)-'c-BBZ- PzA-K13-Fl -T2A-PAC _--2990CD20 - . CD8SP-CD20-7D8-(vL-vH)-M_V'c-BBZ- PzA-Kls-FI -T2A-PAC _-"46CD20 , 299' CDBSP-CDZO-AME(vL-\'H)-Myc- A-K13-FIa-TzA-PAC _-CD22 '2‘"V CD8SP-CD22-hl0F4v2-(vL-VH)-M_Vc- BBz-PzA—K13-FIa—T2A-PAC CD22 CD8SP-CD22-H22Rhov2ACDRKA-(VL- 1248 299aJ VH -M\_’c-BBz-P2A-Kl3-Fla -T2A-PAC —-CD22 CD22-m97 l-(V‘L-VH)-M_V'c-BBZ- "-49 2994 —---BBz—pzA—m—na—m—mcCD22 - - CD85P-CD22-m97 vH-vL)-Myc- _--2996CD30 - CDSSP-CD30-5Fl l-(VL-VH)-NI_VC-BBZ- P2A-Kl3-Fln-T2A-PAC _--2997CD30 - CD8SP-CD30-AclO-(vL-vH)-Myc-BBZ- m—m—n mm CD32 _ CD32-Med9-(vL-vH)-Myc-BBZ- _--2999CD33 - , CDSSP-CD33-AF5-(\-'L-VH)-Myc-BBZ- P2A-Kl3-Fln-T2A-PAC _--300°CD33 - - CD8SP-CD33-huMyc9-(vL-vH)-M_\_"c- BBz-PZA-Kls-Fla -T2A-PAC CD33 CD8SP-CD33-Boehr2800308-(VL-VH)- 1256. 3001 MV'C-BBz-P2A-Kl3-Fl -T2A-PAC —--3°02CD33 - CDSSP-CD33-Him3(vL-VH)-M_\'c- A-K13-FIa—T2A-PAC CD33 CD8SP-CD33-SGNh2Hl 2-(vL-vH)-M)-’c- 1258' 300a3 BBz-PZA-K13-Fla -T2A-PAC _---BBz-pzA—mna—TzA-mcCD33 _ , CD33-15G15(\-'L-vH)-Myc- _-"60CD33 - CDSSP-CD33-33H4-(vL—VH)-Myc-BBZ- PzA-K13-Flw-T2A-PAC _-CD33 CD85P-CD33-9C3(vL-vH)-Myc-BBZ- '26‘ 3°06 PzA-K13-FI -T2A-PAC _-"62CD34 CD8SP-CD34-hu4C7-(VL-VH)-Myc-BBZ- P2A-K13-FI -T2A-PAC _-"63CD44V6 3°08 CD8SP-CD44v6-BiwaS-(vL-vH)-M)-'c- A-K13-Fla-T2A-PAC —-"64CD70 CD8SP-CD70-h l F6-(vL-\-'H)-M)'c-BBZ- pzA—m—n mm CD79b CD8SP~CD79b~2F2-(vL-\-’H)-M_\=‘c-BBZ~ 1265' 3010 P2A-Kl3-Fl -T2A-PAC CD79b CD8SP-huMA79bv28-(vL-vH)-Myc-BBZ- 1266 301 l P2A-Kl3-Fl -T2A-PAC CD99 CD8SP-CD99-hu12E7-(vL-VH)-Myc- 1267 3012 A-K13-F1a-T2A-PAC CD 123 1268 301. CD8SP-CD123-CSL362-(vL-vH)-Myc- BB2-P2A-K13-Fla -T2A-PAC _-"69CD 123 CD8SP-CD123-l l72-(vL-vH)-Myc-BBZ- PzA-K13-FI -T2A-PAC CD 123 CDSSP-CD123-DART(vL-vH)-M_\'C- 1270 3015' BBZ-PZA-K13-Fla -T2A-PAC CD 123 CD8SP-CD123-DART(\-’L-VH)-M}'C- 1271 3016 BBz-PZA-K13-Fla -T2A-PAC CD123 1272 3017 CD8SP-CD123-13RBl8-(VL-VH)-M}-’c- BBz-PZA-K13-F1a - -T2A-PAC CD 123 CD8SP-CD123-11u3E3-(VL-VH)-Myc- 1273 3018 BBz-PZA-K13-F1a -T2A-PAC —-"74CD 123 , CD123-9F6-(vL-\-’H)-Myc-BBZ- 3-F1 -T2A-PAC _--302°CD 123 - CDSSP-CD123-13RBZ-(VL-VH)-Myc- BBz—PzA-K13-Fla-T2A-PAC _-m6CD 123 CD8SP-CD 123-1 176-(VL-\'H)-Myc-BBZ- 3-Fl -T2A-PAC CD123 CDSSP-RitxZ-CD123-SBI 1-(VL-\'H)— 1277 3022 Mvc-BBz-PZA-K 13-F1 ' -T2A-PAC _-"78CD 123 . CDXSP-CD l23-2BX-(VL-VH)-Myc-BBZ- PzA-K13—Fl -T2A-PAC _-CD 123 , CD8SP-CD123-9D7-(vL-vH)-Myc-BBZ- 1279 30" P2A-K13-F1 -T2A-PAC _-"80CD 123 - CD8SP-CD123-3B10-(VL-VH)-M_Vc-BBZ- PZA-Kl3-Fln-T2A-PAC _-CD 138 CD8SP-CD138-(V’L-vH)-Myc-BBz-P2A- "-81 3026 mm -WAC _-"82CD 179b 3°27 CD179b-(vL-vH)—M_\*c—BBz-P2A- K13-FI -T2A-PAC _--3°28CD276 . CDSSP-CDZ76-1 7-(vL-\-’H)-M_\-'c-BBZ- PzA-Kls-FI-TzA-PAC _---BBz-pzA-K13-na-nA-pAcCD324 CD85P-CD324-SC lO(vL-VH)-Myc- CD324 CD8SP-CD324-hSC10-l 7-(vL-vH)-M_VC- 1285 3030 BBz-PZA-Kl3-F1a -T2A-PAC CDH6 CD8SP-CDH6-NOV7 l 0-(vL-VH)-Myc- 1286 303] BBz-PZA-K13-F1a -T2A-PAC CDH6 CD8SP-CDH6-NOV712-(V‘L-VH)-Myc- 1287 30:2‘ BBZ-PZA-K13-F1a -T2A-PAC CDH17 CDXSP-CDHI7-PTA001A4-(vL-VH)- 1288 30"3" z-PZA-Kl3-Fl -T2A-PAC _-CDH19 '289 a, CD8SP-CDH19-16A4-(VL-VH)-M}'C-BBZ- pzA-K13-n-T2A-pAc _-EGFR a - CD8SP-Cetuximab-(vL-vH)-M_vc-BBz- PzA-KI3-FIa-T2A-PAC CLECSA _1291 3036 CD8SP-CLECSA-8H8F5-(vL-vH)-M}-'c- —___BBz-PZA-K13-Fla-T2A-PAC CLECSA CDSSP-CLECSA-3ElZAZ-(vL-vl—I)-Myc- 1292 3037 BBz-PZA-Kl3-Fla -T2A-PAC GR/LHR - SP-CGHb-Gly-Ser-Linker-CGHa-Myc- (Gonadotropm - 1293 3038 BBz-PZA-Kl3-Flag-T2A-PAC Receptor) _-"94CLL1 , a CD8SP-CLLl-MZ6-(vL-vH)-Myc-BBz- 3-F1-T2A-PAC CLL1 - , CLLl-M32-(vL-vI-I)-Myc-BBZ- CLL1 CDSSP-CLL1-2lC9-L2H3-(vL-x-1H)-Myc- 1296 3041 BBz-PZA-K13-Fla -T2A-PAC CLL1 CDSSP-CLL l-6E7L4H -vH)-Myc- 1297 3 042 BBz-PZA-KIB-Fla -T2A-PAC _-"98CLL1 ,1 CLLl-hul075-vl-(vL-vH)-Myc- BBz-PzA-K13-Fla-T2A-PAC _-"99CLL1 CD8SP-CLLl-hu1075-v2-(vL-vH)-Myc- ‘". .

BBz—PzA-K13-FIa-T2A-PAC /MHC CDSSP-CMVpp65-F5-(vL-vH)-M_\=c-BBz- 1300 304i class I complex " P2A~Kl3~Flag~T2A~PAC CS1 (SLAMF7) CD8SP-CS l-huLuc63-(vL-vH)-l\/ch-BBz- 1301 3046 P2A-Kl3-Fl --T2A-PAC CSl (SLAMF7) CD8SP-CSl-HuLuc64-(vL-vH)-Myc- 1302 3047 BBz-PZA-Kl 3-Fla -T2A-PAC CSl (SLAMF7) 1303 3048 CDXSP-CSl-huLuc90-(vL-vH)-Myc-BBZ- P2A-Kl3-Fl -T2A-PAC C51 (SLAMF7) CD8SP-CS l-PDL24l-(vL-vH)-Myc-BBz- 13o4 3049 P2A-Kl3-Fl -T2A-PAC CSl (SLAMF7) CD8SP-CS l-Hu27A-(vL-vH)-Myc-BBZ- 130;* 3050 P2A-K13-Fl. - -T2A-PAC C51 (SLAMF7) CD8SP-CS l-ScHu34C3-(vL-vH)-Myc- "06 3 0 i ‘ ' ' 1 BB2-P2A-Kl3-Fla-T2A-PAC C51 (SLAMF7) CDSSP-CSl-Hu3l-D2-(vL-vH)-l\rlyc- 1307 052 BBz-PZA-K13-Fla -T2A-PAC CSl(SLAMF7) - CDSSP-CSl-Luc34-(vL-vI-I)-Myc-BBz- 1308 0;"0‘3 3-Fl.

C51 (SLAMF7) CD8SP-CSl-Lch2-(vL-vH)-Myc-BBz- 1309 3054 P2A-Kl3-F1 -T2A-PAC CD8SP-CSF2RA-Ab6-(vL-vH)-Myc-BBz- CD8SP-CSF2RA-Ab l -(vL-vH)-Myc-BBZ- PZA-K l 3-Fl - -T2A-PAC I312 CD]23-l-vHH-Myc-BBz-P2A-KI3-Flag- T2A-PAC 1313 3058 CD 1 23Vl—lI-I-Myc-BBz-P2A-K13-Flag- T2A-PAC DLL3 (Delta Like CD8SP-DLL3-hSC l6-l3-(vL-vH)-I\Ilyc- 1314 3059 Ligand 3) BBz-PZA-K13-Flag-T2A-PAC DLL3 (Delta Like DLL3-hSC16(vL-vH)-Myc- 131% 3060 Ligand 3) " BBz-PZA-Kl3-Flag-T2A-PAC EBNA3c/MHC class CD8SP-EBNA3c-3 15-(vL-vH)-Myc-BBz- 1316 3061 1 complex ‘ P2A-Kl3-Flag-T2A-PAC EBV-gp350 EBV-gp350-(vL-vH)-Myc-BBz- 1317 3062 P2A-Kl3-Fl -T2A-PAC EGFR CD8SP-EGFR1 -vHH-Myc-BBz-P2A- 1318- 30m- K l3-Fla AC EGFR & CEA CD8SP-EGFR1-vI~Il-I-Gly-Ser-Linker- 1319 3064 CEAl-vI-II-I-Myc-BBz-PZA-Kl3-Flag- T2A-PAC EGFR & CEA cossp-EGFR33«HH-Gly-Ser-Linker- 1320 3065 CEAS-vI-Hi-Myc-BBz-PZA-Kl3-Flag- TZA-PAC EGFvall CD8SP-EGFvall-l39-(vL-vH)-Myc- 1321‘ 3 066 BBz-PZA-K 1 3-Fla -T2A-PAC EGFRvIlI CDSSP-EGFvall-Zl73-(vL-vH)—M_vc- 1322 3067 BBz-PZA-Kl 3-Fla -T2A-PAC EpCaml - CD8SP-Epcaml-MMl-(vL-vH)-M_\;’c- 1323 3068 BBz-PZA-Kl3-Fla -T2A-PAC EpCam1 , CD8SP-Epcam 1 -D5K5-(vL-vH)-Myc- _--307°FLT3 a - CD8SP—FLT3-NC7-(vL-vH)—Myc-BBz- PzA-K13-F1-T2A-PAC _-FITC CD8SP-FITC-(vL-vH)—Myc—BBz—P2A- '326 307' K13-FI -T2A-PAC FITC CDSSP-FlTC-4M(vL-vH)-M)r'c-BBz- FlTC a CD85P-FlTC-E2-HL-(vH-vL)-Myc-BBz- za A HA CD8SP—FLU-MEDI(vL—vH)—Myc- 1329 3074 BBz-PZA-Kl 3-Fla- -T2A-PAC FRI (Folate CD8SP-FRl-huMov l9—(vL-vH)-Myc- 1330 307i Receptor alpha) ' BBz-PZA-K 1 3-Flag-T2A-PAC FSHR (Follicle - - St1mulat1ng. . CD8SP FSHb G]y-Ser-L'inker—CGHa— 1331 3076 Myc-BBz-PZA-Kl3-Flag-T2A-PAC Hormone Rcccptor) GAD (Glutamic Acid CDSSP-GAD-G3H8-(vL-vH)-Myc-BBz- [332 3077 Decarboxylase)/MH PZA-K I -T2A-PAC C class I x _-GDZ CDSSP-GDZ-hul4-18~(\’L-VH)-M_VC~BBZ- '333 3°78 PzA-KI3-FI--T2A-PAC _-GDZ , CDXSP-GDZ-hu3F8-(VL-VH)-Myc-BBZ- '33" 3°79 PZA-K13'F1"T2A'PAC 0GD3 - CD8SP—GD3-KM-64l-(VL-VH)-Myc-BBZ- PzA-KI3-FI -T2A-PAC GFR34(GDNF CDSSP-GFRAI 1 4 P4 6( L H) M Famll) Receptor -v 1336 308‘ p1a---V m":- A-Kl3-Flag-T2A-PAC Alpha 4) GFRa4(GDNF GFRa4 P4 10( L’- '- H) M- ', V -v -v yc-BBZ- Famll) Receptor '337 3082 P2A-Kl3-Flag-T2A-PAC Alpha 4) GM] CDSSP-GMl-5B2-(vL-VH)-M)’C-BBZ- [3‘83 308"3 PZA-K 1 3-Fl . -T2A-PAC _-GMl CDSSP-GMl-7E5-(vL-vH)-Myc-BBZ- '339 30’", 3-Fl- -T2A-PAC GPRCSD (G-protcin coupled receptor CD8SP-GPRC5D-ET150(vL-vH)-Myc- 1340 308% family C group 5 ' BB2-P2A-Kl3-Flag-T2A-PAC member D) GPRCSD CD8SP-GPRC5D-ET150-l8-(vL-VH)- 1341 3086 Mvc-BBz-PZA-K13-Fl- -T2A-PAC GPRCSD - CD8SP-GPRC5D-ET150-l-(vL-vH)-Myc- 1342 3087 BBz-PZA-K13-Fla -T2A-PAC GPRCSD - CD8SP-GPRC5D-ET150(vL-vH)-Myc- 1343 3088 BBz-PZA-Kl 3-Fla -T2A-PAC gplOO/MHC class I CDSSP-gpl00-(vL-vH)-Myc-BBz-P2A- 1344 3089 complex K l3-Flag-T2A-PAC gplOO/MHC class I 1‘4; CDSSP-gpIOO-GIZD12-(\-’L-vH)-Myc-BBZ- complex ' 3090 P2A-Kl3-Flag-T2A-PAC GPC3 (Glypican 3) CDXSP-GPCS-4E5-(vL-vH)-lVlyc-BBz- 1246‘ ‘2091 - P2A-Kl3-Fla-T2A-PAC gpNMB CD8SP-gpNMB-l lS-(vL-vH)-Myc-BB2- 1347 3092 protein Nmb) PZA-K l 3-Flag-T2A-PAC GRP78 , a CDXSP-GRP78-GClS-(vL-vH)-Myc-BBz- _-1349 3094 CD8-SP-Her2-3F7-vHH-Myc-BBz-P2A- —-- ’ - CD8SP-Hcr3-l7BOSSo-vHH-Myc-BB2— P2A-Kl3-Fl - -T2A-PAC Her3 - CD8SP-Her3-Afﬂ—Myc—BBz-P2A-Kl3- Her2 and Her3 CD8SP-Her3-l7BOSSo-\-'HH-Gly-Ser- 1357 3 102 Linker-Her2—2D3-vHH-Myc-BBz-P2A- Kl3-Fl - -T2A-PAC HIVl-gag/MHC CDSSP-HIVl-E5-(\-'L-\V=H)-M.\-'c-BBz- 13 as 3103 class I complex ‘ PZA-K l 3-Flag-T2A-PAC HIV 1 -cnvclop CD8SP-HIV l-3BNC1 l7-(\'L-VH)-Myc- 1359 3 104 glycoprotein BBz-PZA-Kl 3-Flag-T2A-PAC HIV 1 mwclor) CD8SP-HIV l-PGT(vL-vH)-Myc- 1.603 3 10% glycoprotein " BBz-PZA-K13-Flag-T2A-PAC HIV 1 envelop CD8SP-HIV l-VR-CO l -(vL-\-'H)-Myc- 1361 3 106 glycoprotein BBz-PZA-K13-Flag-T2A-PAC HIVlwveIOP CDSSP-HIVl-X5-(vL-vH)-l\/ch-BBz- 1362 3107 glycoprotein PZA-K l 3-Flag-T2A-PAC HLA-AZ 1363 3 108 CD8SP-I-ILA-AZ-3PBZ-(vL-vH)-Myc- BB2-P2A-K13-Fla -T2A-PAC HMW-MAA CD8SP-HMW-MAA-thD-(vL-vH)-Myc- l 3 64 3 109 BBz-PZA-Kl 3-Fla AC E7/MHC 1365 3 l 10 CDxSP-HPV168-(vL-vH)-Myc-BBz- class 1 complex P2A-Kl3-Flag-T2A-PAC HPV16-E7/MHC CDSSP-HPV l 6(vL-vl—I)-M}_’c-BB2- l366 3 l l l class 1 complex P2A-Kl3-Flag-T2A-PAC HTLV l -TAX/MHC CD8SP-HTLV-TAX-T3F2-(vL-vl-l)-M}_-'c- l 2 67 2 l 12 class I x ‘ ‘ A-Kl3-Flag-T2A-PAC HTLV l -TAX/MHC CDSSP-HTLV-TAX-T3E3-(vL-vH)-Myc- 1%68‘ 3 1 1"» class 1 complex ‘ BBz-PZA-Kl3-Flag-T2A-PAC 1L1 lRa CD8SP—IL1lRa—8E2-Ts107-(vL-vH)-Myc- 1369 31 14 BBz-PZA-Kl 3-Fla- AC IL6Ra IgHSP-IL6RvI-lH-Myc-BBz-P2A- I370 3 l 15‘ K l3-Fl ~ -T2A-PAC lL l3Ra2 CDXSP-lLl3Ra2-hu107-(vL-vH)-M_vc- 1371 31 l 6 A-Kl 3-Fla- -T2A-PAC ILl3Ra2 - CD8SP-IL13Ra2-Hu108-(vL-vI-l)-Myc- 1372 3117 BBz-PZA-Kl 3-Fla -T2A-PAC KSHV-K8.l CDSSP-KSHV-4C3-(vL-vH)-l\lyc-BBZ- l" 73° 3 l 18 PZA-K 1 3-Fl . -T2A-PAC LAMP! omal- LAMPl-humab 1 (vL-vH)-Myc- 1374 3 1 19 associated BBz-PZA-K l 3-Flag-T2A-PAC membrane protein 1) LAMP! (Lysosomal- 1‘75 3120 CD8SP-LAMPl-Mb4-(vL-vH)-Myc-BBz- associated ' P2A-Kl3-Flag-T2A-PAC membrane protein 1) Clone SEQ ID SEQ ID _--CD8SP-LewisY-huS193-(vL-vH)—Myc-BBz-PZA-Kl3-Fla-T2A-PAC BBz-PZA-K13-Fla -T2A-PAC _--SP-LHb-Gly-Scr—Linkcr—CGHa-Myc-BBz-P2A—Kl3-Fl -T2A-PAC Lyml CDSSP-Lym l-(vL-vH)-Myc-BBz-P2A- Lvmz CD8SP-Lvm2- vL-vH -M\'c-BBz-P2A- q _ —--CD8SP-huMA79bv28-(vL-vI-I)-Myc-BBz-P2A-Kl3-Fl -T2A-PAC I complex ‘ ‘ BBz-PZA-Kl3-Flag-T2A-PAC MARTI/MHC class CD8SP-MART1-CLA12-(vL-VH)-Myc- I complex ' ' 3128 BBz-PZA-K13-Flag-T2A-PAC Mesothelin CDSSP-Mesothelin-m9 l 2-(vL-vH)-1\/ch- 1384 3129 BBz-PZA-Kl 3-F1a ~ -T2A-PAC cMet CDSSP-cMet-l71-vHIMyc-BBz-P2A- 1385 3130 K 13-F1 - -T2A-PAC cMet and Her3 CD8SP-cMET- l71-vHH-Gl_\-'-Ser-Linker- 1386 3 13 l Her3-21F06-vl—II-I-Myc-BBz-P2A-Kl3- Fl - AC MPL(\~'L-vH)-1\/lyc-BBz- (Thrombopoietin P2A-Kl3-Flag-T2A-PAC receptor) CDSSP-MPL(vL-vH)-Myc-BBz- bopoietin 3—Flag-T2A-PAC receptor) CD8SP-MPL- l6l-HL-(vH-vL)-Myc-BBZ- ('I'hrombopoietin PZA-K l 3-F1ag-T2A-PAC receptor) CD8SPMPL-1 1 1-(\.'L-VH)-M)-'c-BBz- (Thrombopoietin DJ _. DJ (J- PZA-K l 3—Flag-T2A-PAC receptor) CDSSP-MPL- l78-(vL-vI—1)-1Vch-BBz- (Thrombopoietin P2A-K13-Flag-T2A-PAC receptor) MPL-AB3 l 7-(vL-vH)-Myc-BBZ- (Thrombopoietin PZA-K l 3-Flag-T2A-PAC receptor) CD8SP-MPL-12ElO-(vL-vH)-Myc-BBZ- (Thrombopoietin '393 3138 P2A-Kl3-Flag-T2A-PAC receptor) .— 394 3 139 CD8SP-MPL-huVB22BwS-(vL-vH)-Myc- (Thrombopoietin BBz-PZA-Kl3-Flag-T2A-PAC receptor) Mucl/MHC class I CDXSP-Mucl-D6-M3B8-(vL-vH)-Myc- 139‘ 3140 complex " BBz-PZA-Kl 3-Flag-T2A-PAC Mucl/MHC class I CD8SP-MUG. l-D6-M3Al-(vL-vH)-Myc- l ‘t 96 3141 x ‘ BBz-PZA-Kl3-Flag-T2A-PAC _-Muc l 6 CD8SP-Muc16-4H1 1-(vL-vH)—M_\_-'c-BBz- '397 3 ‘42.

PzA-KI3-FI -T2A-PAC 1398 CD8SP-Ni1not11211n1ab-(\-'L-VH)-Myc-BBZ- ‘ 314.3‘ P2A-Kl3-F] -T2A-PAC NKGZD Ligand CD8SP-NKGZD-(GGGGS-GGGGD)- 1399 3144 Mvc-BBz-PZA-KB-FI -T2A-PAC NKGZD CD8SP-NKGZD-MS-(vL-vH)-Myc-BBz- 1400 314;' P2A-Kl3-Fl --T2A-PAC NY-BRl CDSSP-NYBRI-(vL-vH)-Myc-BBz-P2A- 1401 ‘3146 K l3—Fla -T2A-PAC NY-ESO/MHC class 1402 3147 CDSSP-NYESO-Tl-(vL-vl-I)-l\1yc-BBz- I x PZA-K l 3-Flag-T2A-PAC NY-ESO/MHC Class 1403 3148 CDSSP—NYESO-TZ-(vL-\-'H)—Myc-BBz- I complex PZA-K l -T2A-PAC PD] ligand (0%»: CDSSP-PDl-ECD-Myc-BBz-PZA-KB- 1404 3149 PDLl) Flag-TZA-PAC PDLl - - CDXSP-PDL l -Atezoli-(vL-vH)-Myc-BBZ- PDLl CDSSP-PDLl-SPl42-(vL-vH)-Myc-BBz- 1406 3151 P2A-Kl3-Fl ~ -T2A-PAC —-"07PDLl - PDL 1-1 L—vH)-Myc-BBz- PzA-KI3-FI -T2A-PAC PSCA (Prostate stem CDSSP-PSCA-Hal4-12l-(vL-vH)-Myc- 1408 31s" cell antigen) ' ° BBz-PZA-K13-Flag-T2A-PAC PSCA (Prostate stem CDSSP-PSCA-I-lal4-l l7-(vL-vl—l)-l\/lyc- 1409 3154 cell antigen) BBz-PZA-Kl 3-Flag-T2A-PAC -IC class I CDXSP-PRl-(vL-vH)-Myc-BBz-P2A- 1410 31" complex ' ' K l3-Flag-T2A-PAC PSMA (Prostate CD8SP PSMA 006— ‘- ), -v-v( L H) M BB c Membrane -yc- z , 3136 P2A-Kl3-Flag-T2A-PAC Anttgen) PSMA (Prostate ‘. CDSSP PSMAJ'91- -3 -v-v( L H) M- BB Sp°.c'ﬁ° Mcmbmc z- "12 . , 3137 yc- P2A-Kl3-Flag-T2A-PAC Antlgen) PTK7 (Tyrosine- CDSSP-PTK7-hSC6(vL-vH)-Myc- 1413 3 1 £8 protein kinase-like 7) ' BBz-PZA-K13-Flag-T2A-PAC PTK7 (Tyrosine- CD8SP-PTK7-SC6-l 0(vL-vH)-Myc- 1414 3 159 protein kinase-like 7) A-Kl3-Flag-T2A-PAC IE___3160 CD8SP-R0Rl4A5-(vL-vH)-M.vc-BBz- ——---— k-"‘6 CDSSP-RORI -4C 1 0-(vL-vH)-Myc-BBZ- , 316' PzA-K13-FI -T2A-PAC Mesothelin CD8SP-SDl-vHH-Gly-Ser-Linker-SD2- 1417 3162 vI—H—I-Mvc-BBz-PZA-KB-Fl -T2A-PAC _-"’8SLea . .. CD8SP-SLca-7E3-(vL-vH)—Myc-BBZ- P2A-Kl3-Fl AC _-"'9SLea , , SLea-SBl-(vL-vH)-Myc-BBZ- mix—mm Jim-me SSEA4 (Stage' CDSSP SSEA4- ’- v -v - . .

- BBz-PZA- speciﬁc (:1an 0111c) ‘, 1420, ( L H) Myc- K l3-Flag-T2A-PAC antigen 4) TCRBI (TCR beta 1 CstP-TCRB 1 -CPO 1 -E09-(vL-vH)-Myc- 1421 g ‘ 1 66 constant chain) BBz-PZA-K l 3-Flag-T2A-PAC TCRBI (TCR beta I CDSSP-TCRBl-Jovil-(vL-vH)-M_vc-BBz- 1422 g 167 constant chain) PZA-K l 3-Flag-T2A-PAC TCRB2 (TCR beta 2 CDSSP-TCRBZ-CPO l-DOS-(vL-VH)-Myc- 1423 3 168 constant chain) BBz-PZA-Kl 3-Flag-T2A-PAC TCRB2 (TCR beta 2 CD8SP-TCRB2-CPO l-EOS-(vL-vH)-Myc- 1424 3 169 constant chain) BBz-PZA-Kl 3-Flag—T2A-PAC TCRgd (TCR CD8SP-TCRgd-G5(vL—vH)-Myc-BBz- 1425 3170 gamma/delta) P2A-Kl3—Flag-T2A-PAC hTERT/MHC class I CDSSP-TERT-4A9-T540-(vL-vH)-Myc- 1426 3171 complex BBz-PZA-Kl -T2A-PAC hTERT/MHC class I TERT-3G3-T865—(vL-vH)—Myc- 1427 3172 complex BBz-PZA-Kl 3-Flag-T2A-PAC Tissue -l CD8SP—TGFBR2-Abl-(vL-vH)-Myc-BBz- 1428 317"3 3-Fl- -T2A-PAC TGFBRZ . . CD8SP-TFl(\«'L-vH)-Myc-BBz-P2A- TIMI/HAVCR CDSSP-TIMl-HVCR12-(vLIH)- 1430 317i~ Mvc-BBz-PZA-Kl3-F] -T2A-PAC TIM l/HAVCR CD$SP-TIMl-HVCRl-ARDS-(vL-VH)- 1421‘ 3176 Mvc-BBz-PZA-KB-Fl -T2A-PAC TnAg , CD88P-TnAg-(vL-vH)—Myc-BBz-P2A- Tn-Mucl CDSSP-TnMucl-huSES-RHAS-RKA-Z- 1433 3 178 (vL-vH)-Myc-BBz-P2A-K 1 3-Flag-T2A- ('I'hrlombopoictin - CDSSP-hTPO-IVch-BBz-PZA-Kl3-Flag- 1434 3179 T2A-PAC receptor) TROP2 (Trophoblast CD8SP-TROP2-ARA47-HV3KV3-(VL- gill'surface ant'gen' vI-I)-M_\_-'c-BBz-P2A-K 1 3-Flag-T2A-PAC Clone SEQ ID SEQ ID —--CD8SP-TROPZ-h7E6-SVG-(vL-vH)-Myc-BBz-PZA-Kl3-Fla-T2A-PAC _--SP-TSHb-Gly-Scr-Linkcr-CGHa-Myc-receptor) BBz-PZA—K l 3—Flag-T2A-PAC 3-Fl AC P2A-K13-Fl AC TSHR - . . CD8SP-TSHR-5C9-(vL-vH)-Myc-BBz- TSLPR ('Ilrvmic stromal CDSSP-TSLPR-(vL-vH)-Myc-BBz-P2A- 1441 3186 lymphopoietin K l3-Flag-T2A-PAC receptor) Tyrosinase/MHC CD8SP-Tyros-BZ-(vL-vl-I)—Myc-BBz- 1442 3187 class 1 complex P2A-K13-Flag-T2A-PAC Tyrosinase/MHC CD8SP-Tyros-MC 1 -(vL-vH)-l\lyc-BBz- 1444 4 188 class I complex ‘ ‘ PZA-K l -T2A-PAC Tyrosinase/MHC C08sP-Tyros-TA2-(vL-\.-H)-Myc-BBz- 1 444 3 I 89 class 1 complex PZA-K l 3-Flag-T2A-PAC VEGFR3 --3190 CD8SP-VEGFR3-Abl-(vL-vH)-Myc- ' BBz-PZA-Kl 3-Fla- -T2A-PAC WTl/MHC class I l 446 3191 CD8SP-WT1-Abl-(vL-vH)-Myc-BBz- complex PZA-K 1 3-Flag-T2A-PAC W'Tl/MHC class 1 CDSSP-WTI-Ab5-(vL-VH)-Myc-BB2- 1447 3192 complex PZA-K l 3-Flag-T2A-PAC WTl/MHC Class 1 CD8SP-MYC3-WTl-Ab l3-(vL-vH)-Myc- l 448 3 19"’ complex BBz-PZA-Kl3-Flag-T2A-PAC WTl/MHC Class 1 CD8SP-MYC3-WT1 -Ab lS-(vL-vH)—Myc- 1449 3194 complex BBz-PZA-K l -T2A-PAC CDH19 CDSSP-CDH19-4BlO-(vL-vH)-M_vc-BBz- 1450 3194' P2A-Kl3-Fl -T2A-PAC Folate Receptor beta C08SP-FRbeta-m923-(vL-vH)-Myc-BBZ- 143'" 3196 P2A-Kl3-Fl -T2A-PAC LHR (Luteinizing CDSSP-LHR-SB7-(vL-vH)—Myc-BBz- 1452 3 197 hormone Receptor) P2A-Kl3-Flag-T2A-PAC LHR (Luteinizing CDSSP-LHR-5F4-2l-(vL-vH)—M)»'c-BB2- 1453 3198 honnonc Receptor) 3-Flag-T2A-PAC _--3199B7H4 , -, CD8SP—B7H4—thZCl0—(vL-VH)-Myc- BBz-PzA-K13-Fla -T2A-PAC B7H4 -- CDSSP-B7H4-hulDl l-(vL-vH)-Myc- IgE - , CD8SP-lgE-omalizumab-(vL-vl—I)—Myc- CD23 _-3202 CDSSP-CD23-p5E8-(vL-vH)-Myc-BB2- —___-— GCC (Guanylyl 1458 3202 CD8SP-GCC-5F9-(vL-vH)-Myc-BB2- cyclasc C) * 3 P2A-Kl3-Flag-T2A-PAC GCC (Guanylyl CD8SP-GCC-Ab229-(vL-vH)-Myc-BBz— cyclase C) ' 3204 P2A-Kl3-Flag-T2A-PAC _-"60C0200R - CDZOOR-hqulXZ-(vL-vH)-Myc- BBz—PzA-K13-Fla -T2A-PAC Tn-Mucl CD8SP-Tn-Mucl-5E5-HL-(vH-vL)-Myc- l 461 3206 BBz-PZA-Kl 3-Fla -T2A-PAC _-"62CD22 , CD8SP-CD22HL-(vH-VL)-M_\;‘c-BBZ- PzA-KI3-FI --T2A-PAC _--3208CD22 CD85P-CD22- l 0-HL-(vH-vL)-Myc-BBZ- PzA-KI3-FI-T2A-PAC _-"6"CD22 CDSSP-CD22-3 l —HL-(VH-VL)-Myc-BBZ- , , 3209 PzA-K13-FI -T2A-PAC _--321°CD22 - CD8SP-CD22HL-(vH-\-’L)-M'\_-'c-BBZ- 3-Fl-T2A-PAC C022 - C08SP-C022HL-(vH-vL)-Myc-BBZ- I466 32" P2A-Kl3-Fl -T2A-PAC Table 21: Exemplary CAR constructs containing 4188 costimulatory domain, CD3z activation domain SEQ 10 SEQ 10 CD 19 1 12014- CD8SP-FMC63-(vL-vH)-Myc- C019 - CDSSP-huFMC63-l l-(vL-vl-I)— 1468 3212’ T03 MVc-BBz-TZA-PAC C019 CD8SP-huFMC63-l Q-(VL- C019 082815- - CD19BulZ-(vL-vH)-M_Vc- C019 062915- 2—C0 l9MM-(vL-vH)-1\/I_vc- 1471 3216 003 BBz-TZA-PAC CD19 CDSSP-CDl9-4G7-(vL-vH)—Myc- C019 CDSSP-CDl9-MEDI(VL- 1473* ‘2218 vH)—Mvc-BBz-T2A-PAC , CDSSP-CDl9-Medrex-24Dl-(x-1L- C019 - CDSSP-Ritx-CDl9-MOR0028-(VL- CD19 CD8SP-CDl9-HD37-H2Ll-(vL- C019 _1477 3222 C085P—C019-111113034110101 ——__— CD19 a CDl9-huSJ25Cl-(vL-VH)- ( , CDSSP-Ritx-CDl9-hB4-(\-'L-VH)- CD19 - CDl9-hu-mROOS-I—(VL- CD19 CDSSP-CDl9-hAl9-(vL-vH)—Myc- C class I CD8SP-AFP-6 l-(vL—vH)-Myc— 1482 3227 complex BBz-TZA-PAC AFP/MHC class I A CD8SP-AFP(vL-vH)-M\_'c- AFP/MHC class 1 , CDSSP-AFP(vL-vI—l)-M\jc- HIV 1 —envelop - CDSSP-HIV 1-N6-(vL-vH)-Mvc- ALK (Anaplastic 052616- CD8SP-Alk(vL-vI-I)-I\/ch-BBz- 1486 3231 Lymphoma Kinasc) C05 T2A-PAC ALK (Anaplastic Alk(vL-vI-I)-I\/ch-BBz- 1487 32.23 Lymphoma Kinase) T2A-PAC Amyloid A. SP-Amyloid-l58-(vL-V-‘H)-Myc- CD45 a - CD8SP-BC8-CD45-(vL-vH)-Myc- BCMA a CD8SP-BCMA-J6MO-(vL-VH)- BCMA _ a CDSSP-BCMA-huC12A3-L3H3- BCMA CD8SP-BCMA-ET-4O-(vL-VH)- BCMA CD8SP-BCMA-ET(vL-19'H)- BCMA - , CD8SP-BCMA-ET(vL-vI-I)- BCMA - CDSSP-BCMA-huCll.D5.3L1H3- 1496 3241 (vL-vI—I)-Mvc-BBz-T2A-PAC BCMA CDSSP-BCMA-huCl3-FlZ-(vL- , , CDSSP-CCR4-humAbl567-(vL- HIVlﬂWCIOP 1499 3244 CD8SP-CD4-ECD-Gly-Ser-Linker— glycoprotcin DC-SIGN-Myc-BBz-TZA-PAC CD5 031416- . .

, CD8SP-CD5(vL-vH)-l\/lyc-BBz- —-- .551'333‘22’3X‘5X'Ew'V2' —--—gzséggmg-Ew-w- , , CD8SP-CD20-2F2-(vL-x-rH)-1Vlyc- BBz-TZA-PAC _--§?E?§giﬁi€;¥§:26""L"’"" CD20 CD8SP-CD20-hA20-(vL-vH)-Myc- BBz-TZA-PAC CD20 CDSSP-CDZO-BM-CAv4- (vL-vH -Mvc-BBz-T2A-PAC CD20 CDSSP-CD20-Ubli-v4-(vL-VH)- ‘ MYc-BBz-TZA-PAC CD20 CD8SP-CD20-2H7—(vL-vH)—M_Vc- ‘ BBz-T2A-PAC CD20 _ CDXSP-CDZO-h I F5-(vL-vH)-Myc- ‘ ‘ A-PAC CD20 CD8SP-CD20-7D8-(vL-vH)—Myc- BBz-T2A—PAC CD20 CD8SP-CD20-AME(vL-VH)- Mvc-BBz-T2A-PAC CD8SP-CD22-h10F4v2-(vL-\-'H)- z-TZA-PAC CD22 CD8SP-CD22- H22Rhov2ACDRKA-(vL-vI-I)- Mvc-BBz-TZA-PAC C1322 091515- - CDSSP-CDZZ-m97 l -(\"L-VH)-NI_VC- BBz-TzA-PAC CD22 --- - CD85P-CD22-m97l-HL-(VH-VL} 326‘" Mvc-BBz-TzA-PAC CD85P-CD30-5Fl l-(vL-vH)-Myc- 132' 3266 BBz-TZA-PAC CD30 012216- CD8SP-CD30-Ac lO-(vL-vH)-Myc- 1 522 3267 H04 BBz-TZA-PAC CD32 3268 CD8SP-CD32-Med9-(vL-vH)-Myc- ——__— CDSSP-CD33-AF5-(VL-VH)-Myc- BBz-TZA-PAC CD33 111815- - - CDSSP-CD33-hu1\'1)-'c9-(vL-vI-1)- MYc-BBz-T2A-PAC CD8SP-CD33-Boehr2800308-(VL- VH BBz-T2A-PAC CDXSP-CD33-Him3(VL-VH)- MVC-BBZ-TZA-PAC CDSSP-CD33-SGN112H12-(VL- vH)—M\'c-BBz—T2A-PAC CD8SP-CD33- 150 (vL-VH)- Mvc-BBZ-TZA-PAC CDSSP-CD33-331‘14-(VL-VH)-Myc- BBz-TZA-PAC CD8SP-CD33-9C3—2-(vL-VH)- MVc-BBz-TZA-PAC CD34 052316-103 CDSSP-CD34-hu4C7-(vL-vH)- &052616- 1532 3277 Myc-BBz-TZA-PAC CDSSP-CD44v6-Biwa8-(vL-vH)- MV’C-BBz-TZA-PAC CDXSP-CD70-h 1F6-(vL-\-'H)-1\/lyc- BBz-T2A-PAC CD79b 111815- CD8SP-CD79b-2F2-(vL-vH)-Myc- BBz-TZA-PAC CDSSP-huMA79bv28-(vL-VH)- MVc-BBz-TZA-PAC CDSSP-CD99-hu12E7-(vL-vH)- Mvc-BBz-T2A-PAC CD123 - -.. ., CDSSP-CD l 23-CSL362-(vL-VH)- Mvc-BBz—T2A—PAC CD8SP-CD123-l l72-(vL-v1—I)- Mvc-BBz-TZA-PAC CD8SP-CD123-DART-l -(vL-VH)- MVc-BBz-T2A-PAC CDSSP-CD123-DART(vL-VH)- Mvc-BBz-TZA-PAC CD8SP-CD123-13RBlS-(vL-vH)- Mvc-BBz-TZA-PAC CD8SP-CD123-hu3E3-(VL-VH)- MV’c-BBZ-TZA-PAC CDSSP-CD l -(vL-v1-1)-1\/1yc- BBz-T2A-PAC CDSSP-CD123-13RBZ-(vL-VH)- MVc-BBz-TZA-PAC CD123-l176-(vL-\»‘H)- MVC-BBz-TZA-PAC CD123 _-3292 CDSSP-Ritx2-CD123-8Bl l-(vL- ——___ CD123 -, 1 CDlZ3-2B8-(vL-vH)-Myc- CD 123 - , ( , CDSSP-CDlZ3-9D7-(vL-vH)—Mvc-.

CD123 - - - CDSSP-CD123-3B lO-(vL-vH)- CD138 041715- - - CDSSP-CD 1 38-(vL-vH)-Myc-BB2- CD 17% - - CD8SP-CD179b-(\-’L-\=’H)-Myc- CD276 - . . . CD8SP-CD276- 1 7-(vL-vI-I)-Myc- CD324 052516- - - CD85P-CD324 lO(vL-vH)-Myc- CD324 052516- CDSSP-CD324-hSClO-l7-(vL- 3100 A07 * ’ vH)-Myc-BBz-T2A-PAC CDH6 - - CDSSP-CDH6-NOV7I0-(vL-vH)- CDH6 CDSSP-CDH6-NOV712-(vL-vH)- 1 7 3.023 Mvc-BBz-TZA-PAC CDH17 CstP-CDHl7-PTA001A4-(VL- 1558‘ ' 33023 -Mvc-BBz-T2A-PAC CDH19 - -( , CDSSP-CDH19-l6A4-(vL-vH)- EGFR - - CDSSP-Cetuximab-(vL-vH)-Myc- CLECSA - CD8SP-CLECSA-8H8F5-(vL-VH)- CLEC5A - CDSSP-CLECSA-3ElZAZ-(VL- 1562 3107’ G04 vH)—M\jc-BBz-T2A-PAC GR/LHR (Gonadotropin 1 56.3 mg SP-CGHb-Gly-Ser-Linker-CGHa- Receptor) ‘ ‘ ‘ Myc-BBz-TZA-PAC CLLl 1 10215- . CLLl-M26-(vL-vH)-Myc- CLLl . - - CDSSP-CLLl-M32-(vL-vH)-Myc- CLLl - 1 CDxSP-CLL1-21C9-L2H3-(vL- CLLl . , CDSSP-CLLl-6E7L4Hlc-(vL-VH)- CLLl - , A CDSSP-CLLl-hulO75-vl-(vL-VH)- CLLl - . , CDSSP-CLLl-hu1075-\52-(\'L-VH)- CMVpp65/MHC class I CD8SP-CMVpp65-F5-(x-‘L-VH)- H70 3- H complex ' 3 ‘ Myc-BBz—TZA-PAC CSl (SLAMF7) __- CDSSP-CSl-huLuc63-(vL-vH)- ——__— CS c64-(vL-v1—I)- U03 Mvc-BBz-TZA-PAC —--CDSSP-CS l -Luc90-(vL-vH)-M_vc- BBz-TZA-PAC "-CD8SP-CS 1—PDL24 1-(VL-V’H)- z-TZA-PAC —--CDXSP-CS l -H1127A-(\«'L-VH)-Myc- BBz-TZA-PAC CD8SP-CS1Hu34C3-(vL-vl—1)- Mvc—BBz—T2A—PAC CD8SP-CS 1-Hu3 1-D2-(vL-v1—1)- Mvc-BBz-T2A-PAC —--CDSSP-CS l -Luc34-(vL-vH)-Myc- BBz-TZA-PAC —--CDSSP-CS 1 (vL—vI-1)-Myc- BBz-T2A-PAC CD8SP-CSF2RA-Ab6-(vL-VH)- Mvc-BBz-TZA-PAC —--CDSSP-CSF2RA-Ab l —(VL-vH)-M\-’c-BBZ-TZA-PAC CXCR4 and CD123 CD8SP-CXCR4-l -vHH-G1y-Ser- 1582 3327 Linker-CD123-l-vHH-Myc-BBZ- T2A-PAC CXCR4 and CD123 CDSSP-CXCR4VHH-Gly-Scr— 1583 3328 Linker-CD123VHH-Myc-BBZ- T2A-PAC DLL3 (Delta Like 052516- CDSSP-DLL3-hSCl6(vL—vH)- l 584 3329 Ligand 3) D07 Myc-BBz-T2A-PAC DLL3 (Delta Like 052516- CD8SP-DLL3-hSC16(vL-VH)- .— 'JI GO 1JI Ligand 3) C07 Myc-BBz-T2A-PAC EBNA3c/MHC class I CDSSP-EBNA3c-3 l 5-(vL-v1—I)- ,— UI 0C0 w 9) b) .— complex Myc-BBz-T2A-PAC EBV-gp350 EBV-gp350—(v L-vH)-Myc- 1587 b) L») L») N BBz-TZA-PAC EGFR CD8SP-EGFR 1 Myc-BBz- 1588 3 3 L») U) T2A-PAC EGFR & CEA CD8SP-EGFR1-vH}-I-Gl_\'-Ser— 3334 Linker-CEA 1 -M_\-'c-BBz-T2A- EGFR & CEA CD8SP-EGFR33-VHH-Gly-Ser- Linke r—CEAS-\r'I-lI-l-Myc-BBz-T2APAC EGFRVIII 081115- CD8SP-EGFRVIII- 139-(VL-VH)- A02 Mvc-BBz-T2A-PAC EGFRV‘HI 081115- CDSSP-EGFRVIII-Z l 73-(vL-vI-1)- E01 MVc-BBz-T2A-PAC EpCaml 3338 CD8SP-Epcam l-MM l -(vL-VH)- ——__— 1305 & "94- 3339 CD8SP-Epcam l-D5K5-(vL-vH)— 1 12015- Myc-BBz-TZA-PAC —---BBZ-T2A-pAcFLT3 - - a, CD8SP-FLT3-NC7-(\-’L-\:H)-Myc- —---mmFITC 052316- .

, , CD8SP-FITC-(vL-vI-l)-Myc-BBz- —--3342FlTC - , CD8SP-FlTC-4M(vL-vH)- MVC-BBz-TZA-PAC —--33")FITC - . . CD8SP-FITC-EZ-HL-(vH-VL)- Mvc-BBz-rzA-mc Inﬂuenza A HA CD8SP-FLU-MEDI(vL-vH)- 1599- 3344 Mvc-BBz-TZA-PAC FRI (Folate Receptor 101215— CDSSP-FRl-huMovl9-(vL-vH)— 1600 334% alpha) A01 ' Myc-BBz-TZA-PAC FSHMFOWC - - Stlmulatmg Homlone. . CD8SP FSHb G] s L‘ k , y- cr- m or- 160] 3346 IVC-BBZ'TZA-PAC Receptor) ' GAD (Glummic Add CD8SP H8‘' Dccarbox} lase)/MHC A. "’ 'V , ( L H) M' yc- 1602 3.947 A-PAC class 1 complex _GDZ 100515 a GDZ-llu14(vL-\'H)- F01 Mvc-BBz-TzA-PAC GDZ 0525 l 6- , ., , CDSSP-GDZ-hll3F8-(VL-VH)-Myc- —---Myc—BBz—TzA—mcGD3 052316- - - CD8SP-GD3-KM-64 l H)- CD8SP-GFRAlpha4-P4(vL-vH)- Myc-BBz-TZA-PAC GFRa4 (GDNF Family CD8SP-GFRa4-P4-lO-(vL-vH)- 1 607 3352 Receptor Alpha 4) Myc-BBz-TZA-PAC GM 1 - . CDSSP-GM1-SBZ-(vL-vH)-M}-'c- GM 1 CD8SP-GM l -7E5-(vL-vH)-Myc- BBz-TZA-PAC GPRCSD (G'pmtem CD8SP GPRC'D ETl'O ' - -, 3- a-a-v-( L couplcd receptor famll}; 1610 3 3 3 3-- vH)-Myc-BBz-T2A-PAC C group 3 member D) GPRCSD CD8SP-GPRC5D-ET150-l8-(vL- 1611 3356" -M\,"C-BBz-T2A-PAC GPRCSD - CDSSP-GPRCSD-ETISO-l-(VL— GPRCSD CD8SP-GPRC5D-ET150(VL- 161"3 3'583' vH)-M\_!c-BBz-T2A-PAC gplOO/MHC class I CD8SP-gpl00-(vL-vH)-Myc-BB2- 1614 3359 complex T2A-PAC gplOO/MHC class I CDSSP-gp100-02D12-(vL-v-H)- 161;' 3560’ complex Myc-BBz-TZA-PAC GPC3 (Glypican 3) CD8SP-GPC3-4E5-(vL-vH)-M_vc- 1616 3361 BBz-TZA-PAC gpNMB (Glycoprotein CDSSP-gpNMB-l 15-(vL-vH)- 1617 3a 62’ Nmb) Myc—BBz—TZA—PAC GRP78 . CDSSP-GRP78-GC18-(vL-vH)- q , CDSSP-HerZ-SF7-vI-lI-I-Myc-BBZ- Her2 IgHSP-HerZ-Afﬁ-Myc-BBz-T2A- l 620 3365 HerZ CDSSP-Herz- l n-Myc-BBZ- Her2 ., IgHSP-HerZDarpin-M_\;'c-BBz- Hcr2 Hch-SF7-vHH-Gly-Scr- 1623 3368 Linker-Her2-47DS-VHH-Myc-BBZ- TZA-PAC —-HerZ CD85P-HerZ-Hu4D5-(VL-\-’H)- '624 3369 Mm—BBz—TzA—mc —--337°Her3 - CDXSP-Her3-17B05 SO-\-’HH-M_\_-'c- BBz—rzA—mc HerZ and Her3 CDSSP-Her3- l 7BOSSo-V-‘I-II‘I-Gly- 041316- 1627 33 72 Ser-Linker-Her2-2D3-VHH-Myc- BBz-TZA-PAC HlVl-gag/MHC classl CDSSP-HIVI-E5-(vL-vH)-Myc- 1628 337.’ complex BBz-TZA-PAC HlVlﬂIVCIOP HlVl-3BNCI l7-(vL-vH)- 1629 3574’ glycoprotein Myc-BBz-TZA-PAC HIVl-envelop CDSSP-HIVI-PGT(vL-VH)- 1630 337; glycoprotcin ' Myc-BBz-T2A-PAC nvelop CD8SP-HIVI-VR-C01—(vL—vH)— 1631 3376 glycoprotcin z-TZA-PAC HlVlJenve|0p CDSSP-HIVI-X5-(vL-vH)-Myc- 16523 3.77° glycoprotein BBz-T2A-PAC HLA-AZ .. CDSSP-HLA-A2-3PB2-(vL-\'H)- HMW-MAA 060116- HMW-MAA-thD-(VL- 16543 3‘79’ 302 vH)-l\/Ivc-BBz-T2A-PAC HPVl6-E7/MI-IC classI 1635 3380 CDSSP-HPVl68-(vL-vH)-Myc- complex ' BBz-TZA-PAC HPV l6-E7/MHC class I CD8SP-HPVl6(vL-vH)-l\/lyc- 1636 3381 complex BBz-TZA-PAC HTLVl-TAX/MHC CDSSP-HTLV-TAX-T3F2-(VL- 1627° 3282’ class I x vH)-Myc-BBz-T2A-PAC HTLVl-TAX/MHC HTLV-TAX-T3E3-(VL- 1618 338? class 1 complex ‘ ‘ vH)-Myc-BBz-T2A-PAC 1L1 lRa 052316- CD8SP-IL1lRa-8E2-Tle7-(vL- 16293 3384 002 vH)—Mvc-BBz-T2A-PAC lL6Ra - , IgHSP-IL6RvHH-Myc-BBZ- ILl3RaZ 052616- IL13RaZ-hu lO7-(vL-vH)- 1641 3286’ 303 z-TZA-PAC ILI3Ra2 , a ILI3Ra2-Hu108-(vL-VH)- KSHV—K8.l 042315- CD8SP-KSHV—4C3-(vL-\:'H)-Myc- 1 64., N01 ‘ ‘ms‘ BBz-TZA-PAC LAMPI (Lysosomal' CD8SP LAMP]- - uma - - h b1 2( L- -v assoclated membrane 1644,, 3389 vI-l)-M_vc-BBz-T2A-PAC protein 1) LAMPI (Lysosomal- - - - v - assoclatcd membrane. 1645- CDXSP LAMPI Mb4 3390q I ( L-v 3 , H) Mvc-BBz-TZA-PAC protem l) ' LewisY CD8SP-LewisY-huS l —VH)- LlCAM 052516- CD8SP-L1CAM3-HU3-(VL- 1647 3292’ Q07 vI—I)-l\/Ivc-BBz-T2A-PAC LI-IR SP-LI-lb-Gly-Scr-Linkcr-CGHa- l 648 3 ., 9.‘’ ° Mvc-BBz-TZA-PAC Lyml ( ,, ( CD8SP-Lym l-(V-‘L-VH)-l\/I)-'c-BBZ- LymZ _ - CD8SP-LymZ-(vL-vl-I)-l\«‘l}v'c-BBZ- CD79b - CDSSP-huMA79va8-(vL-VH)- MARTl/MHC class I - CD8SP-MART1-CAGlO-(vL-VH)- 1652 3397 complex Myc-BBz-TZA-PAC MARTl/MHC class I CD8SP-MARTI-CLAlZ-(vL-\'H)- 1653 3‘98 complex " ‘ ‘ 3' Myc-BBz-TZA-PAC Mesothelin - CDSSP-Mesothelin-m9l2-(vL-VH)- 1654 3399 P07 Mvc-BBZ-TZA-PAC cMet - - CDSSP-cMet- l 7 l-vI-lI-I-Myc-BBz- cMet and Her3 CD8SP-cMET- l 7 l-vHH-Gly-Ser- 041316- 1656 3401 Linker-Her3-21 F06-vI-Il-I-Myc- BBz-TZA-PAC MPL(Thrombopoietin 022415— 3402 CD8SP-MPL-l75-(vL-vH)-Myc- ———-BBz—TzA-PAC receptor) BBz-TZA-PAC MPL (Thrombopoietin -- CD8SP-MPL-16l-HL-(vH-vL)— receptor) " Myc-BBz-TZA-PAC MPL b0poietin 041316- CDSSP-Z-MPL-l 1 vH)-Myc- 1660 340i receptor) A03 ' BBz—TZA—PAC MPL(Thrombop0ietin - 1661 3406 CDSSP-MPL-l78-(vL-vH)-Myc- receptor) BBz-TZA-PAC MPLUhrombOpoietin - CDSSP-MPL-AB3l7-(vL-vH)- 1662 3407 receptor) Myc-BBz-TZA-PAC MPL(Thromb0poietin -- CD8SP-MPL-lZElO-(vL-vH)—1\/ch- receptor) A-PAC \APL (Thrombopoietin CD8SP-MPL-huVBZZBwS-(vL- 1664 3409 receptor) vH)-Myc-BBz-T2A-PAC Mucl/MHC class I 052316 CDXSP-Muc l-D6-M3B8-(vL-VH)- 166* 3410 complex A02 ' Myc-BBz-TZA-PAC Mucl/MHC classI - CD8SP-MUCl-D6-M3A1-(VL- 1666 34" complex vH)-l\/ch-BBz-T2A-PAC 111913- 1123?;11112rzsiy-M- NKGZD Ligand - NKGZD-(GGGGS- 1669 3414 GGGGD)-Mvc-BBz-T2A-PAC mm -W NY-ESO/MHC classl - CD8SP-NYESO-Tl-(vL-vI-I)-Myc- 1672 3417 x BBz-TZA-PAC NY-ESO/MI-IC classl -- CDSSP-NYESO-TZ-(vL-vH)-1Vlyc- complex BBz-TZA-PAC PDlligand(e.g.,PDLl) - CDSSP-PDl-ECD-Myc-BBz-T2A- 1674 3419 PSCA Prostate stem cell- CDSSP-PSCA-Ha14(vL-vH)— antigen) Myc-BBz-TZA-PAC PSCA ate stem cell CDSSP-PSCA-Ha14(vL-vH)— 1679 3424 antigen) Myc-BBz—TZA—PAC PRl/MHC class I 052516- PRl-(vL-vl—l)—l\/lyc-BBz- 1680 342i complex K07 ' T2A-PAC PSMA (Prostate c - CD8SP-PSMA(vL-vH)-Myc- 168] 3426 Membrane Antigen) N07 BBz-TZA-PAC PSMA (Prostate Speciﬁc 111815- 1682 3427 CDSSP-PSMA-JSF)l-(vL-vH)—Myc- Membrane Antigen) E06 BBz-TZA-PAC PTK7 (Ti-'rosinc-protcin 052516- CDXSP-PTK7-hSC6(vL-VH)- 168.,‘ ‘"28 kinase-like 7) F07 Myc-BBz-TZA-PAC PTK7 (Tyrosine-protein - CDSSP-PTK76-lO(vL-vH)-Myc- 1 684 2429 kinase-like 7) E07 ‘ BBz-TZA-PAC 082815- . 2 CD8SP-ROR1-4A5-(vL-vH)-M}-c- 082815- 2 CDSSP-RORl-4ClO-(vL-vH)-Myc- Mesothelin CD8SP-SD l -\-’HH-Gly-Ser-Linker- l 687 34., 23 SD2-V:I—IH-M\'c-BBz-T2A-PAC SLea a CDSSP-SLea-7E3-(vL-vI-I)-Myc- SLca a, CDSSP-SLea-SBl-(vL-vH)-M}-'c- SSEA4 (stage-Speciﬁc CD8SP-SSEA4-(vL-vH)-Myc-BBz- 1690 343; embryonic antigen 4) " TZA-PAC TCRB] (TCR beta I CDSSP-TCRBl-CPOl-E09-(vL- 1691 34"63 nt chain) vH)-Myc-BBz-T2A-PAC TCRBl (TCR beta 1 050516- CDXSP-TCRBI-Jovi l-(vL-VH)- 1692 3437 constant chain) A07 z-TZA-PAC TCRBZ (TCR beta 2 050516- CDSSP-TCRBZ-CPOl-DOS-(vL- 169°" 3438 constant chain) C07 vH)-Myc-BBz-T2A-PAC TCRB2 (TCR beta 2 050516- CD8SP-TCRB2-CPO l -E05-(VL- 1694 3439 constant chain) BOS vH)-Myc-BBz-T2A-PAC TCRgd (TCR CDSSP-TCRgd-GS(vL-vI-l)- 1695 3440 gamma/delta) Myc-BBz-TZA-PAC liTERT/MHC class I 0518l6- CD8SP-TERT-4A9-T540-(vL-VH)- 1696 3441 complex H02 z-TZA-PAC hTERT/MHC class I CD8SP-TERT-3G3-T865-(vL-VH)- 1697 3442 complex Myc-BBz-TZA-PAC Tissue Factor-l ,. CDSSP-TGFBRZ-Abl-(vL-vH)- 1698 3443 Mvc-BBz-TZA-PAC TGFBRZ CDSSP-TF l-98—(vL-vH)-lV1yc- 1699 3444, , BBz-TZA-PAC TIM l/HAVCR _ _ CstP-TIMl-HVCRl2-(VL- -Mvc-BBz-T2A-PAC TlMl/HAVCR CDSSP-TIMl-HVCRl-ARDS-(VL- 170' 3446.

-Mvc-BBz-T2A-PAC TnAg 0 2616- I702 CD8SP-TnAg-(vL-vH)-M_vc-BBZ- ——__TZA-PAC l CDSSP-TnMuc l -huSE5-Rl-IA8- 1703 3448 RKA(vL-vH)-Myc-BBz-T2A- MPL Th b ' t' receptor) TROP2 (Trophoblast CDSSP-TROPZ-ARA47-HV3KV3- 170% 3450 cell-surface antigen-2) ‘ " (vL-vH)-M_vc-BBz-T2A-PAC TROP2 (Trophoblast CD88P-TROP2-h7E6-SVG-(VL- 1706 3451 cell-surface antigen-2) vH)—M_vc-BBz-T2A-PAC TSHR tropin SP-TSHb-Gly-Ser-Linker-CGHa- 1707 3442 receptor) ' Myc-BBz-T2A-PAC TSHR (Th\rotropin CDSSP-TSHR-Kl(vL-vH)— 1708 4%"4‘3 receptor) Myc-BBz-TZA-PAC TSHR (Th3rotrop1n 052616-1709 CDSSP-TSHR-KBl-(vL-vl—I)-l\/lyc- receptor) E05 BBz-TZA-PAC TSHR tropin 1710 44%4" " CD8SP-TSHR-5C9-(vL-vH)-Myc- rcccptor) BBz-T2A-PAC TSLPR ("1311110 stromal 052516- CD8SP-TSLPR-(x-rL-VH)-Myc- 171 l 34i6 lymphopoietin receptor) L07 " BBz-TZA-PAC nase/MHC class I 052516- 1712 3457 CD8SP-Tyros-B2-(vL-vI-l)-l\/l_vccomplex D06 BBz-TZA-PAC Tyrosinase/MHC class I Tyros-MCl-(vL-\-=H)-Myc- 171"3 3458 complex BBz-T2A-PAC Tyrosinase/MHC class I 1714 3459 CD8SP-Tyros-TA2-(\-'L-VH)-Myccomplex ' BBz-T2A-PAC VEGFR3 - CD8SP-VEGFR3-Ab1-(vL-vH)- WTl/MHC class I 101415- CDSSP-WTl-Abl-(vL-vH)-Myc- 1716 3461 complcx P01 BBz-T2A-PAC WTl/MHC class I 052516- 1717 3462 CDSSP-WTI-Ab5-(vL-V'H)-M_\-'c- complex M07 BBz-T2A-PAC C class 1 052516- CDSSP-MYC3-WTl-Ab13-(vL- 1718 3463 complex R04 vH)—Myc-BBz-T2A-PAC WTl/MHC class 1 052616- CD8SP-MYC3—WTl-AblS—(vL- 1719 3464 complex 802 vI-l)-l\/ch-BBz-T2A-PAC CDH19 - CDSSP-CDHl9-4B lO-(vL-vl—l)- Folate or beta CDSSP-FRbeta-m923-(vL-VH)- LHR(L(uteinizing CD8SP-LHR—SB7-(vL—vI-l)-Myc- 1722 3467 hormone Receptor) BBz-TZA-PAC LHR (Luteinizing CDSSP-LHR-S F4(vL-vH)- 172,.3 3468 hormone Receptor) Myc-BBz—TZA—PAC —-- 4228244461w- —-- 422827442421M» —-"27CD23 CDSSP-CDZ3-p5E8-(V’L-VH)-M_\'C- BBz-rzA-PAc G)CC lyl cyclase CDSSP-GCC-SF9-(VL-VH)-Myc- G)CC (Guanxlvl cyclase CD8SP-GCC-Ab229-(vL-VH)— 1729 3474 C) Myc-BBz-TZA-PAC —---CDZOOR ., - CDXSP-CDZOOR-huDX l 82-(VL- -~6c-BBz-T2A-PAc Table 22: Exemplary CAR constructs targeting different antigens CLONE 11) SEQ ID SEQ ID CONSTRUCT NAME DNA PRT MPL 021715-207 1731 161- vL-VH -Mvc-CD282-T2A-PAC MPL 090814-C03 CD8SP-l6l- vL-vH -Mvc-CDSTM-BBz-T2A-EGFP MPL 031615-004 CD8SP-l75- vL-vH -Mvc-CD282-T2A-PAC MPL 031615-R04 1734 3479 CDSSP- 1 78-(vL-vH)—Mvc-CD28z-T2A—PAC MPL 031615-T04 3480 CD8SP-AB317- l -M\xc-CD28z-T2A-PAC MPL 031615-503 1736 CD8SP-12E10- vL-VH -M\'c-CD28z-T2A-PAC MPL 032415-M03 1737 CD8SP-huVBZZBw5- vL-VH CD28z-T2A-PAC MPL 03l9lS-U04 1738 hTPO-(l-l87 -M\'c-CD282-T2A-PAC MPL 031915-V03 1739 mTPO-(l-187)-Mvc-CD28z—T2A—PAC CD8SP-16l-(vL-vH)-BBz-P2A-IgHSP-IL6R vHH-Albs-vHH-Fla -T2A-PAC CD88P-l6 l-(vL-vH)-Myc-z-P2A-FKBP-Kl3-FLAG- CD8SP-l6l-(vL-vH)-Myc-z-P2A-FLAG-HTLV2- TAX-RS-TZA-PAC CD8SP-l6l-(vL-vH)-Myc-z-P2A-HTLV2-TAX-RS- TZA-PAC CD8SP-l6l—(vL—vH)-Myc-BBz-PZA-FKBP-Kl3-Flag- TZA-EGFP CDEESP-l61(vL-vH)-BBz-P2A-IgHSP-IL6-19A(vL--T2AEGFP CD8SP-161(\L-vH)--BBZP2A--[gHSP---IL6R-MS3(vL- - -T2A-EGFP CD8SP-l6l-(\=L-\IH)Myc-CDZSz-PZA-KB-Flag- T2A-EGFP l6l-(vL-vH)-BBz-P2A-IgI—ISP-Fx06-Flag- TZA-EGFP CLONE 11) SEQID SEQ ID CONSTRUCT NAME DNA PRT CDSSP-l6 l-(vL-vH)-Myc-BBZ-P2A-MC159-Flag- TZA-Pac CD8SP-FMC63(vL-\-'H)-Myc-z-P2A-MC159-Flag- TZA-PAC CDSSP-FMC63(vL-vl—I)-Myc-BBz-P2A-MC159-Flag— CDSSP-FMC63(vL-vI-I)-Myc-BBz-PZA-Flag-HTLVZ- TAX-RS-TZA-PAC CD8SP-FMC63(vL-vH)-Myc-BBz-PZA-cFLIP-pZZ- Fl -PAC CD8SP-FMC63(vL-vH)-M_\-'c-BBz—P2A-cFLIP-L- F1--T2A-PAC CDSSP-FNIC63(vL-vH)-Myc-z-PZA-FKBP-Kl 3- FLAG-TZA-EGFP CD8SP-FVIC63(vL-vH)-Myc-z-PZA-FKBPxZ-K13- FLAG-TZA-EGFP CDSSP-FMC63(vL-vH)-Myc-z-P2A-Myr-FKBPx2- K13-FLAG-T2A-EGFP CD8SP-FMC63(vL-vH)-Myc-BBz-P2A-FKBPx2- HTLVZ- ax-RS-TZA-EGFP 04 CDSSP-FVIC63-(vL-vH)-Myc-CD282-P2A-K l 3-Flag- TZA-PAC CD8SP-FMC63-(vL-vH)-Myc-CD28z-P2A-MC159- Fl -T2A-PAC CD8SP-FNIC63-(vL-vH)-Myc-CD282-P2A-K l 3-Flag- TZA-EGFP CD88P-FMC63(vL-vH)-BBz-P2A-[gHSP-IL6- l9A(vL-vH)-Fla -T2A-PAC CDXSP-FMC63(vL-vH)-BBz-PZA-lgHSP-IL6R- M83(vL-vH)-Fla -T2A-PAC CD88P—FMC63-(vL-vI—I)—BBz-P2A—IgHSP—Fx06—Flag- TZA-PAC CD8SP-FNIC63-(vL-vH)-M_\'c-z-P2A-HTLV2-TAX2- FLAG-TZA-Pac CDXSP-FMC63-(vL-vI—I)-Myc-z-P2A-HTLV2-TAX- RS-T2A-Pac CD8SP-FMC63-(vL-vH)-M_vc-z-P2A-FLAG-HTLV2- -TZA-Pac FMC63-(vL-vH)-MYC-z-P2A-HTLV2-TAX- TZA-Pac CD8SP-FVIC65- vL-vI—l -M\'c-CD28z-T2A-PAC CD19 112316-806 1770 FMC63(\--L-\-v-H)-Myc-z-P2A-Myr-FKBPx2- AG-T2A-PAC GRP78 031615-005 1771 GRP78-GD4- vL-vH -MYC-CD28z-T2A-Pac CD19 093015-010 1772 7 CDSSP-hCD19—Bul2-(vL-vI-l)-MYC-CD28z-T2A-Pac CDSSP-LVm 1-(vL—vH)—M\_’c-CD282-T2A-Pac CD8SP-L\-'n12(vL-VH)-M\g'c-CD28z-T2A-Pac 3 20 CD8SP-GRP78-GC 18- vL-vI—I -M\,'c-CD282-T2A-Pac CD8SP-FLT3—NC7-(vL-VH -M\’c-CD282-T2A-Pac CLONE 10 SEQID SEQ 10 CONSTRUCT NAME DNA PRT CDSSP-huMA79bv28-(vL-vH)-M\_rc-CD282—T2A-Pac CDSSP-Luc90-(vL-vI-l)-MYC-CD282-T2A-PAC SP-CD403Fl-(vHH)—MYC-BBZ-T2A-Pac KSHV- 031615-W05 CDSSP-4C3-(vL-vH)—M_vc-CD282—T2A—Pac [(8.1 3534 3540 070814-D06 CD8SP-FMC63- vL-vH -Mvc-BBz-T2A-EGFP --- CD8SP-MPL-VB22Bw4-(vL-vI-l)-Myc-CDZSz-TZA- 3536 42 PAC ----CD88P-4C3-(vL-vH)—Myc-BBz-T2A—EGFPK8. 1 3537 3543 CD19 111214-005 CD8SP-FMC63- vL-vH -Mvc-z-T2A-PAC CD19 012315-M03 CD8SP-FMC63(vL-VH)-Myc-BBz-PZA-FKBPXZ- 3539 4 FLAG-TAX2U2RS -T2A-eGFP Table 23. HLA-AZ restricted peptides used for ton of CAR m-—_— -'.l_—— EXAMPLES The following examples are not intended to limit the scope of the claims to the ion, but are rather intended to be exemplary of certain ments. Any variations in the exempliﬁed methods which occur to the skilled artisan are intended to fall within the scope of the present invention.

MPL (TPO Receptor) as targetfor CAR-T cell therapy Thrombopoietin and its cognate receptor, c-Mpl, are the primary molecular regulators of megakaryocytopoiesis(Zhan et al., Monoclonal antibodies in immunodiagnosis and therapy 32:149-61, 2013). Binding of TPO to cell surface c-Mpl activates a cascade of signaling events that result in the proliferation and differentiation of megakaryocytic progenitor cells and ultimately platelet production. TPO and c-Mpl also regulate hematopoietic stem cell self-renewal. Consistent with these biological functions of TP0 in the megakaryocytic lineage, c-Mpl expression is mostly restricted to mphoid poietic tissues. c-Mpl mRNA and cell surface protein have been identified in human CD34+ cells, megakaryocytes, platelets, y leukemia s of myeloid origin, and megakaryocytic and oid leukemia cell lines. c-Mpl mRNA has not been detected in most normal non-hematopoietic tissues or in lymphoid cell lines.

We ed the public gene expression databases for MPL expression. As shown in Fig. 2A-Fig. 2D, we conﬁrmed extremely restrictive expression of MPL in normal s, with only one organ showing low level expression. In contrast, MPL was significantly sed in most patient samples of Acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and chronic myeloid leukemia (CML). These results suggest that MPL is an excellent eutic target for patients with myeloid malignancies, such as AML, MDS and CML, using chimeric antigen receptor modified T cells.

Cell lines and cells The cell lines used in this invention and their growth media are shown in the Table 24. Cells were cultured at 37°C, in a 5% CO2 humidiﬁed incubator. The cell lines were obtained from ATCC, NIH AIDS reagent m or were available in our laboratory or obtained from other laboratories.

Table 24: Cell lines and growth media Cell lines Cell lines BC-l RPM], 20% FCS THP-l RPM], 10% FCS BC-3 RPM], 20% FCS U87MG DMEM, l0% FCS BCBL-l RPM], 20% FCS NCI-H540 DMEM, 10% FCS JSC-l RPM], 20% FCS LoVo DMEM, 10% FCS UMPEL-l RPM], 20% FCS SKOV-3 DMEM, 10% FCS MMlS RPM], 10% FCS NCI-Hl993 DMEM, 10% FCS U266 RPM], 10% FCS Kasumi-l RPM], 20% FCS L363 RPM], 10% FCS Jeko-l RPM], 20% FCS K562 RPM], 10% FCS PC-3 DMEM, 10% FCS BV173 RPM], 10% FCS HeLa DMEM, 10% FCS Nalm6 RPM], 10% FCS NCI-H2452 DMEM, 10% FCS HL60 RPM], 10% FCS LnCap DMEM, 10% FCS U937 RPM], 10% FCS SNUI_' RPM], 20% FCS RS:4ll RPM], 20% FCS OVCAR-3 DMEM, 10% FCS MV24ll RPM]. 10% FCS MEL-624 DMEM. 10% FCS RPM], 10% FCS LSl74-T DMEM, 10% FCS HEL-92.l.7 RPM], 10% FCS MEL-526 DMEM, 10% FCS Ea:U?o RPM], 10% FCS MDA-MB23] DMEM, 10% FCS Jurkat RPM], 10% FCS L1236 RPM], 20% FCS MM 1 DMEM. 10% FCS L428 RPM]. 20% FCS Daudi RPM], 10% FCS Molt-l 6 RPM], 20% FCS REC-l RPM], 10% FCS RPMl8402 RPM], 20% FCS U2932 RPM], 10% FCS KN5-5F2 RPM], 20% FCS H929 RPM], 10% FCS beta CCRF-CEM(ATCC) RPM],10%FCS Mercaptoethanol (BME) KMSZ8 RPM], 10% FCS MG-63 DMEM, 10% FCS EJM RPM], 10% FCS s-299 RPM], 20% FCS MRC-5 DMEM, 10% FCS MCF7 DMEM, 10% FCS CMK RPM], 20% FCS SUDHL-l RPM], 10% FCS TF-l RPM], 10% FCS + AA-Z RPM], 10%FCS GMCSF ML-2 RPM], 20% FCS HL2/3 DMEM, 10% FCS Cell lines Cell lines A20 RPMI. 10% FCS + BME TF228.1.16 DMEM, 10% FCS KMSZ88M RPM], 10% FCS Z.1.1: RPM], 10% FCS KG-l RPM], 20% FCS Sup-Tl RPM], 10% FCS CEM RPMI, 10% FCS HUT-78 RPM], 10% FCS U937 RPM], 10% FCS DMEM, 10% FCS LAMA5 RPM], 10% FCS DMS79 RPM], 10% FCS A549 DMEM, 10% FCS LAN-5 DMEM, 10% FCS HT29 DMEM, 10% FCS PEER] RPM], 10% FCS Molm-l3 RPM], 20% FCS SK-MEL-37 DMEM, 10% FCS A431 DMEM, l % FCS Jurkat-NFAT-GFP RPM], 10% FCS DMEM, l % FCS '11C DMEM, l % FCS ] Jurkat cell line (clone E6—l)engineered with a NFAT-dependent EGFP (or GFP) reporter gene was a gift from Dr. Arthur Weiss at University of California San Francisco and have been described to study CAR-signaling ((Wu, CY et al., Science 350:293- 302,2015). Jurkat cells were maintained in RPMI-1640 medium supplemented with 10% FBS, penicillin and streptomycin.

Generation oflentiviral vectors encoding chimeric antigen receptors against MPL The pLENTI-Blast vector was derived from pLenti6v5gw_lacz vector rogen; ThermoFisher iﬁc) by removal of the LacZ gene. pLenti-MPZ was a gift from Pantelis Tsoulfas (Addgene plasmid # 36097) and was used to generate pLenti-EF la or pLenti-EFla [SEQ ID NO:905] lentiviral vector by replacement of the CMV promoter with human EFla promoter using standard molecular y techniques. pLenti-EFla-DWPRE [SEQ ID NO:906] was d from the pLENTI-EFla vector by deletion of WPRE sequence. The psPAXZ vector was a gift from Didier Trono (Addgene plasmid # 12260). The pLP/VSVG envelope plasmid and 293FT cells were ed from Invitrogen (ThermoFisher Scientiﬁc). The retroviral transfer vector MSCVneo, MSCVhygro, and MSCVpac and the packaging vector pKAT were ed from Dr. Robert Illaria’s laboratory. thGTK Renilla Luciferase plasmid was from Promega.

Several monoclonal antibodies targeting human MPL have been described (U820120269814A1, US 6,342,220 B1, EP1616881A1 and WO 02568109). The nucleotide sequences of the 1.6.1 onal antibody described in U820120269814Al was used for the synthesis of its corresponding scFV fragment ting from 5’ to 3’ ends of a nucleotide sequences encoding a signal peptide d from human CD8 molecule [SEQ ID NO: 1-4], the vL nt, a er)x3 linker and the VB fragment. The sequence of the scFV fragment was codon optimized using GeneArtTM software (Thermo Fisher Scientific) and gene—fragments encoding the zed sequences synthesized by GeneArtTM or Integrated DNA Technologies (IDT). The gene fragment was used as templates in PCR reaction using custom primers containing NheI and MluI restriction enzyme sites and then cloned in a modified pLenti-EFla vector containing the hinge, transmembrane and lic domains of the CAR cassette by standard molecular biology techniques. The final CAR construct named CD8SP-l61-(vL-vH)-Myc-CD8TM-BBz—T2A-EGFP(O908 l4-CO3)[SEQ ID NO: 1732] ted of human CD8 signal peptide (hCD8SP), fused in frame to the scFv (vL-vH) fragment derived from the onal antibody 1.6.1 against human MPL [SEQ ID NO:730], a Myc epitope tag [SEQ ID NO:553], the hinge and transmembrane domain of human CD8 [SEQ ID ] the cytosolic domain of human 41BB (CD137) receptor [SEQ ID NO:845], the cytosolic domain of human CD32, [SEQ ID N01846], a T2A ribosomal skip sequence [SEQ ID N02832] and a cDNA encoding enhanced Green Fluorescent Protein (EGFP). The CAR construct CDSSP-MPL-l6l-(vL-vH)-Myc-BBz-T2A- PAC(021015-R07)[SEQ ID NO:1658] is nearly identical to the above except EGFP cDNA is replaced by cDNA encoding a variant of cin resistance gene (SEQ ID NO: 850). In the CAR construct CDSSP-l61-(vL-vH)-Myc-CD282-T2A-PAC(021715-Z07)[SEQ ID NO:1731], the hinge and transmembrane domain of human CD8 and the cytosolic domain of human 41BB (CD137) receptor are replaced by the human CD28-hinge, -transmembrane and -cytosolic domains (SEQ ID NO: 848). Essentially a similar approach was used to generate several additional CARS against multiple antigens in which the 161 scFv fragment was replaced by the scFv fragments of antibodies targeting different ns. The SEQ ID NOs of the nucleotide and protein sequences of the different scFv fragments and the name of their target antigens are provided in Table 11. Additionally, two constructs targeting MPL were made in which the scFv region was replaced by the extracellular domains of human and mouse thrombopoietin (TPO) (SEQ ID N05: 553 and 554). These constructs, hTPO-(l-187)- Myc-CD28z-T2A-PAC(031915-U04)[SEQ ID 8] and mTPO-( 1-187)-Myc-CD282- T2A-PAC(03I915-V03)[SEQ ID N021739], consisted of the extracellular receptor binding domains of human and mouse thrombopoietin (TPO) timed to the CD28-hinge, - transmembrane and olic domains and CD32 cytosolic domains. The SEQ ID NOS of the nucleotide and protein sequences of the different camelid VHH fragments, non- immunoglobulin binding scaffolds and ligands that can be used to te CAR and the name of their target antigens are provided in Tables 7, 8 and 10.

In the CAR construct CD8SP-l6l-(vL-VH)-Myc-CD28z-P2A-K13-Flag-T2A- EGFP(O22415-T02)[SEQ ID NO:1747], the cDNA encoding LIP from the Kaposi’s sarcoma associated herpesvirus containing a y-terminal Flag epitope tag is inserted downstream of the nucleotide sequence encoding the CD8SP-l6 l-(vL-vH)-Myc-CD282 CAR and is ted from it by nucleotide sequence encoding P2A cleavable . The CAR construct CDSSP-MPL(vL-vH)-Myc-BBz-P2A-Kl3-Flag-T2A-PAC[SEQ ID N021388] is similar in design to SEQ ID NO: 1747 except that it contains the CD8 hinge and transmembrane domains and the 41BB costimulatory . Essentially, a similar approach was used to clone other accessory modules, such as MC 159-VFLIP, cFLIP-L, HTLVl-Tax and I-ITLVZ-Tax, in the CAR-encoding lentiviral vectors. The construct CDSSP-MPL-lol- (vL-vH)-Myc-BBz-P2A—KI3-Flag-T2A-PAC[SEQ ID N021388] comprises a CAR containing a 41BB costimulatory domain (BB) and a CD32 activation domain (2). This construct also co-expresses a Flag-epitope tagged K13-vFLIP downstream of the CAR- encoding sequence and separated from it by a P2A ble . The construct PL-l6l- (vL-vH)-Myc-z-P2A-K13-Flag-T2A-PAC(l l 1516-N05)[SEQ ID N021118] is similar in design to SEQ ID NO: 1388 except that it lacks the 41BB costimulatory domain.

The SEQ ID NOS of the nucleotide and protein sequences of the different CAR constructs and the name of their target antigens are provided in Tables 19-22. All the CAR constructs were generated using standard molecular biology techniques and their sequences conﬁrmed using automated sequencing.

Lentivirus and retrovirus vectors Lentiviruses were generated by calcium phosphate based transfection in 293FT cells ially as described previously (Matta H et al, Cancer biology and therapy. 2(2):206-10. 2003). 293FT cells were grown in DMEM with 10% FCS 4 mM L-Glutamine, 0.1 mM MEM Non-Essential Amino Acids, and 1 mM MEM Sodium Pyruvate (hereby referred to as O). For generation of lentivirus, 293FT cells were plated in 10 ml of DMEM-lO medium without antibiotics in a 10 cm tissue culture plate so that they will be approximately 80 confluent on the day of transfection. The following day, the cells were ected by calcium phosphate transfection method using 10 pg of iral expression plasmid ng different genes, 7.5 pg of PSPAXZ plasmid and 2 pg of PLP/VSVG plasmid. Approximately 15-16 hours ransfection, 9 ml of media was removed and replaced with 5 ml of fresh media. Approximately, 48 hours post- transfection, 5 ml of supernatant was collected (first collection) and replaced with fresh 5 ml media.

Approximately 72 hrs post-transfection, all media was collected (second collection, usually around 6 ml). The collected supernatants were pooled and centrifuged at 1000 rpm for 1 minute to remove any cell debris and non-adherent cells. The cell-free supernatant was ed h 0.45 pm syringe filter. In some cases, the supernatant was further concentrated by ultra-centrifugation at 18500 rpm for 2 hours at 40C. The viral pellet was re- suspended in l/lO of the initial volume in XVIVO medium. The virus was either used fresh to infect the target cells or stored frozen in aliquots at -80°C.

Infection of T cells and PBMC Buffy coat cells were obtained from healthy de-identified adult donors from the Blood Bank at Children Hospital of Los Angeles and used to isolate peripheral blood mononuclear cells (PBMC) by Ficoll-Hypaque gradient centrifugation. PBMC were either used as such or used to isolate T cells using CD3 magnetic microbeads (Miltenyi Biotech) and following the manufacturer’s instructions. PBMC or ed T cells were re-suspended in XVIVO medium (Lonza) supplanted with 10 ng/ml CD3 antibody, lOng/ml CD28 antibody and 100 IU recombinant human-ILZ. Cells were cultured at 37°C, in a 5% C02 humidified incubator. Cells were activated in the above medium for 1 day prior to infection with lentiviral vectors. In general, primary cells (eg. T cells) were infected in the morning using nfection (1800 rpm for 90 minutes at 37°C with 300ul of concentrated virus that had been re—suspended in XVlVO medium in the presence of 8 ug/ml of Polybrene® (Sigma, g no. H9268). The media was changed in the evening and the infection was repeated for two more days for a total of 3 infections. After the 3rd infection, the cells were pelleted and resuspended in fresh XVIVO media containing l CD3 antibody, 10ng/ml CD28 antibody and 100 IU recombinant human-1L2 and supplemented with respective antibiotics (if indicated) and place in the cell culture flask for selection, unless indicated ise. Cells were cultured in the above medium for 10-15 days in case no drug selection was used and for -30 days in case drug-selection was used. In cases, where cells were infected with a lentivirus expressing EGFP, they were expanded without drug-selection or flow-sorted to enrich for EGFP-expressing cells. For infection of cancer cell lines, approximately 0 cells were ed with 2 ml of the un-concentrated viral supernatant in a total volume of 3 ml with ene® (Sigma, Catalog no. H9268). Then next morning, the cells were pelleted and resuspended in the media with respective antibiotics and place in the cell culture flask for selection.

Essentially a similar procedure as described above for lentivirus vector production was used for tion of iral vectors with the exception that 293FT cells were generally transfected in 10 cm tissue culture plates in 10 ml of DMEM-lO medium using 10 pg of retroviral construct, 4ug of pKAT and Zug of VSVG plasmid. The virus collection and infection of target cells was carried out essentially as described above for lentiviral vectors.

Antibodies and drugs Blinatumomab was ed from Amgen. Digitonin was purchased from Sigma (Cat. no D141) and a stock solution of 100mg/ml was made in DMSO. A diluted stock of 1 mg/ml was made in PBS. Final concentration of digitonin used for cell lysis was 30ug/ml unless indicated otherwise.

ELISA Human 1L2, IFNy, 11.6 and TNFa were measured in the cell culture supernatant of CAR-expressing Jurkat-NFAT-GFP effector cells or T cells that had been co- cultured with the c target cell lines for 24 to 96 hours using commercially available ELISA kits from R&D systems (Minneapolis, MN) and BD Biosciences and following the recommendations of the manufacturer.

FACS analysisfor detecting expression ofCAR Mouse Anti-Human c-Myc AFC-conjugated onal Antibody (Catalog # IC3696A) was from R&D Systems (Minneapolis, MN). Biotinylated protein L was sed from GeneScript taway, NJ), reconstituted in phosphate buffered saline (PBS) at 1 mg/ml and stored at 4°C. Streptavidin-APC (SAIOOS) was purchased from ThermoFisher Scientiﬁc.

For detection of CARS using Myc staining, 1 >< 10° cells were harvested and washed three times with 3 ml of ice-cold 1 X PBS containing 4% bovine serum albumin (BSA) wash buffer. After wash, cells were ended in 0.1 ml of the ice-cold wash buffer containing 10 ul of AFC-conjugated Myc antibody and incubated in dark for 1 hour followed by two washings with ice cold wash buffer.

For detection of CARS using Protein L staining, l X 106 cells were harvested and washed three times with 3 ml of ice-cold l X PBS containing 4% bovine serum n (BSA) wash buffer. After wash, cells were resuspended in 0.1 m1 of the ice-cold wash buffer containing 1 pg of protein L at 4°C for 1 hour. Cells were washed three times with ice-cold wash , and then incubated (in the dark) with 10p] of AFC-conjugated streptavidin in 0.1 ml of the wash buffer for 30 minutes followed by two washings with ice cold wash . FACS was done using FACSVerse analyzer from BD Biosciences.

Cell death assay ] To measure cell death, a novel assay based on ectopic cytosolic expression of Gluc, NLuc and other luciferases was utilized as described in US provisional patent application 62/396,650 "A Non-Radioactive Cytotoxicity Assay". The method involves expression of a reporter in a target cells in a manner so that it is entially retained within the healthy cells but is either released from dead and dying cells or whose activity can be preferentially measured in dead and dying cells. The preferred reporter for this assay are l) non-secreted forms of luciferases from the copepods, such as Gaussia princeps, Pleuromamma nalis, Metridia a, Metridia curticauda, Metridia asymmetrica, Metridia okhotensis, Metridia longa, Lucicutia ovaliformis, Heterorhabdus tanneri, and Pleuromamma scutullata 2) engineered luciferase reporters from deep sea shrimp, such as NanoLuc. The sequence of several such exemplary reporter vectors is provided in SEQ ID NO: 912 to SEQ ID NO: 918. The above vectors were used to generate retrovirus and lentiviruses which in turn were used to generate polyclonal population of several target cell lines stably expressing GLuc, NLuc, or TurboLuc following selection with appropriate antibiotics. Unless indicated otherwise, the target cells stably expressing the different luciferases were plated in triplicate in a 384 well plate in the media used for g the target cells. Target cells which grow in suspension were generally plated at a concentration of 2-3 x 104 per well, while target cells which grow as adherent monolayers were plated at a concentration of 1-2 x 104 per well. Unless indicated otherwise, the target cells were cocultured with the genetically modiﬁed T cells (i.e. those sing CAR) at an Effector: Target (EzT) ratio varying from 1: 1 to 10:1 for 4 hours to 96 hours. In the case target cells grow as adherent cells (e.g., HeLa cells), they were allowed to attach to the bottom of the wells overnight before the T cells were added. T cells mediated induction of lysis of target cells was assayed by increase of luciferase activity as measured by BioTek synergy plate reader by ly ing 0.5 x CTZ assay buffer ning native coeloentrazine (Nanaolight) as described below.

CTZ assay A IOOX stock solution of native coelenterazine (CTZ; Nanolight, cat # 303) was made by dissolving 1mg of lyophilized CTZ powder in 1.1 ml of 100% Methanol supplemented with 30pl of 6N HCl to avoid oxidation of CTZ with time. To make CTZ assay buffer, the IOOX stock solution of CTZ was d to 0.5X concentration in PBS.

Unless indicated ise, a total volume of 15p] of the CTZ assay buffer (as prepared above) was added to each well of a 384-well white plate (Greiner, 384 well white plate cat # 781075) containing cells expressing the cretory form of the luciferase in approximately 50-60u1 volume of medium and plates were read for luminescence in endpoint mode using BioTek synergyl—l4 plate reader. For 96 well plates, cells were plated in 200p] of media and approximately 50pl of 0.5X CTZ assay buffer was added. Unless indicated otherwise, the 0.5x CTZ assay buffer was used for ng the ty of GLuc, TurboLucl6, and MLuc7.

The CTZ assay buffer (diluted to O. 125x concentration) was also used for measurement of NLuc activity in some experiments (see below). In general, unless indicated otherwise, the volume of 0.5X CTZ assay buffer added was approximately l/4th of the volume of the liquid in the well containing the cells, although the assay also worked when the 0.5X CTZ assay was added to the media containing the cells in 1:1 volume. Gluc activity in wells containing media alone (Med) and in wells in which target cells were incubated with T cells that were not ed with any SIR construct (T-UI) were used as controls, where indicated.

NLuc assay: Nano-Glo® Luciferase Assay System (Promega) was used for ement of NLuc activity by following the manufacturer’s ctions. , Nano-Glo Reagent was prepared by adding 511] of Nano-Glo substrate in lml of Nano-Glo assay buffer (Promega).

Unless indicated otherwise, lSul of Nano-Glo Reagent prepared above was manually added directly to each well of a 384-well white plate containing cells in 60p] of media and plates.

For 96 well plates, cells were plated in ZOOul of media and approximately SOul of assay buffer was added.

Plates were read for luminescence in endpoint mode using BioTek synerng4 plate reader without prior cell lysis. In some experiments, NLuc activity was measured using CTZ assay buffer but here the buffer was diluted to ﬁnal concentration of 0.125X. When CTZ assay buffer was used for measurement of NLuc activity, a total volume of approximately 15p] (unless indicated otherwise) of the 0.125X CTZ assay buffer was added by injector to each well of a 384-well white plate (Greiner, 384 well white plate cat # 781075) containing cells in approximately 50-60ul volume of medium and plates were read for luminescence in endpoint mode using BioTek synerng4 plate . For 96 well plates, cells were generally plated in 200 pl of media and approximately 50p] of 0.125X CTZ assay buffer was added.

Assay to detect the expression qfantigens on target cells Both CD19 and MPL (also known as Thrombopoietin receptor or TPO-R) are sed on hematopoietic cells but show differential expression in cells of different lineages. FMC63 is a well terized mouse monoclonal antibody that speciﬁcally recognizes human CD19. Similarly, 161 (also designated as 1.6.1) is a monoclonal antibody that recognizes human MPL and is described in US. patent application US 2012/0269814 A1. We generated a FMC63 single chain Fv (scFv) nt based on the known sequence of FMC63 vL and vH fragments. The cDNA encoding FMC63 scFv nt consisted from 5’ to 3’ ends a nucleotide sequences encoding a signal peptide derived from human CD8 molecule, FMC63 vL fragment, a (Gly4Ser)x3 linker and VH nt. The cDNA encoding the FMC63 scFv fragment was then fused me at its 3’ end to cDNA encoding AcVS-tagged NLuc through a Gly-Gly-Ser-Gly linker to generate FMC63-GGSG-NLuc. The resulting fragment was then cloned down-stream of the human EFla promoter into the lentiviral vector pLenti-EF 1a (SEQ ID NO: 905) to make the construct Plenti-EF 1a- FMC63(vL-vH)—GGSG-NLuc-AcV5-UO9. The DNA and PRT sequences of the insert fragment are provided in SEQ ID NO: 923 and SEQ ID NO: 2668, respectively. A uct encoding l6l-GGSG—NLuc was similarly generated using the vL and vH fragment of 1.6.1 monoclonal antibody t MPL. The DNA and PRT sequences of the insert fragment are provided in SEQ ID NO: 924 and SEQ ID NO: 2669, respectively. The pLenti-EFla- vL-vH)-GGSG-NLuc-ACV5 and pLenti-EF1a-161(vL-vH)-GGSG-NLuc-AcV5 plasmids were transfected into 293FT cells by calcium ate co-precipitation method.

Approximately 20h post-transfection, the cell e media was replaced with XVIVO medium. The conditioned media containing the secreted FMC63(vL-vH)-GGSG-NLuc-ACV5 (also referred to as FMC63-GGSG-NLuc-AcV5) and l6l(vL-vH)-GGSG-NLuc-ACV5 (also ed to as l6l-GGSG-NLuc-ACV5) proteins was collected 48-72h later.

The supernatant containing FMC63-GGSG-NLuc-AcV5 and lél-GGSG- NLuc-AcVS proteins were used to detect the expression of CD19 and MPL on the surface of Jurkat, K562, RAJI, RSl 1 (RS411) and HEL.92.1.7 (HEL) cells that had been ered to express a c-MPL cDNA by transducing these cells with a lentiviral vector expressing human c-MPL cDNA or an empty vector. The cells also expressed a humanized Gluc cDNA lacking its signal peptide. The vector- and MPL-expressing Jurkat-Gluc, luc, HEL.92.1.7-Gluc, RAJI-Gluc and RS411-Gluc cells were incubated with the FMC63-GGSG- NLuc-AcVS and 161-GGSG-NLuc-AcV5 supernatants at 4°C for 1h followed by extensive washings with cold PBS supplemented with 0.1% BSA. The cells were pended in cold PBS and 30ul of cell suspension was plated per well in a ﬂat-bottom 384 well plate (Greiner, 384 well white plate cat. # 781075) in triplicate. NLuc assay buffer containing native coelenterazine (CTZ) as NLuc ate (30ul/well of native coelenterazine diluted in PBS) was added to each well by an automatic dispenser in a well mode using a BioTek synergy H4 plate reader and light emission as a measure of NLuc activity was measured. Fig. 3A shows that strong binding with lél-GGSG-NLuc-ACVS was observed on HEL.92.1.7-G1uc-vector cells suggesting significant expression of MPL endogenously. Ectopic expression of MPL in NU-U- .1.7-Gluc-MPL cells led to a modest se in l6l-GGSG-NLuc-AcV5 binding. In contrast, very weak g with lél-GGSG-NLuc-ACVS was ed on vector-expressing Jurkat, RAJI and RS411 cells and was only modestly increased upon ectopic expression of MPL. Finally, weak but stronger binding of 161-GGSG-NLuc-AcV5 was observed on K562- vector cells, and was significantly increased on K562-MPL cells. In contrast tol61-GGSG- NLuc-AcVS, the FMC63-GGSG-NLuc-ACV5 supernatant showed strongest binding on vector- and MPL-expressing RAJI cells, ly strong binding on RS411 cells and very weak to negligible binding on the other cells.

Assay to detect the expression of chimeric antigen receptors targeting CD19 and MPL (Thrombopoietin receptor) ] A frequent problem in the field of chimeric antigen receptors is lack of a sensitive and speciﬁc assay that can detect cells that s chimeric antigen receptors. To detect the sion of CAR targeting CD19 and MPL, we fused the extracellular domains (ECD) of human CD19 and human MPL, including their signal es, in frame with nucleotide ce encoding a Gly-Gly-Ser—Gly linker, NLuc (without a secretory signal) and an AcVS epitope tag. In the case of CD19 construct, a FLAG tag was inserted between the signal peptide and the beginning of the extracellular domain. The whole cassette was cloned downstream of the human EFla promoter into the lentiviral vector pLenti-EF] to make constructs pLenti—EFl—FLAG-CDI9-ECD-GGSG-NLuc-AcV5 and pLenti-EFl-MPL- ECD-GGSG-NLuc-ACVS, respectively. The nucleic acid sequences of the insert fragments are provided in SEQ ID NO: 926 and SEQ ID NO: 925, respectively. The n sequences of the insert fragments are provided in SEQ ID NO: 2671 and SEQ ID NO: 2670, respectively. The constructs were ected into 293FT cells by calcium phosphate co- precipitation method. Approximately 20 h post-transfection, the cell culture media was replaced with fresh medium. The conditioned media containing the secreted FLAG-CD19- ECD-GGSG-NLuc-AcVS (also referred to as CD19-GGSG-NLuc-ACV5) and MPL-ECD- GGSG-NLuc-ACVS (also referred to as SG-NLuc-ACVS) proteins was collected 48- 72 h later. 293FT-cells were transiently transfected (in a 24-well plate, 500p] volume) with lentiviral constructs expressing chimeric antigen receptors CD88P-FMC63-(vL-VH)- Myc-BBz-TZA-PAC(1l2014-A13)[SEQ ID NO:1467], CD8SP-MPL-l61-(vL-vH)-Myc- BBz-T2A-PAC(021015-RO7)[SEQ ID NO:1658] or CD8SP-l61-(vL-vH)-Myc-CD28z-T2APAC (021715-ZO7)[SEQ ID NO:1731] using calcium phosphate co-transfection method or left untransfected. Next day morning, approximately 18 hours post-transfection, cells were collected by pipetting up and down in 1.5 ml tubes. The tubes were spun down at 1500 RPM for 5 minutes. Then the cells were washed once with wash buffer (1% FBS in PBS), followed by tion with lOOpl of indicated secretory forms of GGS NLuc supernatants. The cells were incubated at 4°C for 1 hour.

] After the incubation, cells were washed 5 times with wash buffer (1 ml each wash). y the pellet was resuspended in 200p] wash buffer. ended cells were placed in a 384 well plate in triplicate (25pl each). Luciferase activity was measured using a BioTek synergy H4 plate reader after addition of NLuc assay buffer (Promega) containing native coelenterazine (25pl each well) directly to each well (one at a time).

As shown in the Fig. 4A, 293FT cells expressing the CAR CD88P-FMC63- )—Myc-BBz-T2A-PAC(112014-A13)[SEQ ID NO:1467] that targets CD19 demonstrated strong binding to FLAG-CD19-ECD-GGSG-NLuc-AcV5[SEQ ID NO: 926] as measured by NLuc assay while very little binding was seen on uninfected T cells (U1) or those expressing the control CAR CD8SP-MPL(vL-vH)-Myc-BBz-T2A-PAC(02l015- EQ ID NO:1658]. Similarly, 293FT cells expressing the CAR CD8SP(vL-vH)- Myc-CD282-T2A-PAC(021715-ZO7)[SEQ ID NO:1731] showed strong binding with MPL- ECD-GGSG-NLuc-AcV5[SEQ ID NO: 925] supernatant as compared to un-transfected 293FT cells or those transfected with the CAR CD8SP-FMC63-(vL-vH)-Myc-BBz-T2A- PAC(l 12014-A13)[SEQ ID NO:1467] (Fig. 4B).

Assay to detect the expression of chimeric antigen receptors ing MPL (Thrombopoietin or) on 293FT cells that had been transfected with the different CAR constructs The construct hTPO-(l-l87)-Myc-CD28z-T2A-PAC(031915-UO4)[SEQ ID NO: 1738], mTPO-(l-l87)-Myc-CD282-T2A-PAC(031915-V03)[SEQ ID NO:1739], CD8SP-MPL-l6l-(vL-vH)-Myc-BBz-T2A-PAC(021015-R07)[SEQ ID NO:1658] and a control CAR construct containing a scFv derived from an irrelevant antibody were transiently transfected into 293FT cells and incubated with MPL-ECD-GGSG-NLuc-ACVS[SEQ ID NO: 925] supernatant essentially as described in the preceding example. Fig. 5 shows modest binding of MPL-ECD-GGSG-NLuc-ACVS to 293FT cells transfected with hTPO-(I-I87)- Myc-CD28z-T2A-PAC(O31915-U04)[SEQ ID NO:1738] and mTPO-(l-l87)—Myc-CD282- T2A-PAC(031915-V03)[SEQ ID NO:1739] constructs and strong binding to 293FT cells transfected with CD8SP-MPL-l6l-(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.

MPL-CARs are expressed on the surface of T cells Purified T cells were infected with lentiviruses encoding the different Myc- tagged CAR constructs targeting MPL. The constructs also co-expressed EGFP. The expression of CAR on T cells was examined by EGFP ﬂuorescence and immunofluorescence staining with an APC-conjugated Myc-antibody followed by FACS is on a BD verse ﬂow cytometer.

Fig. 6 shows expression of MPL CARS on the surface of T cells as determined by increased EGFP ﬂuorescence and sed staining with APC-Myc as compared to the uninfected T cells (UI).

T cells expressing MPL CARs are e ofbinding MPL-GGSG-NLucfusion protein T cells expressing the ent MPL CAR constructs were incubated with WL-GGSG-NLuc-ACVS and CD19-GGSG-NLuc-AcV5 atants and after extensive washes assayed for NLuc activity essentially as described in the ing example. Figure 7 shows strong binding of T cells sing CD8SP-MPL(vL-vH)-Myc-BBz-T2A- PAC(021015-R07)[SEQ ID NO: 1658], CD8SP- 1 75-(VL-VH)-Myc-CD282-T2A- PAC(03 1615-Q04)[SEQ ID NO: 1733], CD8SP-MPL-VB22Bw4-(vL-vH)-Myc-CD282-T2A- PAC(O31615-BO6)[SEQ ID NO:3536] CARS and modest binding of T cells expressing 161-(vL-vH)-Myc-CD282-T2A-PAC(O21715-ZO7)[SEQ ID NO:1731], CD8SP- AB317-(vL-vH)-Myc-CDZ82-T2A-PAC(O31615-TO4)[SEQ ID NO:1735] and CD8SP- (vL-vH)-Myc-CD282-T2A-PAC(031615-S03)[SEQ ID NO:1736] to MPL-GGSG- NLuc AcVS supernatant, while no icant binding was observed on uninfected T cells or those expressing the control CAR CD8SP—4C3-(vL-vH)-Myc-CDZSz-TZA-Pac(031615- EQ ID NO:3534]. Similarly, no specific binding was observed on any MPL CAR-T cells with CDl9-GGSG-NLuc-AcV5 supernatant, thereby demonstrating the speciﬁcity of the assay.

MPL CARS te T cell signaling in Jurkat cells upon antigen ation.

Jurkat cells stably expressing an NFAT-EGFP reporter construct were stably transduced with lentiviral vectors expressing the different CAR constructs targeting MPL followed by selection with puromycin. Cells were subsequently co-cultured with MPL- expressing HEL.92.1.7 (HEL) cells. Co—culturing of MPL-CAR expressing Jurkat-NFAT- EGFP cells with HEL cells ed in increase in EGFP expression as determined by FACS is, which was evident as early as 4 h after co-culture. The increase in EGFP expression was strongest in Jurkat-NFAT-EGFP cells expressing the MPL-l6l-(vL-vH)—My3- BBz-TZA-PAC(021015-R07)[SEQ ID NO:1658], CD8SP(vL-vH)-Myc-CD282-T2A- PAC(021715-ZO7)[SEQ ID NO:1731] and CDSSP(vL-vH)-Myc-CD28z-T2A- PAC(031615-QO4)[SEQ ID NO:1733] constructs. In an independent experiment, signiﬁcant EGFP induction was also observed upon culturing of Jurkat—NFAT-EGFP cells expressing the Z-MPL-ll1-(vL-vH)-Myc—BBz-T2A-PAC(041316—A03)[SEQ ID NO: 1660] CAR construct with HEL cells. The experiment was ed by co-culturing the Jurkat- NFAT-EGFP cells with either RAJI cells, which have weak MPL expression, or with RAJI- MPL cells that were engineered to ectopically express human MPL receptor. Jurkat-NFAT- EGFP cells expressing CAR directed against MPL showed more increase in EGFP expression when incubated with PL cells as compared with RAJI-vector cells. The increase in EGFP expression was again strongest in Jurkat-NFAT-EGFP cells expressing the CD8SP- MPL(vL-vH)-Myc-BBz-T2A-PAC(021015-R07)[SEQ ID NO:1658], CD8SP(vL- vH)-Myc-CD282-T2A-PAC(021715-ZO7)[SEQ ID NO:1731] and CD88P(vL-vH)- Myc-CD28z-T2A-PAC(031615-QO4)[SEQ ID NO: 1733] constructs.

Table 25: EGFP induction in Jurkat-NFAT-eGFP cells sing different CARS GFP ion (%) J" 'kat'NFAT'EGFP TARGET CELL LINE RAJI RAJINONE \ector Mp1 =__I--_C-D-SSPMPL-161(V'-L-\H)Mvc-BBz-T2A- _--- GFP induction (%) J"’kdt'NFAT'EGFP‘ TARGET CELL LINE -PAcwzwls-Rwsrsomama] ---_ CDSSP-lél-(\-'L-\'H)-Myc-CD281—T2A-PAC(021715- 207) SE ID NO:173l CDSSP-l75-(vL-VH)-Myc-CD281-T2A-PAC(O31615- 6 33 o 4) SEO ID No:1733 CDSSP-l78-(vL-VH)-Myc-CD282—T2A-PAC(031615- R04 SEO ID NO:1734 ‘ CDSSP-lZEl0-(vL-vH)-Myc-CD28z-T2A- PAC(031615-SO3)[SEQ ID 6] ' CDSSP-AB3 l7-(\'L-vH)-Myc-CD282-T2A- PAC(03 l6lS-TO4) SEQ ID NO: 1735 ---_ T cells expressing lilPL CAR induce cytotoxicity in MPL-expressing target leukemia cells Human peripheral blood T cells isolated using CD3 magnetic beads were infected with lentiviruses expressing the CAR construct CDSSP-l6]-(vL-vH)-Myc-CD8TM- BBz-T2A-EGFP(O90814-C03)[SEQ ID 2] targeting MPL or left uninfected (U1). The indicated number of HEL cells stably expressing GLuc were cocultured with T cells expressing the CDSSP-l6l-(vL-vH)-Myc-CD8TM-BBz-T2A-EGFP(O908l4-CO3)[SEQ ID N021732] in a 384 well plate at an Effector: Target (EzT) ratio of 1:1 for 4 hours. CAR-T cells mediated induction of lysis of target cells was assayed by increase of GLuc activity as measured by BioTek synergy plate reader by directly injecting 0.5X CTZ assay buffer containing native ntrazine (Nanaolight). Fig. 8 shows increase in GLuc activity following ture with T cells expressing MPL-specific 161-(vL-vH)-Myc- CD8TM-BBz—T2A-EGFP(O90814-C03)[SEQ ID N021732] CAR as compared to the uninfected T cells indicating lysis of target cells by the different MPL-CAR-T cells.

PBMC expressing MPL CAR induce lysis of Target Leukemia cells sing lilPL The blood samples to isolate Peripheral blood mononuclear cells ) were obtained from healthy de-identiﬁed adult donors. PBMC were isolated from buffy coats by Ficoll-Hypaque nt centrifugation and re-suspended in XVIVO medium (Lonza) supplanted with 10 ng/ml soluble anti-CD3, 10 ng/ml e anti-CD28 and 100 IU recombinant 1L2. PBMCs were engineered to express CDSSP-MPL-l6l-(vL-vH)- Myc-BBz-TZA-PAC(021015-RO7)[SEQ ID N021658]CAR targeting MPL and CD8SP- FMC63-(vL-vH)-Myc-BBz-T2A-PAC(112014-Al3)[SEQ ID NO:1467] targeting CD19 by infection with the respective lentiviral vectors followed by selection with puromycin. The PBMC expressing the indicated CARS and uninfected PBMC were co-cultured with HEL- GLuc target cells for 4 h and lysis of target cells measured by the single-step homogenous GLuc assay by ing 0.5X CTZ assay buffer as described above. Fig. 9 shows increased killing of HEL-GLuc cells by CD8SP-MPL-l6l-(vL-vH)-Myc-BBz-T2A-PAC(021015- R07)[SEQ ID N021658] CAR-expressing PBMC as compared to the FMC63-(vL-vH)-Myc- BBz-T2A-PAC(112014-Al3)[SEQ ID NO:1467]CAR-expressing PBMC or uninfected PBMC.

PBMC expressing MPL CAR induce lysis of Target Leukemia cells expressing [MPL The blood samples to isolate eral blood mononuclear cells (PBMCs) were ed from healthy de-identified adult donors. PBMC were isolated from buffy coats by Ficoll-Hypaque nt centrifugation and re-suspended in XVIVO medium (Lonza) supplanted with 10 ng/ml soluble anti-CD3, 10 ng/ml soluble anti-CD28 and 100 IU inant human-1L2. PBMCS were engineered to express CDSSP-l75-(vL-VH)-Myc- CD282-T2A-PAC(03l6lS-QO4)[SEQ ID NO: I 733]CAR targeting MPL by infection with the iral vector followed by selection with puromycin. The PBMCs expressing the indicated CAR and uninfected PBMCs were co-cultured with HEL-GLuc target cells for 4 h and lysis of target cells measured by the single-step homogenous GLuc assay by injecting 0.5X CTZ assay buffer as described above. Fig. 10 shows sed killing of HEL-GLuc cells by the CDSSP-l75-(vL—vH)-Myc-CD282-T2A-PAC(031615-QO4)[SEQ ID N021733] CAR expressing PBMC as compared to uninfected PBMC.

T cells expressing MPL CARS expressing CD28 costimulatmy domain induce cytotoxicity in MPL-expressing target leukemia cells Human peripheral blood T cells isolated using CD3 magnetic beads were infected with lentiviruses expressing the CAR constructs CDSSP-l61-(vL-vH)-Myc-CD28z- T2A-PAC(021715-ZO7)[SEQ ID NO:173 1], CD8SP(vL-vH)-Myc-CD28z-T2A- PAC(O31615-Q04)[SEQ ID N011733], CDSSP(vL-vH)-Myc-CD282-T2A- PAC(031615-R04)[SEQ ID NO:1734], and AB3l7-(vL-vH)-Myc-CD28z-T2A- PAC(O31615-T04)[SEQ ID 5] ing NH’L.. Cells were selected with puromycin and expanded. HEL cells stably expressing GLuc were cocultured with T cells expressing the CARS at an Effector: Target (EzT) ratio of 10:1 for 4 hours. CAR-T cells ed induction of lysis of target cells was assayed by increase of GLuc activity. Fig. 11 shows increase in GLuc activity, indicating lysis of target cells, following co-culture with T cells expressing MPL-speciﬁc CARS, which was stronger with the CDSSP-l6l-(VL-VH)-Myc-CD282-T2A- PAC(021715-Z07)[SEQ ID NO:1731], CD8SP- 1 75-(vL-vH)-Myc-CD282-T2A- PAC(031615-Q04)[SEQ ID NO:1733], and CD8SP-AB317-(vL-vH)-Myc-CD282-T2A- PAC(031615-TO4)[SEQ [D NO:1735] constructs and modest with the CD8SP-178—(vL-vH)— Myc-CDZSz-TZA-PAC(O31615-R04)[SEQ [D 4] construct as compared to the uninfected T cells.

T cells expressing MPL CARS expressing CD28 costimulatmy domain induce cytotoxicin in MPL-expressing target leukemia cells Human peripheral blood T cells ed using CD3 magnetic beads were infected with lentiviruses expressing the CAR constructs CDSSP-l61-(vL-vH)-Myc-CD282- C(0217IS-ZO7)[SEQ ID NO:173I], CDSSP-I75-(vL-vI-I)-Myc—CD28z-T2A- PAC(031615-Q04)[SEQ ID NO:1733 , CDSSP(vL-vH)-Myc-CD28z-T2A- 1615-R04)[SEQ ID NO:1734], CDSSP-AB3l7-(vL-vH)—Myc-CD282-T2A- PAC(031615-TO4)[SEQ ID NO:1735], CDSSP-IZElO-(vL-vH)-Myc-CD28z-T2A- PAC(031615-SO3)[SEQ ID NO:1736] and CD8SP-VB22Bw4-(vL-vH)-Myc-CD28z-T2A- PAC(031615-B06)[SEQ ID N023536] targeting MPL. Cells were selected with puromycin and expanded. Jurkat cells stably sing MPL and GLuc were cocultured with T cells expressing the CARS at an or: Target (E:T) ratio of 10:] for 4 hours. CAR-T cells mediated induction of lysis of target cells was assayed by increase of GLuc activity as measured by BioTek synergy plate reader by directly injecting 0.5 x CTZ assay buffer ning native coeloentrazine light). Fig. 12 shows se in GLuc activity, indicating lysis of target cells, following co-culture with T cells expressing MPL-speciﬁc CARs, which was stronger with the CDSSP-l6l-(vL-vH)-Myc-CD28z-T2A-PAC(02l715- ZO7)[SEQ ID NO:1731], CDSSP(vL-vH)-Myc-CD282—T2A-PAC(031615-Q04)[SEQ ID 3 , CDSSP-ABS17-(vL-vH)-Myc-CD28z-T2A-PAC(031615-TO4)[SEQ ID NO:1735], and CDSSP-l2E10-(vL-vH)-Myc—CD28z-T2A-PAC(O31615-SO3)[SEQ lD NO:1736] constructs and modest with the CD8SP(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 N023536] constructs as compared to the uninfected T cells.

NK92MI cells expressing MPL-speciﬁc I 61-CAR induce cytotoxicity in K562 and HEL. 92. I. 7 cells Natural Killer cell derived NK92MI (ATCC) cells were stably transduced with lentiviral vectors expressing the indicated CAR constructs ing CDSSP(vL-VH)- Myc-CD8TM-BBz-T2A-EGFP(O908l4-CO3)[SEQ ID NO:l732] and CD8SP-FMC63-(vL- vH)-Myc-BBz-T2A-EGFP(O708l4-DO6)[SEQ ID NO:3535]. Cells were subsequently co- cultured ght with HEL.92.1.7 cells that had been engineered to express GLuc. CAR- sing NK92MI cells-mediated induction of lysis of target cells was assayed by increase of GLuc activity as measured by directly injecting 0.5X CTZ assay buffer containing native coeloentrazine light). Fig. 13 shows increase in GLuc activity, indicating lysis of target cells, following co-culture with NK92MI cells expressing MPL-specific CDSSP (vL-vH)-Myc-CD8TM-BBz-T2A-EGFP(O908 l4-CO3)[SEQ ID NO: I 732]CAR.

NK92MI cells expressing MPL-speciﬁc CARs induce cytotoxicity in K562-MPL cells l Killer cell derived NK92MI (ATCC) cells were stably transduced with lentiviral vectors expressing the indicated CAR ucts targeting MPL followed by selection with puromycin. Cells were subsequently co-cultured overnight with K562 cells that had been engineered to express MPL and GLuc. The effector to target (EzT) ratio was 5:1.

MPL-CAR-expressing NK92MI cells-mediated induction of lysis of target cells was assayed by increase of GLuc activity as measured by directly injecting 0.5X CTZ assay buffer containing native coeloentrazine (Nanaolight). Fig. 14 shows se in GLuc activity, indicating lysis of target cells, following co-culture with NKQZMI cells expressing MPL- specific CARS. ne Production by T cells expressing ll/IPL-speciﬁc CAR ucts on exposure to target cells sing MPL Human peripheral blood T cells isolated using CD3 magnetic beads were infected with lentiviruses expressing the CDSSP-MPL-l61—(vL-vH)-Myc—BBz-T2A- PAC(021015-R07)[SEQ ID NO:l658] and CDSSP-lol-(vL-vH)—Myc—CD282-T2APAC (021715-ZO7)[SEQ ID NO: 173 1] CAR constructs targeting MPL. Cells were selected with puromycin and expanded. HEL cells were tured with T cells expressing the CARs for approximately 96 hours and secretion of TNFa in the atant measured by ELISA.

Fig. 15 shows increased secretion of TNFa upon co—culture of T cells expressing CAR targeting MPL with HEL cells as compared to uninfected T cells used as controls.

In vivo efficacy ofMPL CAR-expressing immune effector cells against leukemia The in vivo cy of MPL CAR-expressing NK92MI cells against HEL leukemia cell line model was tested in NOD/SCH)/7"' (NSG) mouse model. On day 0, the NSG mice (Jackson Lab) were sub-lethally irradiated at a dose of 275 cGy. 24 hours post irradiation (day 1), mice were injected with 1 x 106 HEL cells intravenously. On day 9 and day 19, the mice were treated with 2 million NK92MI cells expressing the indicated CAR constructs intravenously and followed for survival. Fig. 16 shows that the median survival of mice given CDSSP-l61-(vL-vH)—Myc-CD8TM-BBz-T2A-EGFP(O908l4-CO3)[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 CAR CD8SP-4C3-(vL-vH)-Myc-BBz—T2A-EGFP(100814-K06)[SEQ ID NO:3537]; 28 days; p=.04).

Chimeric Antigen Receptors ing CD19 with scFV nts derived from a humanized CD19 antibody The CD19 CARs in t al trials contain scFV fragments derived from murine monoclonal antibodies. The development of antibodies against the murine antibody fragments not only butes to lack of persistence of T cells expressing such CARS but could also lead to allergic reactions, including anaphylaxis. To overcome this limitation, a CD19 CARS was generated containing an scFv fragment (SEQ ID NO: 566) derived from a humanized CD19 monoclonal antibody huCDl9-Bu 12 (or Bu12) described in PCT/US2008/O80373. Another CAR was constructed containing an scFv (SEQ ID NO: 567) fragment derived from a murine dy described in US7112324 and was designated CD19MM.

The scFV fragments based on CD19-Bu12 and /ﬂ\/I were designed consisting from 5’ to 3’ ends of a nucleotide sequences encoding a signal e derived from human CD8 molecule, the vL fragment, a (Gly4Ser)x3 linker and the vI-l fragment. The sequence of the scFV fragments were codon-optimized using GeneArtTM software (Thermo Fisher Scientific) and gene-fragments encoding the zed ces synthesized using a commercial vendor. The gene nts were used as templates in PCR on using custom primers and then cloned in a modiﬁed pLENTI-EFIa vector containing the hinge, transmembrane and cytosolic domains of the CAR cassette by standard molecular biology techniques. The ﬁnal CAR construct CD8SPCD19MM-(vL-vH)-Myc-BBz-T2APAC (O62915-D03)[SEQ ID NO:1471] consisted of human CD8 signal peptide, fused in frame to the CD19MM scFv fragment (SEQ ID NO: 567), a Myc epitope tag, the hinge and transmembrane domain of human CD8 (SEQ ID NO: 842), the cytosolic domain of human 41BB (CD137) receptor (SEQ ID NO: 845), the cytosolic domain of human CD32 (SEQ ID NO: 846), a T2A ribosomal skip sequence (SEQ ID NO: 832) and a cDNA encoding a variant of cin ance gene (SEQ ID NO: 850). A CAR construct CD8SP- CD19Bu12-(vL-vH)-Myc-BBz-TZA-PAC(082815-PO8)[SEQ ID NO:1470] based on CD19Bu12 scFv fragment (SEQ ID NO: 566) was designed similarly. The CAR construct CD8SP-FMC63-(vL-vH)-Myc-BBz-T2A-PAC(112014-A13)[SEQ ID NO:1467] was designed based on the usly described CD19 CAR containing the scFV fragment (SEQ ID NO: 564) derived from mouse monoclonal antibody FMC63. Finally, the CAR construct CD8SP-KSHV-4C3-(vL-vH)-Myc-BBz-T2A-PAC(042315-N01)[SEQ 1D 3] contains an scFV nt (SEQ ID NO: 718) derived from an antibody against a KSHV encoded K8.1 protein and was used as a negative control. Lentiviruses encoding the above CARS were generated using 293FT and used to infect T cells as described previously for MPL CARs.

CD1 9-CARs are sed on the surface of T cells Purified T cells were infected with lentiviruses encoding the different Myc- tagged CAR constructs targeting MPL (CDSSP-FMC63—(vL-vH)-Myc-BBz-T2A- PAC(112014-A13)[SEQ ID NO:1467], CD8SP-CD19Bu12-(vL-VH)-Myc—BBz-T2A- PAC(O82815-P08)[SEQ ID NO:1470], CD8SPCD191v[M-(vL-vH)-Myc-BBz-T2A- PAC(O62915-D03)[SEQ ID NO:1471] and CD8SP-KSHV-4C3-(vL-vH)-Myc-BBz-T2A- PAC(042315-N01)[SEQ ID NO:1643]) and selected with puromycin. The sion of CAR on T cells (both puromycin selected and unselected) was examined by immunoﬂuorescence staining with an njugated Myc-antibody followed by FACS analysis as described usly. Fig. 17 shows 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). The expression of the different CAR constructs was further increased following puromycin selection.

Jurkat cells expressing CD19 CARS are capable of binding CD19-GGSG-NLuc fusion protein Jurkat-NFAT-Luc cells were stably transduced with the ent CD19 targeted CAR constructs and selected in cin. Cells were incubated with FLAG-CD19- ECD-GGSG-NLuc—AcVS [SEQ ID N022671] supernatant and after extensive washes assayed for NLuc activity essentially as described previously. Fig. 18 shows strong binding of Jurkats expressing CD8SP-CD19Bul2-(vL-vH)-Myc-BBz-T2A-PAC(082815-P08)[SEQ ID NO:1470], CD8SPCDl9MM-(vL-vH)-Myc-BBz-T2A-PAC(062915-D03)[SEQ ID NO:1471] and CD8SP-FMC63-(vL-vH)-Myc-BBz-T2A-PAC(l12014-A13)[SEQ ID NO:1467], and CD19Bu12(vL-vH)-Myc-BBz-PAC-PO8 CAR constructs to D19- ECD-GGSG—NLuc—AcVS [SEQ ID NO:2671] supernatant, while no signiﬁcant binding was observed on parental cells or those expressing CDSSP-KSHV—4C3-(vL-vH)-Myc-BBz-T2A- PAC(042315-N01)[SEQ ID NO:1643] control CAR.

T cells expressing CD19 CARs are capable ofbinding CD19-GGSG-NLucfusi0n protein Puriﬁed T cells were stably transduced with the different CD I eted CAR constructs and ed in puromycin. Cells were incubated with FLAG-CD19-ECD-GGSG- NLuc-AcVS [SEQ ID NO:2671] atant and after ive washes assayed for NLuc activity ially as described previously. Fig. 19 shows strong binding of T cells expressing CD8SP-CD19Bu12-(vL-vH)-Myc-BBz-TZA-PAC(082815-P08)[SEQ ID NO: 1470], CDSSP-Z-CD19MM-(vL-vH)-Myc-BBz-TZA-PAC(062915-D03)[SEQ ID NO:1471] and CD8SP-FMC63-(vL-vH)-Myc-BBz-T2A-PAC(112014-A13)[SEQ ID NO:1467], and CD19Bu12(vL-vH)-Myc-BBz-PAC-POS CAR constructs to FLAG-CD19- ECD-GGSG-NLuc-AcVS [SEQ ID N022671] supernatant, while no signiﬁcant binding was ed on uninfected T cells (UI) or those expressing CD8SP-KSHV-4C3-(vL-vH)-Myc- BBz-TZA-PAC(0423 lS-NOI)[SEQ ID NO:1643] control CAR.

T cells expressing CD19 CARS induce cytotoxicity in CD19-expressing RAJI lymphoma cells Human peripheral blood T cells ed using CD3 magnetic beads were ed with lentiviruses expressing the ted CAR constructs targeting CD19. Cells were left unselected or selected with puromycin and expanded. RAJI cells stably expressing GLuc were cocultured with T cells expressing the CARS at an or: Target (E:T) ratio of :1 for 96 hours. CAR-T cells mediated induction of lysis of target cells was assayed by se of GLuc activity. Fig. 20 shows increase in GLuc activity, indicating lysis of target cells, following ture with T cells expressing CD19-speciﬁc CARS as compared to uninfected T cells (T-Ul) or those expressing the control CAR 4C3. The increased cell death was seen in CD19 CAR-expressing T cells that had been selected with puromycin as well as those that had not been selected.

T cells expressing CD1 9 CARS induce cytotoxiciw in CDI9-expressing RAJI lymphoma cells Human peripheral blood T cells isolated using CD3 magnetic beads were infected with lentiviruses expressing the indicated CAR constructs targeting CD19. Cells were left unselected or selected with puromycin and expanded. RAJI cells stably expressing GLuc were cocultured with T cells expressing the CARS at the indicated Effector: Target (EzT) ratios for 4 hours. CAR-T cells mediated induction of lysis of target cells was assayed by increase of GLuc activity. Fig. 21 shows increase in GLuc activity, indicating lysis of target cells, following co-culture with T cells expressing pecific CARS as comparﬁd to uninfected T cells (T-UI) or those expressing the control 4C3 CAR. In contrast to the results in the preceding section, the increased cell death was seen primarily in CD19 CAR- expressing T cells that had been ed with puromycin. This could be attributable to the fact that cell death was measured after 96 h of co-culture in the preceding example while it was measured after only 4 h in the current example.

In vivo efﬁcacy MM-BBz and CI)!9Bu12-BBz CAR T cells Human eral blood T cells isolated using CD3 magnetic beads were infected with lentiviruses expressing the indicated CAR constructs targeting CD19 or the l CAR (4C3) and expanded in vitro with (with puro) or without (No puro) puromycin selection. NSG mice (Jackson Lab) were thally irradiated at a dose of 175 cGy. 24 hours post irradiation (day2), mice were injected with 2.5 x 104 RAJI cells via tail-vein. On day3, the mice (n =5 for each group) were treated with 1 million T cells that had been infected with the CD8SP-CDl9Bu12-(vL-vH)—Myc-BBz—T2A-PAC(082815-P08)[SEQ ID NO: 1470], CDSSPCD19MM-(vL-vH)-Myc-BBz-TZA-PAC(062915-D03)[SEQ ID 1] encoding lentiviruses and either ed with puromycin (with puro) or left unselected (No puro). Control mice (n =5) were injected with 1 million T cells expressing the CD8SP-KSHV-4C3-(vL-vH)-Myc-BBz—T2A-PAC(042315-N01)[SEQ ID NO: 1643] CAR or given no T cells. Fig. 22 shows the survival of mice in each group. The median survival of mice given RAJI cells alone and CD8SP-KSHV-4C3-(vL-vH)-Myc—BBz-T2A-PAC(042315- N01)[SEQ ID NO:l643]CAR-T cells were 17 days and 21 days respectively. In contrast, the median survival of mice which received CDSSPCD19MM-(vL-vH)—Myc-BBz—T2A- PAC(O62915-D03)[SEQ ID NO:l471] CAR-T cells with and without puromycin selection were 26 and 30 days, respectively. Similarly, the median survival of mice which received CD8SP-CD19Bul2-(vL-vH)-Myc-BBz-T2A-PAC(082815-P08)[SEQ ID NO:l470] CAR-T cells with and without puromycin selection were 30 and 31 days, respectively. Thus, infusion of T cells expressing CDSSP—CD19Bu12-(vL-vH)-Myc-BBz-T2A—PAC(082815—P08)[SEQ ID NO:l470], CD8SPCDl9MM-(vL-vH)-Myc-BBz-T2A-PAC(O62915—DO3)[SEQ ID NO: 1471] CARs lead to tically signiﬁcant improvement in survival of mice in this RAJI xenograft model of lymphoma as compared to mice given no T cells or those given T cells sing the CDSSP-KSHV-4C3-(vL-vI-I)-Myc-BBz-T2A-PAC(O42315-N01)[SEQ ID NO:l643] control CAR.

Applications ofanti-CD19 humanized CAR ucts when used alone or in combination with other gies The humanized CARS of this invention can be expressed in the following way to enhance their safety and efﬁcacy: Although we have used lentiviral methods for expression of the zed CARS, alternate methods such as use of mRNA/DNA transfection by electroporation or by transient perturbation in cell ne can be used to express them in the target cells. The mRNA transfection may involve natural or izing nucleotides to enhance the longevity of expression. Alternate methods for gene delivery, such as use of retroviral, adenoviral and adeno-associated viral vectors have been well described in the literature and are known to those skilled in the art. Alternate promoters for expression of the humanized CAR including CMV, MSCV etc. have been described in the literature.

The CARS of the present invention could be also sed with le selection markers, such as extracellular domain sequences of EGFR, tEGFR, CD19, BCMA (SEQ ID NOs: 853-856), to positively select CAR-expressing cells and to reduce the clonal diversity of the final T cell product before it is used for infusion into the patient. Reduction in clonal-diversity of the CAR-T cell product will lead to reduction in the incidence and ty of Graft versus Host Disease (GVHD) as it will reduce the probability and number of host- reactive T cell clones in the product . Alternate resistance gene, such as CNB3O (SEQ ID NO: 852), which confers resistance to calcineurin inhibitors, can be included to positively select CAR-expressing cells and to reduce the clonal diversity of the final T cell product before it is used for infusion into the patient to reduce the incidence of GVHD.

One of the limitations of CD19 CAR-T cell therapy is erm depletion of normal B cells, which also express CD19 antigen. To circumvent this problem, CD19 CAR-T cell therapy can be combined with out or mutation of endogenous CD19 in normal hematopoietic stem cells using CRIPS/Cas9, TALONS or other suitable gene editing methods which are known in the art. The epitope of CD19 bound by the CD19 CAR-T cells in current clinical use has been mapped to exons 2-4. Thus, missense or se mutations can be generated in exon 2 (or other le exons/regions that are recognized by CDI9Bu12 and CD19MM CAR T-cells) of autologous or allogeneic hematopoietic stem cells using CRISP/Cas9, Zn ﬁnger nucleases, Talons or other s known in the art. The patient can be given CD19 CAR-T cells on to control his/her disease and an autologous or allogeneic lant using CD19 deleted/mutated hematopoietic stem cells. As the B cells that will originate from the modiﬁed stem cells will not be targeted by CD19-CAR-T cells, the patient will escape B cell aplasia which is a common side effect of CD19 CAR-T cells.

The CD19 targeted humanized CARS of the present invention can be also combined with CAR targeting other antigens, such as CD22 and CD20, to overcome ance due to loss of CD19 antigens. Such bispecif1c CARS have been described in the literature and are known to one skilled in the art.

The humanized CAR constructs of the present invention can be expressed not only in T cells but in deﬁned subsets ofT cells such as CD4 and CD8 T cells, central memory T cells, naive T cells and regulatory T cells. They can be also expressed in NK cells, NK92 cell line, hematopoietic stem cells, and iPSC derived stem cells. [007l4] To control their activity, the humanized CAR of the current invention can be expressed in an inducible fashion as is known in the art (Roybal c1611., Cell 164:770-9, 2016; Wu el 0]., Curr Opin Immunol 351123-30, 2015; Wu er (1]., Science 350:aab4077, 2015)).

Alternatively, suicide genes (such as icaspase 9), can be used to control the activity of CAR- T cells in vivo.

] The activity of the CAR-T cells of this invention can be further modulated by use of inhibitors of various signaling pathways, such as mTOR and PI3-AKT ys.

Furthermore, various cytokines, chemokines and their ors, and costimulatory ligands (eg. 4lBB) and their receptor (e.g. eceptor or CD137) can be coexpressed with the humanized CAR of the current invention.

Novel Chimeric Antigen Receptors expressing viral and cellular signalingproteins As discussed previously, the CAR constructs in clinical trials include one or more costimulatory domains d from 4IBB, CD28 or both. These costimulatory domains are generally derived from the cytosolic ing s of TNF (Tumor Necrosis Factor) family receptors and are believed to activate signaling pathways, such as NF-KB and AKT, which are ed to contribute to increased proliferation and survival of CAR-T cells, resulting in their ion and tence in vivo. However, tonic signaling through these costimulatory domains when expressed as part of an artiﬁcial receptor, such as a CAR, is not physiological and is not under the l of feedback regulatory mechanisms that are normally present to control signaling via the TNF family receptors from which they are derived. Therefore, it is conceivable that robust and persistent tion of signaling pathways by the costimulatory s present in the current CAR constructs butes to the cytokine release syndrome and neurological toxicity, which re a major side effect of current CAR-T cells based therapies. It is important to note in this context that cytokine release syndrome was a major side effect in human clinical trials of a CD28 super-agonist antibody.

The present invention circumvents the above limitation of the current CAR s by deleting the ulatory domains. The costimulatory function instead is provided by viral and cellular ns that are known to constitutively activate various pro- survival signaling pathways and/or block cell death. The viral ns suitable for this purpose include viral FLICE Inhibitory Protein (vFLlP) K13 encoded by the Kaposi’s sarcoma associated herpesvirus (also known as human herpesvirus 8), vFLIP MC159 and MC160 from the cum contagiosum virus (MCV), E8 from the equine herpesvirus 2 (EHVZ), vFLIP encoded by the bovine herpesvirus 4, VFLIP encoded by herpesvirus mecaca, VFLIP encoded by herpesvirus saimiri, cFLIP-L isoform or MRIT-a, cFLIP-p22 deletion mutant or MRIT-p22 , HTLVl-Tax, I-ITLV2-Tax and HTLVZ-Tax-RS mutant (Jeang, Cytokine Growth Factor Rev 12:207-17., 2001; Sieburg el al., J Virol 78:10399-409, 2004).

In contrast to artificial and non-physiological nature of the pro-survival signal provided by the costimulatory domains that are components of the CAR constructs in common use today, these ing proteins have d to provide constitutive pro-survival signals to the cells in which they are expressed.

KSHV-encoded viral FLICE inhibitory Protein KI3 imnwrtalizes T cells ] vFLIP K13 was originally believed to protect KSHV-infected cells from death receptor-induced apoptosis (Bertin et al., Proc Natl Acad Sci U S A 94:1172-6, 1997; Hu et al., J Biol Chem 272:9621—4, 1997; Thome et al., Nature 386:517—21, 1997)). However, it was subsequently reported that K13 selectively activates the NF-KB pathway (Chaudhary er al., Oncogene 18:5738-46, 1999; Chugh er. al., Proc Natl Acad Sci U S A 102:12885-90, 2005) by interacting with a ~7OO kDa [KB kinases (IKK) complex which consists of lKKl/IKKoc, lKK2/IKKB and NEMO/IKKy hary et al., Oncogene 18:5738-46, 1999; Liu er al., J Biol Chem 277:13745-51., 2002). HTLVl-encoded Tax protein is also known to activate the NF-KB pathway by binding to NEMO/IKKy. Tax has been also shown to immortalize T cells (Ren et al., J Biol Chem 683-93, 2012; Robek and Ratner, J Virol 73:4856-65, 1999).

Human peripheral blood clear cells (PBMC), CD3+ve T cells and CD3 -ve cells (designated B cells) were cultured in XVIVO medium (Lonza) nted with 10ng/ml soluble anti-CD3, 10 ng/ml e anti-CD28 and 100 IU recombinant human-1L2.

Cells were cultured at 37°C, in a 5% C02 humidiﬁed incubator, and after 1 day infected with an empty lentiviral vector I) carrying blasticidin resistance gene and iral vectors expressing C-terminal Flag-Tagged K13 (pLENTI-K13-FLAG), a deletion mutant of K13 (pLENTI-Kl3A45—48-FLAG) that is known to lack NF-KB activity (Matta et al., Oncogene 27:5243-53, 2008), and HTLVl-Tax protein (pLENTI-Tax). The nucleotide and protein sequences of K13 are provided in SEQ ID NOs: 866 and 2629, respectively.

Approximately 1 day post-infection, cells were selected with blasticidin. After selection, the cells were grown in XVIVO medium and IL2 withdrawn. Table 26 shows that both PBMC and T cells infected with K13-Flag and Tax encoding vectors continued to erate in culture long after 1L2 withdrawal, while no long term proliferating cells were obtained with cultures infected with empty lentiviral vector or those infected with K13A45- 48-FLAG encoding vector. These results demonstrate that similar to HTLVl-Tax, K13 can promote long term proliferation and immortalization of primary T cells and PBMC and its NF-KB activity is ial for this process.

Table 26 ﬁlé§""""é'il‘i'é'ﬁlééi'aﬁiiéésﬁ'"i'éiiiis‘éii‘tléé'iééééiéi‘é"""" {.i?‘ ‘31:... ' Expression of K13-FLAG in the surviving culture of T cells was examined by immunoﬂuorescence staining with FITC-conjugated FLAG antibody followed by FACS is. Fig. 23 shows increased staining with LAG antibody in K13-expressing T cells as compared to uninfected T cells. The immunophenotype of K13 immortalized T cells was examined by staining with CD4 and CD8 antibodies. Fig. 23 shows that K13 immortalized T cells are CD8+/CD4- (49%), D4+ (21%) and CD8+/CD4+ (29%). In contrast, a majority of uninfected T cells persisting in culture were CD8+/CD4- (94%). There was no signiﬁcant difference in the expression of CD69 between cted and K13- immortalized T cells.

FMC63 CAR expressing K13 retain the capacity to induce lysis 0fRS4;11 target cells T cells were infected with lentiviral vectors encoding GFP, CD8SP-FMC63- (vL-vH)-Myc-BBz-T2A-EGFP(070814-DO6)[SEQ ID 5], CD8SP-FMC63-(vL-VH)- Myc-BBz-TZA-PAC(112014-A13)[SEQ ID NO:1467], and CD8SP-FMC63-(vL-vH)-Myc- BBz-P2A-KI3-Flag-T2A-PAC(I I IOI4-Yl l)[SEQ ID NOIl 197]; The cells infected with PAC containing vectors were selected with puromycin or left unselected. Fig. 24 shows that CD8SP-FMC63-(vL-vH)-Myc-BB2-T2A-PAC(112014-A13)[SEQ ID 7], and FMC63-(vL-VH)-Myc-BBz-P2A-K l3-Flag-T2A-PAC(1 l lOl4-Yl l)[SEQ ID NO:1197]-expressing T cells that have been selected with puromycin induce speciﬁc lysis of RS4] 1 cells as measured by Gluc assay.

T cells expressing CAR containing K13, MCI59 or Tax2 induce speciﬁc lysis ofRAJI-Gluc cells Human eral blood T cells isolated using CD3 magnetic beads were infected with lentiviruses expressing the following constructs targeting CD19. The es of the constructs are as follows: CD8SP-FMC63-(vL-vH)-Myc-BBz-T2A-PAC(l 12014-A13)[SEQ ID N021467]: A standard FMC63 based conventional CAR ll containing a 4IBB costimulatory domain and a CD32 domain separated by a T2A sequence from a Puromycin resistance gene.

FMC63—(vL—vH)—Myc—z—T2A—PAC(1 1 1214—D05)[SEQ ID N023538]: A FMC63-based conventional CAR I that lacks a 41BB co-stimulatory domain and contains a CD32 activation domain.

CD8SP-FMC63-(vL-vH)-Myc-BBz-P2A-K13-F1ag-T2A-PAC(1 ] 1014- Y11)[SEQ ID NO:] 197]: A standard FMC63 based conventional CAR 11 ning a 41BB costimulatory domain and a CD32 domain, a P2A cleavable linker, a K13-Flag sequence, a T2A cleavable , and a Puromycin resistance gene.

CD8SP-FMC63-(vL-vH)-MycP2A-K13-Flag-T2A-PAC(1 1 1614- B05)[SEQ ID NO:927]: A standard FMC63 based conventional CAR I containing a CD32 domain, a P2A cleavable linker, a Kl3-Flag sequence, a T2A cleavable linker, and a Puromycin resistance gene.

CD8SP-FMC63(vL-vH)-Myc-BBz-P2A-MC l 59-Flag-T2A-PAC(1 l 1014- Zl3)[SEQ ID 1]: A standard FMC63 based tional CAR 11 containing a 4183 costimulatory domain and a CD32 domain, a P2A cleavable linker, a MC 159-Flag sequence, a T2A cleavable linker, and a Puromycin resistance gene.

] CD8SP-FMC63(vL-vH)-Myc-z-P2A-MC 1 59-F1ag-T2A-PAC( 1 126 I 4- C06)[SEQ ID NO:]750] A standard FMC63 based conventional CAR I containing a CD32 , a P2A cleavable linker, a MC159-Flag sequence, a T2A cleavable linker, and a Puromycin resistance gene.

CD8SP-FMC63(vL-vH)-Myc-BBz-P2A-Flag-HTLVZ-TAX-RS-T2A- PAC(OlOlOO-N01)[SEQ ID NO:]752]: A standard FMC63 based conventional CAR 11 containing a 41BB costimulatory domain and a CD32 domain, a P2A cleavable linker, an RS mutant of HTLV2-encoded TAX, a T2A cleavable linker, and a cin ance gene.

Cells were left unselected or selected with puromycin and expanded. RAJI cells stably expressing Gluc were cocultured with T cells sing the CARs at the Effector: Target (Ez'I‘) ratio of 10:1 for 4 hours. CAR-T cells mediated induction of lysis of target cells was assayed by increase of GLuc activity. Fig. 25 shows increase in GLuc activity, indicating lysis of target cells, following co-culture with T cells expressing CD19- specific CARS as ed to uninfected T cells (T-UI) or those expressing the control CAR targeting 4C3 (SEQ ID NO: 1643). These results demonstrate that incorporation of K13, MC159 or HTLVZ-Tax (Tax2) does not vely impact the killing ability of CARs with or without the presence of4lBB domain.

The above experiment was repeated at E:T ratio of 1:1 and cell death was assayed after 48h. Fig. 26 confirms that incorporation of K13, MC 159 or HTLV2-Tax (Tax2) does not negatively impact the killing ability of CARs with or t the presence of 41BB The experiment was repeated with cells that had been selected with puromycin and were in culture for approximately 2 months. Fig. 27 shows that the T cells expressing CAR containing MC159 and HTLVZ-Tax continue to exert cytotoxicity after 2 months in culture while the T cells expressing the other CARS lost this ability.

In vivo efficacy ofCAR T cells containing vFLIPs and Tax Human peripheral blood T cells isolated using CD3 magnetic beads were infected with lentiviruses expressing the indicated CAR constructs targeting CD19. NSG mice (Jackson Lab) were sub-lethally irradiated at a dose of 175 cGy. 24 hours post irradiation (day2), mice were injected with 2.5 x l04 RAJI cells via tail-vein. On day3, the mice (n =3 for each group) were treated with 1 million T cells that had been infected with the CAR encoding lentiviruses. Table 27 shows the survival of mice in each group. These results demonstrate that highest survival (38 days) is observed in mice injected with T cells expressing FMC63 based CAR CD8SP-FMC63-(vL-vH)-Myc-z-P2A-K13-Flag-T2A- PAC(111614-BOS)[SEQ ID ] that lacks 4lBB ulatory domain but expresses Table 27 Median CD88P-FMC63-(vL-vH)-Myc-BBz-T2A-PAC(l12014-Al3)[SEQ [D b N CDSSP-FMC63-(vL-vI-I)-Myc-z-T2A-PAC(I I 1214-D05)[SEQ ID N023538 CDSSP-FMC63-(vL-VH)-Myc-BBz-P2A-Kl3-FIag-T2A- PAC(111014-Yl 1) SEO ID NO:1197 CDSSP-FMC63-(vL-vH)-Myc-z-P2A-K l 3-Flag-T2A-PAC(] l l6l4- B05) SEO ID NO:927 CD88P-FMC63(vL-\v'I-I)-Myc-BBz-P2A-MC159-Flag-T2A- NA PAC(111014-ZI3 SEO ID 1 CDSSP-FMC63(vL-vI-l)-M}.-'c-z-P2A-MC I 59-Flag-T2A- PAC(1126l4-C06) SEO ID NO:1750 CDSSP-FMC63(vL-vI—I)-M_\_-'c-BBz-PZA-FIag-HTLVZ-TAX-RS-TZA- PAC(OIOIOO-N01 SEO ID NO:1752 NNN OUIO Inducible expression ofK13 by_fusion with FKBP To control the expression of K13, different s were generated expressing K13 in fusion with one or two copies of N-terminal FKBP-based switch or zation s. In addition, a construct was constructed in which 2 copies of FKBP domain were fused at the N-terminal with a myrisotylation signal and at C-terminus with K13. The above constructs of K13 were either expressed alone or in constructs expressing different CAR COI‘I StI'LICIS.

NF-KB reporter assay was carried out essentially as described (Chaudhary er al., Oncogene 18:5738-46, 1999). The indicated ucts were transiently transfected in 293FT cells along with a luciferase reporter construct driven by 4 copies of NF-KB binding site and a RSV-LacZ reporter construct. Cells were left untreated or d with AP20187, which dimerizes the FKBP domains, resulting in activation of K13 signaling. Approximately 24hr later, cells were lysed and NF-KB-Luc and LacZ activities measured in cell lysates. NF- KB-LUC ty was normalized relative to LacZ activity to control for difference in transfection efﬁciency. As shown in Fig. 28A, treatment with AP20187 (100 nM) led to increase in K13-induced NF-KB-Luc activity in all constructs in which K13 was expressed in fusion with FKBP. Essentially similar results were obtained when RS mutant of HTLVZ encoded Tax was expressed in ﬁJsion with FKBP (Fig. 288). Thus, ty of K13 and Tax can be controlled by fusion with FKBP and subsequent administration of AP20187 when they are expressed alone or as part of CAR constructs.

Activation of K13 activity by AP20187 does not impair the ability of CARS to induce cell death Human peripheral blood T cells ed using CD3 magnetic beads were infected with lentiviruses expressing the constructs CD8SP-FMC63(vL-vH)-Myc-z-P2A- FKBP-Kl3-FLAG-T2A-PAC(112316—806) [SEQ ID NO:1770] and CD8SP-161—(vL-vH)— Myc-z-PZA-FKBP-Kl3-FLAG-T2A-PAC(112916-U06) [SEQ ID NO: 174]] and ed with puromycin as described previously. RAJI and HEL cells stably expressing Gluc were ured with uninfected T cells (UI) or T cells expressing the indicated ucts [SEQ ID 0] and [SEQ ID NO: 1741], respectively, at the Effector: Target (EzT) ratio of :1 for 4 hours in the absence and presence of IOOnM AP20187 (AP) compound. CAR-T cells mediated induction of lysis of target cells was assayed by increase of GLuc activity. Fig. 29A and 29B show that activation of K13-mediated NF-KB activity by addition of AP20187 does not adversely affect cell killing d by the two CAR constructs that coexpress FKBP-K13.

Cytotoxic activity of CARS I and II coexpressing K13, MCI59, HTL V2-Tax and HTL V2- Tax RS mutant Human peripheral blood T cells isolated using CD3 magnetic beads were infected with lentiviruses expressing the indicated constructs expressing CAR I and II targeting MPL and coexpressing ent accessory modules (Fig. 30A). Cells were selected with puromycin as described usly. HEL cells stably expressing Gluc were cocultured with uninfected T cells (UI) or T cells expressing the indicated constructs at the Effector: Target (EzT) ratio of 10:1 for 4 hours. CAR-T cells mediated induction of lysis of target cells was assayed by increase of GLuc. Fig. 30A shows induction of target cell death by different MPL-directed CAR constructs that coexpress K13, MC159, HTLVZ-Tax and HTLVZ-Tax RS mutant.

Essentially a similar experiment was conducted with a construct (SEQ ID NO: 983) expressing CAR 1 targeting CD32 and coexpressing K13. Fig. 30B shows effective induction of HEL cell death by T cells sing this construct as measured by Gluc assay.

Cytotoxic activity of Backbone I constructs comprising a conventional CARS I targeting TROP2 and coexpressing K13 ] Human peripheral blood T cells isolated using CD3 magnetic beads were infected with lentiviruses expressing the construct CD8SP-TROP2-h7E6-SVG—(vL-vH)-Myc- z-P2A-KI3-Flag-T2A-PAC(052616-D04)[SEQ ID NO:1166] ing TROP2 and selected with puromycin and tested for the ability to kill PC3 (prostate cancer) cells expressing GLuc using the assay described previously. Fig. 31 shows effective g of PC3 cells by T cells expressing the TROPZ-h7E6-SVG-(vL-vH)-Myc-z-P2A-K 1 3-Flag-T2A- 2616-D04)[SEQ ID N01 166] construct.

Cytotoxic activity of Backbone I ucts comprising a conventional CARS I targeting LAMP] and compressing KI3 Human peripheral blood T cells isolated using CD3 magnetic beads were infected with lentiviruses expressing the indicated constructs targeting LAMPl and ed with cin. Cells were tested for the ability to kill L363 and U266 cells expressing GLuc using the assay described previously. Fig. 32A and 32B shows effective killing of L363 and U266 cells by T cells expressing the CDSSP-LAMPI-humab1(vL-vH)-Myc-z- PZA-Kl3-Flag-T2A-PAC(0517l6-T05)[SEQ ID N011104] and CD8SP-LANlPl-Mb4-(VL- vH)—Myc-z-P2A-Kl3-Flag-T2A-PAC(051916-BO7)[SEQ ID NO:1 105] constructs.

Activation qf K13 signaling by addition Qf AP20187 in Jurkat cells expressing (1 CD19- directed CAR and coexpressing FKBP—KI3fusion n The effect of induction of K13 induced NF-KB activity on CAR-induced NFAT signaling was studied by stably expressing the construct CDSSP-FMC63(vL-vH)- PZA-FKBP-Kl3-FLAG-T2A-PAC(112316-806)[SEQ ID 0], which encodes a conventional CAR 1 targeting CD19 and expresses an l3-FLAG fusion protein, in Jurkat-NFAT-GFP cells (JP). The parental -NFAT-GFP cells (JP) cells and those expressing the construct FMC63(vL-vH)-Myc-z-P2A-FKBP-K13-FLAG-T2A- PAC(112316-SO6)[SEQ ID NO:1770] were cocultured with RAJI cells in the absence and presence of 100 nM AP20187 for approximately 18 hours and induction of NFAT-driven GFP expression examined by FACS analysis as described previously. Fig. 33 shows effective induction of GFP sion in Jurkat cells expressing the CD8SP-FMC63(vL—vH)—Myc-z— P2A-FKBP-Kl3-FLAG-T2A-PAC(1 123 l6-SO6)[SEQ ID NO:1770] construct upon coculture with RAJI cells in the absence (38.7% GFP-expressing cells) and presence (45.85% GFP expressing cells) of AP20187 compound. In contrast, no signiﬁcant GFP ion was observed upon co—culture of parental Jurkat-NFAT-GFP cells (JP) cells with RAJI cells either in the absence or presence of the 7 compound. These results demonstrate 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 duced NFAT signaling.

Generation ofK13 mutants with varying level cB activity It would be useful to have novel mutants of K13 with varying levels of NF-KB activity for the purpose of ve ar therapy and other applications. Site-directed mutagenesis was conducted using Quickchange site-directed kit (Stratagene) to generate several point mutants of K13 in which the different amino acids of K13 (SEQ ID NO:2629) were mutated. For example, in the mutant K19A the amino acid Lysine at position 19 is mutated to alanine. The wild type and the various mutant constructs were transfected in 293FT cells along with a luciferase reporter construct driven by 4 copies of NF-KB binding site and a RSV-LacZ reporter construct essentially as described hary et (11., Oncogene 18:5738-46, 1999) . Approximately 24hr later, cells were lysed and NF-KB-Luc and LacZ ties measured in cell lysates. Luc activity was normalized relative to LacZ activity to control for difference in transfection efﬁciency (Chaudhary et al., Oncogene 18:573 8-46, 1999). Fig. 34 shows that many mutants have either increase (e.g. E48A mutant) or decrease (e.g., E47A mutant) in NF-KB activity as compared to the wild-type K13. These mutants will have application in ve cellular therapy and other ations where varying level of NF-KB activities are required. Thus, mutant E48A can be coexpressed in CAR constructs where strong NF-KB activity is desirable while mutant R50A can be coexpressed in CAR constructs where only modest NF-KB activity is desirable. The activity of these mutants can be also controlled by fusing them with FKBP domains (SEQ ID N05: 2621 and 2622) as described above for wild-type K13.

A method ofcontrolling the activity ofCAR-T cells, Bispeciﬁc T cell engagers (BiTEs) and DARTs (Dual Afﬁnity Re-targeting proteins) with Src Inhibitors Dasatinib, Ponatinib and A- 77004] CARs are synthetic receptors which combine antibody binding city with T cell functionality. CARS are generated by fusing a single chain antibody (scFv) that dictates the c recognition of d tumor n with the intracellular signaling components of CD32 chain of T cell receptor to enable activation of T cell effector function.

The I-ILA- independent recognition of TAA by CAR not only minimize tolerance induction mechanisms, but also allow the versatile design of CARs recognizing virtually any antigen including non-protein moieties on cell surface. Although CARS activate T cells via the CD32 chain, signaling via CARS is different from signaling via the normal T cell receptor (TCR) as they do not engage all the components of the TCR complex. For example, signaling via a normal TCR results in engagement of several molecules, such as TCRa, TCRB, CD37, CD35, CD38, CD3C, and CD4 or CD8 as well as their associated ream signaling proteins, such as Lck, LAT, ZAP7O and SLP-76 (Chakraborty, AK and Weiss, A Nat Immunol :798-807,2014). It is unclear if any of these receptors and signaling intermediates are involved in ing via a chimeric antigen or (CAR). The notion that signaling via CAR is ct from signaling via a normal TCR is evident from the side effects observed following the infusion of CAR versus those observed after on of TCR in patients. While ne release syndrome (CSR) and neurological complications are frequently observed following the infusion of CAR-T cells, they are not seen following the infusion of tumor ating lymphocytes or after infusion of T cells that had been ered to express a TCR against a particular antigen, such as NY-ESO jens, Blood 17-28, 2011; Rapoport et (1]., Nat Med 212914-21, 2015; Robbins et (1]., Clin Cancer Res 21:1019-27, 2015; Sadelain er (7]., Curr. Opin. Immunol. 212215-23, 2009; Turtle et 0]., Curr. Opin.

Immunol. 242633-39, 2012).

Bispeciﬁc Tcell engager (BiTE) Bispeciﬁc T cell engager ) antibody constructs are a type of immunotherapy being investigated for ﬁghting cancer by helping the body's immune system to detect and target malignant cells. The modified antibodies are designed to engage two different s aneously, thereby juxtaposing T cells (a type of white blood cell capable of killing other cells perceived as threats) to cancer cells. BiTE® antibody constructs help place the T cells within reach of the targeted cell, with the intent of allowing T cells to inject toxins and trigger the cancer cell to die (apoptosis). BiTE® antibody constructs are currently being investigated for their potential to treat a wide variety of cancers. BLINCYTO tumomab) is a bispeciﬁc CD19-directed CD3 T cell r (BiTE®) antibody construct that binds speciﬁcally to CD19 expressed on the surface of cells of B-lineage origin and CD3 expressed on the surface of T cells. Although BiTEs, such as Blinatumomab, activate T cells, they bind to the CD3 chain of TCR rather than TCRa and TCRB chains, and therefore signaling through BiTEs is not physiological and is not identical to signaling via the normal T cells.

Limitation ofCAR T cells and Bispeciﬁc antibodies In majority of patients who respond to engineered CAR T cells and Blinatumomab, ive release of proinﬂammatory cytokines causes ms that include fevers, hypotension, hypoxemia, cardiac dysfunction, kidney failure and electrolyte abnormalities, collectively termed as "Cytokine release syndrome’ (CRS). A number of these patients require admission to intensive care units for pressure support and artiﬁcial ventilation. In significant number of cases (up to 50%), CAR T cells and Blinatumomab therapy can lead to neurologic symptoms ing tremor, seizures and can be fatal. For example, approximately 50% of patients receiving Blinatumomab in clinical trials experienced neurological toxicities. Severe, life-threatening, or fatal neurological toxicities occurred in approximately 15% of patients, ing encephalopathy, sions, speech disorders, disturbances in ousness, ion and disorientation, and coordination and balance disorders. Strategies to counteract CRS include treatment with immunosuppressive agents and antibodies to cytokines to block cytokine release and/or activity. Nevertheless, treatment-related deaths have occurred at many institutions. Therefore, there is an unmet and urgent need for new drugs/agents to control the ty of CAR T cells and Blinatumomab.

Dasatinib as an inhibitor ofphysiological T cell and NKfunction The dual BCR-ABL/Src kinase inhibitor dasatinib is FDA approved for the treatment of chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia. Src kinases are known to play an important role in physiological T-cell activation. Consistent with this, dasatinib has been shown to profoundly inhibit antigen speciﬁc physiological T-cell activation, proliferation, cytokine production, and degranulation in a dose-dependent manner e et al., Blood [11:1366-77, 2008; el et (1]., Clin Cancer Res [422484-91, 2008). Dasatinib has been also shown to inhibit the activation of NK cells. However, since the CAR modiﬁed T cells and BiTE do not engage the physiological T cell receptor (see above), it is not clear whether dasatinib can be used to modulate the ty of CAR-T cells and T cells activated via BiTE.

Human peripheral blood T cells ed using CD3 magnetic beads were ed with lentiviruses expressing the indicated CAR uct targeting CDSSP-Z- CD19MM-(vL-vH)-Myc-BBz-T2A-PAC(062915-D03)[SEQ ID NO:1471] and selected with puromycin. CAR-T cells were pre-incubated with the ted concentrations of Dasatinib and Imatinib for approximately 30 min at 37°C. The drug-treated and untreated T cells were plated in a white 384-cell plate. RAJI cells stably expressing GLuc were added to the wells containing the T cells at a concentration of 30K cells/30ul/ well to give an E:T ratio was 5:1.

Dasatinib (Das) and Imatinib (Imat) were added to the wells to maintain the final concentrations as indicated. After 4 h of co-culture, CAR-T cells mediated induction of lysis of target cells was assayed by se of GLuc activity by directly injecting 0.5X CTZ assay buffer containing native coeloentrazine. Fig. 35 shows inhibition of CAR CD8SP CD19MM-(vL-vH)-Myc-BBz-T2A-PAC(0629l5-DO3)[SEQ ID NO:147l]-induced cell death by SOnM and lOOnM Dasatinib, while Imatinib has no effect.

Effect of different Tyrosine Kinase inhibitors on cell death induced by CAR modiﬁed T cells Human peripheral blood T cells isolated using CD3 magnetic beads were infected with iruses expressing the indicated CAR constructs targeting human andCD8SP-FMC63-(vL-vH)-Myc-BBz-T2A-PAC(112014-A13)[SEQ ID NO:1467] and CD8SPCDl9MM-(vL-vH)-Myc-BBz-TZA-PAC(0629l5-DO3)[SEQ ID NO:147l]). RAJI cells stably expressing GLuc were co-cultured for approximately 4 h with T cells expressing the CARS in the presence and absence of indicated concentrations of the tyrosine kinase tors (TKI) nib, Imatinib, Nilotinib, Ponatinib and Axitinib essentially as described above. Among these tors, Dasatinib and Ponatinib have been reported to have ty against Src kinase, while the others lack this activity. CAR-T cells mediated induction of lysis of target cells was assayed by increase of GLuc activity as measured by BioTek synergy plate reader by directly injecting 0.5X CTZ assay buffer containing native coeloentrazine (Nanolight). For measurement of maximum cell death, cells were treated with 30p] of nin (ﬁnal concentration 30 pg/ml) for approximately 90 min prior to measurement of Glue activity. The % speciﬁc lysis was calculated by taking digitonin- induced cell death as 100% and untreated cells as 0%. The formula for % speciﬁc lysis calculation is: % Speciﬁc lysis = 100 x [(experimental data) - (spontaneous cell death))] / [(maximum cell death)-spontaneous cell death)]. Fig. 36 shows increase in GLuc activity following co-culture with T cells expressing CDl9-speciﬁc CARS CD8SP-FMC63-(vL-VH)- Myc-BBz-TZA-PACU12014-Al3)[SEQ [D NO:1467] and CD8SPCD19MM-(vL-VH)- Myc-BBz-TZA-PAC(062915-D03)[SEQ ID NO:1471]as compared to the uninfected T cells (T-UI) indicating lysis of target cells by the ent CD19-CAR-T cells. However, CD19- CAR-T cells d cell death was partially blocked by treatment with SOnM Dasatinib and completely blocked by treatment with lOOnM Dasatinib. Similarly, AR-T cells- induced cell death was partially blocked by treatment with 100 nM nib. However, ent with the indicated doses of Imatinib (50nM and SOOnM), Nilotinib (lOnM and lOOnM), and Axitinib (IM and lOuM) had no effect on CAR-T cell induced cell death.

These results demonstrate that TKI with activity against Src can effectively block CAR-T cell induced cell death. It is also possible that treatment with higher doses of ib and Nilotinib than those used in the above study could block CAR-T cell-induced cell death.

Dasatinib blocks cell death induced by CAR targeting CD22 Human peripheral blood T cells isolated using CD3 magnetic beads were infected with lentivirus expressing the a CAR uct targeting human CD22 - CD22-m971-(vL-vH)-Myc-BBz-T2A-PAC(091515-A02)[SEQ ID N0:]5]9]) and ed with puromycin. The effect of dasatinib on puromycin selected CD22 CAR-T cells induced death of RAJI-GLuc cells was examined after 96 h of co-culture of effector and target cells.

Fig. 37 shows that Dasatinib effectively blocks killing of RAJl-GLuc cells by T cells expressing the CD8SP-CD22-m97l-(vL-vH)-Myc-BBz-T2A-PAC(O91515-A02)[SEQ ID NO: 1519] CAR as determined by GLuc assay.

Dasatinib blocks cytotoxicity induced by parental NK92MI cells and those modiﬁed to s (1 CAR ] NK92 cells are under clinical development for the treatment of a number of cancers. To test if Dasatinib can block the activity of NK92 cells, NK92MI cells (a derivative of NK92 cells sing 1L2) were co-cultured with KS62-Gluc cells in the absence and presence of indicated concentrations of Dasatinib. The target cells were plated a concentration of 100K cells/lOOul/ well in a black 96-cell plate and either left untreated or treated with two different doses of Dasatinib (50nM and 100nM) for 30 min followed by co- culture of target cells with NK92MIMI effector cells at an EzT ratio of 0.521 for 4 h. Fig. 38A shows that Dasatinib blocks NK92 cells mediated death of K562 cells as determined by Gluc assay.

To test if nib would also block cell death induced by NK92 cells which have been engineered to express 8. CAR, NK92MI cells were engineered to express CAR FMC63-(vL-vH)—Myc-BBz-TZA—EGFP(O708l4-D06)[SEQ iD NO:3535] targeting CD19. NK92MI cells expressing the CD8SP-FMC63-(vL-vH)-Myc—BBz-T2A- EGFP(O70814-DO6)[SEQ ID NO:3535] CAR were co—cultured with CD19 positive RAJI- GLuc target cells in the absence and presence of Dasatinib for 4 h and cell death measured by GLuc assay. Fig. 38B shows that Dasatinib blocks death of RAJI-GLuc cells by CD8SP- FMC63-(vL-vH)-Myc-BBz-T2A-EGFP(070814-D06)[SEQ ID NO:3535] CAR-expressing NK92MI cells as determined by Gluc assay.

Dasatinib blocks Blinatumomab (Blincyto) induced cell death and cytokineproduction Effector T-cells (isolated by CD3 microbeads from Miltenyi Biotech) were left untreated or preincubated with SOnM or 100nM Dasatinib for 1 hour at 37°C followed by plating cells in a U-bottom 96-well plates. RAJI-MSCVhygro-Gluc target cells were incubated with a bispeciﬁc antibody (Blinatumomab or BLINCYTO; AMGEN) at 100ng/106 cells for 30 min at 37°C in PBS/2% FBS. Next, Blinatumomab-treated luc or untreated (control) RAJl-Gluc cells were washed three times in PBS, re-suspended in XVIVO medium and 100 pl (100K) cells were plated per well of a 96-well U-bottom plate (EffectorzTarget ratio of 1:1) in the absence and presence of ted concentrations of Dasatinib. Total volume in each well was adjusted to 200 p] with medium. Plates were quickly centrifuged to settle cells, and incubated at 37°C in a 5% C02 incubator for 96 hours. For m cell death, one hour prior to doing GLuc cell death assay, a total volume of 6 pl of Digitonin (stock 1mg/ml, Sigma) was added to wells containing target cells alone to achieve a ﬁnal concentration of 30ug/ml of Digitonin. The plates were centrifuge at 1,000 rpm for 5 min and 50p] of supernatants were transferred to a r 384-well ﬂat bottom white opaque plate. The luciferase activity was measured by BioTek synergy plate reader by directly injecting 0.5X CTZ assay buffer containing native coeloentrazine (Nanaolight) into the 384 well plates in a well mode. Fig. 39A shows that treatment with umomab in the presence of T cells result in strong induction of death of RAJl-GLuc cells which is effectively blocked by 100nM Dasatinib. The supernatant from the above experiment was also assayed for measurement of IFNY production by ELISA. Fig. 398 shows that co-culture of Luc cells with Blinatumomab in the presence of T cells result in strong induction of IFNy production which is ively blocked by 100nM Dasatinib.

Effect of Ruxolitinib, Fosatamatinib and Alisertib on CAR T cells and Blinatumomab induced cell death The effect of Ruxolitinib (JAK-l/Z inhibitor), matinib (syk inhibitor) and Alisertib a Kinase tor) on CDSSP-CD19Bu12-(vL-VH)-Myc-BBz-T2A- PAC(082815-P08)[SEQ ID N021470] -expressing CAR T cells- and Blinatumomab-induced death of RAJI-pLenti-GLuc cells was studied using the assay as described above. Fig. 40A shows no signiﬁcant toxic effect of the s drugs on RAJI-pLenti-Gluc cells that had not been co-cultured with CAR T cells. Fig. 40B shows signiﬁcant induction of cell death, as measured by se in Gluc activity, when RAJ l--pLenti-Gluc cells are ted with the CAR-T cells. Fig. 403 also shows mild to modest inhibition of -CAR-T cell—induced death of RAJI-pLenti-GLuc cells by Ruxolitinib, Fosatamatinib and Alisertib as compared to cells treated with media (Med) alone. Dasatinib was used as a positive control and led to signiﬁcant inhibition of CAR-T cell-induced death of RAJI cells. Fig. 40C shows that the above compound have only a minimal effect on Blinatumomab induced cell death ed by T cells at the indicated concentrations.

Effect qfA-770041, Saracatinib, Avasimibe on CAR T cells- and Blinatunmmab-induced cell death A-77004l is a selective small molecule inhibitor of LCK, one of the 8 members of the src family, that is required for T cell activation and IL2 production at et al., Bioorg Med Chem Lett -22, 2006). A77004l is a 147 nM inhibitor of Lck (1 mM ATP) and is 300 fold selective against Fyn, the other src family kinase involved in T cell signaling. tinib (AZD0530) is a src family that can be administered orally. Avasimibe is an inhibitor of ACAT], a key cholesterol esteriﬁcation enzyme that has been shown to augment anti-tumor response of CD8 cells.

] The effect of A-770041, Saracatinib and Avasimibe on CD19Bu12(vL-vH)- BBz-Pac-P08 expressing CAR T cells- and Blinatumomab-induced death of RAJI-pLenti- GLuc cells was studied next using the assay as described above. Fig. 41A shows the release of Gluc in cells treated with the ted drugs in the presence of media alone without any CAR-T cells. Fig.4lB shows near te inhibition of CDSSP-CDI9Bu12-(vL-vH)-Myc- BB2-T2A-PAC(082815-PO8)[SEQ ID NO:1470] CAR-T cells induced death of RAJI-pLenti- GLuc cells by A-77004l at lOOnM and 200nM while Saracatinib had no signiﬁcant effect. nib was used as a positive control in the above experiment and as expected blocked CAR—T cells induced cell death. Avasimibe had no signiﬁcant effect on CAR-T cells induced cell death at lOOnM but completely blocked it at lpM. Fig. 41C shows that treatment with lOOnM A77004l partially blocked cell death induced by combination of Blinatumomab with T cells while treatment with 200nM A77004l led to complete inhibition. rly, treatment with luM Avasimibe blocked Blinatumomab d cell death mediated by T cells.

Collectively, the above studies demonstrate that inhibitors of LCK kinase can block the effect of CAR-T cells and Blinatumomab and therefore can be used for the treatment of cytokine release syndrome and neurological complications associated with treatment with CAR-T cells and Blinatumomab.

Generation ofCAR ucts essing scFv targeting IL6R and IL6R One of the major limitations of CAR therapy is cytokine release syndrome (CRS). One approach currently being used in clinical practice to control CRS is administration of anti-IL-6R monoclonal antibody zumab. To l CRS associated with CAR, we constructed exemplary CARs against CD19 (CD8SP-FMC63(vL-vH)-BBz- P2A-IgHSP-IL6R-M83(vL-vH)-Flag-T2A-PAC(O11315-K04)[SEQ ID NO:l763]) ) and MPL -I 6| (vL-vI—I)—BBz-P2A-IgHSP-IL6R-M83(vL-vH)—Flag-T2A-EGFP(0I 201 5- 129)[SEQ ID NO:l746])) which expresses a scFv against IL6R, designated M83, along with a C-terminal Flag tag. To control CRS, we also ucted exemplary CARS against CD19 (CD8SP-FMC63(vL-vH)-BBz-P2A-IgHSP-IL6- l 9A(vL-vI-I)-Flag-T2A-PAC(01 13 15- HO6)[SEQ ID N021762]) and MPL (CD8SP-161(vL-vH)-BBz-P2A-IgHSP-IL6-19A(vL- vH)-Flag-T2A-EGFP(011315-F02)[SEQ ID NO:l745]) which co-expresses an scFv against 1L6, designated IL6-19A. The scFv fragments in these constructs are separated from the CAR fragment by a P2A cleavable linker. Jurkat-NFAT-EGFP (J-N-G) cells were stably transduced with the lentivirus encoding the CAR constructs CD8SP-FMC63(vL-VH)-BBz- P2A-IgHSP-IL6-19A(vL-vH)-Flag-T2A-PAC(011315-H06)[SEQ ID NO: 1762] and CDSSP- FMC63(VL—VH)-BBz—P2A-lgHSP-IL6R-MS3(vL-vH)—Flag-T2A-PAC(Ol l3 15-K04)[SEQ ID 3]. The parental and CAR-expressing Jurkats were subsequently cocultured with RAJI cells and induction of EGFP sion red by FACS analysis after 4 h. Fig. 42A shows that coculturing of Jurkat cells expressing CDSSP-FMC63(vL-vH)—BBz-P2A- IgHSP-IL6-19A(vL-vH)-Flag-T2A-PAC(0l1315-H06)[SEQ ID NO:l762] with RAJI led to increase in EGFP expression as compared to cells that had not been exposed to RAM cells.

Essentially r results were obtained with Jurkats expressing the CAR construct CD8SP- FMC63(VL-VH)-BBz-P2A-IgHSP-IL6R-M83(VL-VH)—Flag-T2A-PAC(Ol l3 15-K04)[SEQ ID NO:l763]. No increase in EGFP expression was observed in parental Jurkats (J-N-G—P) without or with culturing with RAJI (Fig. 428). Thus, a CAR can be coexpressed with a scFV against 1L6 and IL6R from a single vector without loss of activity.

] Due to their small size, one of the limitations of the scFv is their short half-life due to their rapid clearance by the kidneys. Therefore, we also constructed a CAR against MPL (16]) that expresses a bispeciﬁc antibody consisting of a camel-derived lL6R single chain antibody (vHH) designated 304 which is fused via a )x3 linker to a single chain camel-derived antibody (vHH) against human serum albumin (Alb8). The lb8 cassette carries a human IgH signal peptide at the N-terminus and a Flag epitope tag at the C- terminus. The secreted bispeciﬁc IL6RAlb8 antibody will bind to human serum albumin through its Ale , which will prevent its excretion via the kidneys and thereby prolong its half-life.

CAR sing Fx06 peptide to block increased vascular Ieakiness (luring CRS Capillary leak syndrome is another major complication of CARS. A natural plasmin digest product of ﬁbrin, peptide B815-42 (also called FX06), has been shown to signiﬁcantly reduce vascular leak (Groger et al., PLoS ONE 42e5391, 2009). We generated vectors that coexpress CARS targeting CD19 or MPL with FX06 e. To facilitate extracellular secretion ofFXO6, a signal e derived from human IgH was fused to its N- terminus. The coexpression of FXO6 peptide with CARS will help ameliorate the capillary leak syndrome seen as part of cytokine release syndrome following on of CAR modiﬁed T cells.

Jurkat-NFAT-EGFP cells were stably transduced with the irus CD8SP- FMC63-(vL-vH)-BBz-P2A-IgHSP-FxO6-Flag—T2A-PAC(0 l 21 15-N04)[SEQ ID NO: 1764] that expresses FMC63 CAR against CD19 along with FXO6. The parental and CAR expressing Jurkats were subsequently cocultured with RAJI cells and induction of EGFP expression monitored by ﬂow try. Coculturing of Jurkat-NFAT-EGFP cells expressing CDSSP-FMC63-(vL-vH)-BBz-P2A-IgHSP-FxO6-Flag-T2A-PAC(0121 15- NO4)[SEQ ID NO:1764] with RAJI led to increase in EGFP expression as compared to cells that had not been exposed to RAJI cells. Essentially similar results were obtained upon culture with Bv173 and NALM6 cells. Thus, 3 CAR can be expressed along with FX06 from a single vector without loss of activity.

CARs targeting multiple diverse ns We next generated lentiviral constructs ng for CARS targeting many diverse ns that are overexpressed in cancer and other diseased causing or disease associated cells. The lentiviruses were generated and used to infect the Jurkat-NFAT-EGFP reporter cell line. The Jurkat-NFAT—GFP cells were engineered to express the different CARS and backbone targeting the different ns and were tested for their ability to activate NFAT-dependent EGFP reporter upon coculture with the target cells expressing their respective target antigen. The Jurkat-NFAT-EGFP (parental) cells were used as control. The results with different CARS are summarized in the following summary Table 28. A CAR is considered positive in the assay in case the CAR-expressing Jurkat-NFAT-GFP cells show greater % GFP positive cells when cultured with the target cell line as compared to parental Jurkat-NFAT—GFP cells. Thus, the Jurkat-NFAT-GFP cells expressing the CAR CD8SP- FMC63(vL-vH)-Myc-BBz-P2A-cFLlP-L-Flag-T2A-PAC(O9221 5-A06)[SEQ [D NO: 1754] showed greater induction of EGFP expression when co-cultured with RAJI cell as compared to EGFP expresson induced in parental Jurkat-NFAT-EGFP cells upon co-culture with RAJI cells. These results also demonstrate that L, an agent known to block activation induced cell death, can be co-expressed with a CAR without adversely affecting its signaling activity. The s in Table 28 demonstrate the onal activity of several conventional CAR II constructs sing 41BB costimualatory domain and CD32 activation domain and targeting novel antigens, such as PTK7, DLL3, cMET, TSHR, CD22, Mucl/MHC class 1 complex, tyrosinase/MHC class I complex. The data in Table 28 also demonstrate the functional ty of several backbone l ucts sing a conventional CAR I and LIP and targeting novel antigens, such as CD34, CD179b, CLECSA, CSF2RA, CDZOOR, LAMP], TROPZ, TnAg, CDH6, CDH17, CDH19, GD3, GFRalpha4, Her2, ILl lRa, IL13Ra2, NYBR-l, Slea, and . Additionally, several bispeciﬁc constructs, such as those targeting EGFR and CEA and those targeting cMet and Her3 were positive in this assay. Essentially a similar experimental approach can be used to test the functional activity of other CARs and backbones described in Tables 19-22.

Table 28: Exemplary CARS positive on Jurkat-NFAT-GFP Assay l-$3f§1€$§1212533";"1’6"3'513'9‘32A'K'3'F'ag' 31151511101121.131::1110K‘3-"ag-m- CD8.5135351032'31‘7‘12f3562‘fs'é‘3’$11633?K‘3' SKOV3 SKOV3 MEL624 Kasumi I CDSSP-EGFR 1 -vHH-Gly-Ser—Linker—CEA l-vHHEGFR & CEA PZA-Kl3-Flag-T2A-PAC(O40716-008)[SEQ Hela ID NO: 1049 CDSSP-GD3-KM-64 l -(vL-\-'H)-Myc-z-P2A-K13-Flag- MEL624 T2A—PAC(051016-D04 SEO ID NOle65 , CDSSP—GFRa4-P4-l0-(vL-vH)-Myc-z-P2A-Kl3—Flag- GFRa4 LAND- TZA-PAC 051316-L02 SE0 ID NO:1067 CDSSP-GFRa4-P4-lO-(vL-vH)-Myc-z-P2A-Kl3-Flag- Gm" - ‘ LAND’TT T2A-PAC 051716-L05 SE0 ID NO:1067 - CD8SP-Herz-Hu4D5-(vL-VH)-Myc-z-I>2A-K13-Flag- T2A-PAC(050516-IO3) SE0 ID N0:1084 CD8SP-IL1 1 Ra-8E2-Tsl07-(vL-vH)-Myc-z-P2A-K 13- ILHR" K362.

-T2A-PAC 050516-D01 SE0 ID NO:1099 CDSSP-ILI3Ra2-Hul08-(vL-vH)-Myc-z-P2A-Kl.)- ILl3Ra U87MG n LAMPI L363, U266 CDSSP-CMET- l 7 l-vHH-Gly-Scr-Linkcr-Hcr3 -2 1F06- cMET-Hcr3 vHH-Myc-z-PZA-Kl3-Flag-T2A-PAC(04l 1 16- U266; MCF7 Bos SEOIDN021116' CD8SPMPL-l 1 \='H)-Myc-z-P2A-Kl 3-Flag- T2A-PAC(0407l6-M02 SE0 ID NO:1120 NYBRI CD8SP-NYBRl-(vL-vH)-Myc-z-PZA-K13-Flag-T2A- L363a PAC(051716—005) SE0 ID 1 ‘ SLea-SBl-(vL-vH)-Myc-z-P2A-Kl3-Flag- LAND- T2A-PAC 051716-X05 SE0 ID N0;1149 Posmve Cell Lme on SEQUENCE NAME co-culture assa 3 CDSSP—TGFBRZ-Abl-(vL-vH)-M_\v'c-z-P2A-Kl3-Flag- TGFB" R2 NALM6 T2A-PAC(0517l6-006 011158 - _ ,. CD8SP-TIMl-HVCRl-ARD5-(vL-vH)-Myc-z-P2A- .

CD88P-TROP2-ARA47-HV3KV3-(\-'L-VH)-Myc-z- P2A-K l 3-Flag-T2A-PAC(05 l7 l6-G05)[SEQ [D CD8SP-CD23-p5E8-(V‘L-vI-I)-M}-'c-BBz-T2A- L1236 PAC SEQ ID NO:l727 RAJ I. NALM6 I-CD8SP-FMC63(vL-vH)-BBz-P2A-IgHSP-[L6-l9A(vL-vH)-Flag-T2A-PAC(0l1315-H06)[SEQ 1D RAJ] N021762 . CD19 CD8SP-FMC63(vL-vH)—Myc-BBz-PZA-cFLIP-L-Flag- RAJ], NALM6, Jekol, T2A-PAC(092215-A06) SE 0 ID N021754 MOLM13 .-CDSSP-FMC63-(vL-vH 2A-I HSP-Fx06-Flag- V173‘NALM6 T2A-PAC(012115-N04 )SEO ID N01gl764 I-CD8SP-FMC63(vL-vH)-BBz-P2A-IgHSP-IL6R-M83(vL-vH)-Flag-T2A-PAC(Ol1315-K04)[SEQ ID RAJ], NALM6,U266 N021763 I-CD8SP-FMC63(vL-vH)—BBz-P2A-IgHSP-IL6-D19 l9A(vL-vH)-Flag-T2A-PAC(01 13 15-H06)[SEQ ID RAJ], NALM6 NO:1762 I-CD88P-CD22-m97l-(vL-vH)-M_vc-BBz-T2A-27 D22 RAJ], NALM6 PAC 091515-A02 SE0 ID N0;15]9 l-27 D324 CD85P-CD324-hSC10-l7-(vL-vH)-Myc-BBz-T2A- 23' PAC(052516-A07) SE01D NO: 1555 CDSSP-DLL3—hSC 1 6—56—(vL—\-'H)—M}-'c—BBz—T2A— SKMEL31, SKMEL3 7, 27 ULL3 PAC(052516-C07) SE 0 ID NO: 1585 LAN5 CDSSP-DLL3-hSCl6(vL-vH)—I\/ch-BBz-T2A- LAN5. 1. 27 DLL3 PAC(0525 l6-D07) SE ID NO: 1584 SKMEL317 I-CD8SP-GCC-SF9-(vL-vH)-Myc-BBz-T2A-PAC[SEQ27 HCTH6 ID N021728 MPL-l6 l-(vL-vH)-Myc-BBz-T2A- 27 MPL HEL, RAJI-MPL PAC 021015-R07 SE0 ID NO:1658 I-CDSSP-Z-MPL-l ] l-(vL-\-'I-I)-Myc-BBz-T2A-27 HEL Muc l /MHC [\J \l SKOV3 class I l-CDSSP-PTK76- 10(vL-vI-I)-Myc-BBz-T2A-27 LANS PAC(052516-E07) SEO ID N021684 CDSSP-TSHR-KB 1-(vL-vH)-M_\g'c-BBz-T2A- 27 1-]SHR RAJI PAC 052616-E05 SEO ID N021709 CD8SP-Tyros-TAZ-(vL-vH)-Myc-BBz—T2A-PAC[SEQ 27 Tyrosinase MEL624 ID NO: I714 Use ofPI3K inhibitors to expand T cells expressing conventional CARS and backbones 1- Human peripheral blood T cells will be isolated using CD3 magnetic beads and infected with lentiviruses expressing the CAR constructs (SEQ ID NO: 111614-B05) targeting CD19. Cells will be left unselected or selected with puromycin and expanded using the standard protocol described previously using CD3/CD28 beads and IL2 but in the absence or presence of dual PI3K/mTOR inhibitor PF-04691502 (0.10 M to 0.5 uM). NSG mice (Jackson Lab) will be thally irradiated at a dose of 175 cGy. 24 hours post irradiation (day2), mice will be injected with 2.5 x 104 RAJI cells via tail-vein. On day 3, the mice (n =5 for each group) will be treated with 5 million T cells that had been infected with the indicated CAR ng lentiviruses and expanded in the absence or presence of PF-04691502. l mice (n =5) will be injected with 5 million uninfected T cells. Mice are given human 1L2 (400 IU i.p.) on alternate days till the death of all mice in control group. The median al of mice which received CAR-T cells expanded in the presence of PI3K/AKT inhibitor is ed to be higher than the mice which received CAR-T cells that had been expanded in the absence of PI3K/AKT inhibitors.

Use ofIL7 along with CAR-T cells The study will be conducted as described in the preceding example with the exception that starting 1 day after the infusion of CAR—T cells, mice will be administered exogenous recombinant human IL-7 at a dosage of 200 ng/mouse by intraperitoneal injection three times, while the control mice receive normal saline. The mice that receive IL-7 are expected to reject their tumor and survive longer than the control mice.

Use ofautologous CAR- T cells-for adoptive cell therapy CAR-T cells of the invention can be used for adoptive cell therapy. As an example, patients with relapsed Acute lymphocytic Leukemia, chronic cytic leukemia, or high-risk intermediate grade B-cell lymphomas may receive immunotherapy with vely transferred autologous CAR-T cells targeting CD19. A leukapheresis t collected from each patient will undergo selection of CD3-positive T lymphocytes using the ACS Prodigy® System from Miltenyi Biotec and following the cturer’s recommendations. Cells will be transduced with clinical-grade CDSSP-CD19Bul2-(vL-VH)- Myc-BBz-TZA-PAC(O82815-P08)[SEQ ID N011470]. Alternatively, clinical grade CD8SPCD1QBul2- (vL-vH)—Myc-z-P2A-Kl3-F1ag-T2A-PAC[SEQ ID NO:930] virus will be used.

The ion and ion of the CAR-T cells will occur in a closed system. After the resulting cell products has undergone quality control testing (including sterility and tumor speciﬁc cytotoxicity tests), they will be cryopreserved. Meanwhile, following leukapheresis, study participants will ce with lymphodepletive chemotherapy (30 mg/mZ/day ﬂudarabine plus 500 mg/mZ/day cyclophosphamide x 3 days). One day after tion of their lymphodepleting regimen, the previously stored CAR—T cell product will be transported, thawed and infused at the patient's bedside. The study participant may also receive CAR- transduced lymphocytes d intravenously followed by ose (720,000 lU/kg) IL-2 (Aldesleukin; Prometheus, San Diego, CA) every 8 hours to tolerance. The dose of CAR-T product will vary from 1 x 104 CAR+ve CD3 cells/kg to 5 x 109 CAR+ve CD3 cells/kg as per the study protocol. The CAR-T product may be administered in a single infusion or split infusions. Research participants will be pre-medicated at least 30 minutes prior to T cell infusion with 15 mg/kg of acetaminophen P.O. (max. 650 mg.) and diphenhydramine 0.5-1 mg/kg I.V. (max dose 50 mg). The study participant will optionally receive daily injections of human IL-2. Clinical and laboratory correlative follow-up studies will then be performed at the physician's discretion, and may e quantitative RT-PCR studies for the presence of CD19-expressing ALL/lymphoma cells and/or the adoptively erred T cells; FDG-PET and/or CT scans; bone marrow examination for disease speciﬁc pathologic tion; lymph node biopsy; and/or long-term follow up per the guidelines set forth by the FDA's Biologic Response Modifiers ry Committee that apply to gene transfer studies. Essentially a similar approach will be used to treat other diseases using autologous immune cells (e.g., T cells) that have been engineered to express the conventional CARS of the invention or conventional CARS with accessory module (i.e. backbones 1-62) where the CAR s an antigen or antigens expressed on the disease causing or disease-associated cells.

Use of autologous T cells expressing tional CARS and backbones 1-62 targeting multiple antigensfor adoptive cell therapy ts with many different diseases, including infectious diseases (e.g., HIV], EBB, CMV, HTLVl, etc), rative diseases (e.g., Alzheimer’s disease), allergic diseases (e.g., chronic idiopathic urticarial) and multiple cancers will be enrolled in an [RE approved phase I clinical trial of immunotherapy with adoptively transferred autologous CAR-T cells coexpressing Kl3-vFLIP (backbone l) targeting different disease-causing or disease-associated antigens. The CAR for different diseases will be selected based on the known expression of their target antigen in the disease-causing or disease-associated cells.

Where possible, the expression of the CAR target on the e causing or disease associated cells will be confirmed by binding with Antigen binding domain-GGS—NLuc fusion n in which the antigen g domain of the CAR is fused to non-secretory form of NLuc protein via a ﬂexible linker. atively, immunohistochemistry or flow cytometry using commercially available antibodies will be used to confirm the expression of the target antigen of the CAR on disease-causing or disease-associated cells. T cells will be collected from the subjects using leukopheresis, transduced with the appropriate lentivirus s and expanded ex vivo using CD3/CD28 beads in a closed system. After the resulting cell products have undergone quality control testing (including sterility and tumor speciﬁc cytotoxicity , they will be cryopreserved. CAR-T cell products will be administered to the ts as described in the preceding example. al and tory correlative follow-up studies can then be performed at the physician's discretion.

Use of an mTOR inhibitor RAD()0] in combination with T cells expressing conventional CARs and backbones 1-62 The study is conducted as described in the preceding examples with the exception that starting 1 day after the infusion of CAR —T cells, study participants will be administered an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RADOOl, at a dosage that provides a target trough level 0.1 to 3 ng/ml, where the trough level" refers to the concentration of a drug in plasma just before the next dose, or the minimum drug concentration between two doses.

Use ofIbrutinib in combination with T cells sing conventional CARS and backbones The study will be conducted as described in the preceding examples with the exception that ng 1 day after the on of CAR—T cells, study participants will be administered oral ibrutinib at close of I40 mg/d to 420 mg/d. It is expected that the study participant receiving ibrutinib will have lower incidence of severe cytokine release me as compared to participants who ed CAR-T cells without nib.

CAR-T Cell c Arterial Infusion In addition to enous infusion, T cells expressing the conventional CARs and backbones 1-62 described in this invention can be infused intra-arterially to provide high concentration of CAR-T cells in a local area or organ involved with a disease. In the following e, this approach is used in case of a patient with hepatic metastases from a gastrointestinal cancer which ses Folate Receptor alpha (FRI). Essentially a similar approach can be used for intra-arterial infusion of T cells expressing conventional CARs and backbones 1-62 targeting other tumor antigens.

A mapping angiogram will be performed via a right common femoral artery approach at baseline. The gastroduodenal and right gastric arteries, in addition to other potential sources of extrahepatic perfusion, will be embolized with microcoils. The same arterial access procedure will be carried out for administration of T cells expressing the construct CD8SP-FR l -huMov l 9-(vL-vH)—Myc-z-P2A-K 1 3-Flag-T2A-PAC[SEQ ID NO:IO60]. The T cells will be collected from the patient on day 0 and will be infected with lentivirus encoding the construct CDSSP-FRI-huMov19-(vL-vH)—Myc-z-P2A-KI3-F1ag- T2A-PAC[SEQ ID NO:IO60] and expanded as described in the previous examples. The CAR-T cells will be given in a dose escalating fashion on day 14 (107 CAR-T cells), day 28 (108 CAR-T cells) and day 44 (109 CAR-T . The CAR- T cells will be injected manually via a 60cc syringe at a rate of <2 cc/second. The total volume of infusion will be approximately 100 cc. Angiography with calibrated contrast rate will be performed after the ﬁrst infusion of 50 cc and at completion of the CAR-T infusion to conﬁrm preserved arterial flow. Infusions will be delivered into the proper hepatic artery when possible. Certain patients may have aberrant hepatic arterial anatomy, where either the right or left c artery does not arise from the proper hepatic artery. In such cases the dose of CAR-T cells will be split based upon lobar volume calculations. In such cases, split doses will be delivered separately into the right and left c es to ensure proportionate CAR-T delivery to both hepatic lobes.

Intruperitoneal stration ofCAR-T cells CAR-T cells can also be administered intraperitoneally, essentially as described in Koneru M et al (Journal of Translational Medicine; 2015; 13:102). In the following example, this ch is used in patients with peritoneal involvement with ovarian cancer which expresses Folate Receptor alpha (FRI). Essentially a similar approach can be used for intra-peritoneal infusion of CAR-T cells targeting other tumor ns.

A screening ed consent will be offered to patients with recurrent high- grade serous n cancer to test their cancer for the expression of FRI. After expression of FRI is conﬁrmed by immunohistochemistry, then ts will have a leukapheresis product obtained from peripheral blood. Excess platelet and red blood cell contamination will be removed from the leukapheresis product and the product will be frozen. In the ent phase of the study, the leukapheresis product will be thawed and washed. Subsequently, CD3+ T cells will be isolated from the thawed leukapheresis product by magnetic separation using CD3/CD28 beads. ted T cells will be lentivirally transduced with the CD8SP- FRI-huMovI9-(vL-vH)—Myc-z—P2A-K13-Flag-T2A-PAC[SEQ ID NO:1060] construct and further expanded using CD3/CD28 bead expansion protocol.

Patients with recurrent high-grade serous ovarian, primary peritoneal or fallopian tube oma shown to express FRI antigen conﬁrmed by immunohistochemistry (II-1C) analysis of banked (parafﬁn embedded) or freshly biopsied tumor will potentially be eligible for the study.

] The phase I dose-escalation dosing will be used in the trial. Cohorts of 3—6 patients will be infused with escalating doses of modiﬁed T cells to ish the maximum tolerated dose (MTD). There will be four planned dose levels: 3 X 105, 1 X 106, 3 X 106, and 1 x 107 CAR-T cells/kg. Cohorts 1 and 11 will be treated with 3 x 105 CAR-T cells/kg but patients in cohort II will also receive lymphodepleting cyclophosphamide. Cohorts II-V will receive escalating doses of the modiﬁed T cells following pretreatment with cyclophosphamide. depleting cyclophosphamide dosed at 750 mg/m2 will be administered 2—4 days prior to the initial T cell infusion. A standard 3 +3 dose escalation schema will be followed.

An IP catheter will be placed prior to T cell infusion. Patients will be ed to the inpatient unit of the al prior to their ﬁrst infusion of CAR T cells and will remain hospitalized until at least 3 days after the second on of CAR T cells. The ﬁrst cohort of patients to be d, and the ﬁrst patient treated in each uent cohort, will be admitted to the intensive care unit (ICU); subsequent patients may be admitted to the medical oncology inpatient e (subject to the clinical judgment of the treating physician).

Patients will receive a single dose of lymphodepleting cyclophosphamide (750 mg/m2 IV) chemotherapy 2 to 4 days prior to initiating treatment with CAR-modiﬁed T cells.

The transduced T cells will be quality tested for number, purity, viability, and ity prior to infusion. All patients will receive 50% of the genetically modiﬁed T cell dose intravenously.

Patients will be closely monitored for toxicities. One to 3 days later, the remaining dose of T cells will be administered as an IP infusion. At least 3 patients will be d at dose level 1, with an accrual of no more than 2 patients per month within each dose level. All patients treated in the ing cohort will be observed for a m of 4 weeks from the day of the initial T cell infusion before escalation to the next cohort occurs. Blood samples will be obtained from all patients prior to and following treatment to assess toxicity, eutic effects, and survival of the genetically modiﬁed T cells.

Use ofCAR-T cellsfor intratumoral injection CAR-T cells can also be administered intra-tumorally, essentially as described in Brown CE, et al, Clin Cancer Res. 2015 September 15; 21(18): 4062—4072. In the following example, this approach will be used in case of patients with recurrent glioblastoma (GBM) which expresses ILl3Ra2. Essentially a similar approach can be used for umoral injection of T cells sing conventional CARS or conventional CARS sing accessory modules (backbones 1-62) targeting other tumor antigens.

A pilot safety and feasibility study will be conducted to test CDSSP-IL13Ra2- Hu108-(vL-vH)-Myc-z-P2A-K13-F1ag-T2A-PAC(0505 16-F04)[SEQ ID NO: 1 102] expressing T cells in recurrent GBM. All participating patients will be required to give written informed consent. The clinical protocol will be approved by the University of Southern California Institutional Review Board and conducted under an Investigational New Drug Application, and registered at ClinicalTrialsgov. Eligible patients will include adults (18-70 yrs) with recurrent or refractory al supratentorial grade III or IV glioma whose tumors do not show communication with ventricles/CSF pathways and are amenable to resection. ipation in this trial will be independent of ILl3Ra2 (or ILl3Ra2) tumor antigen status. Patients will be enrolled following initial diagnosis of high-grade glioma (WHO grade III or IV), at which time they will undergo leukapheresis for collection of peripheral blood clear cells (PBMC). These cells will be used to engineer T cells to express the construct CDSSP-ILl3Ra2-HulO8-(vL—vH)-Myc-z-P2A-Kl3-Flag-T2A- PAC(050516-F04)[SEQ ID N021102] containing the puromycin resistance gene (PAC) following infection with the corresponding lentiviral vector as described in the previous es. Alternatively, the CAR-T cells could be generated ing infection with a retroviral vector or using sleeping beauty transposon or by transfection of IVT mRNA.

Subsequently, the release tested therapeutic CAR-T cells will be cryopreserved and stored for later use. At the time of first recurrence of the tumor, the research participant will undergo resection of tumor along with placement of a Rickham oir/catheter. Concurrently, the eutic CAR-T cells will be thawed, re-expanded in vitro using CD3/CD28 beads based rapid expansion protocol. Following recovery from surgery and post baseline MR imaging, the CAR-T cells will be administered directly into the resection cavity via the indwelling catheter, essentially as described (Brown CE, et al, Clin Cancer Res. 2015 21(18): 4062— 4072). Cells will be manually injected into the Rickham reservoir using a 21 gauge butterfly needle to deliver a 2 mL volume over 5-10 s, ed by 2 mL ﬂush with preservative free normal saline over 5 minutes. The protocol ent plan will specify an intra-patient dose escalation schedule with a target of 12 CAR T cell doses administered intracranially over a 5 week period comprised of weekly treatment cycles. During cycles 1, 2, 4 and 5, T cell infusions will be performed on days 1, 3 and 5 of the cycle week, and week 3 will be a rest cycle. For , in cycle 1 an intrapatient dose escalation strategy, with CAR T cell doses of 107, 5 X 107 and 108 cells per inﬁJsion administered on days 1, 3 and 5 respectively, will be used and this will be followed by 9 additional CAR T cell infusions of 108 cells over 4 weeks. Imaging to assess response will be performed during the week 3 rest cycle and after week 5, The ines provided in the NCI Common Toxicity Criteria version 2.0 (https://ctep.ifo.nih.gov/l) will be followed for the monitoring of toxicity and adverse event reporting Use of CAR-T cells for ex-vivo purging of bone marrow or peripheral blood stem cell preparation prior to transplant ] CAR T cells can be used to purge the bone marrow or eral blood stem cell preparation of cancer cells prior to stem cell transplant. In the following example, CD8SP-CS l -HuLuc64-(VL-vH)-Myc-z-P2A-K l 3-Flag-T2A-PAC[SEQ ID NO: 1032] expressing T cells will be used to purge bone marrow or peripheral blood stem cells obtained from a patient with multiple myeloma prior to gous stem cell (or bone marrow) transplant.

Patient will undergo leukopheresis to t peripheral blood mononuclear cells (PBMC). T cells will be puriﬁed using CD3 beads. These cells will be used to engineer T cells to express the CD8SP-CSl-HuLuc64-(vL-vH)-Myc-z-P2A-K13-Flag-T2A-PAC[SEQ ID 2] CAR following infection with the corresponding iral vector as described in the previous examples. Subsequently, the release-tested therapeutic CAR-T cells will be cryopreserved and stored for later use or used fresh. Bone marrow cells and peripheral blood itor cell products will be collected from a patient with multiple myeloma following standard procedures. For mobilization of peripheral blood stem cells, patients will receive cyclophosphamide, 3 gm/m2 followed by G-CSF, 10 pig/kg subcutaneously each day beginning 24 h after cyclophosphamide until pheresis is complete. Peripheral blood stem cells will be collected once the peripheral blood CD34+—cell count is 15 cells/pl. The collection goal will be to process three blood volumes per day until a minimum of 2.0 times 106 CD34+ cells/kg are reached after processing. The bone marrow and peripheral blood stem cell products will be optionally depleted of Red Blood Cells and/or enriched for CD34 expressing cells using CliniMACS Prodigy® System from Miltenyi Biotec and following the cturer’s recommendations. The ts will be used for ex-w'vo purging fresh or cryopreserved. For purging, the bone marrow or eral blood stem cell products will be cocultured with thawed CAR-T cells at an effector to target ratio ranging from 5: 1 to 30:1 for 4 to 24 hours in XVIVO medium (Lonza) supplanted with 100 IU recombinant human- lL2. Cells will be cultured at 37°C, in a 5% C02 humidified incubator. At the end of the coculture period, an aliquot of the cells will be taken for sterility and quality testing (including measurement of CFU-GM and ﬂow cytometry for CD34 and CD138 ve cells). The remaining sample will be administered intravenously to the patient who has previously received myeloablative chemotherapy (e.g., high dose Melphalan in two divided doses of 70 mg/m2 for a total dose of 140 mg/mz).

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Chugh, P., H. Matta, S. Schamus, S. Zachariah, A. Kumar, J. A. Richardson, A. L. Smith, and P. M. Chaudhary (2005), tutive NF-kappaB activation, normal Fas-induced apoptosis, and increased incidence of lymphoma in human herpes virus 8 K13 transgenic mice, Proc NatlAcad Sci U SA, 102(36), 12885-90.

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The various methods and techniques described above e a number of ways to carry out the application. Of course, it is to be understood that not arily all objectives or advantages described can be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the s can be med in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A y of alternatives are mentioned herein. It is to be understood that some preferred embodiments speciﬁcally include one, another, or several features, while others speciﬁcally exclude one, another, or several features, while still others mitigate a particular feature by inclusion of one, another, or several ageous features.

Furthermore, the skilled artisan will recognize the applicability of various es from different ments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, es, and steps some will be speciﬁcally included and others speciﬁcally excluded in diverse ments. gh the ation has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the application extend beyond the speciﬁcally disclosed embodiments to other alternative embodiments and/or uses and modiﬁcations and equivalents f.

Preferred embodiments of this application are described , including the best mode known to the inventors for carrying out the application. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be ced otherwise than speciﬁcally described .

Accordingly, many embodiments of this application e all modiﬁcations and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law.

Moreover, any combination of the above-described elements in all possible ions thereof is encompassed by the application unless ise indicated herein or otherwise clearly contradicted by context.

All patents, patent applications, publications of patent applications, and other material, such as es, books, speciﬁcations, publications, documents, things, and/or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution ﬁle history ated with same, any of same that is inconsistent with or in conflict with the present nt, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the t document. By way of example, should there be any inconsistency or conﬂict between the description. ion, and/or the use of a term associated with any of the incorporated material and that associated with the present document, the description, deﬁnition, and/or the use of the term in the present nt shall prevail.

It is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the application. Other modiﬁcations that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative conﬁgurations of the embodiments of the ation can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.

Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modiﬁcations and/or variations to the c embodiments shown and described herein. Any such modiﬁcations or variations that fall within the purview of this descn'ption are intended to be included therein as well. Unless speciﬁcally noted, it is the intention of the inventors that the words and phrases in the speciﬁcation and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

] The foregoing description of various embodiments of the invention known to the applicant at this time of ﬁling the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modiﬁcations and variations are possible in the light of the above teachings. The embodiments described serve to explain the ples of the invention and its cal application and to enable others skilled in the art to utilize the invention in various embodiments and with s modiﬁcations as are suited to the particular use contemplated. Therefore, it is ed that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

While particular embodiments of the present ion have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings , changes and modiﬁcations may be made without departing from this invention and its broader aspects and, ore, the appended claims are to encompass within their scope all such changes and modiﬁcations as are within the true spirit and scope of this invention.

The present application and invention further includes the subject matter of the following numbered clauses. 1. A chimeric antigen receptor comprising: an antigen-speciﬁc targeting domain; a hinge region; a transmembrane domain; and an intracellular signaling domain, n each antigen-speciﬁc targeting region comprises an antigen-speciﬁc single chain Fv (scFv) fragment or an antigen speciﬁc ligand . ] 2. The chimeric antigen receptor of clausel, further comprising one or more co- atory domains. ] 3. The chimeric antigen receptor of clause 1, wherein the antigen-speciﬁc targeting domain targets any one or more of MPL receptor, CD19, GRP-78, CD79b, Lym-l, Lym-2, FLT3 or combinations thereof. 4. The chimeric antigen receptor of clause 1, wherein the hinge region comprises any one or more of human CD8 hinge region, an Fc fragment of an antibody or a functional equivalent, fragment or derivative thereof, a hinge region of an antibody or a functional equivalent, fragment or tive f, a CH2 region of an antibody, a CH3 region of an antibody, an artiﬁcial spacer sequence and combinations thereof. 5. The chimeric antigen receptor of clause 4, wherein 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 CD8a, (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. 6. The chimeric antigen receptor of clause 1, n the embrane domain comprises any one or more of a transmembrane domain of a zeta chain of a T cell receptor complex, CD28, CD8a, and combinations thereof. 7. The chimeric n receptor of clause 2, wherein the co-stimulatory domain comprises a signaling domain from any one or more of CD28, CD137 (4-1BB), CD134 , DaplO, CD27, CD2, CD5, ICAM-l, LFA-l, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40 and combinations thereof. 8. The chimeric antigen receptor of clause 1, n the intracellular signaling domain comprises a signaling domain of one or more of a human CD3 zeta chain, chRIII, FceRl, a cytoplasmic tail of 3 Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, and combinations thereof. 9. The ic antigen receptor of clause 1, further coexpressed with scFv or vHH or bispeciflc antibody specific for nes or cytokine receptor. 10. The chimeric antigen receptor of clause 9, wherein the coexpressed cytokines or cytokine receptor are any one or more of, IL-6, IL6R, [FNy, TNFa or combinations thereof. 11. The chimeric antigen receptor of clause 1, further essing the peptide FX06 so as to mitigate capillary leak associated with CAR therapy. 12. The chimeric antigen receptor of clause 1, further coexpressing a viral or cellular signaling protein to ate T cell activation, proliferation and/or to block T cell activation induced cell death. 13. The chimeric antigen receptor of clause 12, wherein the signaling protein is any one or more of vFLIP K13, vFLIP MC159, VFLIP MC160, VFLIP E8, HSV vFLIP, BHV VFLIP, Herpes virus Mecaca vFLIP, cFLIP/MRITa, cFLIP-p22, HTLVl Tax, HTLV2 Tax, HTLVl- Tax RS mutant, HTLVZ Tax RS mutant, Tio, StpC, Tip or mutants or combinations thereof. ] 14. The chimeric antigen receptor of clause 12, wherein the signaling protein is expressed in a constitutive or ble . 15. The chimeric antigen receptor of clause 12, where activity of the signaling protein is controlled at the post-translational level by administration of a compound. 16. The chimeric n or of clause 12, where the signaling protein is expressed in fusion with one or more copies of FKBP domain. ] 17. The chimeric antigen receptor clause 16, where the activity of the protein is controlled at the post-translational level by administration of therapeutically ive amount of a chemical compound that induces dimerization of the FKBP domain. 18. A ic antigen receptor of clauses 1 or 3, where the antigen speciﬁc targeting domain targeting MPL comprises a scFV fragment of 161, 175, 178, AB317, 12E10, or VB22 or human TPO or mouse TPO or mutants thereof. 19. A chimeric antigen receptor of clauses 1 or 3, where the antigen speciﬁc targeting domain targeting CD19 comprises a scFV fragment of CD19Bu12 or CD19MM or mutants thereof. 20. A chimeric antigen receptor of clauses 1 5 or .3, where the antigen c targeting domain targeting CD79b comprises a scFV fragment of huMA79bv28 or CD79b- 2F2 or mutants thereof. 21. A chimeric n receptor of s 1 ’V or 3, wherein the antigen speciﬁc targeting domain targeting FLT3 comprises a scFV fragment of NC7 or mutants thereof. 22. A chimeric antigen receptor of clauses 1 or 3, n the antigen speciﬁc targeting domain targeting Lyml antigen comprises a scFV nt of Lyml or mutants thereof. 23. A chimeric antigen receptor of clauses 1 or 3, wherein the antigen speciﬁc ing domain targeting Lym2 antigen comprises a scFV nt of Lym2 or mutants thereof. 24. A chimeric n receptor of clauses l or 3, wherein the antigen speciﬁc targeting domain ing GRP-78 comprises a scFV fragment of GRP78-GCI8 or mutants thereof. 25. A polynucleotide ng the chimeric antigen receptor of any one of clauses 1, 12 and 18-24. 26. A polypeptide encoding the polynucleotide of clause 25. 27. A vector comprising the polynucleotide of clause 25. 28. A virus comprising the polynucleotide of clause 25. 29. The virus of clause 25, wherein the virus is a retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, a pox virus or a herpes virus. 30. A genetically engineered cell, comprising the polynucleotide of clause 25 or the chimeric antigen receptor of clause 1. 31. The genetically ered cell of clause 30, n the cell is a T-lymphocyte (T-cell). 32. The genetically ered cell of clause 31, wherein the cell is a naive T cells, a central memory T cells, an effector memory T cell, Treg or a combination thereof. 33. The genetically engineered cell of clause 21, wherein the cell is a natural killer (NK) cell, a hematopoietic stem cell (HSC), an embryonic stem cell, or a pluripotent stem cell. 34. A ceutical composition comprising: any one or more of the chimeric antigen receptor of clause 1, the polynucleotide of clause 25, the polypeptide of clause 26, the vector of clause 27, the virus of clause 28, the genetically engineered cell of clause 30 and combination thereof; and a pharmaceutically acceptable carrier. 35. A method for treating cancer comprising: providing the composition of clause 34; and administering a eutically effective amount of the ition to the subject so as to treat cancer. 36. The method of clause 35, n the cancer is blood cancer. 37. The method of clause 36, wherein the blood cancer is any one or more of acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, lymphoma, multiple myleoma and acute lymphocytic leukemia. 38. A method of lling the activity of T cells expressing the CAR of clause 1, comprising administering a therapeutically effective amount of an inhibitor of tyrosine kinases wherein the kinase are selected from the src family of kinases. ] 39. A method of treating CRS associated with treatment with CAR-T cells comprising administering a eutically effective amount of an inhibitor of tyrosine kinases wherein the kinase are selected from the src family of kinases. ] 40. A method of treating neurological complications associated with treatment with CAR-T cells comprising administering a therapeutically effective amount of an inhibitor of tyrosine kinases wherein the kinase are selected from the src family of s. 41. The method of clauses 38, 39 or 40, wherein the inhibitor inhibits the src kinase 42. The method of clause 41, wherein the inhibitor is Dasatinib. 43. The method of clause 41, wherein the inhibitor is Ponatinib. 44. The method of clause 41, wherein the inhibitor is A-77004l. 45. A method of controlling the activity of T cells activated by a BiTE comprising administering a therapeutically ive amount of an inhibitor of tyrosine kinases wherein the kinase are selected from the src family of kinases. 46. A method of treating CRS caused by treatment with BiTE comprising administering a therapeutically effective amount of an tor of tyrosine kinases wherein the kinase are selected from the src family of kinases. 47. A method of treating neurological complications caused by treatment with BiTE comprising administering a therapeutically effective amount of an inhibitor of tyrosine kinases wherein the kinase are ed from the src family of kinases. ] 48. The method of clauses 45, 46 or 47, wherein the BiTE is Blinatumomab (Blincyto). 49. The method clauses 45, 46 or 47, wherein the inhibitor inhibits the src kinase 50. The method of clause 49, wherein the inhibitor is Dasatinib. 51. The method of clause 49, wherein the tor is Ponatinib. 52. The method of clause 49, wherein the inhibitor is A-77004 1. 53. A method of treating complications of CAR and BiTE comprising stering a therapeutically effective amount of an inhibitor of tyrosine kinases wherein the kinase are selected from the src family of kinases. ] 54. The method of clause 53, wherein the BiTE is Blinatumomab (Blincyto). 55. The method of clause 53, wherein the inhibitor inhibits the src kinase Lck. 56. The method of clause 55, wherein the inhibitor is Dasatinib. 57. The method of clause 55, wherein the inhibitor is Ponatinib. 58. The method of clause 55, wherein the inhibitor is A-7704l. 59. A method of treating CRS, neurological complications and other complications of CAR-T cells and BiTE by administration of therapeutic effective amount of Avasimibe. 60. A method of immortalizing human peripheral blood clear cells and T cells by expressing vFLIP [(13 in the cells. 61. The method clause 60, wherein vFLIP K13 is sed constitutively. ] 62. The method clause 60, wherein vFLIP K13 is expressed in a manner that allows control of its activity at the transcriptional or post-translational level. 63. The method clause 60, wherein vFLIP K13 is expressed in fusion with one or more domains of FKBP. 64. The method of clause 63, where FKBP-vFLIP-Kl3 fusion protein further comprises an N-terminal Myristoylation sequence. 65. The method of clause 64, wherein the activity of FKBP-vFLIP-Kl3 fusion protein is lled by a chemical that induces dimerization of FKBP domain and activation of K13 induced NF-kB signaling. 66. The method of clause 65, wherein the peripheral blood mononuclear cells and/or T cells expressing K13 or FKBP-K13 fusion protein are administered to a patient. 67. The method of clause 65, wherein the peripheral blood mononuclear cells and/or T cells expressing K13 or FKBP-K13 fusion protein also co-express a chimeric antigen receptor, a native T cell receptor or an artiﬁcial T cell receptor. 68. A method of immortalizing human peripheral blood mononuclear cells and T cells by expressing HTLV2 Tax protein in the cells. 69. A method of immortalizing human peripheral blood mononuclear cells and T cells by expressing HTLV2 Tax RS mutant n in the cells. ] 70. The method clauses 68 or 69, wherein the HTLV2 Tax and its RS mutants are expressed in fusion with one or more domains of FKBP. 71. A method of clause 70, wherein FKBP-Tax fusion proteins further comprises an N-terminal Myristoylation sequence. 72. The method of clause 71, wherein the activity of FKBP-Tax and ax-RS fusion ns is controlled by a chemical that induces dimerization of FKBP domain and activation of Tax induced NF-kB signaling. 73. The method of clause72, wherein the peripheral blood mononuclear cells and/or T cells sing HTLV2 Tax HTLV2 Tax-RS mutant, FKBP-Tax, or FKBP-Tax-RS mutant proteins are administered to a patient. 74. The method of clause 72, wherein the peripheral blood mononuclear cells and/or T cells expressing HTLV-Tax or ax fusion protein further co-express a chimeric antigen receptor, a native T cell receptor or an artiﬁcial T cell receptor.

Claims

1.What is claimed is: A 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 ing domain comprising an immunoreceptor tyrosine-based activation motif (ITAM); wherein c) is d at the C-terminus of the ic receptor. The cell of claim 1, further comprising c acid encoding VFLIP signaling protein. The cell of claim 1, n the CAR further comprises one or more co-stimulatory domains. The cell of claim 1, wherein the cell further comprises nucleic acids encoding scFvs targeting 1L6 and/or 1L6 receptor alpha. The cell of any of claims 1-4, further comprising nucleic acid encoding e FX06 so as to mitigate capillary leak associated with CAR therapy. The cells of any of claims 1-4, wherein the signaling protein is expressed in fusion with one or more copies of FKBP domain. The cells of claim 6, wherein the ty of the ing protein is controlled post- tranlationally by dimerization of the FKBP domain in the presence of a dimerizing agent. The cell of claim 7, wherein the dimerizing agent is AP20187. The cell of claim 1, wherein the antigen speciﬁc domain of the CAR targets MPL. 10. The cell of claim I, wherein the antigen speciﬁc domain targets two antigens. 11. The cell of claim 10, wherein the two antigens are MPL and CD123. 12. The cell of claim 1, wherein the antigen speciﬁc domain of the CAR targets CD19, CD23, Lyml, Lym2, CLECSA, CDH179b, FLT3, GCC, Mucl, CSFZRA, GFRa4, CD32, ILl lRa, ILl3Ra, NYBRl, SLea, CDZOOR, TGFBetaRZ, CD276, TROPZ, LAMP], PTK7, DLL3, CDHl, CDH6, CDH17, CDH19, TSHR and tyrosinase. 13. The cell of claim 9, wherein the antigen speciﬁc domain of the CAR targets MPL and comprises one or more scFv nts selected from 161 (SEQ ID NO: 2500 and 2501), I75 (SEQ ID NO: 2499), I78 (SEQ ID NO: 2503), III (SEQ ID NO: 2502), AB317 (SEQ ID NO: 2404), l2ElO (SEQ H) 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). 14. The cell of claim 12, wherein the antigen specific domain of the CAR targets CD19 and comprises one or more scFv nts selected from CD19Bu12 or CD19MM. 15. The cell of claim 1, wherein the cell is a T-lymphocyte (T-cell). 16. The cells of claim 1, wherein the cells is a Natural Killer (NK) cell. 17. Nucleic acids comprising a ﬁrst polynucleotide encoding the CAR of claim 1 and a second polynucleotide encoding the signaling protein of claim 1. 18. The nucleic acid of claim 17, further comprising a third polynucleotide encoding the MC159-VFLIP ing protein. I9. ptides encoded by the nucleic acids of claims 17 or 18. 20. Vectors comprising c acids of claims 17 or 18. 21. A pharmaceutical composition, comprising the cell of any of claims 1-16, nucleic acid of any of claims 17-18, polypeptides of any of claim 19 or a vector of claims 20, and a pharmaceutically acceptable carrier. 22. A method of treating a pressing cancer comprising administering to the t, a therapeutically effective amount of the cell of claim 13. 23. The method of claim 23, wherein the cancer is a blood cancer. 24. The method of claim 23, wherein the blood cancer is any one or more of acute myeloid leukemia, chronic myeloid ia, myelodyplastic syndrome, lymphoma, multiple myeloma and acute lymphocytic leukemia. The method of claim 22, further comprising administering to the subject a tyrosine kinase inhibitor, wherein the inhibitor inhibits the src family of kinases. 26. The method of claim 25, wherein the inhibitor ts Lck. 27. The method of claim 26, wherein the inhibitor is any one or more of Dasatinib, nib or A-77004l. Proliferation/c okine reduction Reference: Casucci. Monica; Attilio Bondanza (2011). Journal of Cancer. 2: 378-382. ’«3333337333333333333'\33333'333333'3333333’33333333 _ 3 3 m... .3V3333333333333333333:3333333333333333333333333333333333333333333~33333333~ 5133333333333333333» R3...“3“3““.3“3H.“33“m“...wvw3n3w“: 33 33333333333«3333333333333333333333333333 33333 .33. . m. .33433.3.33333.333333.333333333333333333-«3333-«3333333 - mm«33313133333133!33313311133311313113331333331113}:‘13}33333333' '3333-«4333333333333v3333W333.33w333w~33333~3333333333w3 3333wwwv3 W“Ww‘m\\§\|:\muW‘uwui\u=w§\u:uw\\\\wu\\u=§m\\u=\w|\A\=MW\A\:\WA\AA\M\\\\A\=\A‘~8 ~ \«x33:3V«33333a33\3I.v.33m\«3333v.33333s3333.3\«3\33as3\33as53s3v.I43333s33333as\\~3\««\-«s\\\\«s\3~\s «m.- m-W-m-W m 46 “333333333333333333333333‘33353‘33333333‘33333333‘“333333333“333333333‘ 3m3m3m33m3m3m3.m.m m.m. . ‘ \M\\w 33:3333333333333333333:3333333333333333333333333333333333333333333v.333333333333333333333333333333 amhv» verV n». E¢M ~33 »a. N». a 33 ~ 3.3.» 2A 333333333333333333333'333333'«333333333333333333333'33'33'3 3'33 33333333 33' \\\\\ \\\\\\\\\\\\ \\\\\\ 3 ”,,.,W. 3333.3333333333333:333333333333333333333333 “W333“~33“W333~33333-3~33~33w33333~33~33333~33«3333333333333.»